# Patent

## Title
Methods for testing anti-thrombotic agents

## Abstract

The invention provides a transgenic non-human animal expressing von Willebrand Factor A1 protein containing at least one mutation selected from the group consisting of: 1263P>S, 1269N>D, 1274K>R, 1287M>R, 1302G>D, 1308H>R, 1313R>W, 1314I>V, 1326R>H, 1329L>I, 1330E>G, 1333A>D, 1344T>A, 1347I>V, 1350T>A, 1370G>S, 1379H>R, 1381T>A, 1385T>M 1391P>Q, 1394A>S, 1397L>F, 1421S>N, 1439L>V, 1442G>S, 1449R>Q, 1466A>P, 1469Q>L, 1472Q>H, 1473V>M, 1475H>Q, 1479S>G, and any combination thereof.

## Background
This application is continuation of U.S. patent application Ser. No. 12/345,363, filed Dec. 29, 2008, which is a continuation-in-part of International Application No. PCT/US2007/015043 filed on Jun. 28, 2007, which claims the benefit of priority of U.S. Application No. 60/817,600 filed on Jun. 29, 2006, the contents of which are hereby incorporated in their entirety.
Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.


GOVERNMENT INTERESTS
This invention was made with support from the U.S. Federal Government under Grant No. 5RO1HL63244-7 awarded by the National Heart, Lung, and Blood Institute (NHLBI). As such, the United States government has certain rights in this invention.


BACKGROUND OF THE INVENTION
The ability of platelets to rapidly stick to the damaged wall of arterial blood vessels is critical for preventing blood loss (hemorrhage). Inappropriate deposition of these hemostatic cells in arterial blood vessels due to pathological disease processes such as artherosclerosis can result in lack of blood flow to vital organs such as the heart and brain. Thus a delicate balance exists between providing adequate hemostasis without causing blockage of blood vessels by excessive platelet deposition (a.k.a. thrombus formation).
von Willebrand Factor (VWF) is a multidomain, plasma glycoprotein of complex multimeric structure which is synthesized by vascular endothelial cells and megakaryocytes (1-3) (FIG. 1A). Its presence in the blood is vital to maintaining the integrity of the vasculature. To accomplish this task, VWF forms a “bridge” between the injured vessel wall and platelets by virtue of its ability to interact with extracellular matrix components, such as collagen, and receptors expressed on platelets, such as glycoprotein Ib alpha (4-9). It also binds to and confers stability to factor VIII (10). The importance of this glycoprotein in hemostasis is underscored by the occurrence of clinical bleeding when the plasma VWF levels fall below 50 IU/dL (type I von Willebrand's disease, VWD), or when functional defects in the protein occur (type 2 VWD) (11,12).
Upon surface immobilization of VWF at sites of vascular injury, it is the role of the A1 domain of VWF (residues 1260-1480) to initiate the process of platelet deposition at sites of vascular injury and under conditions of high rates of shear flow (>1,000 s−1; Ruggeri, Z. M. et al., Blood. 108, 1903-1910 (2006)). The critical nature of this interaction is exemplified by the bleeding disorder, termed type 2M VWD, which results from the incorporation of loss-of-function mutations within this domain that perturb interactions with GPIb alpha (Sadler, J. E. et al. (2006) J. Thromb. Haemost. 4, 2103-2114; Rabinowitz, I. et al. (1992) Proc. Natl. Acad. Sci. USA 89, 9846-9849; Cruz et al., (2000) J. Biol. Chem. 275, 19098-19105). In addition, recombinant VWF multimers lacking the A1 domain cannot support platelet adhesion at high rates of flow despite retaining the ability to interact with collagen (Sixma et al., (1991) Eur J Biochem. 196:369-75).
The structure of the A1 domain includes the α/β fold with a central β-sheet flanked by α-helices on each side as well as one intra-disulfide bond (Cys1272-Cys 1458), but no MIDAS motif (Emsley et al., (1998) J Biol Chem. 273:10396-401). Its overall shape is cuboid, with the top and bottom faces forming the major and minor binding sites, respectively, that interact with the concave surface of GPIbα. The most extensive contact site buries  ̃1700 Å2 of surface area, interacting with LRR five to eight and the C-terminal flank of the GPIbα (Huizinga, E. G. et al. (2002) Science 297, 1176-1179). For this to occur, the β-switch region of this platelet receptor undergoes a conformation change so that it aligns itself with the central beta sheet of the A1 domain. The smaller site ( ̃900 Å2) accommodates the binding of the β-finger and the first LRR of GPIbα, an event that appears to require the displacement of the amino-terminal extension of the A1 domain. Based on these findings as well as the preferential localization of mutations in humans within this region, which enhance GPIbα binding, it is speculated that the amino-terminal extension regulates the adhesive properties of this domain. This is also supported by the fact that recombinant A1 proteins lacking this extension have a higher affinity for this platelet receptor (Sugimoto et al., (1993) J Biol Chem. 268:12185-92). Despite these observations, the physiological relevance of such structural changes in this receptor-ligand pair remains to be determined as well as the contribution of other domains to this process.
In addition to its role in hemostasis, VWF also contributes to pathological thrombus formation on the arterial side of the circulation. This may be the consequence of injury to the blood vessel wall from inflammatory disease states and/or medical/surgical interventions. Pathological thrombus formation is the leading cause of death in the Western world. Thus, pharmaceutical companies have committed considerable resources towards the research and design of drugs to prevent or treat thrombosis. However, there remains an urgent need to develop new and improved therapies such as those aimed at reducing platelet and/or VWF interactions with the injured arterial wall. One major hurdle hindering drug development in this field is the lack of an appropriate small animal model of thrombosis to test promising therapies. For instance, differences in the structure or isoform of protein receptors or ligands on mouse vs. human platelets that are critical for the activation and/or binding of these cells to the injured vessel wall preclude testing of drugs developed against human platelets in a mouse model of thrombosis. Moreover, this issue cannot be overcome by simply transfusing mice with human platelets as we have observed that mouse VWF does not support significant interactions with human cells (see below). Thus, the development of a “humanized” mouse model of hemostasis and thrombosis would potentially expedite drug discovery and testing.
That said, we have discovered that only one amino acid difference between mouse and human VWF-A1 domains accounts for most of the inability of the former to interact with human platelets and vice versa. With this knowledge in hand, we have genetically altered a mouse to express VWF that contains this amino acid found in human VWF-A1, imparting on it the ability to support adhesion of human platelets to a level observed for its human counterpart. As a result, not only are we uniquely poised to better understand the molecular mechanisms governing human platelet binding at sites of vascular injury in vivo, but now have the capability to perform pre-clinical testing of anti-thrombotic agents and targeted molecular imaging agents directed against human platelet cells in a living animal. The material contained within this document describes the features of this unique biological platform for drug testing the testing of drugs and targeted molecular imaging agents.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1A is a schematic representation of the prepro form of VWF. From top to bottom: repeated homologous regions; A1 and A3 disulfide loops and functional domains of the mature VWF subunit.
FIG. 1B is an illustration depicting the sequential adhesive and activation events that promote platelet deposition at sites of vascular injury.
FIG. 2 is a model depicting the location of residues in the human VWF-A1 domain that if mutated, diminish GPIb alpha-mediated platelet binding in flow. Type 2M mutations are in red, residues identified by alanine scanning mutagenesis are in blue, and residues identified by crystal structure are in green.
FIG. 3A shows a structure model depicting residues associated with type 2M (red) or type 2B (yellow) VWD.
FIG. 3B represents a silver stained gel that clinically depicts a type 2B VWD disease state individual, which is characterized by a loss of circulating high molecular weight VWF multimers (HMWM, FIG. 3B, Lane 2).
FIG. 3C shows a legend correlating standard nomenclature with nomenclature in the image of FIG. 3A.
FIG. 4A shows structure models of the human VWF-A1 domain representing the location of the Ile1309 mutation and its proposed effects on residues critical for GPIb binding.
FIG. 4B shows the loss of the isoleucine methyl group allows a water molecule to enter, which ultimately results in changes in orientation of the G1324 peptide plane and the side chain of H1326 as depicted, residues critical for GPIb binding.
FIG. 4C shows a legend correlating standard nomenclature with nomenclature in the image of FIG. 4A.
FIGS. 5A and 5B show a space-filling model of the botrocetin-A1 complex with sites involved in GPIb alpha binding and location of type 2B mutations indicated in FIG. 5A, wherein botrocetin does not alter the conformation of VWF-A1 and minor conformational changes in the A1 domain being represented by FIG. 5B with uncomplexed (blue) and complexed (green) mutant domains superimposed onto the WT structure (red).
FIG. 6 is schematic wherein the uncomplexed A1 domain, an amino-terminal extension (pink) appears to block a binding site for the amino-terminal β-hairpin (orange arrows) of GPIb alpha. Binding requires the amino-terminal extension of A1 to move, and also induces the β-switch (yellow loop) of GPIb alpha to form a β-strand motif.
FIGS. 7A and 7B depict microscope images wherein the use of platelets in lieu of recombinant proteins or transfected cells as the immobilized substrate enables evaluation of GPIb alpha in its native form (i.e. correct orientation and proper post-translational modification). Platelet coverage of <10% can be bound in this manner and can remain relatively unactivated for up to 30 min as evident by morphology on light microscopic examination (FIG. 7A) and lack of expression of P-selectin by fluorescence microscopy (FIG. 7B).
FIGS. 8A through 8D represent quantitations of bead-platelet interactions under flow. FIGS. 8A and 8B demonstrates the direct visualization of bead-platelet interaction under flow (60×DIC microscopy). An approaching bead moving at a velocity of 609±97 um/sec (wall shear stress of 1.5 dyn cm−2) is captured by a surface-immobilized platelet at (t=12.8 msec), pivots a distance of less than 3 μm in under 40 msec, and is then released after a pause time of tp=228.2 msec into the flow stream (escape velocity=288+/−90.4 μm/sec). FIG. 8C depicts representative experiments of koff values for WT human VWF-A1 coated beads based on a distribution of interaction (pause) times. FIG. 8D shows that the kinetics of the GPIb alpha tether bond are identical whether platelets are metabolically inactivated or fixed in paraformaldehyde to prevent activation upon surface-immobilization.
FIG. 9 represents the deduced single-letter amino acid sequence of mouse VWF-A1 domain (M VWF) compared to its human counterpart (H VWF) from amino acid 1260 to 1480. The locations of cysteines forming the loop structure are numbered (1238 and 1472) and differences in residues are highlighted in red. Conversion of the arginine (R) in the mouse A1 domain to histidine (H) as found in its human counterpart (blue χ) enables mouse VWF to bind human platelets.
FIGS. 10A through 10D represent graphs of ristocetin-induced platelet aggregation assays (RIPA). Concentrations of the ristocetin modulator known to cause agglutination of human platelets ( ̃1.0 mg/ml) had no effect using murine platelet rich plasma (FIG. 10B, FIG. 10D). Incubation of murine platelet rich plasma (PRP) with thrombin resulted in >90% platelet aggregation (FIG. 10A). Concentrations of ≧2.5 mg/ml of modulator resulted in murine platelet aggregation ( ̃30%, FIG. 10C).
FIGS. 11A and 11B depict a multimer gel analysis of purified VWF from human (lane 1, FIG. 11A) and mouse (lane 2, FIG. 11A) plasma. The ability of human and mouse VWF to mediate platelet adhesion in flow was determined in order to evaluate platelet interactions between human and murine VWF with GPIb alpha, as depicted in the bar graph of FIG. 11B. Surface-immobilized murine VWF supports adhesion of syngeneic platelets (1×108/ml) at a shear rate encountered in the arterial circulation (1600 s−1) as observed for the human plasma protein (FIG. 11B). In contrast, murine VWF did not support significant interactions with human platelets and vice versa.
FIG. 12 is an image of a gel of mouse and human VWF-A1 highly purified protein, which was dialyzed against 25 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20, pH 7.8. SDS-PAGE analysis revealed a prominent protein band of  ̃34,000 Da for mouse VWF-A1 under non-reducing conditions.
FIGS. 13A and 13B depict bar graphs of a series of in vitro flow chamber assays performed to assess platelet adhesion, wherein human or murine platelets (5×107/ml) were infused through a parallel plate flow chamber containing glass cover slips coated with either human (H) VWF-A1 or murine (M) VWF-A1 protein (100 μg/ml final concentration) at a shear rate of 800 s−1. M VWF-A1 protein supported platelet adhesion as efficiently as its human counterpart under physiological flow conditions (FIG. 13A). The translocation of mouse platelets occurred to a similar degree as its human counterpart under physiological flow conditions (FIG. 13B). However, human platelets had a reduced capacity to interact with M VWF-A1 protein and mouse platelets had a reduced capacity to interact with H VWF-A1 protein in flow.
FIGS. 14A and 14B represent purified bacterial His-tagged VWF-A1 protein and non-His tagged VWF-A1 protein that was analyzed by SDS-PAGE (12.5%) under non-reducing and reducing conditions. FIG. 14B depicts a bar graph of a human platelet adhesion assay to recombinant VWF proteins with and without the presence of a His-tag at a shear rate of 800 s−1.
FIGS. 15A through 15C represents models of the crystal structure of VWF-A1 domains solved using a recombinant protein. The main chain schematic of the mouse VWF-A1 domain, with β-strands (arrows) and helices (coils), is shown in FIG. 15A. The two cysteines involved in the disulfide bridge are shown as yellow spheres. FIG. 15B demonstrates that the C-alpha atoms of human (red) and mouse (blue) VWF-A1 domains closely overlap. FIG. 15C shows the model of the murine VWF-A1 domain and the residues that purportedly interact with GPIb alpha, wherein amino acid residue 1326 of mouse (M) VWF-A1 was mutated to the corresponding amino acid at the identical location in its human counterpart (from Arg to His).
FIGS. 16A and 16B show graphs that depict platelet adhesion assays (FIG. 16A) and platelet translocation measurements (FIG. 16B). The ability of murine and human platelets to interact with a mutant protein substrate (human VWF-A1 domain wherein amino acid residue 1326 was mutated from His to Arg and mouse VWF-A1 domain wherein amino acid residue 1326 was mutated from Arg to His) was evaluated at a wall shear rate of 800 s−1. FIG. 16C identifies the amino acid position designations in FIGS. 16A and 16B relative to the standard vWF nomenclature.
FIG. 17 represents data from an ELISA assay. Following several injections of mouse (M) VWF-A1, serum was collected from rats and screened by ELISA for anti-VWF-A1 antibodies. Spleens from animals with the highest antibody titers were harvested and splenocytes fused with Sp2/0 mouse myeloma cells (54). Supernatants of hybridomas were screened for reactivity to murine (M) VWF-A1 by ELISA. Pre-immune rat serum was used as control. Mabs to M VWF-A1 not only reacted with WT and mutant proteins (1324G>S) but also recognized native VWF purified from mouse plasma.
FIGS. 18A, 18B, and 18C show representative graphs depicting the distribution of interaction times for more than 35 individual transient attachment events at various times. Analysis of the distribution of interactions times between human or murine VWF-A1 coated beads and their respective platelet substrates, as measured by high temporal resolution video microscopy, indicate that >95% of all transient tether bonds events fit a straight line, the regressed slope of which corresponded to a single koff, wherein the cellular off-rates of these quantal units of adhesion for the wild type human (H) and murine (M) proteins are found in FIGS. 18A, 18B, and 18C and M VWF-A1 protein containing the type 2B mutation 11309V (1309I>V) corresponds to FIG. 18C.
FIGS. 19A and 19B show graphs that represent an assessment of transient tether events (FIG. 19A) and analysis of the distribution of interactions times (FIG. 19B) between human VWF-A1 coated microspheres and human immobilized platelets. The type 2B mutation Ile1309Val (1309I>V) was incorporated into recombinant human (H) VWF-A1 containing either the type 2M mutation Gly1324Ser (1324G>S) or the function reducing mutation His1326Arg (1326H>R).
FIGS. 20A and 20B show a scheme for generating transgenic mice with mutant VWF-A1 domains. FIG. 20A is a diagram of a knock-in construct for proposed mutations in the VWF-A1 domain of mice. FIG. 20B represents Southern blot hybridization with probe “a” or “b”, respectively, to determine if the construct was appropriately targeted.
FIG. 21 represents Southern blot analysis wherein heterozygous and homozygous mice for the amino acid substitution at residue 1326 (R1326H; 1326R>H) display the Arg1326His mutation (lanes 2 and 3 respectively) while wild-type animals did not (lane 1).
FIGS. 22A through 22C represent sequence analysis of purified PCR products of WT, heterozygous, or homozygous VWF-A1 domains wherein the red-boxed area denotes the conversion of Arg to His (CGT in FIG. 22A wherein the codon corresponds to Arg, CNT in FIG. 22B, and CAT in FIG. 22C wherein the codon corresponds to the amino acid His).
FIG. 23 is a graph of an ELISA assay which demonstrated that conversion of Arg to His in the mouse A1 domain did not alter plasma protein levels of VWF in mutant mice nor its ability to form multimers. The ELISA assay detected mouse VWF in plasma obtained from WT and homozygous (KI) animals, but not from plasma obtained from animals deficient in VWF (VWF KO).
FIG. 24 is a gel image of multimer gel analysis of plasma VWF that revealed an identical banding pattern between mouse and human VWF. Incorporation of His at position 1326 in the mouse A1 domain had no effect on multimerization of VWF in mutant mice.
FIG. 25 is a graphical representation of the bleeding times (s) observed in the mutant VWF-A1 mice that are either heterozygous or homozygous for the 1326R>H mutation. Results are compared to normal counterparts and VWF-deficient mice. Tail cut=1 cm.
FIG. 26 is a bar graph depicting thrombus formation induced by perfusion of whole blood from either wild type (WT) or homozygous mutant mice over surface-immobilized collagen in vitro wherein an  ̃80% reduction in thrombus formation was observed compared to WT controls.
FIG. 27 are micrographs that demonstrate reduced thrombus formation occurring when whole blood from either the knock-in animals (homozygous for the R1326H mutation) or WT is perfused over collagen-coated cover slips at a shear rate of 1600 s−1 indicating a 70% reduction in thrombi formed on collagen as compared to WT controls.
FIGS. 28A and 28B demonstrate a platelet adhesion assay in flow. R1326H mutant mouse VWF promotes interactions with human platelets under physiologic flow conditions, wherein anticoagulated human blood was infused over surface-immobilized WT or mutant mouse plasma VWF at 1600 s−1 as shown in the micrographs of FIG. 28A. FIG. 28B is a graph that depicts the amount of human platelets that bound to WT murine VWF or R1326H mutant murine VWF.
FIGS. 29A and 29B are transmitted light micrographs demonstrating that homozygous mutant mice infused with human (FIG. 29A) but not mouse platelets (FIG. 29B) were able to generate an arterial thrombus that occludes the vessel lumen in response to laser-induced vascular injury as depicted by intravital microscopy.
FIG. 30 is a bar graph that depicts the average bleeding time for mice receiving blood-banked human platelets ( ̃3 minutes for a 1 cm tail cut) or given an intravenous infusion of a physiological buffered saline solution (10 minutes (end point)).
FIG. 31 is a schematic representing the isolation of the cremaster muscle and the catheter set-up used in intravital microscopy assays to assess thrombus formation.
FIG. 32 is a schematic of an intravital microscopy method.
FIGS. 33A and 33B shows images of mouse platelet interactions and a bar graph of such interactions in a wild type animal. FIG. 33A are representative intravital photomicrographs that depict the range of platelet interactions that occur at a site of vascular injury (60×). Platelets were observed to either transiently pause (*) or rapidly tether to and translocate (TP) on damaged arterial endothelium. A composite image demonstrates translocation of two platelets over a 3 s interval of time (panel 6). FIG. 33B depicts interacting platelets at the site of arterial injury that were classified as either undergoing translocation or firm adhesion (sticking) during an observation period of 1 min.
FIGS. 34A and 34B represents photomicrographs that depict the vessel wall in a wild type mouse in the (FIG. 34A) absence of injury or (FIG. 34B) post-laser-induced injury as visualized under transillumination (40× objective). Thrombus is indicated by the arrows.
FIGS. 35A and 35B are graphical representations of the bleeding phenotype observed in the mutant VWF-A1 1326R>H heterozygous or homozgygous mouse compared to its WT counterpart when tails were cut either 5 mm (FIG. 35A) or 15 mm (FIG. 35B) from the tip of the tail.
FIGS. 36A and 36B are graphs that depict ex vivo analysis of human platelet interactions with plasma VWF or recombinant VWF-A1 proteins. Accumulation of human platelets on surface-immobilized plasma human or mouse VWF (FIG. 36A) or recombinant human or mouse A1 domain proteins (FIG. 36B) after 4 min of perfusion with whole blood (shear rate of 1600 s−1) is shown. Data are representative of three separate experiments performed in triplicate (mean±s.e.m.).
FIGS. 36C and 36D are graphs that depict ex vivo analysis of mouse platelet interactions with plasma VWF or recombinant VWF-A1 proteins. Accumulation of murine platelets on surface-immobilized human or mouse plasma VWF (FIG. 36C) or recombinant human or mouse A1 domain proteins (FIG. 36D) after 4 min of perfusion with whole blood (shear rate of 1600 s−1) is shown. Data are representative of three separate experiments performed in triplicate (mean±s.e.m.).
FIGS. 37A and 37B are structural representations of human and murine VWF-A1 domains. FIG. 37A depicts the alignment of Ca atoms for human (blue) and murine (red) A1 domains. Key residues described in EXAMPLE 4 are shown as red spheres or as ball- and stick side-chains. FIG. 37B is a 90° rotation about a horizontal axis to reveal the packing of residue 1397 (Phe in human, Leu in mouse) that results in a 3 Å shift (blue arrow) of helix α4.
FIGS. 37C and 37D are structural representations of human and murine GPIbα-VWF-A1 complexes. FIG. 37C depicts the model of the murine-murine complex. FIG. 37D depicts the crystal structure of the human-human complex. Salt bridges are circled and key residue differences are boxed. Zooms reveal details of the electrostatic interactions at the β-switch contact region. The region of contact involving helix α3 of the A1 domain and one face of the LRR repeats of GPIbα is highly conserved betweens species, except for two residue changes that do not participate in bond formation: GPIbαE151K and VWF-A1 G1370S (human:mouse). Thus, minor differences in this region are unlikely to contribute to a reduction in binding between the murine and human proteins. This is also the case with the contact area located at the bottom of the A1 domain, which is invariant in both species and participates in salt-bridge formation (red circle).
FIG. 37E is a model of the human GPIbα-murine A1 complex, showing the loss (green arrow) and gain (blue circle) of salt-bridges. The upper zoom shows the interspecies interface at the β-switch region, revealing the electrostatic clash. The lower zoom shows the murine VWF-A1 point mutant 1326R>H, which removes the electrostatic clash and now closely resembles the human-human complex.
FIG. 37F is a model of the murine GPIbα-human VWF-A1 complex. Two salt-bridges are lost as compared to the murine complex; murine GPIbαD238 with residue 1326 due to the R>H change in human VWF-A1, and murine GPIbαK237 with residue 1330 owing to the E>G change in the human protein. Moreover, neither the chimeric nor murine complex forms a salt-bridge between residues 225 and 1395 on GPIbα and VWF-A1, respectively, as compared to its human counterpart (green circle). The upper zoom shows the interspecies interface at the β-switch region; there is no electrostatic clash but no salt-bridge can form with H1326. The lower zoom shows the human point mutant 1326H>R, which adds a salt-bridge and now closely resembles the murine-murine complex.
FIG. 37G is a graph that shows the accumulation of human platelets on surface-immobilized recombinant WT murine VWF-A1 domain proteins, those containing the selected mutations 1326R>H, 1330E>G and 1370S>G, or WT human VWF-A1 protein (shear rate of 1600 s−1). Data are representative of three separate experiments performed in triplicate (mean±s.e.m.).
FIG. 38A is schematic for the generation of the VWF1326R>H mouse that represents the targeting strategy for insertion of exon 28 containing adenine in lieu of guanine at position 3977 of the cDNA for murine VWF. RI, EcoRI; RV; EcoRV; B, BamHI; X, XhoI; pGK-TK, pGK-Neo, thymidine kinase/neomycin resistance cassette; , loxP sites.
FIG. 38B is a blot of a Southern analysis of tailed DNA digested with EcoR1. Wild-type (WT) allele, 14 kb; mutant allele, 6 kb using Probe A.
FIG. 38C represents the DNA sequencing of the tailed DNA demonstrating successful incorporation of adenine at position 3977 in heterozygous and homozygous animals (CGT>CAT). Sequence analysis of genomic DNA from these animals, 2 kb upstream and 6 kb downstream of exon 28, did not reveal any other alterations in nucleotide base pairs that would affect VWF production and/or function.
FIGS. 39A and 39B represent the analysis of VWF gene transcription and translation. FIG. 39A is a gel of RT-PCR of lung tissue from WT or mutant VWF mice to detect for A1, A2, and/or A3 domain message. β-actin analyzed to demonstrate use of equivalent amounts of mRNA. FIG. 39B is a graph demonstrating VWF antigen levels in plasma obtained from WT littermates (pooled) or six individual mice homozygous for 1326R>H mutation as detected by ELISA. Data are representative of two independent experiments performed in triplicate.
FIG. 39C is a gel showing the analysis of VWF multimers in plasma from WT or homozygous mutant animals. Normal human plasma as well as that obtained from a patient with type 2B VWD is shown for comparison.
FIG. 40 depicts representative photomicrographs showing murine platelet accumulation at sites of laser-induced arteriolar injury in WT or homozygous mutant animals 20 s and 2 min post-injury. White lines demarcate the extent of the thrombus.
FIG. 41A is a graphical representation of the tail bleeding times (s) for heterozygous and homozygous VWF1326R>H and WT mice when tails were cut 1 cm from the tip of the tail. Each point represents one individual mouse and experiments were performed on five separate days.
FIG. 41B is an ex vivo analysis of human platelet interactions with surface-immobilized plasma VWF1326R>H at a shear rate of 1,600 s−1. A role for GPIb alpha on human platelets is demonstrated by the function-blocking antibody to this platelet receptor (mAb 6D1) to prevent adhesion in flow.
FIG. 41C are microscopy images of in vivo analysis of human platelet interactions with murine plasma VWF1326R>H using infused fluorescently labeled human platelets into the vasculature of the cremaster muscle of mice. Human platelet accumulation was examined at sites of laser-induced arteriolar injury in WT (n=10) or homozygous mutant animals (n=12) using 2 channel confocal microscopy with excitation wavelengths of 488 nm (BCECF) and 561 nm (rhodamine 6G). Representative composite images of fluorescent images depicting human thrombus formation in homozygous mutant (upper panels) or WT (lower panels) mice (V=venule; A=arteriole). Rhodamine and BCECF are depicted in red and green, respectively, and merged is presented in yellow.
FIG. 41D is a bar graph depicting the composition of thrombi (% of total area) in WT or homozygous mutant animals.
FIG. 41E is a bar graph measuring thrombus size during an in vivo study of human platelet interactions with plasma VWF1326R>H to determine the effect of GPIbα or αIIbβ3 blockade on human platelet adhesion in vivo. The requirement for GPIb alpha-mediated adhesion is shown by the ability of a function-blocking antibody (mAB 6D1 or mAb 7E3) to GPIb alpha to prevent human platelet thrombus formation in vivo. Fluorescently labeled human platelet accumulation was examined at sites of laser-induced arteriolar injury in WT (n=6) or homozygous mutant animals (n=8). Data represent the mean±s.e.m.
FIG. 41F is a graphical representation of tail bleeding times (s) for homozygous VWF1326R>H that received an infusion of either normal saline or human platelets prior to severing 10 mm of distal tail, wherein the ability of human platelets to restore hemostasis in homozygous VWF1326R>H and the effect of PLAVIX or ReoPro® on this process was examined. Each point represents one individual mouse and experiments were performed on 3 separate days.
FIG. 42 is a schematic depicting a perfluorocarbon nanoparticle capable of incorporating imaging agents (Gd+3, 99m Tc) and chemotherapeutics into the outer layers. Antibodies complexed to the surface of the particle can target the agent to specific sites within the body.
FIG. 43 is a photographic image depicting the accumulation of fluorescent PNP, coupled to an antibody that recognizes human alphaIIb beta 3 on the surface of human platelets, at a site of vascular injury in homozygous 1326R>H mutant mice infused with human platelets.
FIGS. 44A and 44B graphical representations of the structure of the VWF-A1-GPIb alpha-botrocetin ternary complex. FIG. 44A is a ribbon representation; GPIb alpha, green; botrocetin, red; A1, cyan. FIG. 44B demonstrates the location of the previously unknown interface between GPIb alpha and botrocetin.
FIG. 45A is a schematic representation showing that Recombinant GPIb alpha is surface-immobilized in a 96 well format. After blocking potential non-specific binding sites, recombinant VWF-A1 containing a His tag is added to the wells and allowed to interact with GPIb alpha for a specified period of time. The unbound material is removed by washing the wells and the complex formed between the 2 proteins detected by the addition of a HRP-conjugated antibody that binds to the His tag on A1. The amount of bound A1 can then be quantified by either fluorescence (addition of LumiGlow) or by color change.
FIG. 45B is an image representing that the specificity of the interaction can be determined by the addition of the GPIb function blocking antibody 6D1 prior to the addition of recombinant VWF-A1. DMSO (0.5%) was added to illustrate that this reagent does not interfere with the assay.
FIGS. 46A and 46B are graphs depicting the effect of Plavix (FIG. 46A) or ReoPro (FIG. 46B) on human platelet-induced hemostasis in homozygous VWF1326R>H mice.
FIG. 47 is a graph showing the efficacy of anti-platelet drugs administered to patients by studying the ability of platelets harvested from patients on therapies in the VWF1326R>H mouse.



SUMMARY OF THE INVENTION
The invention provides for a mouse model for pre-clinical screening and testing of candidate compounds, wherein the model comprises a mouse expressing plasma von Willebrand Factor protein that contains a mutation or combination of mutations in its A1 domain that change the mouse protein's binding specificity from being specific for mouse platelets to being specific for human platelets. Thus, the mutant A1 domain contained within mouse plasma VWF particularly supports the binding of human platelets in vivo and ex-vivo. In one embodiment, the mouse model is infused with human platelets. In another embodiment, human platelets are labeled ex-vivo or in vivo so as to be detected while in the animal.
The invention provides for where the mutation in the A1 domain of mouse VWF comprises 1263P>S, 1269N>D, 1274K>R, 1287M>R, 1302G>D, 1308H>R, 1313R>W, 1314I>V, 1326R>H, 1329L>I, 1330E>G, 1333A>D, 1344T>A, 1347I>V, 1350T>A, 1370G>S, 1379H>R, 1381T>A, 1385T>M 1391P>Q, 1394A>S, 1397L>F, 1421S>N, 1439L>V, 1442G>S, 1449R>Q, 1466A>P, 1469Q>L, 1472Q>H, 1473V>M, 1475H>Q, 1479S>G, and any combination thereof, wherein the mutation corresponds to an amino acid position of human von Willebrand Factor A1 protein shown in SEQ ID NO: 6. In addition, the mouse VWF A1 domain can be fully or partially replaced with the human VWF A1 domain. In one embodiment, the mutation in mouse VWF-A1 is 1326R>H. In another embodiment, the mouse model comprises a device within a vessel, such as a stent or a graft, or mechanical, chemical, or heat-induced disruption of vascular endothelium in vivo. This model system is useful for testing compounds in an in vivo environment. The compounds can be tested for an effect on the interaction between human platelets and human-like (the mutant VWF-A1, 1326R>H, for example), or the actual human VWF-A1 domain. For example, the animal model can be used for pre-clinical testing of drugs in order to determine whether 1) there is a desired effect on hemostasis and/or thrombus formation or anti-thrombotic effect by the test drug or 2) there is an undesired effect on hemostasis and/or thrombus formation or anti-thrombotic effect by a test drug not specifically designed to alter hemostasis and/or thrombus formation. In the latter case, many drugs are only identified as having an effect on clotting or bleeding once they are in human clinical trials, this animal model will fill an unmet need, which is to test such effects prior to clinical trials. The invention also permits testing of compounds targeted to the VWF-A1 domain that can correct the bleeding phenotype associated with a loss-of-function mutations (1326R>H, for example) by altering the kinetics of the interaction between GPIbα and VWF-A1 (for example, enhancing the on-rate and/or prolonging the bond lifetime as shown for the snake venom protein botrocetin.
The invention also provides for an isolated mutant human von Willebrand Factor A1 protein comprising one or more mutations selected from the group consisting of: 1263S>P, 1269D>N, 1274R>K, 1287R>M, 1302D>G, 1308R>H, 1313W>R, 1314V>I, 1326H>R, 1329I>L, 1330G>E, 1333D>A, 1344A>T, 1347V>I, 1350A>T, 1370S>G, 1379R>H, 1381A>T, 1385M>T, 1391Q>P, 1394S>A, 1397F>L, 1421N>S, 1439V>L, 1442S>G, 1449Q>R, 1466P>A, 1469L>Q, 1472H>Q, 1473M>V, 1475Q>H, 1479G>S, wherein each amino acid position corresponds to a position in SEQ ID NO: 6. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1263S>P mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1269D>N mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1274R>K mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1287R>M mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1302D>G mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1308R>H mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1313W>R mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1314V>I mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1326H>R mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1329I>L mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1330G>E mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1333D>A mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1344A>T mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1347V>I mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1350A>T mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1370S>G mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1379R>H mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1381A>T mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1385M>T mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1391Q>P mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1394S>A mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1397F>L mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1421N>S mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1439V>L mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1442S>G mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1449Q>R mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1466P>A mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1469L>Q mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1472H>Q mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1473M>V mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1475Q>H mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1479G>S mutation in an amino acid sequence of SEQ ID NO: 1.
The invention also provides for an isolated mutant human von Willebrand Factor A1 protein having SEQ ID NO: 6, wherein the protein comprises a mutation selected from the group consisting of: 1263S>P, 1269D>N, 1274R>K, 1287R>M, 1302D>G, 1308R>H, 1313W>R, 1314V>I, 1326H>R, 1329I>L, 1330G>E, 1333D>A, 1344A>T, 1347V>I, 1350A>T, 1370S>G, 1379R>H, 1381A>T, 1385M>T, 1391Q>P, 1394S>A, 1397F>L, 1421N>S, 1439V>L, 1442S>G, 1449Q>R, 1466P>A, 1469L>Q, 1472H>Q, 1473M>V, 1475Q>H, or a 1479G>S.
The invention provides for a transgenic non-human animal expressing von Willebrand Factor A1 protein containing mutation(s) at one of more amino acid position selected from the group consisting of: 1263, 1269, 1274, 1287, 1302, 1308, 1313, 1314, 1326, 1329, 1330, 1333, 1344, 1347, 1350, 1370, 1379, 1381, 1385, 1391, 1394, 1397, 1421, 1439, 1442, 1449, 1466, 1469, 1472, 1473, 1475, and 1479, wherein the position corresponds to an amino acid position of human von Willebrand Factor A1 protein shown in SEQ ID NO: 6. In one embodiment, the animal is a murine, a porcine, a canine, a feline, a rabbit, or a primate. In another embodiment, the animal is a mouse, a rat, a dog, a sheep, a goat, a horse, a cow, a cat, a monkey, a primate, a pig, a llama, an alpaca, a chicken, etc. In other embodiments, the protein comprises a single mutation. In further embodiments, the protein comprises two or more mutations. In yet another embodiment, the protein comprises at least one mutation selected from the group consisting of: 1263>S, 1269>D, 1274>R, 1287>R, 1302>D, 1308>R, 1313R>W, 1314>V, 1326>H, 1329>1, 1330>G, 1333>D, 1344>A, 1347>V, 1350>A, 1370>S, 1379>R, 1381>A, 1385>M 1391>Q, 1394>S, 1397>F, 1421>N, 1439>V, 1442>S, 1449>Q, 1466>P, 1469>L, 1472>H, 1473>M, 1475>Q, 1479>G, and any combination thereof. In particular embodiments, the protein comprises a 1326R>H mutation. In other embodiments, the protein comprises a 1314I>V mutation. In yet other embodiments, the protein comprises a 1326R>H mutation, a 1314I>V mutation, or a combination of the two mutations listed previously. In some embodiments of the invention, the animal is a mouse. In further embodiments, the protein comprises SEQ ID NO: 5. In other embodiments, the VWF protein is at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the A1 domain of human VWF protein as shown in SEQ ID NO: 1. In particular embodiments, the von Willebrand Factor A1 protein of the transgenic animal comprises the human A1 domain shown in SEQ ID NO: 1. In some embodiments, the von Willebrand Factor A1 protein is partially or completely replaced with a human von Willebrand Factor A1 protein comprising SEQ ID NO: 1. In other embodiments, the animal is a model for pre-clinical testing of compounds that expresses a mutant von Willebrand Factor (VWF) A1 protein containing one or more mutations, wherein the binding specificity of the mutant VWF-A1 protein changes from being specific for the animal platelets to being specific for human platelets. In further embodiments the mutant VWF-A1 protein in the animal binds to human platelets.
The invention provides a method for identifying a compound that modulates binding of VWF-A1 protein to GPIb-alpha protein. The method comprises: providing an electronic library of test compounds; providing atomic coordinates listed in Table 8 for at least 10 amino acid residues for the A1 domain of the VWF protein, wherein the coordinates have a root mean square deviation therefrom, with respect to at least 50% of Ca atoms, of not greater than about 2.5 Å, in a computer readable format; converting the atomic coordinates into electrical signals readable by a computer processor to generate a three dimensional model of the VWF-A1 domain; performing a data processing method, wherein electronic test compounds from the library are superimposed upon the three dimensional model of the VWF-A1 domain; and determining which test compound fits into the binding pocket of the three dimensional model of the VWF-A1 protein, thereby identifying which compound would modulate the binding of VWF-A1 protein to GPIb-alpha protein. Alternatively, the method can comprise: providing an electronic library of test compounds; providing atomic coordinates listed in Table 8 in a computer readable format for at least 10, 15, 20, 25, 30, 35, or 40 amino acid residues for the A1 domain of the VWF protein, wherein the residues comprise two or more of the following residues: Pro1391, Arg1392, Arg1395, Val1398, Arg1399, Gln1402, Lys1406, Lys1423, Gln1424, Leu1427, Lys1430, or Glu1431; converting the atomic coordinates into electrical signals readable by a computer processor to generate a three dimensional model of the VWF-A1 domain; performing a data processing method, wherein electronic test compounds from the library are superimposed upon the three dimensional model of the VWF-A1 domain; and determining which test compound fits into the binding pocket of the three dimensional model of the VWF-A1 protein, thereby identifying which compound would modulate the binding of VWF-A1 protein to GPIb-alpha protein.
In one embodiment, determining comprises detecting an IC50 of less than about 7.5 μg/ml for a test compound. In another embodiment, the method can further comprise: obtaining or synthesizing a compound; contacting VWF-A1 protein with the compound under a condition suitable for GPIb-alpha-VWF-A1 binding; and determining whether the compound modulates GPIb-alpha-VWF-A1 protein binding using a diagnostic assay. In a further embodiment, contacting comprises perfusing platelets into a flow chamber at a shear flow rate of at least 100 s−1, wherein mutant murine VWF-A1 protein is immobilized on a bottom surface of the chamber, while in another embodiment contacting comprises perfusing platelets into the transgenic non-human animal described above. In some embodiments, contacting occurs sequentially. In other embodiments, the perfusing of platelets occurs prior to administration of the compound. In one embodiment, the platelets are human platelets, while in other embodiments, the platelets are not murine platelets. In further embodiments, the determining comprises detecting an increase or decrease in the dissociation rate between VWF-A1 protein and GPIb-alpha protein by at least two-fold. In other embodiments, the determining comprises detecting an increase or decrease of platelet adhesion to a surface expressing VWF-A1 protein, while in some embodiments the determining comprises detecting an increase or decrease in a stabilization of an interaction between VWF-A1 protein and GPIb-alpha protein. In particular embodiments, the determining comprises detecting thrombosis formation. In some embodiments, the determining comprises identifying an occurrence of an abnormal thrombotic event in the subject. In further embodiments of the invention, an abnormal thrombotic event comprises abnormal bleeding, abnormal clotting, death, or a combination of the events listed. In some embodiments, the determining comprises dynamic force microscopy, a coagulation factor assay, a platelet adhesion assay, thrombus imaging, a bleeding time assay, aggregometry, review of real-time video of blood flow, a Doppler ultrasound vessel occlusion assay, or a combination thereof. In particular embodiments of the invention, perfusing platelets is followed by perfusion of a labeled agent. In some embodiments, the labeled agent comprises one or more of a nanoparticle, a fluorophore, a quantum dot, a microcrystal, a radiolabel, a dye, a gold biolabel, an antibody, or a small molecule ligand. In other embodiments, the agent targets a platelet receptor, a VWF protein, or a portion thereof. In another embodiment, the animal is injected with nanoparticles, and/or peptides, and/or small molecules, which label the human platelets, at some time prior testing, wherein the nanoparticles, and/or peptides, and/or small molecules are capable of being imaged while in the animal. In another embodiment, the testing comprises tracking of human platelet deposition in the animal. In another embodiment, the compound or agent is an anti-thrombotic, such as an anti-platelet drug, e.g., PLAVIX, an ADP inhibitor, and/or a humanized antibody and/or small molecule that inhibits human alpha IIb and/or beta 3 integrin function, human alpha2 and/or beta 1 integrin function, human glycoprotein VI (GPVI) function, human thrombin receptors function, and/or intracellular signaling pathways (for example, phosphoinositide 3-kinases (PI3K)) vital to platelet function in hemostasis and thrombosis.
The invention provides for a method for testing a compound or agent, the method comprising: (a) providing a candidate agent or compound to be tested; (b) administering the agent or compound to an animal in an effective amount, wherein the animal expresses a mutant von Willebrand Factor A1 protein containing a mutation, combination of mutations that change the animal protein's binding specificity from being specific for animal platelets to being specific for human platelets, so that the mutant VWF-A1 protein in the animal binds to human platelets, and wherein the animal is perfused with human platelets; (c) testing the animal to determine whether the animal experiences any abnormal hemostatic and/or thrombotic events, thereby testing the compound or agent.
The invention provides for a nucleic acid encoding the mutant von Willebrand Factor A1 protein of the invention. The invention provides for a vector containing such a nucleic acid. The invention provides for an animal expressing such a nucleic acid to express the mutant VWF protein.
The invention also provides a method for treating von Willebrand Disease (VWD) in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound that promotes platelet adhesion in the subject, wherein the compound increases the dissociation rate between VWF-A1 protein and GPIb-alpha protein by at least two-fold, thereby administration of the compound increases blood coagulation in the subject. In one embodiment, coagulation is measured by a coagulation factor assay, an ex-vivo flow chamber assay, or a combination thereof.
The invention provides a method for rapidly detecting an internal vascular injury site in a subject. The method comprises: administering to a subject a targeted molecular imaging agent, wherein the molecule circulates for an effective period of time in order to bind to the injury site within the subject; tracking a deposition of the labeled targeted molecular imaging agent in the subject; and identifying the site of a thrombus formation in the subject by imaging the targeted molecular imaging agent, thereby the deposition of the targeted molecular imaging agent at the internal vascular injury site is indicative of internal bleeding within a subject. In one embodiment, the targeted molecular imaging agent is administered by subcutaneous, intra-muscular, intra-peritoneal, or intravenous injection; infusion; by oral, nasal, or topical delivery; or a combination of the routes listed. In another embodiment, the targeted molecular imaging agent comprises a nanoparticle, a fluorophore, a quantum dot, a microcrystal, a radiolabel, a dye, a gold biolabel, an antibody, a peptide, a small molecule ligand, or a combination of the agents listed. In a further embodiment, the nanoparticle comprises a perfluorocarbon, while in particular embodiments, the nanoparticle is coupled to an antibody, a small molecule, a peptide, or a receptor trap. In some embodiments, the targeted molecular imaging agent specifically binds to a platelet receptor, or a VWF protein, or a portion thereof. In other embodiments, the targeted molecular imaging agent has a T1/2 of at least 30 minutes. In further embodiments, imaging comprises a PET scan, MRI, IR scan, ultrasound, nuclear imaging, or a combination of the methods listed. In a particular embodiment, the subject is further administered a prothrombotic compound. In a further embodiment, the compound increases the dissociation rate between VWF-A1 protein and GPIb-alpha protein by at least two-fold.
Considerable emphasis has been placed on elucidating the role of structural changes in the A1 domain of VWF in order to gain insight into mechanism(s) that may regulate its binding to platelet GPIb alpha. This invention concerns the contribution of the biophysical properties of this interaction in governing platelet adhesion under hydrodynamic conditions. It has been demonstrated that flow-dependent adhesion and rapid and force-driven kinetic properties define the GPIb alpha-VWF-A1 bond. The invention provides classification of subtypes of von Willebrand disease (vWD), such as platelet-type, type 2B or type 2M in terms of similarities in the alterations in the biophysical properties of bonds to better understand the clinical phenotypes associated with these bleeding disorders. The invention is directed to understanding the biomechanical and molecular mechanisms by which the VWF-A1 domain mediates adhesive interactions with GPIb alpha both in vitro and in vivo. This invention provides for mice with mutant A1 domains to demonstrate the importance of the intrinsic kinetic and mechanical properties of this receptor-ligand pair in preventing inappropriate platelet aggregation in circulating blood, to facilitate the study of human platelet biology, as well as to generate a humanized animal model of hemostasis and thrombosis. The animal model is also useful for the generation and testing of novel anti-thrombotic therapies designed to inhibit platelet-VWF interactions as well as those designed to correct the bleeding phenotype associated with a reduction in adhesion between this receptor-ligand pair.
The invention is directed to understanding the effect that alterations in platelet size and shape have on the force-driven kinetics of the GPIb alpha-VWF-A1 tether bond. Platelets can bind to and translocate on surface-immobilized VWF under shear forces that preclude selectin-dependent adhesion of leukocytes to the vessel wall, due to their small discoid shape and not as a consequence of the unique kinetic properties of the GPIb alpha-VWF-A1 tether bond. The contribution of particle geometry in supporting the interactions between this receptor-ligand pair, we is performed by evaluating the interaction between vWF-A1 coated microspheres, ranging from 4 to 12 μm in diameter (platelet to leukocyte size), with surface-immobilized platelets under physiologic flow conditions. The utility of using polystyrene microspheres with a uniform shape and size to permit determination of the relationship between wall shear stress and the force acting on the GPIb alpha-vWF-A1 tether bond has been demonstrated. Moreover, it has been shown that the “sidedness” of the receptor or ligand does not alter the kinetic properties of this bond. β1-tubulin deficient mice have a defect in the cytoskeleton of platelets that changes the shape of these cells from discoid to spherical. Preliminary data demonstrates that the “spherical” platelets have a >60% reduction in attachment at high shear rates as compare to wild-type (WT) platelets. No differences in the kinetics of the GPIb alpha-vWF-A1 tether bond for WT and mutant platelets have been found using our microsphere assay system. Thus, platelet shape and not alterations in the biophysical properties of the GPIb alpha-vWF-A1 tether bond are responsible for the defect in adhesion. Data generated from these experiments will be use to develop a computational algorithm designed to stimulate the adhesion of platelets to surface-immobilized vWF under various hydrodynamic conditions. Thus, the invention provides an in vivo method to test for defects in hemostasis and thrombus formation that result from abnormalities in platelet shape and size. It also provides for a method to test the ability of synthetic platelet substitutes, which may be of varying shapes and sizes, to support hemostasis.
The invention provides a method for determining whether platelet function or morphology in a subject is abnormal. The method comprises: affixing a molecule comprising a murine VWF-A1 domain to a surface of a flow chamber, wherein the domain comprises at least one mutation at a position selected from the group consisting of 1263>S, 1269>D, 1274>R, 1287>R, 1302>D, 1308>R, 1313R>W, 1314>V, 1326>H, 1329>1, 1330>G, 1333>D, 1344>A, 1347>V, 1350>A, 1370>S, 1379>R, 1381>A, 1385>M 1391>Q, 1394>S, 1397>F, 1421>N, 1439>V, 1442>S, 1449>Q, 1466>P, 1469>L, 1472>H, 1473>M, 1475>Q, 1479>G, and any combination thereof, where the position corresponds to an amino acid position of human von Willebrand Factor A1 protein shown in SEQ ID NO: 6; perfusing through the flow chamber a volume of blood or plasma from a subject at a shear flow rate of at least about 100 s−1; perfusing a targeted molecular imaging agent into the flow chamber; and comparing the flow rate of the blood or plasma from the subject as compared to a normal flow rate, so as to determine whether the subject's platelet function or morphology is abnormal.
In one embodiment, affixing comprises (i) affixing an antibody which specifically binds VWF-A1 domain, and (ii) perfusing murine mutant VWF-A1 protein in the flow chamber at a shear flow rate of at least 100 s−1. In another embodiment, the targeted molecular imaging agent comprises a nanoparticle, a fluorophore, a quantum dot, a microcrystal, a radiolabel, a dye, a gold biolabel, an antibody, a peptide, a small molecule ligand, or a combination of the agents listed. In a further embodiment, the targeted molecular imaging agent binds to a platelet receptor, a platelet ligand, or any region of a VWF protein or a portion thereof. In a particular embodiment, the targeted molecular imaging agent comprises horseradish peroxidase (HRP) coupled to an antibody directed at VWF-A1. In other embodiments, the comparing comprises a platelet adhesion assay, fluorescence imaging, a chromogenic indicator assay, a microscopy morphology analysis, or any combination of the listed modes. In some embodiments, platelets bound to VWF-A1 are less than about 500 cells/mm2. In particular embodiments, the platelets are substantially spherical. In further embodiments, the subject is a human, a canine, a feline, a murine, a porcine, an equine, or a bovine. In other embodiments, the VWF molecule is an antibody, a peptide, or a Fab fragment directed to a VWF polypeptide or a portion thereof.
The invention also provides for a method for producing von Willebrand Factor A1 protein that specifically binds human platelets, the method comprising: (a) providing an animal expressing a plasma VWF containing a mutant von Willebrand Factor A1 protein, wherein the mutation causes the animal's von Willebrand Factor A1 protein to bind preferentially to for human platelets; and (b) harvesting the mutant animal von Willebrand Factor A1 so as to produce von Willebrand Factor A1 protein that specifically binds human platelets. In one embodiment, the mutant animal von Willebrand Factor A1 protein comprises at least one mutation comprising 1263P>S, 1269N>D, 1274K>R, 1287M>R, 1302G>D, 1308H>R, 1313R>W, 1314I>V, 1326R>H, 1329L>I, 1330E>G, 1333A>D, 1344T>A, 1347I>V, 1350T>A, 1370G>S, 1379H>R, 1381T>A, 1385T>M 1391P>Q, 1394A>S, 1397L>F, 1421S>N, 1439L>V, 1442G>S, 1449R>Q, 1466A>P, 1469Q>L, 1472Q>H, 1473V>M, 1475H>Q, 1479S>G, or any combination thereof.
The invention provides a method for testing efficacy and toxicity of a gene therapy vector, wherein the method comprises: (a) introducing a gene therapy vector into the animal of claim 4, allowing sufficient time for expression of the vector; (b) perfusing platelets from a subject into the animal under a condition suitable for GPIb-alpha-VWF-A1 protein binding; and (c) determining whether or not a thrombotic event occurs in the animal. In one embodiment, the vector comprises a nucleic acid encoding a platelet receptor polypeptide, a platelet ligand polypeptide, or a VWF polypeptide, or a portion thereof. In another embodiment, the subject is a human, a dog, a cat, a horse, a pig, or a primate. In a particular embodiment, the platelets are not murine platelets. In a further embodiment, the thrombotic event comprises blood clotting, abnormal bleeding, abnormal clotting, death, or a combination thereof. In some embodiments, the determining comprises dynamic force microscopy, a coagulation factor assay, a platelet adhesion assay, thrombus imaging, a bleeding time assay, aggregometry, review of real-time video of blood flow, a Doppler ultrasound vessel occlusion assay, or a combination thereof. In other embodiments, perfusing platelets is followed by perfusion of a labeled agent. In further embodiments, the labeled agent comprises one or more of a nanoparticle, a fluorophore, a quantum dot, a microcrystal, a radiolabel, a dye, a gold biolabel, an antibody, or a small molecule ligand. In particular embodiments, the agent targets a platelet receptor, a VWF protein, or a portion thereof.
The invention also provides a method for calibrating an aggregometry device or a device for measuring clot formation or retraction, wherein the method comprises: (a) providing hematologic data obtained from a subject, wherein blood or platelets from the subject is assessed by the device; (b) determining whether or not a thrombotic event occurs in the animal described above, wherein the animal is perfused with a sample of blood or platelets from the subject; and (c) correlating data obtained from (b) with the data obtained in (a) so as to calibrate the device, wherein a certain data obtained from the device is indicative of the corresponding thrombotic outcome determined in the animal described above. In one embodiment, the thrombotic event comprises blood clotting, abnormal bleeding, abnormal clotting, death, or a combination thereof.
The invention provides for in vivo determination of intrinsic and mechanical properties of the GPIb alpha-vWF-A1 tether bond and to determine if they are indeed critical for regulating the adhesion between platelets and vWF and how they may be manipulated to impair or enhance hemostasis and/or thrombosis. The invention provides determination of whether animals that posses gain-of-function mutations in VWF-A1, for example, those associated with type 2B vWD, have a defect in platelet deposition at sites of vascular injury and/or a loss of high molecular weight multimers of vWF. The invention provides determination of whether animals that possess loss-of-function mutations in VWF-A1, for example, those associated type 2M vWD, have a defect in platelet deposition at sites of vascular injury. Based on the results obtained herein, mice are genetically engineered with 1) mutant A1 domains that increase or decrease the on- and/or off-rate of this receptor-ligand pair by varying degrees, 2) A1 domains containing both types of mutations to confirm whether specific regions within this domain are essential for the stabilization of GPIb alpha binding, and 3) mutations within the A1 domain that favor binding to human but not murine GPIb alpha to enable the study of human platelet behavior in an animal model of hemostasis or thrombosis.
The invention provides methods for determining the impact of altering the intrinsic bond kinetics and/or its mechanical properties of the GPIb alpha-VWF-A1 interaction on hemostasis and thrombosis, which comprises: measuring platelet counts, plasma levels of vWF, and bleeding times; performing multimer gel analysis of mutant vWF; measuring the affinity of mutant vWF for platelets using a fluid phase binding assay; evaluating in vitro platelet tethering, rolling, and thrombus formation on surface-immobilized murine plasma vWF containing mutant A1 domains under physiological flow conditions; determining the ability of thrombi to form at sites of vascular injury in vivo in mutant VWF mice using epifluorescent intravital microscopy in; measuring platelet tethering frequency and rolling velocities in vivo.
In certain embodiments, the subject is a human. In other embodiments, the subject is a non-human animal such as a canine, equine, feline, porcine, murine, bovine, foul, sheep, or any other animal in need of treatment. In certain embodiments, the pharmaceutical composition further comprises another active agent. The additional active agent can be, but is not limited to, an analgesic, an antioxidant, diuretic, or a combination thereof. In certain embodiments, the composition is in a capsule form, a granule form, a powder form, a solution form, a suspension form, a tablet form, or any other form suitable for use by the method of the present invention. In certain embodiments, the composition is administered via oral, sublingual, buccal, parenteral, intravenous, transdermal, inhalation, intranasal, vaginal, intramuscular, rectal administration or any other route of administration that is suitable for delivery of the compound.
DETAILED DESCRIPTION OF THE INVENTION
The invention provides methods for identifying and evaluating potential anti-thrombotic reagents and compounds. The invention provides methods for testing for undesirable thrombotic or bleeding side effects of reagents in the setting of preclinical testing. The invention provides an in vivo model to test the efficacy of potential anti-thrombotic drugs directed against receptors, ligands, and/or intracellular signaling pathways on or in human platelets prior to FDA approval. To date, in vitro models of thrombosis do not accurately recapitulate the hemodynamic conditions, cell-cell interactions, or cell-protein interactions that occur at sites of vascular injury in a living animal. Thus, the system provided by this invention provides a method to test drugs directed at inhibiting or altering human platelet function other than directly testing them in humans. The invention provides a great advantage of being able to test directly compounds that target human platelets in an in vivo system. The invention provides a method to test contrast agents for imaging of human platelets at sites of thrombosis. For instance, one could test the ability of nanoparticle contrast agents targeted to human platelets to identify areas of thrombosis or occult bleeding. The invention provides a method to test compounds that correct the bleeding phenotype associated with a reduction in interactions between GPIb alpha and VWF-A1. The invention provides a method to test gene therapies directed at correcting genetic mutations associated with von Willebrand disease. The invention provides a method to correlate results obtained with an in vitro assay designed to measure the effects of antithrombotics or markers of platelet activation in patients.
Terms
A “pharmaceutical composition” refers to a mixture of one or more of the compounds, or pharmaceutically acceptable salts, hydrates, polymorphs, or pro-drugs thereof, with other chemical components, such as physiologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
As used herein von Willdebrand factor is abbreviated “VWF” and, alternatively, “vWF”.
A “pro-drug” refers to an agent which is converted into the parent drug in vivo. Pro-drugs are often useful because, in some situations, they are easier to administer than the parent drug. They are bioavailable, for instance, by oral administration whereas the parent drug is not. The pro-drug also has improved solubility in pharmaceutical compositions over the parent drug. For example, the compound carries protective groups which are split off by hydrolysis in body fluids, e.g., in the bloodstream, thus releasing active compound or is oxidized or reduced in body fluids to release the compound.
A compound of the present invention also can be formulated as a pharmaceutically acceptable salt, e.g., acid addition salt, and complexes thereof. The preparation of such salts can facilitate the pharmacological use by altering the physical characteristics of the agent without preventing its physiological effect. Examples of useful alterations in physical properties include, but are not limited to, lowering the melting point to facilitate transmucosal administration and increasing the solubility to facilitate administering higher concentrations of the drug.
The term “pharmaceutically acceptable salt” means a salt, which is suitable for or compatible with the treatment of a patient or a subject such as a human patient or an animal.
The term “pharmaceutically acceptable acid addition salt” as used herein means any non-toxic organic or inorganic salt of any base compounds of the invention or any of their intermediates. Illustrative inorganic acids which form suitable acid addition salts include hydrochloric, hydrobromic, sulfuric and phosphoric acids, as well as metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids that form suitable acid addition salts include mono-, di-, and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic and salicylic acids, as well as sulfonic acids such as p-toluene sulfonic and methanesulfonic acids. Either the mono or di-acid salts can be formed and such salts exist in either a hydrated, solvated or substantially anhydrous form. In general, the acid addition salts of compounds of the invention are more soluble in water and various hydrophilic organic solvents, and generally demonstrate higher melting points in comparison to their free base forms. The selection of the appropriate salt will be known to one skilled in the art. Other non-pharmaceutically acceptable salts, e.g., oxalates, are used, for example, in the isolation of compounds of the invention for laboratory use or for subsequent conversion to a pharmaceutically acceptable acid addition salt.
As used herein and as well understood in the art, “treatment” is an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.
Arterial thrombosis is a pathological consequence of disease states such as atherosclerosis and remains a major cause of morbidity and mortality in the Western world with healthcare cost ranging in the billions of dollars in the USA alone (Circulation 2006; 113:e85). Central to this process is the inappropriate deposition and activation of platelets in diseased vessels that can ultimately occlude the lumen, thus impeding blood flow to vital organ such as the heart and brain. Another key player is VWF, a large plasma glycoprotein of complex multimeric structure, which under normal physiological conditions prevents excessive bleeding by promoting platelet deposition at sites of vascular injury, thus “sealing off” leaky blood vessels. In order for this event to occur, VWF must form a “bridge” between receptors expressed on circulating platelets and exposed components of the injured vessel wall. This is the function of the A1 and A3 domains of this plasma protein, respectively. Each is folded into a disulfide-bonded loop structure that is critical for optimal biological activity (FIG. 1A).
It is the A1 domain that contains residues that compose the binding site for its receptor on platelets known as GPIb alpha, an adhesive event essential for the ability of these cells to rapidly attach to the injured vessel wall. The critical nature of this interaction is exemplified by the bleeding disorder, termed type 2M von Willebrand Disease (VWD), which results from the incorporation of loss-of-function point mutations within this domain that reduce the interaction between VWF-A1 and GPIb alpha (Sadler J E et al. (2006) J. Thromb. Haemost. 4: 2103-14). The A3 domain, on the other hand, is believed to be important in anchoring plasma VWF to sites where extracellular matrix components (i.e., collagen) are exposed as a result of disruption of the overlying vasculature endothelium (Wu D. et al. Blood 2002). Once in contact with exposed elements of the damaged vessel wall, platelets become “activated” through various signaling pathways (i.e. GPVI) enabling other adhesion molecules, such as α2β1 (collagen receptor) and αIIbβ3 (fibrinogen and VWF receptor) integrins, to firmly anchor these cells at the site of injury and to each other (FIG. 1B). In addition, ADP released from adherent platelets serves to amplify the activation of integrin receptors as well as other platelets leading to thrombus growth. Considerable emphasis has been placed on understanding the mechanism(s) that govern the interaction between GPIb alpha and the A1 domain of VWF and how it can be perturbed by point mutations associated with von Willebrand Disease, information relevant to the development of anti-thrombotic therapies.
von Willebrand Factor (VWF), the A1 Domain, and Related Diseases
During the past two decades, there has been considerable progress in understanding how VWF mediates platelet adhesion. Both the VWF cDNA and gene have been cloned and the primary structure of the VWF subunit (FIG. 1A) has been determined (13-16). It has been reported that  ̃59% of the mature VWF consists of repeated segments which are 29% to 43% homologous (17). These regions consist of domains that are triplicated (domains “A” and “B”), duplicated (domain “C”) or quadruplicated (domain “D”). The triplicated A repeats encode for the central region of each VWF subunit. The A1 and A3 domains contain the sequences that mediate VWF's interaction with receptors on platelets or components of subendothelial extracellular matrix, respectively. Each is folded into a disulfide-bonded loop structure that is critical for optimal biological activity. The sequences of the amino terminal halves of each loop and the location of the cysteines forming the loop structure of each domain are highly conserved. It is the VWF-A1 domain (1260-1480) that contains sequences that provide binding sites for the platelet glycoprotein receptor Ib alpha, an interaction critical for the ability of these cells to rapidly attach and translocate at sites vascular injury (18,19). The role of the A3 domain, however, is believed to be in anchoring plasma VWF at sites where extracellular matrix components (i.e., collagen) are exposed as a result of disruption of the endothelium (20-25).
With regard to mediating adhesive interactions with platelets, it has become increasingly evident that the VWF-A1 domain plays a crucial role in this process based on molecular genetic studies of individuals with type 2M or 2B VWD (26-31). In the majority of cases, patients with these designated genotypes have single point mutations contained within the disulfide loop (between Cys 1272 and Cys 1458) of this domain. With regard to type 2M VWD, afflicted individuals have significant impairments in hemostasis that appears to result from a lack of or reduced adhesive interactions between GPIb alpha and VWF at sites of vascular injury, and not from an alteration in VWF multimer structure. Structural and functional evidence has been provided in that type 2M mutations, such as 1324G>S, are localized within a region of the A1 domain (FIG. 5B), which is critical for supporting GPIb alpha-mediated platelet adhesion at physiological flow rates. Confirmation that this residue, as well as others predicted by our analysis of the crystal structure of the A1 domain, does contribute to GPIb alpha binding is suggested by studies evaluating the structure of the complex formed between this receptor-ligand pair (32, 33). Thus, it is possible to make accurate predictions about protein function from the three-dimensional protein structure and to confirm these postulates by site-specific mutagenesis and analysis under physiologically relevant flow conditions. The localization of some of the residues within the A1 domain that when mutated disrupt GPIb alpha binding is shown below (FIG. 2). The invention provides methods for evaluating the effect that loss-of-function mutations have on hemostasis and thrombus formation.
In contrast type 2M VWD, mutations associated with type 2B VWD are known to enhance the interaction between VWF-A1 and GPIb alpha, that is, they mitigate the requirement for exogenous modulators such as ristocetin or botrocetin to induce platelet agglutination (30). Moreover, these altered residues are localized in a region remote from the major GPIb alpha binding site that has been identified by mutagenesis (26; FIG. 3A-yellow). Clinically, this disease state is characterized by a loss of circulating high molecular weight VWF multimers (HMWM, FIG. 3B) together with a mild to moderate thrombocytopenia, which ultimately results in bleeding but not thrombosis (30, 31). It is the clearance of the HMWM of VWF from plasma that is believed to be responsible for the increased bleeding tendencies in patients with this disorder as they contribute to the majority of the hemostatic function associated with this plasma glycoprotein (34). The invention provides methods for evaluating the effect that gain-of-function mutations have on hemostasis, thrombus formation, and plasma levels of VWF.
Surface-immobilization of VWF and subsequent exposure to physiologically relevant shear forces appears to be a prerequisite for its ability to support interactions with platelets as this multimeric protein does not bind appreciably to these cells in the circulation. These hydrodynamic conditions are believed to promote structural changes within the A1 domain that in turn increases its affinity for GPIb alpha (35-37). Evidence suggested to support the existence of such an alteration in structure includes the ability of non-physiologic modulators such as the antibiotic ristocetin or the snake venom protein botrocetin to promote platelet agglutination in solution-based assays (38, 39). Moreover, this “on” and “off” conformation is exemplified by type 2B VWD. For instance, it was initially hypothesized that incorporation of type 2B mutations into the A1 domain shifted the equilibrium between two distinct tertiary conformations, analogous to those seen in crystal structures of the integrin I domain in ligand-free and collagen-bound states (40). The location of the type 2B mutants at sites distinct from the GPIb alpha binding site suggest that they disrupt a region responsible for regulation of binding affinity, thus affecting ligand binding allosterically. The crystal structure of a type 2B mutant A1 domain, 1309I>V, was determined and compared to its wild-type counterpart (41). A change was discovered in the structure of a loop, thought to be involved in GPIb alpha binding, lying on the surface distal to the mutation site. A similar finding has been observed for the VWF-A1 crystal structure containing the identical mutation (FIGS. 4A-4C).
This altered conformation represents a high affinity binding state of the A1 domain. However, the pathway of allosteric change proposed previously, involving the burial of a water molecule, cannot be a general feature of type 2B mutants, and the structural rearrangements appear too subtle to explain the altered kinetics. Interestingly, complex formation between botrocetin and VWF-A1 in which the type 2B mutation 1309I>V has been incorporated, and has demonstrated that most of these structural differences are reversed including: (1) loss of the buried water molecule at the mutation site; (2) the peptide plan between Asp 1323 and Gly 1324 flips back to a conformation similar to that in the WT structure; and (3) the side chain of His 1326 remains in the “mutant” position, although there is some evidence from electron density of an alternative conformation similar to WT. However, this reversion in structure does not correlate with a loss in the function-enhancing activity associated with the type 2B mutation. In fact, the addition of botrocetin further augments the interaction between the mutant A1 domain and platelets in flow (42). Thus, an alternative mechanism must account for function-enhancing nature of type 2B mutations. Moreover, these subtle alterations in structure did not compare to the large conformational changes in homologous integrin I-type domains that occur on ligand binding.
Recent findings provide support that type 2B mutations may stabilize the binding of a region of GPIb alpha known as the β-hairpin to an area near the location of these altered residues, distinct from that identified by site-directed mutagenesis. Type 2B mutations have been suggested to destabilize a network of interactions observed between the bottom face of the A1 domain and its terminal peptides in the wild type A1 structure, thereby making the binding site accessible (FIG. 6).
Progress has been made in understanding the structure of this receptor-ligand pair and potential alterations in conformation that may regulate this interaction. This model is non-limiting. However, the model permits determination of the kinetic and biomechanical basis for 1) the regulation of VWF-A1 domain activity in response to hydrodynamic forces, 2) the alterations in bond kinetics that result from incorporation of type 2B mutations into the VWF-A1 domain, and 3) the susceptibility of the kinetics of the GPIb alpha-VWF-A1 bond to an applied force.
Transgenic Animals
The invention provides for transgenic non-human animals that comprise a genome contains a nucleotide sequence (such as a gene) encoding a modified form of the A1 domain of VWF. The modification can be an amino acid residue substitution at a position involved with binding to GPIb alpha or in close proximity to this region such as, but not limited to, positions 1263, 1269, 1274, 1287, 1302, 1308, 1313, 1314, 1326, 1329, 1330, 1333, 1344, 1347, 1350, 1370, 1379, 1381, 1385 1391, 1394, 1397, 1421, 1439, 1442, 1449, 1466, 1469, 1472, 1473, 1475, 1479. It can also comprise a partial or full replacement of the animal A1 domain of VWF with the A1 domain of human VWF. It can also comprise a partial or full replacement of the entire VWF gene in an animal with the human VWF gene. Such non-human animals include vertebrates such as ovines, bovines, rodents, non-human primates, porcines, caprines, equines, ruminants, lagomorphs, canines, felines, ayes, and the like. In one embodiment, non-human animals are selected from the order Rodentia that includes murines (such as rats and mice). In another embodiment, mice are particularly useful.
The transgenic non-human animals of the current invention are produced by experimental manipulation of the genome of the germline of the non-human animal (such as those animals described above). These genetically engineered non-human animals may be produced by several methods well known in the art which include the introduction of a “transgene” that comprises a nucleic acid (for example, DNA such as the A1 domain of VWF) integrated into a chromosome of the somatic and/or germ line cells of a non-human animal via methods known to one skilled in the art or into an embryonal target cell. A transgenic animal is an animal whose genome has been altered by the introduction of a transgene.
The term “transgene” as used herein refers to a foreign gene that is placed into an organism by introducing the foreign gene into newly fertilized eggs, embryonic stem (ES) cells, or early embryos. The term “foreign gene” refers to any nucleic acid (for example, a gene sequence) that is introduced into the genome of an animal by experimental manipulations. These nucleic acids may include gene sequences found in that animal so long as the introduced gene contains some modification (for example, the presence of a selectable marker gene, a point mutation—such as the base pair substitution mutant that contributes to the amino acid change at amino acid residue 1326 of the A1 domain of VWF—the presence of a loxP site, and the like) relative to the naturally-occurring gene.
The term “loxP site” refers to a short (34 bp) DNA sequence that is recognized by the Cre recombinase of the E. coli bacteriophage P1. In the presence of Cre recombinase, placement of two loxP sites in the same orientation on either side of a DNA segment can result in efficient excision of the intervening DNA segment, leaving behind only a single copy of the loxP site (Sauer and Henderson (1988) Proc. Natl. Acad. Sci. USA 85:5166).
In one embodiment, the invention provides for a targeting construct or vector that comprises a selectable marker gene flanked on either side by a modified A1 domain of VWF. The modification of the A1 domain can comprise an amino acid residue substitution at a position involved with binding to GPIb alpha (such as, but not limited to, positions 1263, 1269, 1274, 1287, 1302, 1308, 1313, 1314, 1326, 1329, 1330, 1333, 1344, 1347, 1350, 1370, 1379, 1381, 1385 1391, 1394, 1397, 1421, 1439, 1442, 1449, 1466, 1469, 1472, 1473, 1475, 1479) or can be a partial or full replacement of an animal (for example, a mouse) A1 domain of VWF with the A1 domain of human VWF. The targeting vector contains the modified A1 domain of VWF gene sequence sufficient to permit the homologous recombination of the targeting vector into at least one allele of the A1 domain of the VWF gene resident in the chromosomes of the target or recipient cell (for example, ES cells). The targeting vector will usually harbor 10 to 15 kb of DNA homologous to the A1 domain of the VWF gene, wherein this 10 to 15 kb of DNA will be divided more or less equally on each side of the selectable marker gene. The targeting vector can contain more than one selectable maker gene and when multiple selectable marker genes are utilized, the targeting vector usually contains a negative selectable marker (for example, the Herpes simplex virus tk (HSV-tk) gene) and a positive selectable marker (such as G418 or the neo gene). The positive selectable marker permits the selection of recipient cells containing an integrated copy of the targeting vector and but does not enable one skilled in the art to determine whether this integration occurred at the target site or at a random site. The presence of the negative selectable marker permits the identification of recipient cells containing the targeting vector at the targeted site (for example, which has integrated by virtue of homologous recombination into the target site). Cells growing in medium that selects against the expression of the negative selectable marker represents that the cells do not contain a copy of the negative selectable marker.
Targeting vectors can also be of the replacement-type wherein the integration of a replacement-type vector results in the insertion of a selectable marker into the target gene. Replacement-type targeting vectors may be employed to disrupt a gene (such as the VWF gene or the A1 domain of the VWF gene). This can result in the generation of a null allele; for example, an allele not capable of expressing a functional protein wherein the null alleles may be generated by deleting a portion of the coding region, deleting the entire gene, introducing an insertion and/or a frameshift mutation, and the like.
A selectable marker can include a gene which encodes an enzymatic activity that confers resistance to an antibiotic or drug upon the cell in which the selectable marker is expressed. Selectable markers may be positive. A positive selectable marker is usually a dominant selectable marker wherein the genes encode an enzymatic activity that can be detected in a mammalian cell or a cell line (including ES cells). Some non-limiting examples of dominant selectable markers include the bacterial xanthine-guanine phosphoribosyl transferase gene (also referred to as the gpt gene) which confers the ability to grow in the presence of mycophenolic acid, the bacterial hygromycin G phosphotransferase (hyg) gene which confers resistance to the antibiotic hygromycin, and the bacterial aminoglycoside 3′ phosphotransferase gene (also referred to as the neo gene) which confers resistance to the drug G418 in mammalian cells. Selectable markers may also be negative. Negative selectable markers encode an enzymatic activity whose expression is toxic to the cell when grown in an appropriate selective medium. One non-limiting example of a negative selectable marker is the HSV-tk gene wherein HSV-tk expression in cells grown in the presence of gancyclovir or acyclovir is catatonic. Growth of cells in selective medium containing acyclovir or gancyclovir therefore selects against cells capable of expressing a functional HSV TK enzyme.
The ES cells suitable of the present invention utilized to generate transgenic animals can harbor introduced expression vectors (constructs), such as plasmids and the like. The expression vector constructs can be introduced via transfection, lipofection, transformation, injection, electroporation, or infection. The expression vectors can contain coding sequences, or portions thereof, encoding proteins for expression. Such expression vectors can include the required components for the transcription and translation of the inserted coding sequence. Expression vectors containing sequences encoding the produced proteins and polypeptides, as well as the appropriate transcriptional and translational control elements, can be generated using methods well known to and practiced by those skilled in the art. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination which are described in J. Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y. and in F. M. Ausubel et al., 1989, Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y. In one embodiment, loxP expressing targeting vectors are used for transfection methods (such as pDNR-1r vector, pACD4K-C vector, and the like). In other embodiments, Cre-recombinase-expressing plasmids are also utilized (for example, crAVE cre recombinase vectors).
Introducing targeting vectors into embryonic stem (ES) cells can generate the mutant VWF-A1 transgenic animals of the present invention. ES cells are obtained by culturing pre-implantation embryos in vitro under appropriate conditions (Evans, et al. (1981) Nature 292:154-156; Bradley, et al. (1984) Nature 309:255-258; Gossler, et al. (1986) Proc. Acad. Sci. USA 83:9065-9069; and Robertson, et al. (1986) Nature 322:445-448). Using a variety of methods known to those skilled in the art, transgenes can be efficiently introduced into the ES cells via DNA transfection methods, which include (but are not limited to), protoplast or spheroplast fusion, electroporation, retrovirus-mediated transduction, calcium phosphate co-precipitation, lipofection, microinjection, and DEAE-dextran-mediated transfection. Following the introduction into the blastocoel of a blastocyst-stage embryo, transfected ES cells can thereafter colonize an embryo and contribute to the germ line of the resulting chimeric animal (see Jaenisch, (1988) Science 240:1468-1474). Assuming that the transgene provides a means for selection, the transfected ES cells may be subjected to various selection protocols to enrich for ES cells that have integrated the transgene prior to the introduction of transfected ES cells into the blastocoel. Alternatively, the polymerase chain reaction (PCR) may be used to screen for ES cells that have integrated the transgene and precludes the need for growth of the transfected ES cells under appropriate selective conditions prior to transfer into the blastocoel.
Alternative methods for the generation of transgenic animals (such as transgenic mice) containing an altered A1 domain of the VWF gene are established in the art. For example, embryonic cells at various stages of development can be used to introduce transgenes for the production of transgenic animals and different methods are used that depend on the stage of embryonic cell development. For microinjection methods, the zygote is best suited. In the mouse, the male pronucleus reaches the size of approximately 20 microns in diameter, which allows for reproducible injection of 1-2 picoliters (pl) of suspended DNA solution. A major advantage in using zygotes as a gene transfer target is that in most cases the injected DNA will be incorporated into the host genome before the first cleavage (Brinster, et al. (1985) Proc. Natl. Acad. Sci. USA 82:4438-4442). Thus, all cells of the transgenic non-human animal (such as a mouse) will carry the incorporated transgene (mutant A1 domain of the VWF gene), which can result in the efficient transmission of the transgene to the offspring of the founder since 50% of the germ cells will harbor the transgene (see U.S. Pat. No. 4,873,191).
Another method known in the art that can be used to introduce transgenes into a non-human animal is retroviral infection. The developing non-human embryo can be cultured in vitro to the blastocyst stage wherein during this time, the blastomeres can be targets for retroviral infection (Janenich (1976) Proc. Natl. Acad. Sci. USA 73:1260-1264). Enzymatic treatment to remove the zona pellucida can increase infection efficiency of the blastomeres (Hogan et al. (1986) in Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Plainview, N.Y.). The viral vector system used by one skilled in the art in order to introduce the transgene is usually a replication-defective retrovirus that harbors the transgene (Jahner, D. et al. (1985) Proc. Natl. Acad Sci. USA 82:6927-6931; Van der Putten, et al. (1985) Proc. Natl. Acad Sci. USA 82:6148-6152). Transfection can be easily and efficiently obtained via culturing blastomeres on a monolayer of virus-producing cells (Van der Putten, supra; Stewart, et al. (1987) EMBO J. 6:383-388). Infection can also be performed at a later stage whereby virus or virus-producing cells are injected into the blastocoele (Jahner, D. et al. (1982) Nature 298:623-628). Most of the founder non-human animals will be mosaic for the transgene since incorporation occurs only in a subset of cells that form the transgenic animal and the founder may additionally contain various retroviral insertions of the transgene at different positions in the genome that generally will segregate in the offspring. Additional methods of using retroviruses or retroviral vectors to create transgenic animals known to those skilled in the art involves microinjecting mitomycin C-treated cells or retroviral particles producing retrovirus into the perivitelline space of fertilized eggs or early embryos (see Haskell and Bowen (1995) Mol. Reprod. Dev. 40:386).
In one embodiment of the invention, non-human transgenic animals can be generated that express a modified A1 domain of the VWF sequence. The modified A1 domain can contain an amino acid residue substitution at a position involved with binding of the VWF protein to GPIb alpha (such as, but not limited to, positions 1263, 1269, 1274, 1287, 1302, 1308, 1313, 1314, 1326, 1329, 1330, 1333, 1344, 1347, 1350, 1370, 1379, 1381, 1385 1391, 1394, 1397, 1421, 1439, 1442, 1449, 1466, 1469, 1472, 1473, 1475, 1479). In some embodiments, the VWF-A1 domain can comprise a single mutation, while in other embodiments, it can comprise 2 or more mutations.
In one embodiment of the invention, the modification of the A1 domain can be a partial or full replacement of an animal (for example, a mouse) A1 domain of VWF with the A1 domain of human VWF. In other words, the A1 domain in the animal VWF is removed and replaced with the human A1 sequence. In another embodiment, the animal VWF A1 domain may be partially replaced so that some portion of the human A1 domain replaces a portion of the animal A1 domain. For example, human A1 domain sequence could comprise at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 99%, or 100% of the animal A1 domain. The A1 domain of human VWF protein comprises SEQ ID NO: 1. In another embodiment, the animal can be a model for pre-clinical testing of compounds, wherein the animal expresses a mutant von Willebrand Factor (VWF) A1 protein containing one or more mutations, such that the binding specificity of the mutant VWF-A1 protein changes from being specific for the animal platelets to being specific for human platelets. In another embodiment, the mutation occurs in the VWF-A1 domain of a mouse. In particular embodiments, the murine mutant VWF-A1 protein comprises at least one mutation comprising 1263P>S, 1269N>D, 1274K>R, 1287M>R, 1302G>D, 1308H>R, 1313R>W, 1314I>V, 1326R>H, 1329L>I, 1330E>G, 1333A>D, 1344T>A, 1347I>V, 1350T>A, 1370G>S, 1379H>R, 1381T>A, 1385T>M 1391P>Q, 1394A>S, 1397L>F, 1421S>N, 1439L>V, 1442G>S, 1449R>Q, 1466A>P, 1469Q>L, 1472Q>H, 1473V>M, 1475H>Q, 1479S>G, or any combination thereof. In a particularly useful embodiment, the murine mutant VWF-A1 protein comprises a 1326R>H mutation, a 1314I>V mutation, or a combination thereof.
The modification of the A1 domain can be an amino acid substitution at residue 1326 (for example Arg for His in the mouse). In some embodiments, the non-human transgenic animal harbors a mutant construct wherein an amino acid residue substitution at a position involved affects binding to GPIb alpha (such as, but not limited to, positions 1263, 1269, 1274, 1287, 1302, 1308, 1313, 1314, 1326, 1329, 1330, 1333, 1344, 1347, 1350, 1370, 1379, 1381, 1385 1391, 1394, 1397, 1421, 1439, 1442, 1449, 1466, 1469, 1472, 1473, 1475, 1479). In yet other embodiments, non-human transgenic animals can successfully harbor a type 2B (Ile1309Val; 1309I>V) mutation and/or an Arg1326His (1326R>H) mutant construct. In another embodiment, the non-human transgenic animal expresses an Arg1326His (1326R>H) mutation wherein the mutant VWF-A1 domain comprises SEQ ID NO: 5, which corresponds to the His amino acid at the same position in humans, canines, chimpanzees, rat, porcine, felines, equines, bovine, and the like (Jenkins et al., (1998) Blood 91(6): 2032-44). In further embodiments of the invention, the non-human transgenic animal is a mouse. Example 3 below describes the transgenic animal of the current invention.
Molecular Manipulations of VWF-A1 and its Corresponding Mutants
The present invention utilizes conventional molecular biology, microbiology, and recombinant DNA techniques available to one of ordinary skill in the art. Such techniques are well known to the skilled worker and are explained fully in the literature. See, e.g., Maniatis, Fritsch & Sambrook, “Molecular Cloning: A Laboratory Manual” (1982): “DNA Cloning: A Practical Approach,” Volumes I and II (D. N. Glover, ed., 1985); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Nucleic Acid Hybridization” (B. D. Hames & S. J. Higgins, eds., 1985); “Transcription and Translation” (B. D. Hames & S. J. Higgins, eds., 1984); “Animal Cell Culture” (R. I. Freshney, ed., 1986); “Immobilized Cells and Enzymes” (IRL Press, 1986): B. Perbal, “A Practical Guide to Molecular Cloning” (1984), and Sambrook, et al., “Molecular Cloning: a Laboratory Manual” (1989).
The VWF sequences from mouse, human, have been aligned as shown in Jenkins et al. (1998) “Molecular Modeling of Ligand and Mutation Sites of the Type A Domains of Human von Willebrand Factor and Their Relevance to von Willebrand's Disease” Vol. 91, No. 6, Blood, pp. 2032-2044.
The DNA and polypeptide sequences of human VWF are readily available to those skilled in the art, under Genbank Accession No. X04385. The polypeptide sequence of the A1 domain of human VWF, which runs from amino acid residue number 1260 to amino acid residue number 1480 of the nucleotide sequence of SEQ ID NO:6, is shown in SEQ ID NO: 1. The polypeptide sequence of the A1 domain of mouse VWF, which runs from amino acid residue number 1260 to amino acid residue number 1480 of the of SEQ ID NO:8, is shown in SEQ ID NO: 2.
SEQ ID NO: 1 is the human wild type amino acid sequence corresponding to the A1 domain of VWF. The residues shown in SEQ ID NO: 1 are residues 1260-1480, the A1 domain, of SEQ ID NO: 6.







SEQ ID NO: 1:


EDISEPPLHDFYCSRLLDLVFLLDGSSRLSEAEFEVLKAFVVDMMERLRI


 


SQKWVRVAVVEYHDGSHAYIGLKDRKRPSELRRIASQVKYAGSQVASTSE


 


VLKYTLFQIFSKIDRPEASRIALLLMASQEPQRMSRNFVRYVQGLKKKKV


 


IVIPVGIGPHANLKQIRLIEKQAPENKAFVLSSVDELEQQRDEIVSYLCD


 


LAPEAPPPTLPPHMAQVTVGP






SEQ ID NO: 2 is the mouse wild type amino acid sequence corresponding to the A1 domain of VWF. The residues shown in SEQ ID NO:2 are residue numbers 1260-1480 from the full length mouse VWF shown in SEQ ID NO: 8.







SEQ ID NO: 2:


EDTPEPPLHNFYCSKLLDLVFLLDGSSMLSEAEFEVLKAFVVGMMERLH


 


ISQKRIRVAVVEYHDGSRAYLELKARKRPSELRRITSQIKYTGSQVAST


 


SEVLKYTLFQIFGKIDRPEASHITLLLTASQEPPRMARNLVRYVQGLKK


 


KKVIVIPVGIGPHASLKQIRLIEKQAPENKAFLLSGVDELEQRRDEIVS


 


YLCDLAPEAPAPTQPPQVAHVTVSP






The nucleotide sequence of the A1 domain of human VWF corresponding to amino acid residues 1260-1480 is shown in SEQ ID NO: 3 and of mouse VWF is shown in SEQ ID NO: 4 below.







SEQ ID NO: 3 is the human wild type nucleotide


 


sequence corresponding to the A1 domain of VWF:


 


GAGGACATCTCGGAACCGCCGTTGCACGATTTCTACTGCAGCAGGCTAC


 


TGGACCTGGTCTTCCTGCTGGATGGCTCCTCCAGGCTGTCCGAGGCTGA


 


GTTTGAAGTGCTGAAGGCCTTTGTGGTGGACATGATGGAGCGGCTGCGC


 


ATCTCCCAGAAGTGGGTCCGCGTGGCCGTGGTGGAGTACCACGACGGCT


 


CCCACGCCTACATCGGGCTCAAGGACCGGAAGCGACCGTCAGAGCTGCG


 


GCGCATTGCCAGCCAGGTGAAGTATGCGGGCAGCCAGGTGGCCTCCACC


 


AGCGAGGTCTTGAAATACACACTGTTCCAAATCTTCAGCAAGATCGACC


 


GCCCTGAAGCCTCCCGCATCGCCCTGCTCCTGATGGCCAGCCAGGAGCC


 


CCAACGGATGTCCCGGAACTTTGTCCGCTACGTCCAGGGCCTGAAGAAG


 


AAGAAGGTCATTGTGATCCCGGTGGGCATTGGGCCCCATGCCAACCTCA


 


AGCAGATCCGCCTCATCGAGAAGCAGGCCCCTGAGAACAAGGCCTTCGT


 


GCTGAGCAGTGTGGATGAGCTGGAGCAGCAAAGGGACGAGATCGTTAGC


 


TACCTCTGTGACCTTGCCCCTGAAGCCCCTCCTCCTACTCTGCCCCCCC


 


ACATGGCACAAGTCACTGTGGGCCCG


 


SEQ ID NO: 4 is the mouse wild type nucleotide


 


sequence corresponding to the A1 domain of VWF:


 


GAGGATACCCCCGAGCCCCCCCTGCACAACTTCTACTGCAGCAAGCTGC


 


TGGATCTTGTCTTCCTGCTGGATGGCTCCTCTATGTTGTCCGAGGCTGA


 


GTTTGAAGTGCTCAAAGCTTTTGTGGTGGGCATGATGGAGAGGTTACAC


 


ATCTCTCAGAAGCGCATCCGCGTGGCAGTGGTAGAGTACCATGATGGCT


 


CCCGTGCCTACCTTGAGCTCAAGGCCCGGAAGCGACCCTCAGAGCTTCG


 


GCGCATCACCAGCCAGATTAAGTATACAGGCAGCCAGGTGGCCTCTACC


 


AGTGAGGTTTTGAAGTACACACTGTTCCAGATCTTTGGCAAAATTGACC


 


GCCCTGAAGCCTCCCATATCACTCTGCTCCTGACTGCTAGCCAGGAGCC


 


CCCACGGATGGCTAGGAATTTGGTCCGCTATGTCCAAGGTCTGAAGAAG


 


AAGAAGGTTATCGTGATCCCTGTGGGCATTGGGCCCCACGCCAGCCTCA


 


AACAGATCCGCCTCATCGAGAAGCAGGCCCCTGAAAACAAGGCTTTTCT


 


GCTCAGTGGGGTGGATGAGCTGGAGCAGAGAAGAGATGAGATAGTCAGC


 


TACCTCTGTGACCTTGCTCCCGAGGCCCCAGCCCCAACTCAGCCTCCAC


 


AGGTAGCCCACGTCACCGTGAGTCCA






Human mRNA for pre-pro-von Willebrand factor:








SEQ ID NO: 6: Amino Acid Seguence Human VWF-(residue 1 to residue 2813)










1
MIPARFAGVL LALALILPGT LCAEGTRGRS STARCSLFGS DFVNTFDGSM YSFAGYCSYL



 


61
LAGGCQKRSF SIIGDFQNGK RVSLSVYLGE FFDIHLFVNG TVTQGDQRVS MPYASKGLYL


 


121
ETEAGYYKLS GEAYGFVARI DGSGNFQVLL SDRYFNKTCG LCGNFNIFAE DDFMTQEGTL


 


181
TSDPYDFANS WALSSGEQWC ERASPPSSSC NISSGEMQKG LWEQCQLLKS TSVFARCHPL


 


241
VDPEPFVALC EKTLCECAGG LECACPALLE YARTCAQEGM VLYGWTDHSA CSPVCPAGME


 


301
YRQCVSPCAR TCQSLHINEM CQERCVDGCS CPEGQLLDEG LCVESTECPC VHSGKRYPPG


 


361
TSLSRDCNTC ICRNSQWICS NEECPGECLV TGQSHFKSFD NRYFTFSGIC QYLLARDCQD


 


421
HSFSIVIETV QCADDRDAVC TRSVTVRLPG LHNSLVKLKH GAGVAMDGQD IQLPLLKGDL


 


481
RIQHTVTASV RLSYGEDLQM DWDGRGRLLV KLSPVYAGKT CGLCGNYNGN QGDDFLTPSG


 


541
LAEPRVEDFG NAWKLHGDCQ DLQKQHSDPC ALNPRMTRFS EEACAVLTSP TFEACHRAVS


 


601
PLPYLRNCRY DVCSCSDGRE CLCGALASYA AACAGRGVRV AWREPGRCEL NCPKGQVYLQ


 


661
CGTPCNLTCR SLSYPDEECN EACLEGCFCP PGLYMDERGD CVPKAQCPCY YDGEIFQPED


 


721
IFSDHHTMCY CEDGFMHCTM SGVPGSLLPD AVLSSPLSHR SKRSLSCRPP MVKLVCPADN


 


781
LRAEGLECTK TCQNYDLECM SMGCVSGCLC PPGMVRHENR CVALERCPCF HQGKEYAPGE


 


841
TVKIGCNTCV CRDRKWNCTD HVCDATCSTI GMAHYLTFDG LKYLFPGECQ YVLVQDYCGS


 


901
NPGTFRILVG NKGCSHPSVK CKKRVTILVE GGEIELFDGE VNVKRPMKDE THFEVVESGR


 


961
YIILLLGKAL SVVWDRHLSI SVVLKQTYQE KVCGLCGNFD GIQNNDLTSS NLQVEEDPVD


 


1021
FGNSWKVSSQ CADTRKVPLD SSPATCHNNI MKQTMVDSSC RILTSDVFQD CNKLVDPEPY


 


1081
LDVCIYDTCS CESIGDCACF CDTIAAYAHV CAQHGKVVTW RTATLCPQSC EERNLRENGY


 


1141
ECEWRYNSCA PACQVTCQHP EPLACPVQCV EGCHAHCPPG KILDELLQTC VDPEDCPVCE


 


1201
VAGRRFASGK KVTLNPSDPE HCQICHCDVV NLTCEACQEP GGLVVPPTDA PVSPTTLYVE


 


1261
DISEPPLHDF YCSRLLDLVF LLDGSSRLSE AEFEVLKAFV VDMMERLRIS QKWVRVAVVE


 


1321
YHDGSHAYIG LKDRKRPSEL RRIASQVKYA GSQVASTSEV LKYTLFQIFS KIDRPEASRI


 


1381
ALLLMASQEP QRMSRNFVRY VQGLKKKKVI VIPVGIGPHA NLKQIRLIEK QAPENKAFVL


 


1441
SSVDELEQQR DEIVSYLCDL APEAPPPTLP PHMAQVTVGP GLLGVSTLGP KRNSMVLDVA


 


1501
FVLEGSDKIG EADFNRSKEF MEEVIQRMDV GQDSIHVTVL QYSYMVTVEY PFSEAQSKGD


 


1561
ILQRVREIRY QGGNRTNTGL ALRYLSDHSF LVSQGDREQA PNLVYMVTGN PASDEIKRLP


 


1621
GDIQVVPIGV GPNANVQELE RIGWPNAPIL IQDFETLPRE APDLVLQRCC SGEGLQIPTL


 


1681
SPAPDCSQPL DVILLLDGSS SFPASYFDEM KSFAKAFISK ANIGPRLTQV SVLQYGSITT


 


1741
IDVPWNVVPE KAHLLSLVDV MQREGGPSQI GDALGFAVRY LTSEMHGARP GASKAVVILV


 


1801
TDVSVDSVDA AADAARSNRV TVFPIGIGDR YDAAQLRILA GPAGDSNVVK LQRIEDLPTM


 


1861
VTLGNSFLHK LCSGFVRICM DEDGNEKRPG DVWTLPDQCH TVTCQPDGQT LLKSHRVNCD


 


1921
RGLRPSCPNS QSPVKVEETC GCRWTCPCVC TGSSTRHIVT FDGQNFKLTG SCSYVLFQNK


 


1981
EQDLEVILHN GACSPGARQG CMKSIEVKHS ALSVELHSDM EVTVNGRLVS VPYVGGNMEV


 


2041
NVYGAIMHEV RFNHLGHIFT FTPQNNEFQL QLSPKTFASK TYGLCGICDE NGANDFMLRD


 


2101
GTVTTDWKTL VQEWTVQRPG QTCQPILEEQ CLVPDSSHCQ VLLLPLFAEC HKVLAPATFY


 


2161
AICQQDSCHQ EQVCEVIASY AHLCRTNGVC VDWRTPDFCA MSCPPSLVYN HCEHGCPRHC


 


2221
DGNVSSCGDH PSEGCFCPPD KVMLEGSCVP EEACTQCIGE DGVQHQFLEA WVPDHQPCQI


 


2281
CTCLSGRKVN CTTQPCPTAK APTCGLCEVA RLRQNADQCC PEYECVCDPV SCDLPPVPHC


 


2341
ERGLQPTLTN PGECRPNFTC ACRKEECKRV SPPSCPPHRL PTLRKTQCCD EYECACNCVN


 


2401
STVSCPLGYL ASTATNDCGC TTTTCLPDKV CVHRSTIYPV GQFWEEGCDV CTCTDMEDAV


 


2461
MGLRVAQCSQ KPCEDSCRSG FTYVLHEGEC CGRCLPSACE VVTGSPRODS QSSWKSVGSQ


 


2521
WASPENPCLI NECVRVKEEV FIQQRNVSCP QLEVPVCPSG FQLSCKTSAC CPSCRCERME


 


2581
ACMLNGTVIG PGKTVMIDVC TTCRCMVQVG VISGFKLECR KTTCNPCPLG YKEENNTGEC


 


2641
CGRCLPTACT IQLRGGQIMT LKRDETLQDG CDTHFCKVNE RGEYFWEKRV TGCPPFDEHK


 


2701
CLAEGGKIMK IPGTCCDTCE EPECNDITAR LQYVKVGSCK SEVEVDIHYC QGKCASKAMY


 


2761
SIDINDVQDQ CSCCSPTRTE PMQVALHCTN GSVVYHEVLN AMECKCSPRK CSK


 








VWF mature peptide (AA 763-2790)



VWF pro polypeptide (AA 1-2790)


 


SEQ ID NO: 7 Nucleic acid seguence-human VWF









1
agctcacagc tattgtggtg ggaaagggag ggtggttggt ggatgtcaca gcttgggctt



 


61
tatctccccc agcagtgggg actccacagc ccctgggcta cataacagca agacagtccg


 


121
gagctgtagc agacctgatt gagcctttgc agcagctgag agcatggcct agggtgggcg


 


181
gcaccattgt ccagcagctg agtttcccag ggaccttgga gatagccgca gccctcattt


 


241
gcaggggaag gcaccattgt ccagcagctg agtttcccag ggaccttgga gatagccgca


 


301
gccctcattt atgattcctg ccagatttgc cggggtgctg cttgctctgg ccctcatttt


 


361
gccagggacc ctttgtgcag aaggaactcg cggcaggtca tccacggccc gatgcagcct


 


421
tttcggaagt gacttcgtca acacctttga tgggagcatg tacagctttg cgggatactg


 


481
cagttacctc ctggcagggg gctgccagaa acgctccttc tcgattattg gggacttcca


 


541
gaatggcaag agagtgagcc tctccgtgta tcttggggaa ttttttgaca tccatttgtt


 


601
tgtcaatggt accgtgacac agggggacca aagagtctcc atgccctatg cctccaaagg


 


661
gctgtatcta gaaactgagg ctgggtacta caagctgtcc ggtgaggcct atggctttgt


 


721
ggccaggatc gatggcagcg gcaactttca agtcctgctg tcagacagat acttcaacaa


 


781
gacctgcggg ctgtgtggca actttaacat ctttgctgaa gatgacttta tgacccaaga


 


841
agggaccttg acctcggacc cttatgactt tgccaactca tgggctctga gcagtggaga


 


901
acagtggtgt gaacgggcat ctcctcccag cagctcatgc aacatctcct ctggggaaat


 


961
gcagaagggc ctgtgggagc agtgccagct tctgaagagc acctcggtgt ttgcccgctg


 


1021
ccaccctctg gtggaccccg agccttttgt ggccctgtgt gagaagactt tgtgtgagtg


 


1081
tgctgggggg ctggagtgcg cctgccctgc cctcctggag tacgcccgga cctgtgccca


 


1141
ggagggaatg gtgctgtacg gctggaccga ccacagcgcg tgcagcccag tgtgccctgc


 


1201
tggtatggag tataggcagt gtgtgtcccc ttgcgccagg acctgccaga gcctgcacat


 


1261
caatgaaatg tgtcaggagc gatgcgtgga tggctgcagc tgccctgagg gacagctcct


 


1321
ggatgaaggc ctctgcgtgg agagcaccga gtgtccctgc gtgcattccg gaaagcgcta


 


1381
ccctcccggc acctccctct ctcgagactg caacacctgc atttgccgaa acagccagtg


 


1441
gatctgcagc aatgaagaat gtccagggga gtgccttgtc actggtcaat cccacttcaa


 


1501
gagctttgac aacagatact tcaccttcag tgggatctgc cagtacctgc tggcccggga


 


1561
ttgccaggac cactccttct ccattgtcat tgagactgtc cagtgtgctg atgaccgcga


 


1621
cgctgtgtgc acccgctccg tcaccgtccg gctgcctggc ctgcacaaca gccttgtgaa


 


1681
actgaagcat ggggcaggag ttgccatgga tggccaggac atccagctcc ccctcctgaa


 


1741
aggtgacctc cgcatccagc atacagtgac ggcctccgtg cgcctcagct acggggagga


 


1801
cctgcagatg gactgggatg gccgcgggag gctgctggtg aagctgtccc ccgtctacgc


 


1861
cgggaagacc tgcggcctgt gtgggaatta caatggcaac cagggcgacg acttccttac


 


1921
cccctctggg ctggcagagc cccgggtgga ggacttcggg aacgcctgga agctgcacgg


 


1981
ggactgccag gacctgcaga agcagcacag cgatccctgc gccctcaacc cgcgcatgac


 


2041
caggttctcc gaggaggcgt gcgcggtcct gacgtccccc acattcgagg cctgccatcg


 


2101
tgccgtcagc ccgctgccct acctgcggaa ctgccgctac gacgtgtgct cctgctcgga


 


2161
cggccgcgag tgcctgtgcg gcgccctggc cagctatgcc gcggcctgcg cggggagagg


 


2221
cgtgcgcgtc gcgtggcgcg agccaggccg ctgtgagctg aactgcccga aaggccaggt


 


2281
gtacctgcag tgcgggaccc cctgcaacct gacctgccgc tctctctctt acccggatga


 


2341
ggaatgcaat gaggcctgcc tggagggctg cttctgcccc ccagggctct acatggatga


 


2401
gaggggggac tgcgtgccca aggcccagtg cccctgttac tatgacggtg agatcttcca


 


2461
gccagaagac atcttctcag accatcacac catgtgctac tgtgaggatg gcttcatgca


 


2521
ctgtaccatg agtggagtcc ccggaagctt gctgcctgac gctgtcctca gcagtcccct


 


2581
gtctcatcgc agcaaaagga gcctatcctg tcggcccccc atggtcaagc tggtgtgtcc


 


2641
cgctgacaac ctgcgggctg aagggctcga gtgtaccaaa acgtgccaga actatgacct


 


2701
ggagtgcatg agcatgggct gtgtctctgg ctgcctctgc cccccgggca tggtccggca


 


2761
tgagaacaga tgtgtggccc tggaaaggtg tccctgcttc catcagggca aggagtatgc


 


2821
ccctggagaa acagtgaaga ttggctgcaa cacttgtgtc tgtcgggacc ggaagtggaa


 


2881
ctgcacagac catgtgtgtg atgccacgtg ctccacgatc ggcatggccc actacctcac


 


2941
cttcgacggg ctcaaatacc tgttccccgg ggagtgccag tacgttctgg tgcaggatta


 


3001
ctgcggcagt aaccctggga cctttcggat cctagtgggg aataagggat gcagccaccc


 


3061
ctcagtgaaa tgcaagaaac gggtcaccat cctggtggag ggaggagaga ttgagctgtt


 


3121
tgacggggag gtgaatgtga agaggcccat gaaggatgag actcactttg aggtggtgga


 


3181
gtctggccgg tacatcattc tgctgctggg caaagccctc tccgtggtct gggaccgcca


 


3241
cctgagcatc tccgtggtcc tgaagcagac ataccaggag aaagtgtgtg gcctgtgtgg


 


3301
gaattttgat ggcatccaga acaatgacct caccagcagc aacctccaag tggaggaaga


 


3361
ccctgtggac tttgggaact cctggaaagt gagctcgcag tgtgctgaca ccagaaaagt


 


3421
gcctctggac tcatcccctg ccacctgcca taacaacatc atgaagcaga cgatggtgga


 


3481
ttcctcctgt agaatcctta ccagtgacgt cttccaggac tgcaacaagc tggtggaccc


 


3541
cgagccatat ctggatgtct gcatttacga cacctgctcc tgtgagtcca ttggggactg


 


3601
cgcctgcttc tgcgacacca ttgctgccta tgcccacgtg tgtgcccagc atggcaaggt


 


3661
ggtgacctgg aggacggcca cattgtgccc ccagagctgc gaggagagga atctccggga


 


3721
gaacgggtat gagtgtgagt ggcgctataa cagctgtgca cctgcctgtc aagtcacgtg


 


3781
tcagcaccct gagccactgg cctgccctgt gcagtgtgtg gagggctgcc atgcccactg


 


3841
ccctccaggg aaaatcctgg atgagctttt gcagacctgc gttgaccctg aagactgtcc


 


3901
agtgtgtgag gtggctggcc ggcgttttgc ctcaggaaag aaagtcacct tgaatcccag


 


3961
tgaccctgag cactgccaga tttgccactg tgatgttgtc aacctcacct gtgaagcctg


 


4021
ccaggagccg ggaggcctgg tggtgcctcc cacagatgcc ccggtgagcc ccaccactct


 


4081
gtatgtggag gacatctcgg aaccgccgtt gcacgatttc tactgcagca ggctactgga


 


4141
cctggtcttc ctgctggatg gctcctccag gctgtccgag gctgagtttg aagtgctgaa


 


4201
ggcctttgtg gtggacatga tggagcggct gcgcatctcc cagaagtggg tccgcgtggc


 


4261
cgtggtggag taccacgacg gctcccacgc ctacatcggg ctcaaggacc ggaagcgacc


 


4321
gtcagagctg cggcgcattg ccagccaggt gaagtatgcg ggcagccagg tggcctccac


 


4381
cagcgaggtc ttgaaataca cactgttcca aatcttcagc aagatcgacc gccctgaagc


 


4441
ctcccgcatc gccctgctcc tgatggccag ccaggagccc caacggatgt cccggaactt


 


4501
tgtccgctac gtccagggcc tgaagaagaa gaaggtcatt gtgatcccgg tgggcattgg


 


4561
gccccatgcc aacctcaagc agatccgcct catcgagaag caggcccctg agaacaaggc


 


4621
cttcgtgctg agcagtgtgg atgagctgga gcagcaaagg gacgagatcg ttagctacct


 


4681
ctgtgacctt gcccctgaag cccctcctcc tactctgccc ccccacatgg cacaagtcac


 


4741
tgtgggcccg gggctcttgg gggtttcgac cctggggccc aagaggaact ccatggttct


 


4801
ggatgtggcg ttcgtcctgg aaggatcgga caaaattggt gaagccgact tcaacaggag


 


4861
caaggagttc atggaggagg tgattcagcg gatggatgtg ggccaggaca gcatccacgt


 


4921
cacggtgctg cagtactcct acatggtgac cgtggagtac cccttcagcg aggcacagtc


 


4981
caaaggggac atcctgcagc gggtgcgaga gatccgctac cagggcggca acaggaccaa


 


5041
cactgggctg gccctgcggt acctctctga ccacagcttc ttggtcagcc agggtgaccg


 


5101
ggagcaggcg cccaacctgg tctacatggt caccggaaat cctgcctctg atgagatcaa


 


5161
gaggctgcct ggagacatcc aggtggtgcc cattggagtg ggccctaatg ccaacgtgca


 


5221
ggagctggag aggattggct ggcccaatgc ccctatcctc atccaggact ttgagacgct


 


5281
cccccgagag gctcctgacc tggtgctgca gaggtgctgc tccggagagg ggctgcagat


 


5341
ccccaccctc tcccctgcac ctgactgcag ccagcccctg gacgtgatcc ttctcctgga


 


5401
tggctcctcc agtttcccag cttcttattt tgatgaaatg aagagtttcg ccaaggcttt


 


5461
catttcaaaa gccaatatag ggcctcgtct cactcaggtg tcagtgctgc agtatggaag


 


5521
catcaccacc attgacgtgc catggaacgt ggtcccggag aaagcccatt tgctgagcct


 


5581
tgtggacgtc atgcagcggg agggaggccc cagccaaatc ggggatgcct tgggctttgc


 


5641
tgtgcgatac ttgacttcag aaatgcatgg tgccaggccg ggagcctcaa aggcggtggt


 


5701
catcctggtc acggacgtct ctgtggattc agtggatgca gcagctgatg ccgccaggtc


 


5761
caacagagtg acagtgttcc ctattggaat tggagatcgc tacgatgcag cccagctacg


 


5821
gatcttggca ggcccagcag gcgactccaa cgtggtgaag ctccagcgaa tcgaagacct


 


5881
ccctaccatg gtcaccttgg gcaattcctt cctccacaaa ctgtgctctg gatttgttag


 


5941
gatttgcatg gatgaggatg ggaatgagaa gaggcccggg gacgtctgga ccttgccaga


 


6001
ccagtgccac accgtgactt gccagccaga tggccagacc ttgctgaaga gtcatcgggt


 


6061
caactgtgac cgggggctga ggccttcgtg ccctaacagc cagtcccctg ttaaagtgga


 


6121
agagacctgt ggctgccgct ggacctgccc ctgcgtgtgc acaggcagct ccactcggca


 


6181
catcgtgacc tttgatgggc agaatttcaa gctgactggc agctgttctt atgtcctatt


 


6241
tcaaaacaag gagcaggacc tggaggtgat tctccataat ggtgcctgca gccctggagc


 


6301
aaggcagggc tgcatgaaat ccatcgaggt gaagcacagt gccctctccg tcgagctgca


 


6361
cagtgacatg gaggtgacgg tgaatgggag actggtctct gttccttacg tgggtgggaa


 


6421
catggaagtc aacgtttatg gtgccatcat gcatgaggtc agattcaatc accttggtca


 


6481
catcttcaca ttcactccac aaaacaatga gttccaactg cagctcagcc ccaagacttt


 


6541
tgcttcaaag acgtatggtc tgtgtgggat ctgtgatgag aacggagcca atgacttcat


 


6601
gctgagggat ggcacagtca ccacagactg gaaaacactt gttcaggaat ggactgtgca


 


6661
gcggccaggg cagacgtgcc agcccatcct ggaggagcag tgtcttgtcc ccgacagctc


 


6721
ccactgccag gtcctcctct taccactgtt tgctgaatgc cacaaggtcc tggctccagc


 


6781
cacattctat gccatctgcc agcaggacag ttgccaccag gagcaagtgt gtgaggtgat


 


6841
cgcctcttat gcccacctct gtcggaccaa cggggtctgc gttgactgga ggacacctga


 


6901
tttctgtgct atgtcatgcc caccatctct ggtctacaac cactgtgagc atggctgtcc


 


6961
ccggcactgt gatggcaacg tgagctcctg tggggaccat ccctccgaag gctgtttctg


 


7021
ccctccagat aaagtcatgt tggaaggcag ctgtgtccct gaagaggcct gcactcagtg


 


7081
cattggtgag gatggagtcc agcaccagtt cctggaagcc tgggtcccgg accaccagcc


 


7141
ctgtcagatc tgcacatgcc tcagcgggcg gaaggtcaac tgcacaacgc agccctgccc


 


7201
cacggccaaa gctcccacgt gtggcctgtg tgaagtagcc cgcctccgcc agaatgcaga


 


7261
ccagtgctgc cccgagtatg agtgtgtgtg tgacccagtg agctgtgacc tgcccccagt


 


7321
gcctcactgt gaacgtggcc tccagcccac actgaccaac cctggcgagt gcagacccaa


 


7381
cttcacctgc gcctgcagga aggaggagtg caaaagagtg tccccaccct cctgcccccc


 


7441
gcaccgtttg cccacccttc ggaagaccca gtgctgtgat gagtatgagt gtgcctgcaa


 


7501
ctgtgtcaac tccacagtga gctgtcccct tgggtacttg gcctcaaccg ccaccaatga


 


7561
ctgtggctgt accacaacca cctgccttcc cgacaaggtg tgtgtccacc gaagcaccat


 


7621
ctaccctgtg ggccagttct gggaggaggg ctgcgatgtg tgcacctgca ccgacatgga


 


7681
ggatgccgtg atgggcctcc gcgtggccca gtgctcccag aagccctgtg aggacagctg


 


7741
tcggtcgggc ttcacttacg ttctgcatga aggcgagtgc tgtggaaggt gcctgccatc


 


7801
tgcctgtgag gtggtgactg gctcaccgcg gggggactcc cagtcttcct ggaagagtgt


 


7861
cggctcccag tgggcctccc cggagaaccc ctgcctcatc aatgagtgtg tccgagtgaa


 


7921
ggaggaggtc tttatacaac aaaggaacgt ctcctgcccc cagctggagg tccctgtctg


 


7981
cccctcgggc tttcagctga gctgtaagac ctcagcgtgc tgcccaagct gtcgctgtga


 


8041
gcgcatggag gcctgcatgc tcaatggcac tgtcattggg cccgggaaga ctgtgatgat


 


8101
cgatgtgtgc acgacctgcc gctgcatggt gcaggtgggg gtcatctctg gattcaagct


 


8161
ggagtgcagg aagaccacct gcaacccctg ccccctgggt tacaaggaag aaaataacac


 


8221
aggtgaatgt tgtgggagat gtttgcctac ggcttgcacc attcagctaa gaggaggaca


 


8281
gatcatgaca ctgaagcgtg atgagacgct ccaggatggc tgtgatactc acttctgcaa


 


8341
ggtcaatgag agaggagagt acttctggga gaagagggtc acaggctgcc caccctttga


 


8401
tgaacacaag tgtctggctg agggaggtaa aattatgaaa attccaggca cctgctgtga


 


8461
cacatgtgag gagcctgagt gcaacgacat cactgccagg ctgcagtatg tcaaggtggg


 


8521
aagctgtaag tctgaagtag aggtggatat ccactactgc cagggcaaat gtgccagcaa


 


8581
agccatgtac tccattgaca tcaacgatgt gcaggaccag tgctcctgct gctctccgac


 


8641
acggacggag cccatgcagg tggccctgca ctgcaccaat ggctctgttg tgtaccatga


 


8701
ggttctcaat gccatggagt gcaaatgctc ccccaggaag tgcagcaagt gaggctgctg


 


8761
cagctgcatg ggtgcctgct gctgcctgcc ttggcctgat ggccaggcca gagtgctgcc


 


8821
agtcctctgc atgttctgct cttgtgccct tctgagccca caataaaggc tgagctctta


 


8881
tcttgctgca tgttctgctc ttgtgccctt ctgagcccac aat


 








Mus musculus strain CASA/RkJ VWF (Vwf) mRNA



SEQ ID NO: 8 Amino Acid Seguence of Mouse VWF (residue no. 1 to residue no. 2813)









1
MNPFRYEICL LVLALTWPGT LCTEKPRDRP STARCSLFGD DFINTFDETM YSFAGGCSYL



 


61
LAGDCQKRSF SILGNFQDGK RMSLSVYLGE FFDIHLFANG TVTQGDQSIS MPYASQGLYL


 


121
EREAGYYKLS SETFGFAARI DONGNFQVLM SDRHFNKTCG LCGDFNIFAE DDFRTQEGTL


 


181
TSDPYDFANS WALSSEEQRC KRASPPSRNC ESSSGDMHQA MWEQCQLLKT ASVFARCHPL


 


241
VDPESFVALC EKILCTCATG PECACPVLLE YARTCAQEGM VLYGWTDHSA CRPACPAGME


 


301
YKECVSPCPR TCQSLSINEV CQQQCVDGCS CPEGELLDED RCVQSSDCPC VHAGKRYPPG


 


361
TSLSQDCNTC ICRNSLWICS NEECPGECLV TGQSHEKSED NRYFTFSGIC QYLLARDCED


 


421
HTFSIVIETM QCADDPDAVC TRSVSVRLSA LHNSLVKLKH GGAVGIDGQD VQLPFLQGDL


 


481
RIQHTVMASV RLSYAEDLQM DWDGRGRLLV KLSPVYSGKT CGLCGNYNGN KGDDFLTPAG


 


541
LVEPLVVDFG NAWKLQGDCS DLRRQHSDPC SLNPRLTRFA EEACALLTSS KFEACHHAVS


 


601
PLPYLQNCRY DVCSCSDSRD CLCNAVANYA AECARKGVHI GWREPGFCAL GCPQGQVYLQ


 


661
CGNSCNLTCR SLSLPDEECS EVCLEGCYCP PGLYQDERGD CVPKAQCPCY YDGELFQPAD


 


721
IFSDHHTMCY CEDGFMHCTT SGTLGSLLPD TVLSSPLSHR SKRSLSCRPP MVKLVCPADN


 


781
PRAQGLECAK TCQNYDLECM SLGCVSGCLC PPGMVRHENK CVALERCPCF HQGAEYAPGD


 


841
TVKIGCNTCV CRERKWNCTN HVCDATCSAI GMAHYLTFDG LKYLFPGECQ YVLVQDYCGS


 


901
NPGTFQILVG NEGCSYPSVK CRKRVTILVD GGELELFDGE VNVKRPLRDE SHFEVVESGR


 


961
YVILLLGQAL SVVWDHHLSI SVVLKHTYQE QVCGLCGNFD GIQNNDFTTS SLQVEEDPVN


 


1021
FGNSWKVSSQ CADTRKLSLD VSPATCHNNI MKQTMVDSAC RILTSDVFQG CNRLVDPEPY


 


1081
LDICIYDTCS CESIGDCACF CDTIAAYAHV CAQHGQVVAW RTPTLCPQSC EEKNVRENGY


 


1141
ECEWRYNSCA PACPVTCQHP EPLACPVQCV EGCHAHCPPG RILDELLQTC VDPQDCPVCE


 


1201
VAGRRLAPGK KITLSPDDPA HCQNCHCDGV NLTCEACQEP GGLVAPPTDA PVSSTTPYVE


 


1261
DTPEPPLHNF YCSKLLDLVF LLDGSSMLSE AEFEVLKAFV VGMMERLHIS QKRIRVAVVE


 


1321
YHDGSRAYLE LKARKRPSEL RRITSQIKYT GSQVASTSEV LKYTLFQIFG KIDRPEASHI


 


1381
TLLLTASQEP PRMARNLVRY VQGLKKKKVI VIPVGIGPHA SLKQIRLIEK QAPENKAFLL


 


1441
SGVDELEQRR DEIVSYLCDL APEAPAPTQP PQVAHVTVSP GIAGISSPGP KRKSMVLDVV


 


1501
FVLEGSDEVG EANFNKSKEF VEEVIQRMDV SPDATRISVL QYSYTVTMEY AFNGAQSKEE


 


1561
VLRHVREIRY QGGNRTNTGQ ALQYLSEHSF SPSQGDRVEA PNLVYMVTGN PASDEIKRLP


 


1621
GDIQVVPIGV GPHANMQELE RISRPIAPIF IRDFETLPRE APDLVLQTCC SKEGLQLPTL


 


1681
PPLPDCSQPL DVVLLLDGSS SLPESSFDKM KSFAKAFISK ANIGPHLTQV SVIQYGSINT


 


1741
IDVPWNVVQE KAHLQSLVDL MQQEGGPSQI GDALAFAVRY VTSQIHGARP GASKAVVIII


 


1801
MDTSLDPVDT AADAARSNRV AVFPVGVGDR YDEAQLRILA GPGASSNVVK LQQVEDLSTM


 


1861
ATLGNSFFHK LCSGFSGVCV DEDGNEKRPG DVWTLPDQCH TVTCLANGQT LLQSHRVNCD


 


1921
HGPRPSCANS QSPVRVEETC GCRWTCPCVC TGSSTRHIVT FDGQNFKLTG SCSYVIFQNK


 


1981
EQDLEVLLHN GACSPGAKQA CMKSIEIKHA GVSAELHSNM EMAVDGRLVL APYVGENMEV


 


2041
SIYGAIMYEV RFTHLGHILT YTPQNNEFQL QLSPKTFASK MHGLCGICDE NGANDFTLRD


 


2101
GTVTTDWKRL VQEWTVQQPG YTCQAVPEEQ CPVSDSSHCQ VLLSASFAEC HKVIAPATFH


 


2161
TICQQDSCHQ ERVCEVIASY AHLCRTSGVC VDWRTTDFCA MSCPPSLVYN HCERGCPRHC


 


2221
DGNTSFCGDH PSEGCFCPQH QVFLEGSCVP EEACTQCVGE DGVRHQFLET WVPDHQPCQI


 


2281
CMCLSGRKIN CTAQPCPTAR APTCGPCEVA RLKQSTNLCC PEYECVCDLF NCNLPPVPPC


 


2341
EGGLQPTLTN PGECRPTFTC ACRKEECKRV SPPSCPPHRT PTLRKTQCCD EYECACSCVN


 


2401
STLSCPLGYL ASATTNDCGC TTTTCLPDKV CVHRGTVYPV GQFWEEGCDT CTCTDMEDTV


 


2461
VGLRVVQCSQ RPCEDSCQPG FSYVLHEGEC CGRCLPSACK VVAGSLRGDS HSSWKSVGSR


 


2521
WAVPENPCLV NECVRVEDAV FVQQRNISCP QLAVPTCPTG FQLNCETSEC CPSCHCEPVE


 


2581
ACLLNGTIIG PGKSVMVDLC TTCRCIVQTD AISRFKLECR KTTCEACPMG YREEKSQGEC


 


2641
CGRCLPTACT IQLRGGRIMT LKQDETFQDG CDSHLCRVNE RGEYIWEKRV TGCPPFDEHK


 


2701
CLAEGGKIVK IPGTCCDTCE EPDCKDITAK VQYIKVGDCK SQEEVDIHYC QGKCASKAVY


 


2761
SIDIEDVQEQ CSCCLPSRTE PMRVPLHCTN GSVVYHEVIN AMQCRCSPRN CSK


 








SEQ ID NO: 9-Nucleic Acid Seguence of Mouse VWF










1
agtagcggct gggtttcctc aagggacctt ggagatacag cccctgtttg tatgggcaag



 


61
atgaaccctt tcaggtatga gatctgcctg cttgttctgg ccctcacctg gccagggacc


 


121
ctctgcacag aaaagccccg tgacaggccg tcgacggccc gatgcagcct ctttggggac


 


181
gacttcatca acacgtttga tgagaccatg tacagctttg cagggggctg cagttatctc


 


241
ctggctgggg actgccagaa acgttccttc tccattctcg ggaacttcca agatggcaag


 


301
agaatgagcc tgtctgtgta tcttggggag ttttttgaca tccatttgtt tgccaatggc


 


361
accgtaacgc agggtgacca aagcatctcc atgccctacg cctcccaagg actctaccta


 


421
gaacgcgagg ctgggtacta taagctctcc agtgagacct ttggctttgc ggccagaatc


 


481
gatggcaatg gcaacttcca agtcctgatg tcagacagac acttcaacaa gacctgtggg


 


541
ctgtgcggtg attttaacat cttcgcggaa gatgatttta ggacgcagga ggggaccttg


 


601
acctcagacc cctatgattt tgccaactcc tgggccctga gcagtgagga acagcggtgt


 


661
aaacgggcat ctcctcccag caggaactgc gagagctctt ctggggacat gcatcaggcc


 


721
atgtgggagc aatgccagct actgaagacg gcatcggtgt ttgcccgctg ccaccctctg


 


781
gtggatcccg agtcctttgt ggctctgtgt gagaagattt tgtgtacgtg tgctacgggg


 


841
ccagagtgcg catgtcctgt actccttgag tatgcccgaa cctgcgccca ggaagggatg


 


901
gtgctgtacg gctggactga ccacagtgcc tgtcgtccag cttgcccagc tggcatggaa


 


961
tataaggagt gtgtgtctcc ttgccccaga acctgccaga gcctgtctat caatgaagtg


 


1021
tgtcagcagc aatgtgtaga cggctgtagc tgccctgagg gagagctctt ggatgaagac


 


1081
cgatgtgtgc agagctccga ctgtccttgc gtgcacgctg ggaagcggta ccctcctggc


 


1141
acctccctct ctcaggactg caacacttgt atctgcagaa acagcctatg gatctgcagc


 


1201
aatgaggaat gcccagggga gtgtcttgtc acaggccaat cgcacttcaa gagcttcgac


 


1261
aacaggtact tcaccttcag tgggatctgc caatatctgc tggcccggga ctgcgaggat


 


1321
cacactttct ccattgtcat agagaccatg cagtgtgccg atgaccctga tgctgtctgc


 


1381
acccgctcgg tcagtgtgcg gctctctgcc ctgcacaaca gcctggtgaa actgaagcac


 


1441
gggggagcag tgggcatcga tggtcaggat gtccagctcc ccttcctgca aggtgacctc


 


1501
cgcatccagc acacagtgat ggcttctgta cgcctcagct atgcggagga cctgcagatg


 


1561
gactgggatg gccgtgggcg gctactggtt aagctgtccc cagtctattc tgggaagacc


 


1621
tgtggcttgt gtgggaatta caacggcaac aagggagacg acttcctcac gccggccggc


 


1681
ttggtggagc ccctggtggt agacttcgga aacgcctgga agcttcaagg ggactgttcg


 


1741
gacctgcgca ggcaacacag cgacccctgc agcctgaatc cacgcttgac caggtttgca


 


1801
gaggaggctt gtgcgctcct gacgtcctcc aagttcgagg cctgccacca cgcagtcagc


 


1861
cctctgccct atctgcagaa ctgccgttat gatgtttgct cctgctccga cagccgggat


 


1921
tgcctgtgta acgcagtagc taactatgct gccgagtgtg cccgaaaagg cgtgcacatc


 


1981
gggtggcggg agcctggctt ctgtgctctg ggctgtccac agggccaggt gtacctgcag


 


2041
tgtgggaatt cctgcaacct gacctgccgc tccctctccc tcccggatga agaatgcagt


 


2101
gaagtctgtc ttgaaggctg ctactgccca ccagggctct accaggatga aagaggggac


 


2161
tgtgtgccca aggcccagtg cccctgctac tacgatggtg agctcttcca gcctgcggac


 


2221
attttctcag accaccatac catgtgttac tgtgaagatg gcttcatgca ctgtaccaca


 


2281
agtggcaccc tggggagcct gttgcctgac actgtcctca gcagtcccct gtctcaccgt


 


2341
agcaaaagga gcctttcctg ccggccaccc atggtcaagc tggtgtgtcc tgctgacaac


 


2401
ccacgggctc aagggctgga gtgtgctaag acgtgccaga actacgacct ggagtgtatg


 


2461
agcctgggct gtgtgtctgg ctgcctctgt cccccaggca tggtccggca cgaaaacaag


 


2521
tgtgtggcct tggagcggtg tccctgcttc catcagggtg cagagtacgc cccgggagac


 


2581
acagtgaaga ttggctgcaa cacctgtgtc tgccgggagc ggaagtggaa ctgcacgaac


 


2641
catgtgtgtg acgccacttg ctctgccatt ggtatggccc actacctcac cttcgatgga


 


2701
ctcaagtacc tgttcccggg ggagtgccag tatgttctgg tgcaggatta ctgtggcagt


 


2761
aaccctggga cctttcagat cctggtggga aatgagggtt gcagctatcc ctcggtgaag


 


2821
tgcaggaagc gggtgaccat cctggtggat ggaggggagc ttgaactgtt tgacggagag


 


2881
gtgaacgtta agaggcccct gagagatgaa tctcactttg aggtggtgga gtcgggccgg


 


2941
tacgtcatcc tgctgctggg tcaggccctt tctgtggtct gggaccacca cctcagcatc


 


3001
tctgtggtcc tgaagcacac ataccaggaa caggtgtgtg gcctctgcgg gaactttgat


 


3061
ggcatccaga acaatgactt caccactagc agcctccagg tggaggaaga ccccgtcaac


 


3121
tttgggaact cctggaaagt gagctcacag tgtgctgaca cgagaaagct gtcactagat


 


3181
gtttcccctg ccacttgcca caacaacatc atgaaacaga cgatggtgga ctcagcctgc


 


3241
agaatcctta ccagtgacgt cttccagggc tgcaacaggc tggtggaccc tgagccatac


 


3301
ctggacatct gtatttatga cacttgctcc tgtgagtcca tcggggactg cgcctgtttc


 


3361
tgtgacacca ttgctgccta tgcccacgtg tgtgcccagc atggccaggt ggtagcctgg


 


3421
aggacaccca cactgtgccc ccagagctgt gaagaaaaga atgttcggga aaatggctat


 


3481
gagtgtgagt ggcgttataa cagctgtgcg cctgcttgcc cagtcacgtg tcagcaccct


 


3541
gagcctctgg cttgccctgt gcagtgtgtg gagggttgtc atgcacattg ccctccaggg


 


3601
agaatcctgg atgaacttct gcagacctgc gtagaccccc aagactgccc cgtgtgtgag


 


3661
gtggctggtc ggcgcttggc tcctggaaag aaaatcacct tgagtcctga tgaccctgca


 


3721
cactgtcaga attgtcactg tgatggtgtg aaccttacgt gtgaagcctg ccaagagccc


 


3781
ggaggcctgg tggcaccccc aactgatgcc ccagtcagct ctaccacccc atatgttgag


 


3841
gatacccccg agccccccct gcacaacttc tactgcagca agctgctgga tcttgtcttc


 


3901
ctgctggatg gctcctctat gttgtccgag gctgagtttg aagtgctcaa agcttttgtg


 


3961
gtgggcatga tggagaggtt acacatctct cagaagcgca tccgcgtggc agtggtagag


 


4021
taccatgatg gctcccgtgc ctaccttgag ctcaaggccc ggaagcgacc ctcagagctt


 


4081
cggcgcatca ccagccagat taagtataca ggcagccagg tggcctctac cagtgaggtt


 


4141
ttgaagtaca cactgttcca gatctttggc aaaattgacc gccctgaagc ctcccatatc


 


4201
actctgctcc tgactgctag ccaggagccc ccacggatgg ctaggaattt ggtccgctat


 


4261
gtccaaggtc tgaagaagaa gaaggttatc gtgatccctg tgggcattgg gccccacgcc


 


4321
agcctcaaac agatccgcct catcgagaag caggcccctg aaaacaaggc ttttctgctc


 


4381
agtggggtgg atgagctgga gcagagaaga gatgagatag tcagctacct ctgtgacctt


 


4441
gctcccgagg ccccagcccc aactcagcct ccacaggtag cccacgtcac cgtgagtcca


 


4501
gggatcgctg ggatctcgtc accgggacca aaacggaagt ccatggttct ggatgtggtg


 


4561
tttgtcctgg aggggtcaga cgaagttggt gaagccaact tcaataagag caaggagttc


 


4621
gtggaggagg taatccagcg catggacgtg agcccggatg caacgcgcat ctcagtactg


 


4681
cagtattcct acacggtaac catggagtat gccttcaatg gggcccagtc caaggaggag


 


4741
gtgctgcggc acgtgcgaga gatccgctac cagggcggca ataggacaaa cactgggcag


 


4801
gccctgcagt acctttctga gcacagcttc tctcccagcc aaggggaccg ggtagaggca


 


4861
cctaacctgg tctacatggt cacggggaac cccgcctctg atgagatcaa gaggttgcct


 


4921
ggagacatcc aggtggtacc cattggggtg ggcccccatg ccaacatgca ggaactggag


 


4981
aggatcagca ggcccatcgc tcccatcttc atccgggact ttgagacact tccccgagag


 


5041
gctcctgacc tggtcctgca gacatgttgc tccaaggagg gtctgcaact gcccaccctc


 


5101
ccccctctcc ctgactgcag ccaacccctg gatgtggtcc tgctcctgga tggctcctct


 


5161
agcttgccag agtcttcctt tgataaaatg aagagttttg ccaaggcttt catttcaaag


 


5221
gccaacattg ggccccacct cacacaggtg tccgtgatac agtatggaag catcaatacc


 


5281
attgatgtac catggaatgt ggttcaggag aaagcccatc tacagagttt ggtggacctc


 


5341
atgcagcagg agggtggccc cagccagatt ggggatgctc tggcctttgc cgtgcgctat


 


5401
gtaacttcac aaatccacgg agccaggcct ggggcctcca aagcagtggt catcatcatc


 


5461
atggatacct ccttggatcc cgtggacaca gcagcagatg ctgccagatc caaccgagtg


 


5521
gcagtgtttc ccgttggggt tggggatcgg tatgatgaag cccagctgag gatcttggca


 


5581
ggccctgggg ccagctccaa tgtggtaaag ctccagcaag ttgaagacct ctccaccatg


 


5641
gccaccctgg gcaactcctt cttccacaaa ctgtgttctg ggttttctgg agtttgtgtg


 


5701
gatgaagatg ggaatgagaa gaggcctggg gatgtctgga ccttgccgga tcagtgccac


 


5761
acagtgactt gcttggcaaa tggccagacc ttgctgcaga gtcatcgtgt caattgtgac


 


5821
catggacccc ggccttcatg tgccaacagc cagtctcctg ttcgggtgga ggagacgtgt


 


5881
ggctgccgct ggacctgccc ttgtgtgtgc acgggcagtt ccactcggca catcgtcacc


 


5941
ttcgatgggc agaatttcaa gcttactggt agctgctcct atgtcatctt tcaaaacaag


 


6001
gagcaggacc tggaagtgct cctccacaat ggggcctgca gccccggggc aaaacaagcc


 


6061
tgcatgaagt ccattgagat taagcatgct ggcgtctctg ctgagctgca cagtaacatg


 


6121
gagatggcag tggatgggag actggtcctt gccccgtacg ttggtgaaaa catggaagtc


 


6181
agcatctacg gcgctatcat gtatgaagtc aggtttaccc atcttggcca catcctcaca


 


6241
tacacgccac aaaacaacga gttccaactg cagcttagcc ccaagacctt tgcttcgaag


 


6301
atgcatggtc tttgcggaat ctgtgatgaa aacggggcca atgacttcac gttgcgagat


 


6361
ggcacggtca ccacagactg gaaaaggctt gtccaggaat ggacggtgca gcagccaggg


 


6421
tacacatgcc aggctgttcc cgaggagcag tgtcccgtct ctgacagctc ccactgccag


 


6481
gtcctcctct cagcgtcgtt tgctgaatgc cacaaggtca tcgctccagc cacattccat


 


6541
accatctgcc agcaagacag ttgccaccag gagcgagtgt gtgaggtgat tgcttcttac


 


6601
gcccatctct gtcggaccag tggggtctgt gttgattgga ggacaactga tttctgtgct


 


6661
atgtcatgcc caccgtccct ggtgtataac cactgtgagc gtggctgccc tcggcactgc


 


6721
gatgggaaca ctagcttctg tggggaccat ccctcagaag gctgcttctg tccccaacac


 


6781
caagtttttc tggaaggcag ctgtgtcccc gaggaggcct gcactcagtg tgttggcgag


 


6841
gatggagttc gacatcagtt cctggagacc tgggtcccag accatcagcc ctgtcagatc


 


6901
tgtatgtgcc tcagtgggag aaagattaac tgcactgccc agccgtgtcc cacagcccga


 


6961
gctcccacgt gtggcccatg tgaagtggct cgcctcaagc agagcacaaa cctgtgctgc


 


7021
ccagagtatg agtgtgtgtg tgacctgttc aactgcaact tgcctccagt gcctccgtgt


 


7081
gaaggagggc tccagccaac cctgaccaac cctggagaat gcagacccac ctttacctgt


 


7141
gcctgcagga aagaagagtg caaaagagtg tccccaccct cctgcccccc tcaccggaca


 


7201
cccactctcc ggaagaccca gtgctgtgat gaatacgagt gtgcttgcag ctgtgtcaac


 


7261
tccacgctga gctgcccact tggctacctg gcctcagcca ctaccaatga ctgtggctgc


 


7321
accacgacca cctgtctccc tgacaaggtt tgtgtccacc gaggcaccgt ctaccctgtg


 


7381
ggccagttct gggaggaggg ctgtgacacg tgcacctgta cggacatgga ggatactgtc


 


7441
gtgggcctgc gtgtggtcca gtgctctcaa aggccctgtg aagacagctg tcagccaggt


 


7501
ttttcttatg ttctccacga aggcgagtgc tgtggaaggt gcctgccctc tgcttgcaag


 


7561
gtggtggctg gctcactgcg gggcgattcc cactcttcct ggaaaagtgt tggatctcgg


 


7621
tgggctgttc ctgagaaccc ctgcctcgtc aacgagtgtg tccgcgtgga ggatgcagtg


 


7681
tttgtgcagc agaggaacat ctcctgccca cagctggctg tccctacctg tcccacaggc


 


7741
ttccaactga actgtgagac ctcagagtgc tgtcctagct gccactgtga gcctgtggag


 


7801
gcctgcctgc tcaatggcac catcattggg cccgggaaga gtgtgatggt tgacctatgc


 


7861
acgacctgcc gctgcatcgt gcagacagac gccatctcca gattcaagct ggagtgcagg


 


7921
aagactacct gtgaggcctg ccccatgggc tatcgggaag agaagagcca gggtgaatgc


 


7981
tgtgggagat gcttgcctac agcttgcact attcagctaa gaggaggacg gatcatgacc


 


8041
ctgaagcaag atgagacatt ccaggatggc tgtgacagtc atttgtgcag ggtcaacgag


 


8101
agaggagagt acatctggga gaagagggtc acgggctgcc caccatttga tgaacacaag


 


8161
tgtctggctg aaggaggcaa aatcgtgaaa attccaggca cctgctgtga cacatgtgag


 


8221
gagcctgatt gcaaagacat cacagccaag gtgcagtaca tcaaagtggg agattgtaag


 


8281
tcccaagagg aagtggacat tcattactgc cagggaaagt gtgccagcaa agctgtgtac


 


8341
tccattgaca tcgaggatgt gcaggagcaa tgctcctgct gcctgccctc gaggacggag


 


8401
cccatgcgcg tgcccttgca ctgcaccaat ggctctgtcg tgtaccacga ggtcatcaac


 


8461
gccatgcagt gcaggtgttc tccccggaac tgcagcaagt gaggcctgtg cagctacagc


 


8521
ggattcctac tgatacc






DNA sequences or oligonucleotides having specific sequences can be synthesized chemically or isolated by one of several approaches established in art. The basic strategies for identifying, amplifying, and isolated desired DNA sequences as well as assembling them into larger DNA molecules containing the desired sequence domains in the desired order, are well known to those of ordinary skill in the art. See, e.g., Sambrook, et al., (1989) Nature November 16; 342(6247):224-5; Perbal, B. et al., (1983) J Virol. March; 45(3):925-40. DNA segments corresponding to all or a portion of the VWF sequence may be isolated individually using the polymerase chain reaction (M. A. Innis, et al., “PCR Protocols: A Guide To Methods and Applications,” Academic Press, 1990). A complete sequence may be assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge (1981), Nature, 292:756; Nambiar, et al. (1984), Science, 223:1299; Jay et al. (1984), J. Biol. Chem., 259:6311.
The assembled nucleotide sequence can be cloned into a suitable vector or replicon and maintained in said carrier in a composition that is substantially free of vectors that do not contain the assembled sequence, thus providing a reservoir of the assembled sequence wherein the entire sequence can be extracted from the reservoir via excising it from DNA material with restriction enzymes or by PCR amplification. Those of ordinary skill in the art are familiar with numerous cloning vectors, and the selection of an appropriate cloning vector is a matter of choice. The construction of vectors containing desired nucleotide sequences linked by appropriate DNA sequences is accomplished by discussed above. These vectors may be constructed to contain additional DNA sequences, such as bacterial origins of replication to make shuttle vectors in order to shuttle between prokaryotic hosts and mammalian hosts.
Procedures for construction and expression of mutant proteins of defined sequence are well known in the art. A DNA sequence encoding a mutant form of VWF or a fragment thereof can be synthesized chemically or prepared from the wild-type sequence by one of several approaches, including primer extension, linker insertion and PCR (see, e.g., Sambrook, et al., (1989) Nature November 16; 342(6247):224-5; Maniatis, Fritsch & Sambrook, “Molecular Cloning: A Laboratory Manual” (1982): “DNA Cloning: A Practical Approach,” Volumes I and II (D. N. Glover, ed., 1985); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Nucleic Acid Hybridization” (B. D. Hames & S. J. Higgins, eds., 1985); “Transcription and Translation” (B. D. Hames & S. J. Higgins, eds., 1984); “Animal Cell Culture” (R. I. Freshney, ed., 1986); “Immobilized Cells and Enzymes” (IRL Press, 1986): B. Perbal, “A Practical Guide to Molecular Cloning” (1984), and Sambrook, et al., “Molecular Cloning: a Laboratory Manual” (1989)). Mutants can be prepared by these techniques having additions, deletions, and substitutions in the wild-type sequence (for example, the mouse VWF-A1 1326R>H mutant of SEQ ID NO: 5). To confirm that the mutant contains the desired changes, one skilled in the art can confirm the changes of interest via sequence-by-sequence analysis and/or by methods available to one skilled in the art.
In one embodiment, modification of the A1 domain can contain an amino acid residue substitution at a position involved with binding to GPIb alpha (such as, but not limited to, positions 1263, 1269, 1274, 1287, 1302, 1308, 1313, 1314, 1326, 1329, 1330, 1333, 1344, 1347, 1350, 1370, 1379, 1381, 1385 1391, 1394, 1397, 1421, 1439, 1442, 1449, 1466, 1469, 1472, 1473, 1475, 1479). In further embodiments, the modification of the A1 domain can be a partial or full replacement of an animal (such as a mouse) A1 domain of VWF with the A1 domain of human VWF. In other embodiments of the invention, the modification of the A1 domain can be an amino acid substitution at residue 1326 (for example HIS for ARG) such as depicted in SEQ ID NO: 5.
SEQ ID NO: 5 is the sequence for the mouse VWF-A1 1326 R>H mutant, wherein the modified amino acid sequence corresponds to the A1 domain of mouse VWF (residues 1260-1480) having an amino acid substitution at residue 1326 of HIS for ARG (in Bold):







EDTPEPPLHNFYCSKLLDLVFLLDGSSMLSEAEFEVLKAFVVGMMERLH


 


ISQKRIRVAVVEYHDGSHAYLELKARKRPSELRRITSQIKYTGSQVAST


 


SEVLKYTLFQIFGKIDRPEASHITLLLTASQEPPRMARNLVRYVQGLKK


 


KKVIVIPVGIGPHASLKQIRLIEKQAPENKAFLLSGVDELEQRRDEIVS


 


YLCDLAPEAPAPTQPPQVAHVTVSP






An expression vector containing a nucleotide sequence encoding a protein of interest, such as a mutant VWF-A1 molecule described above, is transfected into a host cell, either eukaryotic (for example, yeast, mammalian, or insect cells) or prokaryotic, by conventional techniques well established in the art. Transfection techniques carried out depend on the host cell used. For example, mammalian cell transfection can be accomplished using lipofection, protoplast fusion, DEAE-dextran mediated transfection, CaPO4 co-precipitation, electroporation, direct microinjection, as well as other methods known in the art which can comprise: scraping, direct uptake, osmotic or sucrose shock, lysozyme fusion or erythrocyte fusion, indirect microinjection such as via erythrocyte-mediated techniques, and/or by subjecting host cells to electric currents. Some of the techniques mentioned above are also applicable to unicellular organisms, such as bacteria or yeast. As other techniques for introducing genetic information into host cells will be developed, the above-mentioned list of transfection methods is not considered to be exhaustive. The transfected cells are then cultured by conventional techniques to produce a mutant VWF-A1 molecule harboring at least one of the mutations previously described.
One skilled in the art understands that expression of desired protein products in prokaryotes is most often carried out in E. coli with vectors that contain constitutive or inducible promoters. Some non-limiting examples of bacterial cells for transformation include the bacterial cell line E. coli strains DH5a or MC1061/p3 (Invitrogen Corp., San Diego, Calif.), which can be transformed using standard procedures practiced in the art, and colonies can then be screened for the appropriate plasmid expression. Some E. coli expression vectors (also known in the art as fusion-vectors) are designed to add a number of amino acid residues, usually to the N-terminus of the expressed recombinant protein. Such fusion vectors can serve three functions: 1) to increase the solubility of the desired recombinant protein; 2) to increase expression of the recombinant protein of interest; and 3) to aid in recombinant protein purification by acting as a ligand in affinity purification. In some instances, vectors, which direct the expression of high levels of fusion protein products that are readily purified, may also be used. Some non-limiting examples of fusion expression vectors include pGEX, which fuse glutathione S-tranferase to desired protein; pcDNA 3.1/V5-His A B & C (Invitrogen Corp, Carlsbad, Calif.) which fuse 6×-His to the recombinant proteins of interest; pMAL (New England Biolabs, MA) which fuse maltose E binding protein to the target recombinant protein; the E. coli expression vector pUR278 (Ruther et al., (1983) EMBO 12:1791), wherein the coding sequence may be ligated individually into the vector in frame with the lac Z coding region in order to generate a fusion protein; and pIN vectors (Inouye et al., (1985) Nucleic Acids Res. 13:3101-3109; Van Heeke et al., (1989) J. Biol. Chem. 24:5503-5509. Fusion proteins generated by the likes of the above-mentioned vectors are generally soluble and can be purified easily from lysed cells via adsorption and binding to matrix glutathione agarose beads subsequently followed by elution in the presence of free glutathione. For example, the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target can be released from the GST moiety.
Other suitable cell lines, in addition to microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing coding sequences for a mutant VWF-A1 molecule described above, may alternatively be used to produce the molecule of interest. Non-limiting examples include plant cell systems infected with recombinant virus expression vectors (for example, tobacco mosaic virus, TMV; cauliflower mosaic virus, CaMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing coding sequences for a mutant VWF-A1 molecule described above; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing coding sequences for a mutant VWF-A1 molecule described above; yeast (for example, Saccharomyces sp., Pichia sp.) transformed with recombinant yeast expression vectors containing coding sequences for a mutant VWF-A1 molecule described above; or mammalian cell lines harboring a vector that contains coding sequences for a mutant VWF-A1 molecule described above.
Mammalian cells (such as BHK cells, VERO cells, CHO cells and the like) can also contain an expression vector (for example, one that harbors a nucleotide sequence encoding a mutant VWF-A1 molecule described above) for expression of a desired product. Expression vectors containing such a nucleic acid sequence linked to at least one regulatory sequence in a manner that allows expression of the nucleotide sequence in a host cell can be introduced via methods known in the art, as described above. To those skilled in the art, regulatory sequences are well known and can be selected to direct the expression of a protein of interest in an appropriate host cell as described in Goeddel, Gene Expression Technology (1990) Methods in Enzymology 185, Academic Press, San Diego, Calif.). Regulatory sequences can comprise the following: enhancers, promoters, polyadenylation signals, and other expression control elements. Practitioners in the art understand that designing an expression vector can depend on factors, such as the choice of host cell to be transfected and/or the type and/or amount of desired protein to be expressed.
Animal or mammalian host cells capable of harboring, expressing, and secreting large quantities of a mutant VWF-A1 molecule (described above) of interest into the culture medium for subsequent isolation and/or purification include, but are not limited to, Chinese hamster ovary cells (CHO), such as CHO-K1 (ATCC CCL-61), DG44 (Chasin et al., (1986) Som. Cell Molec. Genet, 12:555-556; Kolkekar et al., (1997) Biochemistry, 36:10901-10909; and WO 01/92337 A2), dihydrofolate reductase negative CHO cells (CHO/dhfr-, Urlaub et al., (1980) Proc. Natl. Acad. Sci. U.S.A., 77:4216), and dp12.CHO cells (U.S. Pat. No. 5,721,121); monkey kidney CV1 cells transformed by SV40 (COS cells, COS-7, ATCC CRL-1651); human embryonic kidney cells (e.g., 293 cells, or 293 cells subcloned for growth in suspension culture, Graham et al., (1977) J. Gen. Virol., 36:59); baby hamster kidney cells (BHK, ATCC CCL-10); monkey kidney cells (CV1, ATCC CCL-70); African green monkey kidney cells (VERO-76, ATCC CRL-1587; VERO, ATCC CCL-81); mouse sertoli cells (TM4; Mather (1980) Biol. Reprod., 23:243-251); human cervical carcinoma cells (HELA, ATCC CCL-2); canine kidney cells (MDCK, ATCC CCL-34); human lung cells (W138, ATCC CCL-75); human hepatoma cells (HEP-G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL-51); buffalo rat liver cells (BRL 3A, ATCC CRL-1442); TRI cells (Mather (1982) Annals NY Acad. Sci., 383:44-68); MCR 5 cells; FS4 cells. A cell line transformed to produce a mutant VWF-A1 molecule described above can also be an immortalized mammalian cell line of lymphoid origin, which include but are not limited to, a myeloma, hybridoma, trioma or quadroma cell line. The cell line can also comprise a normal lymphoid cell, such as a B cell, which has been immortalized by transformation with a virus, such as the Epstein Barr virus (such as a myeloma cell line or a derivative thereof).
A host cell strain, which modulates the expression of the inserted sequences, or modifies and processes the nucleic acid in a specific fashion desired also may be chosen. Such modifications (for example, glycosylation and other post-translational modifications) and processing (for example, cleavage) of protein products may be important for the function of the protein. Different host cell strains have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. As such, appropriate host systems or cell lines can be chosen to ensure the correct modification and processing of the foreign protein expressed, such as a mutant VWF-A1 molecule described above. Thus, eukaryotic host cells possessing the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Non-limiting examples of mammalian host cells include 3T3, W138, BT483, Hs578T, CHO, VERY, BHK, Hela, COS, BT2O, T47D, NSO (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, MDCK, 293, HTB2, and HsS78Bst cells.
For protein recovery, isolation and/or purification, the cell culture medium or cell lysate is centrifuged to remove particulate cells and cell debris. The desired polypeptide molecule (for example, a mutant VWF-A1 protein) is isolated or purified away from contaminating soluble proteins and polypeptides by suitable purification techniques. Non-limiting purification methods for proteins include: separation or fractionation on immunoaffinity or ion-exchange columns; ethanol precipitation; reverse phase HPLC; chromatography on a resin, such as silica, or cation exchange resin, e.g., DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, e.g., Sephadex G-75, Sepharose; protein A sepharose chromatography for removal of immunoglobulin contaminants; and the like. Other additives, such as protease inhibitors (e.g., PMSF or proteinase K) can be used to inhibit proteolytic degradation during purification. Purification procedures that can select for carbohydrates can also be used, e.g., ion-exchange soft gel chromatography, or HPLC using cation- or anion-exchange resins, in which the more acidic fraction(s) is/are collected.
In one embodiment, the protein isolated is a mutant human von Willebrand Factor A1 protein comprising one or more of the following mutations: 1263S>P, 1269D>N, 1274R>K, 1287R>M, 1302D>G, 1308R>H, 1313W>R, 1314V>I, 1326H>R, 1329I>L, 1330G>E, 1333D>A, 1344A>T, 1347V>I, 1350A>T, 1370S>G, 1379R>H, 1381A>T, 1385M>T 1391Q>P, 1394S>A, 1397F>L, 1421N>S, 1439V>L, 1442S>G, 1449Q>R, 1466P>A, 1469L>Q, 1472H>Q, 1473M>V, 1475Q>H, or 1479G>S. In another embodiment, the protein isolated is a mutant human von Willebrand Factor A1 protein comprising a 1263S>P, 1269D>N, 1274R>K, 1287R>M, 1302D>G, 1308R>H, 1313W>R, 1314V>I, 1326H>R, 1329I>L, 1330G>E, 1333D>A, 1344A>T, 1347V>I, 1350A>T, 1370S>G, 1379R>H, 1381A>T, 1385M>T, 1391Q>P, 1394S>A, 1397F>L, 1421N>S, 1439V>L, 1442S>G, 1449Q>R, 1466P>A, 1469L>Q, 1472H>Q, 1473M>V, 1475Q>H, or a 1479G>S mutation. In a particular embodiment, the protein isolated is a mutant human von Willebrand Factor A1 protein comprising a 1326H>R mutation.
The invention also provides a method for producing mutant von Willebrand Factor A1 protein that specifically binds human platelets. For example, an animal expressing a mutant von Willebrand Factor A1 (VWF-A1) protein can be provided, wherein the mutation causes the platelet binding specificity of the animal VWF-A1 protein to change to be specific for human platelets. VWF plasma protein containing the mutant A1 domain from an animal (such as from a mouse) can then be subsequently harvested. In one embodiment, the animal von Willebrand Factor A1 protein contains at least one mutation at amino acid position 1263, 1269, 1274, 1287, 1302, 1308, 1313, 1314, 1326, 1329, 1330, 1333, 1344, 1347, 1350, 1370, 1379, 1381, 1385, 1391, 1394, 1397, 1421, 1439, 1442, 1449, 1466, 1469, 1472, 1473, 1475, or 1479. In another embodiment, the mutations occur in a murine VWF-A1 protein. In particular embodiments, the mutant murine von Willebrand Factor A1 protein comprises at least one mutation comprising 1263P>S, 1269N>D, 1274K>R, 1287M>R, 1302G>D, 1308H>R, 1313R>W, 1314I>V, 1326R>H, 1329L>I, 1330E>G, 1333A>D, 1344T>A, 1347I>V, 1350T>A, 1370G>S, 1379H>R, 1381T>A, 1385T>M 1391P>Q, 1394A>S, 1397L>F, 1421S>N, 1439L>V, 1442G>S, 1449R>Q, 1466A>P, 1469Q>L, 1472Q>H, 1473V>M, 1475H>Q, 1479S>G, or any combination thereof.
Pre-Screening Evaluation of Anti-Thrombotics and Associated Diseases
Diversity libraries, such as random or combinatorial peptide or non-peptide libraries can be screened for small molecules and compounds that specifically bind to a VWF-A1 protein. Many libraries are known in the art that can be used such as, e.g., chemically synthesized libraries, recombinant (e.g., phage display) libraries, and in vitro translation-based libraries.
Any screening technique known in the art can be used to screen for agonist (i.e., compounds that promote platelet adhesion) or antagonist molecules (such as anti-thrombotics) directed at a target of interest (e.g., VWF-A1). The present invention contemplates screens for small molecule ligands or ligand analogs and mimics, as well as screens for natural ligands that bind to and modulate VWF-A1 binding to GPIb-alpha, such as via examining the degree of thrombus formation, platelet adhesion, coagulation, blood flow, vessel occlusion, or bleeding times. For example, natural products libraries can be screened using assays of the invention for molecules that modulate the activity of a molecule of interest, such as a VWF-A1 binding to GPIb-alpha.
Knowledge of the primary sequence of a molecule of interest, such as a VWF-A1, can provide an initial clue as to proteins that can modulate VWF-A1 binding to GPIb-alpha. Identification and screening of modulators is further facilitated by determining structural features of the protein, e.g., using X-ray crystallography, neutron diffraction, nuclear magnetic resonance spectrometry, and other techniques for structure determination. These techniques provide for the rational design or identification of such modulators.
Test compounds, such as test modulators of VWF-A1 binding to GPIb-alpha, are screened from large libraries of synthetic or natural compounds. Numerous means are currently used for random and directed synthesis of saccharide, peptide, and nucleic acid based compounds. Synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, N.J.), Brandon Associates (Merrimack, N.H.), and Microsource (New Milford, Conn.). A rare chemical library is available from Aldrich (Milwaukee, Wis.). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from e.g. Pan Laboratories (Bothell, Wash.) or MycoSearch (N.C.), or are readily producible. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means (Blondelle et al., (1996) Tib Tech 14:60).
Methods for preparing libraries of molecules are well known in the art and many libraries are commercially available. Libraries of interest in the invention include peptide libraries, randomized oligonucleotide libraries, synthetic organic combinatorial libraries, and the like. Degenerate peptide libraries can be readily prepared in solution, in immobilized form as bacterial flagella peptide display libraries or as phage display libraries. Peptide ligands can be selected from combinatorial libraries of peptides containing at least one amino acid. Libraries can be synthesized of peptoids and non-peptide synthetic moieties. Such libraries can further be synthesized which contain non-peptide synthetic moieties, which are less subject to enzymatic degradation compared to their naturally-occurring counterparts. Libraries are also meant to include for example but are not limited to peptide-on-plasmid libraries, polysome libraries, aptamer libraries, synthetic peptide libraries, synthetic small molecule libraries and chemical libraries. The libraries can also comprise cyclic carbon or heterocyclic structure and/or aromatic or polyaromatic structures substituted with one or more of the above-identified functional groups. Screening compound libraries listed above [also see EXAMPLE 6 and U.S. Patent Application Publication No. 2005/0009163, which is hereby incorporated by reference], in combination with dynamic force microscopy, a coagulation factor assay, a platelet adhesion assay, thrombus imaging, a bleeding time assay, aggregometry, review of real-time video of blood flow, a Doppler ultrasound vessel occlusion assay, or a combination of these assays (for example, those assays described in EXAMPLES 1-4) can be used to identify modulators of VWF-A1 binding to GPIb-alpha, wherein the compound abbreviates or increases off-rate (koff) binding kinetics between VWF-A1 and GPIb-alpha by at least two-fold (Lew et al., (2000) Curr. Med. Chem. 7(6):663-72; Werner et al., (2006) Brief Funct. Genomic Proteomic 5(1):32-6).
Small molecule combinatorial libraries may also be generated. A combinatorial library of small organic compounds is a collection of closely related analogs that differ from each other in one or more points of diversity and are synthesized by organic techniques using multi-step processes. Combinatorial libraries include a vast number of small organic compounds. One type of combinatorial library is prepared by means of parallel synthesis methods to produce a compound array. A compound array can be a collection of compounds identifiable by their spatial addresses in Cartesian coordinates and arranged such that each compound has a common molecular core and one or more variable structural diversity elements. The compounds in such a compound array are produced in parallel in separate reaction vessels, with each compound identified and tracked by its spatial address. Examples of parallel synthesis mixtures and parallel synthesis methods are provided in U.S. Ser. No. 08/177,497, filed Jan. 5, 1994 and its corresponding PCT published patent application WO95/18972, published Jul. 13, 1995 and U.S. Pat. No. 5,712,171 granted Jan. 27, 1998 and its corresponding PCT published patent application WO96/22529, which are hereby incorporated by reference.
Examples of chemically synthesized libraries are described in Fodor et al., (1991) Science 251:767-773; Houghten et al., (1991) Nature 354:84-86; Lam et al., (1991) Nature 354:82-84; Medynski, (1994) BioTechnology 12:709-710; Gallop et al., (1994) J. Medicinal Chemistry 37(9):1233-1251; Ohlmeyer et al., (1993) Proc. Natl. Acad. Sci. USA 90:10922-10926; Erb et al., (1994) Proc. Natl. Acad. Sci. USA 91:11422-11426; Houghten et al., (1992) Biotechniques 13:412; Jayawickreme et al., (1994) Proc. Natl. Acad. Sci. USA 91:1614-1618; Salmon et al., (1993) Proc. Natl. Acad. Sci. USA 90:11708-11712; PCT Publication No. WO 93/20242, dated Oct. 14, 1993; and Brenner et al., (1992) Proc. Natl. Acad. Sci. USA 89:5381-5383.
Screening methods of the invention allowed for the identification of potential compounds that modulate VWF-A1 binding to GPIb-alpha. In some embodiments of the invention, the compound comprises one or more compounds having a structure depicted in Table 8 below.
Examples of phage display libraries are described in Scott et al., (1990) Science 249:386-390; Devlin et al., (1990) Science, 249:404-406; Christian, et al., (1992) J. Mol. Biol. 227:711-718; Lenstra, (1992) J. Immunol. Meth. 152:149-157; Kay et al., (1993) Gene 128:59-65; and PCT Publication No. WO 94/18318.
In vitro translation-based libraries include but are not limited to those described in PCT Publication No. WO 91/05058; and Mattheakis et al., (1994) Proc. Natl. Acad. Sci. USA 91:9022-9026.
In one non-limiting example, non-peptide libraries, such as a benzodiazepine library (see e.g., Bunin et al., (1994) Proc. Natl. Acad. Sci. USA 91:4708-4712), can be screened. Peptoid libraries, such as that described by Simon et al., (1992) Proc. Natl. Acad. Sci. USA 89:9367-9371, can also be used. Another example of a library that can be used, in which the amide functionalities in peptides have been permethylated to generate a chemically transformed combinatorial library, is described by Ostresh et al. (1994), Proc. Natl. Acad. Sci. USA 91:11138-11142.
Screening the libraries can be accomplished by any variety of commonly known methods. See, for example, the following references, which disclose screening of peptide libraries: Parmley and Smith, (1989) Adv. Exp. Med. Biol. 251:215-218; Scott and Smith, (1990) Science 249:386-390; Fowlkes et al., (1992) BioTechniques 13:422-427; Oldenburg et al., (1992) Proc. Natl. Acad. Sci. USA 89:5393-5397; Yu et al., (1994) Cell 76:933-945; Staudt et al., (1988) Science 241:577-580; Bock et al., (1992) Nature 355:564-566; Tuerk et al., (1992) Proc. Natl. Acad. Sci. USA 89:6988-6992; Ellington et al., (1992) Nature 355:850-852; U.S. Pat. Nos. 5,096,815; 5,223,409; and 5,198,346, all to Ladner et al.; Rebar et al., (1993) Science 263:671-673; and PCT Pub. WO 94/18318.
The invention provides a method for identifying a compound that modulates VWF-A1 binding to GPIb-alpha. In one embodiment, the method can comprise providing an electronic library of test compounds stored on a computer (such as those libraries described above); providing atomic coordinates for at least 10 amino acid residues of the A1 domain of the VWF protein listed in Table 8, where the coordinates having a root mean square deviation therefrom, with respect to at least 50% of the Ca atoms, of not greater than about 2.5 Å, in a computer readable format; converting the atomic coordinates into electrical signals readable by a computer processor to generate a three dimensional model of the VWF-A1 domain; performing a data processing method, wherein electronic test compounds from the library are superimposed upon the three dimensional model of the A1 domain of VWF; and determining which test compound fits within the binding pocket of the three dimensional model of the VWF-A1 protein. Thus, compounds can be identified that would modulate the binding of VWF-A1 to GPIb-alpha.
In another embodiment, the method can comprise providing an electronic library of test compounds stored on a computer; and providing atomic coordinates listed in Table 8 in a computer readable format for at least 10, 15, 20, 25, 30, 35, or 40 amino acid residues of the A1 domain of the VWF protein, wherein the residues can comprise 2 or more of the following residues: Pro1391, Arg1392, Arg1395, Val1398, Arg1399, Gln1402, Lys1406, Lys1423, Gln1424, Leu1427, Lys1430, or Glu1431; converting the atomic coordinates into electrical signals readable by a computer processor to generate a three dimensional model of the A1 domain of the VWF protein; performing a data processing method, wherein electronic test compounds from the library are superimposed upon the three dimensional model of the A1 domain of the VWF protein; and determining which test compound fits within the binding pocket of the three dimensional model of the VWF-A1 protein. Thus, compounds can be identified that would modulate the binding of VWF-A1 to GPIb-alpha.
In a further embodiment, the method can comprise providing an electronic library of test compounds stored on a computer (such as those libraries described above); providing atomic coordinates for at least 10 amino acid residues of the Botrocetin-VWF-A1 complex listed in accession entry 1IJK (http://www.rcsb.org/pdb/explore.do?structureId=1IJK), where coordinates having a root mean square deviation therefrom, with respect to at least 50% of the Cα atoms, not more than about 3 Å, in a computer readable format; converting the atomic coordinates into electrical signals readable by a computer processor to generate a three dimensional model of the Botrocetin-VWF-A1 complex; performing a data processing method, wherein electronic test compounds from the library are superimposed upon Botrocetin within the three dimensional model of the Botrocetin-VWF-A1 complex; and determining which test compound fits within the binding pocket of the three dimensional model of the VWF-A1 protein and best overlays the three-dimensional model generated above. Thus, compounds can be identified that would modulate the binding of VWF-A1 to GPIb-alpha.
In other embodiments, the method can comprise providing an electronic library of test compounds stored on a computer; providing atomic coordinates listed in accession entry 1IJK (http://www.rcsb.org/pdb/explore.do?structureId=1IJK) in a computer readable format for at least 10, 15, 20, 25, 30, 35, or 40 amino acid residues of the Botrocetin-murine VWF-A1 complex, wherein the residues comprise 2 or more of the following residues: Pro1391, Arg1392, Arg1395, Val1398, Arg1399, Gln1402, Lys1406, Lys1423, Gln1424, Leu1427, Lys1430, or Glu1431; converting the atomic coordinates into electrical signals readable by a computer processor to generate a three dimensional model of the Botrocetin-VWF-A1 complex; performing a data processing method, wherein electronic test compounds from the library are superimposed upon Botrocetin within the three dimensional model of the Botrocetin-VWF-A1 complex; and determining which test compound fits within the binding pocket of the three dimensional model of the VWF-A1 protein and best overlays the three-dimensional model generated above. Thus, compounds can be identified that would modulate the binding of VWF-A1 to GPIb-alpha.
The invention also provides for a compound identified by the method described above. In one embodiment, the compound inhibits thrombosis formation or promotes platelet adhesion.
The present invention provides methods for evaluating potential anti-thrombotic reagents in pre-clinical testing using a non-human transgenic animal (for example, non-human animals include, but are not limited to, vertebrates such as ovines, bovines, rodents, non-human primates, porcines, caprines, equines, ruminants, lagomorphs, canines, felines, ayes, and the like). There are three main classes of antithrombotic drugs that can be screened using the transgenic mouse model of the invention: Anticoagulant drugs (such as Heparins; Vitamin K antagonists, which are currently the only anticoagulants that can be administered orally; and direct thrombin inhibitors), Antiplatelet drugs (such as cyclooxygenase inhibitors like aspirin; phosphodiesterase inhibitors like ticlopidine (Ticlid); adenosine diphosphate receptor inhibitors like clopidogrel (Plavix); tirofiban (Aggrastat); adenosine reuptake inhibitors, and inhibitors of integrins on platelets (for example, alpha IIb Beta3) like eptifibatide (Integrilin)), and Thrombolytic or fibrinolytic drugs (such as t-PA (alteplase Activase); reteplase (Retavase); urokinase (Abbokinase); streptokinase (Kabikinase, Streptase); tenectaplase; lanoteplase; and anistreplase (Eminase)).
The invention provides an in vivo model to test the efficacy of potential anti-thrombotic drugs against human platelets prior to FDA approval. To date, in vitro models of thrombosis do not accurately recapitulate the hemodynamic conditions, cell-cell interactions, or cell-protein interactions that occur at sites of vascular injury in a living animal. Thus, anti-thrombotics can be identified and their potential therapeutic effects can be assessed for treatment of abnormal thrombotic events associated with atherothrombotic arterial diseases and venous thrombotic diseases (such as abnormal bleeding and/or abnormal clotting).
Atherothrombotic arterial diseases can include, but is not limited to, coronary artery disease, (for example, acute myocardial infarction, acute coronary syndromes (such as unstable angina pectoris) and stable angina pectoris); mesenteric ischemia, “abdominal angina,” and mesenteric infarction; cerebral vascular disease, including acute stroke and transient ischemic attack; mesenteric arterial disease; as well as peripheral arterial disease, including acute peripheral arterial occlusion and intermittent claudication. Anti-thrombotic compounds identified by the pre-clinical testing method of the present invention can also be useful for the treatment of coronary artery disease (which includes, but is not limited to anti-thrombotic therapy during coronary angioplasty, anti-thrombotic therapy during cardiopulmonary bypass, and limiting of platelet activation during ischemia reperfusion) as well as venous thrombotic diseases (which include, but are not limited to deep venous thrombosis and pulmonary thromboembolism). Anti-thrombotic compounds identified by the pre-clinical testing method of the present invention can also be useful in anti-thrombotic therapy for pulmonary hypertension.
The invention provides a method for testing a compound that modulates VWF-A1 binding to GPIb-alpha. The method can entail obtaining or synthesizing a compound identified in the screens previously described above; contacting VWF-A1 with the compound under a condition suitable for GPIb-alpha-VWF-A1 binding; and determining whether the compound can modulate GPIb-alpha-VWF-A1 binding using a diagnostic assay. In one embodiment, contacting can comprise perfusing platelets into a flow chamber at a shear flow rate of at least 100 s−1, wherein mutant murine VWF-A1 protein is immobilized on a bottom surface of the chamber. In another embodiment, contacting can comprise perfusing platelets into a transgenic non-human, for example the transgenic mouse described in EXAMPLE 3. In some embodiments, of the invention, contacting first occurs in vitro by way of the flow chamber described above, subsequently followed by in vivo testing of the compound's efficiency to modulate GPIb-alpha binding to VWF-A1 after the compound was determined to have a purported effect in modulating such binding in vitro. Thus, the invention provides a great advantage of being able to test directly compounds that target human platelets in an in vivo system. The transgenic mouse (which, for example, can harbor the 1326R>H mutation in the A1 domain of VWF of SEQ ID NO: 5) displays a bleeding phenotype, thus serves as a model for screening potential anti-thrombotic compounds useful for humans when the mouse is perfused with human platelets. Since the 1326R>H mutation in VWF-A1 in the mouse model (SEQ ID NO: 5) has been shown to support human platelet binding and it corresponds to the His amino acid at the same position in human VWF-A1 (as well as in canines, chimpanzees, rat, porcine, felines, equines, bovine, and the like (Jenkins et al., (1998) Blood 91(6): 2032-44)), the test compounds screened using this mouse model (while subject to perfusion with platelets from human, dog, cat, or other relevant organism) will be applicable to multiple species.
After perfusion with human platelets, a test compound (such as a purported anti-thrombotic that would minimize blood clotting or a compound that could promote platelet adhesion) can be administered to the animal subsequent to vessel injury in order to determine whether blood clotting is minimized or if it is enhanced. In one embodiment, the platelets infused are human platelets while in other embodiments the platelets infused are not murine platelets. In some embodiments, the compound can slow the on-rate, and/or increase the off-rate (koff) binding kinetics, and/or reduce bond strength of the interaction between VWF-A1 and GPIb-alpha by at least two-fold, thus resulting in a decreased lifetime of the bond(s). Such compounds could reduce thrombosis formation. In other embodiments, the compound can abbreviate off-rate (koff) binding kinetics between VWF-A1 and GPIb-alpha by at least two-fold, thus resulting in a prolongation in the lifetime of the bond(s). Such compounds could promote platelet adhesion due to the compound stabilizing an interaction between VWF-A1 and GPIb-alpha. To assess binding efficiency between VWF-A1 and GPIb-alpha, binding kinetics can be determined by measuring translocation velocity, tethering frequency, and bond strength (Fukuda, K., et al., (2005) Nat. Struct. Mol. Biol. 12:152-159; Doggett, et al., (2003) Blood 102(10): 152-60; Doggett, T. A. et al. (2002) Biophys. J. 83, 194-205; Schmidtke and Diamond (2000) J Cell Bio 149(3): 719-29; Mody et al., (2005) Biophys. J. 88: 1432-43, all of which are incorporated by reference in their entirety).
The compound identified and tested using the methods described above can be an anti-platelet drug. In one embodiment, the anti-platelet drug can be a cyclooxygenase inhibitor, a phosphodiesterase inhibitor, an adenosine diphosphate receptor inhibitor, a PI3K inhibitor, an adenosine reuptake inhibitors, thrombin receptor inhibitor or inhibitor of any intracellular signaling pathway in platelets, an alphaIIb beta3 inhibitor, an alpha2 beta1 inhibitor, a glycoprotein V inhibitor, a glycoprotein VI inhibitor, a PECAM-1 inhibitor or any adhesion molecule and/or activation pathway critical for human platelet function
The diagnostic assay used in this method for testing a compound that modulates VWF-A1 binding to GPIb-alpha can assess whether an abnormal thrombotic event occurred in the subject. An abnormal thrombotic event can comprise abnormal bleeding, abnormal clotting, death, or a combination thereof. The assay can comprise dynamic force microscopy, a coagulation factor assay, a platelet adhesion assay, thrombus imaging, a bleeding time assay, aggregometry, review of real-time video of blood flow, a Doppler ultrasound vessel occlusion assay, or a combination thereof.
After perfusion with a purported anti-thrombotic or a compound that could promote platelet adhesion, a labeled agent can subsequently be perfused either into the flow chamber or to the animal. Such an agent would enable the visualization of either the presence or absence of a thrombus. In one embodiment, the labeled agent can comprise one or more of a nanoparticle, a fluorophore, a quantum dot, a microcrystal, a radiolabel, a dye, a gold biolabel, an antibody, or a small molecule ligand. In some embodiments, the agent can target a platelet receptor, a VWF protein, or a portion thereof.
Methods for Assessing Thrombotic Events In Vivo
The invention provides methods for detecting an internal vascular injury site (occult bleeding) in a subject. This could be useful in ER settings or on the battlefield in order to quickly identify sites of internal bleeding. For instance, the method can entail: administering to a subject a targeted molecular imaging agent, wherein the molecule circulates for an effective period of time in order to bind to the injury site within the subject; tracking a deposition of the labeled thrombosis-indicating-molecule in the subject; and identifying the site of a thrombus formation in the subject by imaging the labeled targeted molecular imaging agent. Thus, the deposition of the targeted molecular imaging agent at the internal vascular injury site can be indicative of internal bleeding within a subject. For example, a targeted molecular imaging agent can recognize constituents of thrombi that comprise a lipid, a protein, a cellular molecule, or a combination thereof. In one embodiment, the targeted molecular imaging agent is administered by subcutaneous, intra-muscular, intra-peritoneal, or intravenous injection; infusion; by oral, nasal, or topical delivery; or a combination of the listed routes of administration. In other embodiments, the targeted molecular imaging agent has a T1/2 of at least 30 min. In some embodiments, the targeted molecular imaging agent can comprise an antibody, peptide, or Fab fragment directed to a platelet receptor, a VWF protein, or a portion thereof. In particular embodiments, the targeted molecular imaging agent can comprise a VWF-A1 or GPIb-alpha receptor trap. For example, a receptor trap is a decoy receptor that can comprise fusions between two distinct receptor components and the Fc region of an antibody molecule, which can result in the generation of a molecule with an increased affinity over single component reagents. This technology is available from Regeneron (Tarrytown, N.Y.) and is described in Wachsberger et al., (2007) Int J Radiat Oncol Biol Phys. 67(5):1526-37; Holash et al., (2002) Proc Natl Acad Sci USA. 2002 99(17):11393-8; Davis et al., (1996) Cell. 87(7):1161-9; U.S. Pat. No. 7,087,411; and in United States Publication Applications 2004/0014667, 2005/0175610, 2005/0260203, 2006/0030529, 2006/0058234, which are all hereby incorporated by reference in their entirety.
To aid in the visualization of a site of thrombus formation, the targeted molecular imaging agent can further comprise a label. In one embodiment, the labeled thrombosis-indicating-molecule comprises a nanoparticle, a fluorophore, a quantum dot, a microcrystal, a radiolabel, a dye, a gold biolabel, an antibody, a peptide, a small molecule ligand, or a combination thereof. A fluorophore, for example green fluorescent protein, (such as GFP, RFP, YFP and the like; see Johnson and Johnson, (2007) ACS Chem Biol. 2(1):31-8) can be used as a biomarker. A quantum dot is a semiconductor nanocrystal, that can be as small as 2 to 10 nm or can 15-20 nm (for example, Q-dot nanocrystals; also see Kaji et al., (2007) Anal Sci. 23(1):21-4). Quantum dot fluorescence can be induced by exposure to ultraviolet light. Both a fluorescent protein and a quantum dot can be obtained commercially (for example, Molecular Probes-Invitrogen, Carlsbad, Calif. or Evident Technologies, Troy N.Y.). A fluorophore can also be generated in the laboratory according to molecular biology methods practiced in the art. A radiolabel is a radioactive isotope that can be used as a tracer. Non-limiting examples of radiolabels include: Technetium-99m, Iodine-123 and 131, Thallium-201, Gallium-67, Fluorine-18, -19, Indium-111, Xenon-133, and Krypton-81m. Radiolabels can be obtained commercially, for example, from SRI International (Menlo Park, Calif.). In one embodiment, the nanoparticle can comprise a perfluorocarbon (PFC). Non-limiting examples of perfluorocarbons include perfluorobutane, perfluorohexane, perfluorooctane, perfluorodecalin, perfluoromethyldecalin, and perfluoroperhydrophenanthrene. These can be synthesized according to the method described in EXAMPLE 5 or according to Partlow et al. (FASEB J (2007) Feb. 6 on-line publication, fj.06-6505com). The perfluorocarbon molecules can also be obtained commercially (F2 Chemicals Ltd.; Lancashire, UK). In another embodiment, the PFC nanoparticle can be coupled to a platelet receptor antibody (such as platelet receptor alpha-IIb beta3). In some embodiments, imaging can comprise a PET scan, a CT scan, an MRI, an IR scan, an ultrasound, nuclear imaging, or a combination thereof.
Since the usefulness of the method pertains to the swiftness in identifying sites of internal bleeding (for example in an ER setting or on the battlefield), the subject can be further administered a compound aid in the cessation of such bleeding. In one embodiment, the subject is further administered a thrombotic compound (for example, a compound identified in the screens described above or a compound comprising a structure depicted in Table 8). In some embodiments, the compound can abbreviate off-rate (koff) binding kinetics, and/or slow the on-rate, and/or reduce the bond strength between VWF-A1 and GPIb-alpha by at least two-fold.
The invention also provides a method to test contrast agents for imaging of human platelets at sites of thrombosis. For instance, one could test the ability of nanoparticle contrast agents targeted to human platelets to identify areas of thrombosis or occult bleeding. In some embodiments, the prevention or reduction of thrombus formation at site of injury upon administration of a compound can be visually examined via tracking the localization of labeled platelets (such as with high resolution in vivo microscopy or MRI). In further embodiments, the platelets can be labeled with a nanoparticle, fluorophore, quantum dot, microcrystal, radiolabel, dye, or gold biolabel. The prevention or reduction of thrombus formation also can be readily determined by methods known to one skilled in the art, which include but are not limited to aggregometry, review of real-time video of blood flow in the animal, and determination of vessel occlusion, as well as by the examples provided below.
Additionally, the transgenic mouse's bleeding phenotype can be exploited to screen potential prothrombotic compounds, in addition to anti-thrombotics discussed above. A test compound (such as an alleged thrombotic that would induce and/or stimulate blood clotting) can be administered to the animal perfused with human platelets subsequent to vessel injury in order to determine whether blood clotting occurs. In some embodiments, the induction or stimulation of thrombus formation at site of injury upon administration of a compound can be visually examined via tracking the localization of labeled platelets (such as with high resolution in vivo microscopy or MRI). In further embodiments, the platelets can be labeled with a nanoparticle, fluorophore, quantum dot, microcrystal, radiolabel, dye, or gold biolabel. The prevention or reduction of thrombus formation also can be readily determined by methods known to one skilled in the art, which include but are not limited to aggregometry, ex-vivo flow chamber studies, review of real-time video of blood flow in the animal, and determination of vessel occlusion.
One of ordinary skill in the art can assess that the VWF-A1 mutants of this invention have the required properties of competitive binding to the GPIb platelet receptor in a manner that competes with the native VWF. Suitable assays are set forth in detail in the examples below, including ristocetin-induced platelet aggregation, platelet aggregation induced by ADP, thrombin, collagen, and platelet adhesion in a flow model.
In one embodiment of the invention, the non-human transgenic animal that expresses a modified A1 domain of the VWF sequence (for example, an amino acid residue substitution at a position involved with binding to GPIb alpha, such as, but not limited to, positions 1263, 1269, 1274, 1287, 1302, 1308, 1313, 1314, 1326, 1329, 1330, 1333, 1344, 1347, 1350, 1370, 1379, 1381, 1385 1391, 1394, 1397, 1421, 1439, 1442, 1449, 1466, 1469, 1472, 1473, 1475, 1479) can be used to validate new devices aimed at determining the effectiveness of antithrombotics in humans.
The non-human transgenic animal may also be used for determining the effectiveness of gene therapy (for example, assessing whether VWF-A1 protein targeting and protein expression was successful). Gene therapy refers to the insertion of genes into an individual's cells and tissues to treat a disease. For example, in a hereditary disease, a defective mutant allele is replaced with a functional one. The efficiency of VWF-A1 gene transfer by nonviral methodologies (i.e. lipofection) or viral methodologies (such as adenovirus infection described in U.S. Pat. No. 6,927,278 or United States Application Publication No. 2005/0169899) can be assessed using the non-transgenic mouse model described above via examining whether replacement of a portion or the whole VWF gene in a subject (such as a mutant VWF mouse of the invention) affects clot formation in vivo. Results obtained from such a mouse model can then be correlated with the likely effect to be observed in human subjects. For gene therapy reviews, see Zuckerbraun et al., (2002) Arch Surg. 137(7):854-61; Melo et al., (2004) Arterioscler Thromb Vasc Biol. 24(10):1761-74; and Dulak et al., (2006) Cell Biochem Biophys. 44(1):31-42, which are incorporated by reference in their entirety.
The invention provides a method for testing the efficiency of gene therapy in regulating thrombus formation in a subject. It also provides a method to test gene therapies directed at correcting genetic mutations associated with von Willebrand disease. The method can comprise the following steps: introducing a vector into the non-human transgenic animal of the invention described above, wherein the vector comprises a nucleic acid encoding a platelet receptor polypeptide, a platelet ligand polypeptide, or a VWF polypeptide, or a portion thereof; allowing sufficient time for expression of the polypeptide; perfusing platelets into the non-human transgenic animal that has one or more mutations in the VWF-A1 domain as previously described under a condition suitable for GPIb-alpha-VWF-A1 protein binding; and identifying an occurrence of a thrombotic event in the animal. For example, the vector introduced into the subject can be an adenovirus or DNA vector described in earlier sections utilizing methods discussed previously (see also Zuckerbraun et al., (2002) Arch Surg. 137(7):854-61; Melo et al., (2004) Arterioscler Thromb Vasc Biol. 24(10):1761-74; and Dulak et al., (2006) Cell Biochem Biophys. 44(1):31-42). For example, the non-human animal that has one or more mutations in the VWF-A1 domain can be the murine model homozygous for the VWF-A11326R>H mutation.
In one embodiment of the invention, the platelets can be human platelets. In particular, the platelets are not murine platelets. In some embodiments, the thrombotic event comprises blood clotting, abnormal bleeding, abnormal clotting, death, or a combination thereof. Such an event can be identified using dynamic force microscopy, a coagulation factor assay, a platelet adhesion assay, thrombus imaging, a bleeding time assay, aggregometry, review of real-time video of blood flow, a Doppler ultrasound vessel occlusion assay, or a combination of the techniques previously described. In a further embodiment, perfusing platelets can be followed by a perfusion of a labeled agent. Non-limiting examples of a labeled agent comprises one or more of a nanoparticle, a fluorophore, a quantum dot, a microcrystal, a radiolabel, a dye, a gold biolabel, an antibody, or a small molecule ligand. In some embodiments, the agent targets a platelet receptor, a VWF protein, or a portion thereof.
The invention also provides a method to correlate results obtained with an in vitro assay designed to measure the effects of antithrombotics or biomarkers of platelet activation in patients. For example, a biomarker is an indicator of a particular disease state or a particular state of an organism, such as when the subject experiences vascular vessel wall injury. Upon injury to the vessel wall and subsequent damage to the endothelial lining, exposure of the subendothelial matrix to blood flow results in deposition of platelets at the site of injury via binding to the collagen with the surface collagen-specific glycoprotein Ia/IIa receptor. This adhesion is strengthened further by the large multimeric circulating protein VWF, which forms links between the platelet glycoprotein Ib/IX/V and collagen fibrils. The platelets are then activated and release the contents of their granules into the plasma, in turn activating other platelets. For example, Glycoprotein VI (GP6) is a 58-kD platelet membrane glycoprotein that plays a crucial role in the collagen-induced activation and aggregation of platelets. The shedding of GP6 can act as a marker representing that a person is at risk of myocardial infarction. In one embodiment, platelets obtained from a subject determined to have an elevated biomarker level (for example, GP6) can be infused into the non-human transgenic animal described above according to previously described methods, wherein the occurrence of a thrombotic event can be evaluated. In another embodiment, platelets obtained from a subject undergoing an anti-thrombotic treatment can be infused into the non-human transgenic animal described above according to previously described methods, wherein the occurrence of a thrombotic event can then be evaluated.
Method of Screening and Treating Subjects with Abnormalities of Platelet Function
The invention provides methods for treating subject with platelet function abnormalities, such as Von Willebrand disease (VWD), Bernard-Soulier syndrome, May-Hegglin anomaly, Chediak Higashi syndrome, and the like. In addition, the invention also provides methods for detecting abnormal platelet function or morphology in a subject.
VWD is a common hereditary coagulation abnormality that arises from a quantitative or qualitative deficiency of VWF). VWD affects humans, in addition to dogs and cats. There are three types of VWD: type 1, type 2, and type 3. Type 1 VWD is a quantitative defect, wherein decreased levels of VWF are detected but subjects may not have clearly impaired clotting, Type 2 VWD is a qualitative defect, wherein subjects have normal VWF levels but VWF multimers are structurally abnormal, or subgroups of large or small multimers are absent. Four subtypes exist: Type 2A, Type 2B, Type 2M, and Type 2N. Type 3 is rare and the most severe form of VWD (homozygous for the defective gene). (Braunwald et al., Harrison's Principle of Internal Medicine, 15th ed., (Chapter 116) 2001, McGraw Hill, Columbus, Ohio).
Bernard-Soulier Syndrome is a rare disorder caused by a deficiency of the surface platelet receptor GPIb alpha. As a result, platelets fail to stick and clump together at the site of the injury. Functional abnormalities have also been observed in some hereditary platelet disorders wherein the platelets are of abnormal size or shape, such as in May-Hegglin Anomaly and Chediak Higashi syndrome. (Braunwald et al., Harrison's Principle of Internal Medicine, 15th ed., (Chapter 116) 2001, McGraw Hill, Columbus, Ohio).
According to the invention, abnormal platelet function or morphology can be screened in a subject. The method can comprise the following steps: affixing a VWF-A1 molecule to a bottom surface of a flow chamber, or chip (such as a BIAcore chip), wherein the VWF-A1 molecule comprises at least one mutation at a position selected from the group consisting of 1263>S, 1269>D, 1274>R, 1287>R, 1302>D, 1308>R, 1313R>W, 1314>V, 1326>H, 1329>1, 1330>G, 1333>D, 1344>A, 1347>V, 1350>A, 1370>S, 1379>R, 1381>A, 1385>M 1391>Q, 1394>S, 1397>F, 1421>N, 1439>V, 1442>S, 1449>Q, 1466>P, 1469>L, 1472>H, 1473>M, 1475>Q, 1479>G, and any combination thereof, where the position corresponds to an amino acid position of human von Willebrand Factor A1 protein shown in SEQ ID NO: 6; perfusing a volume of whole blood or plasma over the surface-immobilized VWF-A1 molecule complexed to the murine mutant VWF-A1 protein in the flow chamber at a shear flow rate of at least 100 s−1; perfusing a targeted molecular imaging agent into the flow chamber at a shear flow rate of at least 100 s−1; determining whether platelets bind to the surface-immobilized-mutant-murine-VWF-A1 using a diagnostic assay; and comparing diagnostic assay results to a standard control, wherein the standard control sample was subjected to the steps described above. For example, the VWF molecule can be an antibody, a peptide, or a Fab fragment directed to a VWF polypeptide or a portion thereof. In one embodiment, the molecule can comprise a native or mutant VWF-A1 molecule, a purified native VWF or a mutant plasma VWF.
In one embodiment, whole blood or plasma sample can be perfused into the chamber or onto the chip, wherein the sample is obtained from the subject. For example, approximately 50 μl of whole blood can be perfused according to the method, or about 100 μl to about 150 μl of plasma can be perfused. As a standard control, the steps of the method described above can be performed using lyophilized non-self platelets, and can be subsequently compared to results obtained using the subjects' platelets. Here, the subject can be a human, a canine, a feline, a murine, a porcine, an equine, or a bovine.
In one embodiment, the targeted molecular imaging agent can comprise a nanoparticle, a fluorophore, a quantum dot, a microcrystal, a radiolabel, a dye, a gold biolabel, an antibody, a peptide, a small molecule ligand, or a combination thereof. In another embodiment, the targeted molecular imaging agent can bind to a platelet receptor, a platelet ligand, or any region of a VWF protein or a portion thereof. In a further embodiment, the targeted molecular imaging agent can comprise horseradish peroxidase (HRP) coupled to an antibody directed at VWF-A1. Following binding to VWF-A1, a reaction with diaminobenzadine (DAB) can be performed where DAB is reduced by HRP to produce a brown precipitate at the site of binding. This technique allows for enzymatic, colorimetric detection of binding that can be visualized by transmitted light microscopy. For example, if the antibody is directed at a platelet receptor, and colorimetric detection represents whether the antibody bound to the platelet-VWF-A1 complex, the absence of color would denote the lack of a complex formation, thus suggesting that platelets were unable to bind to VWF-A1. The lack of platelet binding could suggest functional defects in the subject's platelets. In one embodiment, platelets bound to VWF-A1 are less than about 500 cells/mm2.
The normal platelet morphology is discoid with some spherical shaping. In one embodiment of the invention, the platelets obtained from the subject and that are subsequently screened are substantially spherical in shape. To further analyze platelet morphology, gross platelet histology can be assessed via light microscopy or electron microscopy. In another embodiment, platelets having an abnormal morphology are greater than about 2 μm in diameter. (Ross M H, Histology: A text and atlas, 3rd edition, Williams and Wilkins, 1995: Chapter 9). Various assays can be used to assess whether platelet function is normal, such as a platelet adhesion assay, fluorescence imaging, a chromogenic indication, microscopy morphology analysis, or those listed in Harrison's Principle of Internal Medicine, 15th ed., ((Chapter 116) 2001, McGraw Hill, Columbus, Ohio), which are hereby incorporated by reference.
The invention also provides a method of treating abnormalities in clotting due to a defect in the interaction between GPIb alpha and the A1 domain of VWF as occurs in certain types of von Willebrand Disease (VWD), where the method entails administering to the subject an effective amount of a compound that promotes platelet adhesion in the subject, wherein the compound abbreviates off-rate (koff) and/or enhances the on-rate binding kinetics, and or strengthens the bond between between VWF-A1 and GPIb-alpha by at least two-fold. Thus, administration of the compound increases blood coagulation in the subject, for example, subjects diagnosed with VWD. In one embodiment, VWD is Type 1 or Type 2. In another embodiment, the compound is one identified by the screening methods described above. Coagulation can be measured by a coagulation factor assay, an ex-vivo flow chamber assay, a platelet adhesion [see EXAMPLES section] or those assays listed in Harrison's Principle of Internal Medicine, 15th ed., ((Chapter 116) 2001, McGraw Hill, Columbus, Ohio).
Therapeutic Formulations
Therapeutic compounds according to this invention are formulated in pharmaceutical compositions containing the compound and a pharmaceutically acceptable carrier. The pharmaceutical composition may contain other components so long as the other components do not reduce the effectiveness of the compound according to this invention so much that the therapy is negated. Some other components may have independent therapeutic effects. Pharmaceutically acceptable carriers are well known, and one skilled in the pharmaceutical art can easily select carriers suitable for particular routes of administration (see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985).
The pharmaceutical compositions containing any of the compounds of this invention may be administered by a topical, oral, rectal, parenteral (such as subcutaneously, intramuscularly, intravenously, intraperitoneally, intrapleurally, intravesicularly or intrathecally), or nasal route, as compelled by the choice of drug and disease. One skilled in the pharmaceutical art can discern the optimal route of administration.
These compounds may also be applied topically or locally, in liposomes, solutions, gels, ointments, biodegradable microcapsules, or impregnated bandages. Compositions or dosage forms for topical application may include suspensions, dusting powder, solutions, lotions, suppositories, sprays, aerosols, biodegradable polymers, ointments, creams, gels, impregnated bandages and dressings, liposomes, and artificial skin.
Pharmaceutical carriers utilized by one skilled in the art which make up the foregoing compositions include petrolatum, polyethylene glycol, alginates, carboxymethylcellulose, methylcellulose, agarose, pectins, gelatins, collagen, vegetable oils, phospholipids, stearic acid, stearyl alcohol, polysorbate, mineral oils, polylactate, polyglycolate, polyanhydrides, polyvinylpyrrolidone, and the like.
Therapy dose and duration will depend on a variety of factors, such as the disease type, patient age, therapeutic index of the drugs, patient weight, and tolerance of toxicity. Initial dose levels will be selected based on their ability to achieve ambient concentrations shown to be effective in in vitro models (for example, a dose level used to determine therapeutic index), in vivo models, and in clinical trials. The skilled clinician using standard pharmacological approaches can determine the dose of a particular drug and duration of therapy for a particular patient in view of the above stated factors. The response to treatment can be monitored by analysis of body fluid or blood levels of the compound and the skilled clinician will adjust the dose and duration of therapy based on the response to treatment revealed by these measurements.
EXAMPLES
A number of Examples are provided below to facilitate a more complete understanding of the present invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only.
Biophysical and molecular approaches are essential for understanding the structure:function relationship between a receptor and its ligand. Thus, the ability to study such interactions in an appropriate physiological and/or pathological setting is desirable. To do so, one requires an animal model that is amenable to genetic manipulation and has receptor-ligand interactions that closely resemble those found in humans. Thrombosis models in hamsters and guinea pigs have proven useful in pharmacological studies, but a mouse model would prove to be more beneficial based on the ability to insert or delete genes of interest, accessibility of tissues for study, and cost and ease of handling (49, 50). Regarding GPIb alpha-VWF interactions, two groups have significantly advanced the understanding of the importance of these interactions in mediating thrombosis by generating mice deficient in these proteins (51, 52). Yet, no information regarding the role of the biophysical properties of the GPIb alpha-VWF-A1 in regulating the processes of thrombosis and hemostasis will be obtained. Thus, the next logical approach is to generate animals with mutations within the VWF-A1 domain that change its kinetic properties in a desired manner and to correlate these biophysical alterations with the ability of these mice to maintain adequate hemostasis and to develop thrombi in response to vascular injury. Such information will be useful in designing therapies that reduce or enhance these processes.
Example 1
VWF Characterization
VWF Microsphere Studies
The association and dissociation kinetics of the GPIb alpha-VWF-A1 bond and the impact of fluid shear and particle size on these parameters can be determined by measuring the frequency and duration of transient adhesive events, known as transient tethers, that represent the smallest unit of interaction observable in a parallel-platelet flow chamber.
Production of Recombinant VWF-A1 Protein and Coating of Microspheres.
The generation of recombinant VWF-A1 protein (residues 1238 to 1472 of the mature, recombinant VWF) and its subsequent coupling to microspheres is performed as previously described (Doggett, T. A. et al. (2002) Biophys. J. 83, 194-205). Proper size, purity, and disulfide bonding of all proteins is assessed by Coomasie-blue staining of SDS-PAGE gels run under reducing and non-reducing conditions. Mass spectrometry is also employed to evaluate size and disulfide bonding pattern.
The resulting recombinant proteins are bound to polystyrene microspheres (goat anti-mouse IgG (FC); Bangs Lab, Inc., Fishers, Ind.) that were initially coated with a saturating concentration of mouse anti-6-HIS antibody as previously described in our publications. We have found this coating method to be superior to direct covalent coupling of the VWF-A1 to the beads as it prevents significant loss in protein function. Estimation of the amount of VWF-A1 bound to the beads is determined using a monoclonal antibody generated in our lab against the human and murine A1 domains, mAb AMD-1 and mAb AMD-2, respectively, and a calibrated microbead system (Quantum Simply Cellular; Flow Cytometry Standards Corp., San Juan, P.R.) following the manufacturer's instructions.
Laminar Flow Assays.
In flow assays involving protein-coated microspheres, human or murine platelets purified by gel filtration are incubated with 10 mM sodium azide (NaN3), 50 ng/ml prostaglandin E1, and 10 μm indomethacin (Sigma Immunochemicals, St. Louis, Mo.) to reduce the possibility of activation and potential alterations in expression and/or distribution of GPIb alpha on their surface. Platelets are subsequently allowed to settle in stasis on Fab fragments of monoclonal antibodies that recognize either human (i.e., mAb 7E3) or murine (i.e., mAb NAD-1) alpha IIb/β3 in order to form a reactive substrate. The use of platelets in lieu of recombinant proteins or transfected cells as the immobilized substrate enables evaluation of GPIb alpha in its native form (i.e. correct orientation and proper post-translational modification). Platelet coverage of <10% will be bound in this manner can remain relatively unactivated for up to 30 min as evident by morphology on light microscopic examination (FIG. 7A) and lack of expression of P-selectin by fluorescence microscopy (FIG. 7B).
To reduce the possibility of multiple bond formation that would result in a prolongation in interaction times between the beads and immobilized platelets, the lowest site densities of VWF-A1 capable of supporting these brief interactions is used, a value we found to correspond to  ̃30 molecules μm2. At this site density, we have shown that the formation of transient tethers between this receptor-ligand pair has distribution of bond lifetimes that obey first order dissociation kinetics. The duration of these interactions are measured by recording images from a Nikon X60 DIC objective (oil immersion) viewed at a frame rate of 235 fps (Speed Vision Technologies, San Diego, Calif.) and subjected to wall shear stresses (WSS) ranging from 0.5 to 3.0 dyn cm−2. The cellular off-rates are determined by plotting the natural log of the number of VWF-A1 coated microspheres that interacted as a function of time after the initiation of tethering, the slope of the line=−koff (s−1) which is the inverse of the bond lifetime. The force acting on the tether bond was calculated from force balance equations as stated above and koff plotted as a function of these forces. An example of the measurement of the duration of a transient tether and estimation of off-rates as a function of WSS is demonstrated below for WT human VWF-A1 (FIGS. 8A-8C). To demonstrate that our method for surface immobilization of platelets does not result in an alteration in the kinetics of the GPIb alpha-VWF-A1 tether bond, resting platelets were first fixed in paraformaldehyde prior to immobilization. As these platelets cannot activate, the kinetics should be reflective of GPIb alpha in the resting state. Indeed, analysis of the koff for this interaction using fixed platelets was identical to that observed for platelets treated with metabolic inhibitors (FIG. 8D).
The Structure-Function of Murine VWF-A1
To determine the structure and function of murine VWF-A1, its adhesive interactions with murine and human GPIb alpha, and whether the kinetics of this interaction mimic those reported in studies of its human counterpart, the domain was initially cloned by PCR from purified mouse genomic DNA. For the purpose of generating a mouse with a genetically modified VWF-A1 domain, a100-kb P1 clone was obtained from screening a 129/Svj DNA genomic library (Genomic Systems, St. Louis, Mo.) by polymerase chain reaction (PCR) using primers directed against a 200 bp region of exon 28. Sequence analysis of flanking regions (10 kb in size) as well as the A1 domain itself was performed and compared to those obtained from a BLAST search to confirm the fidelity of the clone. The deduced single-letter amino acid sequence of mouse VWF-A1 domain (M VWF) is shown compared to its human counterpart (H VWF) and encompasses amino acids 1260 to 1480 (FIG. 9). The locations of cysteines forming the loop structure are numbered (1272 and 1458) and differences in residues are highlighted in red. Conversion of the arginine (R) in the mouse A1 domain to histidine (H) as found in its human counterpart (blue χ) has been shown to enable mouse VWF to bind human platelets and simultaneously reduce the binding of mouse platelets. Locations of some, but not all, mutations known to affect human VWF-A1 function are also depicted.
Although the amino acid sequence homology is  ̃85%, and preliminary studies suggest that functional differences do exist between human and murine VWF-A1 domains. In a Ristocetin-induced platelet aggregation assays, platelet GPIb alpha binding to wild type human VWF or mouse VWF was analyzed in the absence or presence of ristocetin as described by Inbal, et al. (1993, Thromb. Haemost., 70:1058-1062). In this method, platelet rich plasma (PRP) is placed in a clear cuvette containing a stir bar and inserted into the aggregometer. Platelet aggregation is induced by the addition of ristocetin. In this ristocetin-induced platelet aggregation assay (RIPA), we observe that concentrations of this modulator that are known to cause agglutination of human platelets ( ̃1.0 mg/ml) had no such effect using murine PRP (FIG. 10B). In fact, only at concentrations of ≧2.5 mg/ml was there any evidence of murine platelet aggregation observed ( ̃30%, FIG. 10C). In comparison, incubation of murine PRP with thrombin resulted in >90% platelet aggregation (FIG. 10A).
To better evaluate the above interactions and to compare functional relationships between human and murine VWF with GPIb alpha, VWF from human and mouse plasma was purified and its ability to mediate platelet adhesion in flow was determined. Multimer gel analysis did not reveal any differences between the two species, especially with regard to high molecular weight components (FIG. 11A). Moreover, surface-immobilized murine VWF could support adhesion of syngeneic platelets (1×108/ml) at a shear rate encountered in the arterial circulation (1600 s−1) as observed for the human plasma protein (FIG. 11B). In contrast, murine VWF did not support significant interactions with human platelets and vice versa. These results suggest that functional and possibly significant structural differences do exist between the A1 domains of murine and human VWF as primary attachment of platelets at this wall shear rate is dependent on its function. Thus, generation of a recombinant murine VWF-A1 domain is required to fully evaluate similarities and/or differences from its human counterpart.
Recombinant protein was expressed using a bacterial expression vector under the control of an inducible promoter (pQE9, Qiagen). Insertion of the murine fragment containing the majority of the VWF A1-domain (encoding for amino acids 1233 to 1471) into pQE9 produces an amino-terminal fusion protein containing 10 amino acids (including 6× histidine) contributed by the vector. After induction, inclusion bodies were harvested, washed, and solubilized according to previously published methods (32). The solubilized protein was diluted 40-fold in 50 mM Tris-HCl, 500 mM NaCl, 0.2% Tween 20, pH 7.8 and initially purified over a Ni2+-chelated Sepharose (Pharmacia) column. To increase the yield of functional protein, the material purified from the Ni2+ column was absorbed to and eluted from a Heparin-Sepharose column (Amersham Pharmacia Biotech).
The highly purified protein was dialyzed against 25 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20, pH 7.8. SDS-PAGE analysis revealed a prominent protein band of  ̃34,000 Da under non-reducing conditions (FIG. 12). The overall yield of protein obtained using the purification methods described above is  ̃2 mg/l of bacterial cells.
The protein was subsequently used in a series of in vitro flow chamber assays to assess function. Washed human or murine platelets (5×107/ml) were infused through a parallel plate flow chamber containing glass cover slips coated with either human VWF-A1 or mouse VWF-A1 protein (100 μg/ml final concentration) at a shear rate of 800 s−1. After 5 min of continuous flow, adherent platelets were quantified.
As shown in FIG. 13A, mouse VWF-A1 protein supported platelet adhesion as efficiently as its human counterpart under physiological flow conditions. To demonstrate the importance of the single disulfide bond formed by C1272 and C1458, reduced (DTT) and alkylated (iodoacetamide) mouse VWF-A1 was prepared and tested in flow. Reduction and alkylation of the protein abrogated attachment of murine platelets in flow. In addition, the limited ability of the native form of the protein to mediate adhesion of human platelets and lack of interaction between human VWF-A1 and mouse platelets suggests that structural/conformational differences exist between the species. However, this does not preclude the study of GPIb alpha-VWF-A1 interactions in mice as both proteins must share common kinetic attributes as they support rapid attachment and translocation of platelets to a similar degree under physiological flow conditions (FIG. 13B).
To demonstrate that the presence of the N-terminus His tag does not appear to affect the function of the recombinant protein, we compared the ability of a tagged vs. a non-tagged M VWF-A1 to support mouse platelet adhesion in flow. In the case of the latter, the murine A1 fragment was inserted into pET-11b (Stratagene) and purified as previously described (53). The purified bacterial non-His tag protein was analyzed by SDS-PAGE (12.5%) and found to migrate in an analogous manner to its tagged counterpart under non-reducing and reducing conditions (FIG. 14A). In addition, no differences were observed in the number of platelets that adhered to and translocated on either protein (449±53 platelets/mm2 His-tag vs. 423±17 platelets/mm2 non-His tag) at a shear rate of 800 s−1 (FIG. 14B).
Characterization of the M VWF-A1 Domain.
The A1 domains of human and mouse serve an identical purpose: to mediate primary attachment and translocation of platelets in flow. The crystal structure of the mouse A1 domain was solved using recombinant proteins (Fukuda, K., et al., (2005) Nat. Struct. Mol. Biol. 12:152-159). The main chain schematic of this domain, with β-strands (arrows) and helices (coils), is shown in FIG. 15A. The model was built from residues 1270 to 1463 of the murine VWF-A1 crystal. The two cysteines involved in the disulfide bridge are shown as yellow spheres (involving residues 1272 and 1458). The mouse and human A1 domains appear to overlap very closely, which suggests that only minor structural differences may account for the preferential binding of platelets from mice or man to their respective VWF-A1 proteins (FIG. 15B). In fact, the β-sheets of both species are identical within experimental error (a root mean square difference of 0.33 Å for Cα atoms). Thus, minor differences in residues, but not structure, most probably account for the inability of human platelets to interact with mouse VWF-A1 and vice versa.
Support for this hypothesis is provided by mutagenesis studies. By analyzing the data obtained from the crystal structure of the murine VWF-A1 domain, we have identified several residues that may participate in interactions with GPIb alpha (FIG. 15C). Residue 1326 was initially chosen for study and was mutated to the corresponding amino acid at the identical location in its human counterpart (from Arg to His). Subsequently, the ability of murine and human platelets to interact with this mutant protein substrate was evaluated at a wall shear rate of 800 s−1. Incorporation of a histidine for arginine at position 1326 in murine VWF-A1 reduced murine platelet adhesion by  ̃5-fold and increased translocation velocities of cells by  ̃7-fold as compared to the WT mouse protein (FIGS. 13A, 13B, 16A, and 16B). Interestingly, human platelet interactions with the mutated murine protein were comparable to that of WT human VWF-A1. Conversely, substitution of Arg for His in the human VWF-A1 protein resulted in an increased ability of murine platelets to attach and translocate in a manner similar to that observed for WT murine VWF-A1. These studies support the hypothesis that from a structural and functional standpoint, mouse and human VWF-A1 are very similar.
Thus, all that remains is to demonstrate that the kinetics of the interaction between the murine GPIb alpha and murine VWF-A1 are similar to its human counterpart and that mutations in man that cause functional alterations in platelet adhesion with VWF have the identical impact on the biophysical properties of the murine receptor-ligand pair.
The Kinetics of Murine VWF-A1
To determine whether the kinetics of the murine GPIb alpha interactions with the murine VWF-A1 domain is similar to that of the human receptor-ligand pair, we measured the dissociation of transient tethering events using VWF-A1 coated beads (7 μm diameter) interacting with surface-immobilized platelets. The use of beads with one uniform size and shape permits determination of the relationship between wall shear stress and the force directly acting on the GPIb alpha-VWF-A1 tether bond (Fb), a parameter difficult to estimate for discoid-shaped objects such as platelets. A coating concentration of VWF-A1 was chosen (5 μg/ml corresponding to 30 molecules/m2) that supported tether bond formation at wall shear stresses ranging from 0.5 to 3 dyn cm−2. Estimation of the site density of murine VWF-A1 on beads was performed using a monoclonal antibody generated in our laboratory designated as AMD-2. This antibody was made by immunizing Fischer 344 rats (3-4 months old) with recombinant WT protein. Following several injections of murine VWF-A1, serum was collected and screened by ELISA for anti-VWF-A1 antibodies. Spleens from animals with the highest antibody titers were harvested and splenocytes fused with Sp2/0 mouse myeloma cells (54).
Supernatants of hybridomas were screened for reactivity to mouse VWF-A1 by ELISA (FIG. 17). Pre-immune rat serum was used as control. Monoclonal antibodies (Mabs) to murine VWF-A1 not only reacted with WT and mutant proteins (1324G>S) but also recognized native VWF purified from mouse plasma. Antibodies are currently being tested for function blocking capabilities to use in both in vitro and in vivo assays. Antibodies will also be used for epitope mapping.
Analysis of the distribution of interactions times between human or murine VWF-A1 coated beads and their respective platelet substrates, as measured by high temporal resolution videomicroscopy, indicate that >95% of all transient tether bonds events fit a straight line, the regressed slope of which corresponded to a single koff (FIGS. 18A-C). Notably, the cellular off-rates of these quantal units of adhesion for the WT human and mouse proteins (FIGS. 18A and B) were quite similar, but were significantly higher than those observed for the murine VWF-A1 containing the type 2B mutation 11309V (1309I>V) (FIG. 18C). This is consistent with previous results obtained using the same mutation in the human protein (Table 1).














TABLE 1







SINGLE
H1326R*
G1330E*
R1287M*
Q1391P*
A1350T*
S1370G*
D1333A*









Based on these preliminary results, it appears that the dissociation kinetics of murine GPIb alpha interactions with murine VWF-A1 are nearly identical to its human counterpart and that type 2B mutations also prolongs the bond lifetime of this interaction as seen in man. A complete biophysical analysis is underway and in order to determine values for the intrinsic koff and susceptibility of the bond to force drive dissociation as performed previously for its human counterpart.
Example 2
VWF-A1 Mutagenesis
Preliminary results indicate that minor differences may exist between murine and human VWF that would preclude one from studying human platelet behavior in a mouse model of thrombosis. However, our findings that the estimated off-rate values and structure of these domains are similar suggest that one can investigate the role of the biophysical properties of the GPIb alpha-VWF-A1 bond in regulating platelet-VWF interactions in vivo using a mouse model. However, neither a delineation of the binding region for GPIb alpha within the murine VWF-A1 domain nor determination of the impact of mutations on the kinetics of this interaction has been performed to date. Thus, both murine and human A1 crystal structures can be exploited to 1) identify candidate residues involved in the binding site for murine GPIb alpha and to determine their impact on the kinetic properties of this receptor-ligand pair, 2) identify residues that confer species specificity, and 3) ascertain whether insertion of known point mutations that cause type 2M and 2B VWD in man alter the kinetic properties of the murine A1 domain in a similar manner. Critical residues can be classified in terms of their impact on the cellular association and dissociation rate constants. Information obtained from these studies can be used to generate mice with mutant A1 domains in order to establish the degree in alteration in the kinetics of the GPIb alpha-VWF-A1 bond that is necessary to perturb hemostasis.
Similar critical structural elements exist in murine A1 domain to those identified in its human counter-part that contribute to the biophysical properties of the bond formed with GPIb alpha. Thus, to identify structural elements within the murine VWF-A1 domain that impact on the kinetics of interaction with GPIb alpha, the hypothesis that only minor structural alterations in this domain are responsible for its reduced ability to support interactions with GPIb alpha receptor on human platelets will be tested.
Site-Specific Mutagenesis of Murine VWF-A1 Domain
Site-specific mutagenesis of murine VWF-A1 domain will be performed to define residues that contribute to GPIb alpha binding as well as those in that regulate this interaction. Studies will initially focus on amino acids that differ between human and mouse A1 that lie within the vicinity of the proposed GPIb alpha binding pocket.
To better define residues within murine VWF-A1 that are critical for binding of GPIb alpha on mouse platelets, mutations into VWF-A1 cDNA using a PCR-based strategy will be introduced and the resulting DNA will be sequenced to confirm the presence of the desired mutation(s). Mutations will be based on the murine A1 crystal structure and amino acid substitutions known to affect human VWF function such as those associated with Type 2M or 2B vWD (Tables 2-4). Several surface exposed residues have been identified within the murine A1 domain likely to participate in GPIb alpha binding. These are non-conserved residues in comparison to the human domain. Thus, we will convert these residues at first singly (then doubly and triply), into the murine VWF-A1 to those found in human VWF-A1*. Residues are chosen based on surface-exposure on the front and upper surfaces of the domain as understood by modeling and crystal structure analysis.














TABLE 2







SINGLE
R1326 H*
E1350G*
M1287R*
P 1391 Q*
T1350A*
G1370S*
A1333D*








DOUBLE
R 1287 M* + Q 1391 P*


TRIPLE
H 1326 R* + G 1350 E* + A1333D*






















TABLE 3









SINGLE
S1289R*
D1323R*
K1348E*
R1392E*










Residues that perturb but do not abrogate platelet binding in the human VWF-A1 protein (Table 3, FIG. 5B).












TABLE 4







TYPE 2M
G1324S*
Q1367R*
I1369F*
I1425F*


TYPE 2B
R1306L* 
I1309V*
V1316M*
R1341L*









Residues chosen based on their ability to abrogate or enhance interactions between human VWF and GPIb alpha (Table 4).

Type 2B mutations will also be combined with those that dramatically shorten the bond lifetime to determine (increase koff) if these function-enhancing mutations can restore adhesion to that observed for WT VWF-A1.
Laminar flow assays will be performed to assess the impact of various mutations on platelet adhesion as well as the degree in alteration in the kinetic properties of the GPIb alpha-VWF-A1 bond.
Murine platelets will be purified as described above and stored in Tyrode's buffer containing 0.25% BSA, pH 7.4. For studies requiring human platelets, blood will be collected by venopuncture from healthy donors and cells obtained from centrifugation of PRP. All platelets will be used within 2 hours of purification.
To evaluate the impact of mutations on platelet adhesion, both human and murine platelets are perfused over high concentrations of murine VWF-A1 proteins (100 μg per ml) absorbed to glass cover slips in a parallel plate flow chamber at wall shear rates ranging from 20 to 1600 s−1. An enzyme-linked immunosorbent assay is utilized to ensure that an equivalent amount of recombinant WT or mutant protein is immobilized. The number of platelets that attach per unit area per min and their velocity of forward motion (μm/s), termed translocation, is recorded on Hi-8 videotape using an inverted Nikon microscope with a plan 10× or 40× objective, respectively. In addition, whether the incorporated mutations alter the requirement for a critical level of hydrodynamic flow, termed the shear threshold phenomenon, to support interactions of platelets with the reactive substrate can be determined. It has been previously demonstrated that human attachment to immobilized VWF-A1 requires a minimum of >85 s−1 of WSR to initiate and sustain this interaction. This phenomenon has also been well described for selectin-dependent adhesion and is believed to rely on a balance between the number of times a receptor encounters it ligand over a defined period of time and the rate at which a bond can form, parameters affected by shear rate, association rate constant, and receptor-ligand concentrations (59, 60). Once attached, however, the bond lifetime influences the velocity at which the cell will move on the reactive substrate in response to shear-induced force (61). Thus, it is likely that several mutations may perturb platelet accumulation on surface-bound VWF-A1 by altering the level of shear flow required to promote platelet attachment as well as the translocation velocities of these cells. For example, it has been shown that the type 2B mutation, Ile1309Val (1309I>V), promotes greater platelet attachment at low shear rates and reduces their translocation velocities as compared to the WT substrate. Similar results were observed upon incorporation of the identical mutation into murine VWF-A1. Demonstration that GPIb alpha on murine platelets is responsible for mediating interactions with recombinant A1 domains can be confirmed by antibody blocking experiments.
Determination of Tethering Frequencies, Translocation Velocities, Detachment Profiles, and Dissociation Rate Constants Using VWF-A1 Coated Microspheres.
To better ascertain the alteration in kinetics associated with the proposed mutations, the tethering frequency, translocation velocities, detachment profile, and off-rates of VWF-A1 coated beads (7 μm diameter) interacting with surface-immobilized platelets can be measured. As stated before, the use of beads with one uniform size and shape will permit determination of the relationship between wall shear stress and the force directly acting on the GPIb alpha-VWF-A1 tether bond (Fb), a parameter difficult to estimate for discoid shaped objects such as platelets. Platelet coverage >90% of the glass surface area is used in determining the tethering frequency (on rate driven phenomenon), translocation velocities (correlates with off-rate) and resistance to detachment forces (measure of bond strength) of VWF-A1 coated beads in flow. Recently, we have demonstrated that comparison of cellular on-rates and apparent bond strengths between WT and mutant forms of VWF-A1 can be achieved by limiting the concentration of these molecules as to prevent multiple bond formation, a process that can mimic an enhancement in either of these kinetic parameters. By using a similar strategy, we can determine whether the proposed mutations will alter the apparent on-rate of the GPIb alpha-VWF-A1 bond by evaluating the frequency of transient tethering events between microspheres coated with low site densities of VWF-A1 proteins and a platelet substrate. Results are expressed as the percentage of beads (per 10× field) that paused, but did not translocate, on over a range of wall shear rates that support such interactions (20 to 400 s−1). Tethers per minute are divided by the flux of beads near the wall per minute to obtain the frequency of this adhesive interaction. Only one tethering event per bead is counted during the observation period.
For determining translocation velocities, beads (1×106/ml), coated with a saturating concentration of VWF-A1 protein are infused into the parallel-plate flow chamber at 1.0 dyn cm−2 and allowed to accumulate for 5 min. Subsequently, the wall shear stress is increased every 10 s to a maximum 36 dyn cm−2 and the velocities of the beads determined.
For detachment assays, beads (1×106/ml), coated with the minimum but equal amounts of VWF-A1 required to support translocation, are infused into the parallel-plate flow chamber at 1.0 dyn cm−2 and allowed to accumulate for 5 min. Subsequently, the wall shear stress is increased every 10 s to a maximum 36 dyn cm−2. The number of beads remaining bound at the end of each incremental increase in wall shear stress is determined and expressed as the percentage of the total number of beads originally bound. Using this strategy, we were able to support our claim that type 2B mutations do not strengthen, and in fact may even weaken the interaction between GPIb alpha and VWF-A1 as suggested by the increase in reactive compliance as compared to the native complex (Table 1). In all studies, video images are recorded using a Hi8 VCR (Sony, Boston, Mass.) and analysis of performed using a PC-based image analysis system (Image Pro Plus).
For determining the kinetics of dissociation, we measure the duration of transient tethers between murine VWF-A1 coated microspheres and immobilized murine platelets as described herein. MC simulations are run and estimates of koff fit to the Bell model by standard linear regression to obtain the intrinsic off-rate (k0off) and the reactive compliance σ. Results are compared for all mutations to determine their impact on these kinetic parameters (i.e., increase or shorten the bond lifetime (k0off) and/or increase or decrease the susceptibility of the bond to hydrodynamic forces (σ).
These experiments complement recent work on identifying residues in human VWF-A1 domain critical for interacting with the GPIb alpha. Moreover, they allow for delineation of its binding site in murine VWF-A1. We believe this to be important, as this work will be essential for elucidating the role of the biophysical properties of this receptor-ligand pair in regulating platelet-VWF interactions in vivo. Furthermore, it will pave the way for the generation of mice with comparable types of human vWD (i.e., type 2B) and may even permit the study of human platelets in a mouse model of thrombosis.
Although our approach to mapping the GPIb alpha binding site is reasonable based on our previous studies, there is no guarantee that the introduction of mutations will not significantly perturb protein structure and thus function. The ability of murine VWF-A1 specific mAbs to recognize mutant proteins should clarify this matter. Similarly, our proposed gain/loss of function experiments involving swapping of residues between human and mouse VWF-A1 will also prove useful in avoiding this pitfall.
It is important to know whether the regions flanking the mouse A1 domain are important in mediating interactions with GPIb alpha. To this end, we plan to express full-length mouse VWF by inserting it into a mammalian expression vector and transfecting it into COS-7 cells (62). Mutations found to be critical for binding, will be inserted into the full-length construct. As we are in the process of generating the full-length cDNA, we will initially attempt to generate a recombinant protein containing the A1-A2-A3 domains to use in our studies. This will be accomplished using a baculovirus expression system as demonstrated for GPIb alpha.
The Relationship Between the Major and Minor Binding Sites for GPIb Alpha.
The recent results on the structure of GPIb alpha and its complex with VWF-A1 domain has not only confirmed our work as well as others with regard to the major binding site for this platelet receptor, but has shed new light into the mechanism by which type 2B mutations may enhance this critical interaction. As shown in FIG. 6, the concave face of GPIb alpha embraces the A1 domain in two distinct regions. The C-terminal loop of this receptor binds near the top of the domain (major binding site) and the N-terminal region known as the β-finger, at the bottom face (minor binding site) adjacent to the site where type 2B mutations are clustered. Based on these results that type 2B mutations appear to enhance the on-rate (reduced shear rate needed for formation of transient tethers in flow) and prolong the lifetime (5-6 fold) of the interaction between VWF-A1 and GPIb alpha, it is interesting to speculate whether similar alterations in bond kinetics would be observed with type 2B mutations if one interfered with the primary site. For instance, would inclusion of a type 2B with a type 2M mutation reconstitute adhesion, or is some finite interaction time required in the primary binding pocket for GPIb alpha before the effects of these mutations can be observed? These are important questions as they will guide the development of reagents that can either enhance or reduce the interaction between GPIb alpha and VWF-A1.
The type 2B mutation 1309I>V was incorporated into recombinant human VWF-A1 containing either the type 2M mutation Gly1324Ser (1324G>S) that completely abolishes adhesion or the function reducing mutation His1326Arg (1326H>R) and determined the ability of these doubly mutated proteins to support human platelet adhesion in flow. In comparison to WT, a  ̃3-fold increase in wall shear rate is required to promote platelet attachment to human VWF-A1 containing Arg at 1326 (FIG. 19A). Incorporation of the type 2B mutation, however, appeared to enhance the on-rate of this interaction as manifested by an increase in platelet binding at lower levels of shear flow, but not to levels observed for the type 2B mutation alone.
To determine whether the 1309 mutation would also prolong the lifetime of the interaction with GPIb alpha, the distribution of interactions times was analyzed between VWF-A1 coated beads and surface-immobilized platelets at a wall shear stress of 1 dyn cm−2. Remarkably, a 2-fold reduction in koff was noted (from 15.6 to 8.5 s−1) as compared to the single, function-diminishing mutation (FIG. 19B). This value is similar to that of the native receptor-ligand interaction with a koff of 6.5 s−1 under identical flow conditions. By contrast, incorporation of Val for Be at residue 1309 in an A1 domain containing the type 2M mutation 1324G>S did not reconstitute platelet adhesion. Thus, these results suggest that it is essential for bond formation to occur in the primary GPIb alpha binding site (top face of the A1 domain). Moreover, the region of the A1 domain where type 2B mutations are clustered appears to be critical for stabilizing interactions with GPIb alpha. Similar findings were observed for murine VWF-A1 containing the identical type 2B mutation but with a change in Arg to His at residue 1326. A complete biophysical analysis is underway and in order to determine the full extent of the 1326 mutation on the intrinsic koff, the susceptibility of the bond to force drive dissociation, and whether type 2B mutations can restore these parameters to levels obtained for the native receptor-ligand bond. In addition, it would be interesting to determine in vivo whether the enhancement in binding and increase in bond lifetime imparted by the type 2B mutation would correct any perturbation in hemostasis that may occur as a result of impacting on the function of the primary binding site for GPIb alpha.
Example 3
Genetically Modified VWF-A1 Mice
Recent kinetic evaluation of mutations associated with type 2B and platelet-type vWD suggests that the intrinsic properties of the GPIb alpha-VWF-A1 tether bond contribute to the regulation of platelet interactions with VWF. This is also supported by our preliminary studies investigating the impact of botrocetin on the biophysical properties of this receptor-ligand pair. Thus, by using the information obtained in Example 2, mutations can be incorporated into the murine A1 domain of the VWF gene that increase or decrease the intrinsic on- and off-rates by varying degrees in order to truly understand the importance of these kinetic parameters in controlling platelet adhesion. Moreover, the role of the minor binding site, where the majority of type 2B mutations have been identified, can be further delineated by combining such mutations with those that significantly shorten the lifetime of interaction between GPIb alpha and VWF-A1. Results indicate that substitution of the murine residue Arg at position 1326 for His at the same location in the human A1 domain results in a diminished on-rate as manifested by the increased requirement for shear flow to promote attachment and a significant increase in koff (shortening of bond lifetime). Subsequent incorporation of the type 2B mutation 1309I>V into this mutant domain significantly reverses the functional defect in adhesion and returns the off-rate closer to that observed for the WT domain. Similar results have been obtained with murine VWF-A1 in which Arg was replaced by His at residue 1326. Thus, introduction of these two mutations separately and then together into the mouse VWF gene will be the initial focus. This single amino acid substitution enables the mouse VWF-A1 domain to bind platelets at levels equivalent to its human counterpart. Thus, introduction of the two mutations separately (1326R>H or 1309I>V) and then together into the mouse VWF gene has been the target of recent studies. In addition, replacing >90% of the entire mouse A1 domain with its human counterpart is also central to current investigations. This model will enable one to test all potential therapies directed against this human domain in a mouse model of thrombosis.
Generation of mice that incorporate mutations into their A1 domain that significantly shorten the bond lifetime will be present with prolonged bleeding times and will be resistant to thrombus formation, while the additional incorporation of a type 2B mutation will correct these abnormalities by prolonging the tether bond lifetime to that observed for the WT domain. This should allow for sufficient time to form multiple bonds between platelets and VWF deposited at sites of vascular injury. Moreover, mice possessing the 1326R>H mutation should be able to support human platelet adhesion at sites of vascular injury. Thus, it will be demonstrated in vivo that the intrinsic properties of the GPIb alpha-VWF-A1 tether bond are indeed critical for regulating the interactions between platelets and VWF at sites of vascular injury.
By performing a detailed kinetic analysis of mutant VWF-A1 domains prior to the generation of animals with the identical substitutions in amino acids, we have greatly increased the likelihood of altering the interaction between platelets and VWF in a similar manner. We will be able to study the role of the intrinsic properties of the bonds formed between this receptor-ligand pair under complex hemodynamic conditions (i.e., in vivo). The initial choice of the 1326R>H mutation will be of benefit in two ways. First, it allows us to test our hypothesis that a critical interaction time between platelets and VWF is essential to maintaining adequate hemostasis. Secondly, the ability of this mutation to enhance human platelet adhesion will permit us to study their behavior in a mouse model of thrombosis. This is an intriguing concept as it may pave the way to test the impact of various pharmacological agents on human platelet adhesion at sites of vascular injury. For example, a substitution in which a single mutation in murine VWF-A1 (1260 to 1480) can be made in order to achieve human platelet bonding. In addition, a combination of two or more mutations that further perturb the kinetics of the interaction to achieve human platelet binding can also be made. In some instances, the entire mouse A1 domain in the mouse VWF gene with the human A1 domain found in the human VWF gene can be replaced.
Generation and Characterization of Mice Expressing a Mutant VWF-A1 Domain
Mutant Mouse.
As mentioned previously, a 100-kb P1 clone containing the majority of the VWF gene (Genomic Systems, St. Louis, Mo.) was obtained. Digestion with Bam H1 resulted in a  ̃5.3 kb fragment containing part of intron 28 (including the splice sites), all of exon 28 and part of intron 29 which was the inserted into the pSP72 vector (Promega, Wis.). This was subsequently digested with Bam H1 and Eco R5 to yield a 2.9 kb (including exon 28) and a 2.4 kb fragment, designated Arm 1 and 2, respectively, both of which were subcloned back into pSP72 vector. This facilitated site-directed mutagenesis of the A1 domain contained within Arm 1. In addition, the 3′ end of Arm 1 was extended 2 kb by PCR. Subsequently, Arms 1 and 2 were inserted into a lox P-targeting vector as shown below (FIGS. 20A and 20B). The fidelity of three constructs containing either the 1309I>V or 1326R>H substitutions or both mutations was confirmed by sequence analysis.
R1 embryonic stem cells derived from a 129/Sv×129/Sv-CP Fl 3.5-day blastocysts were electroporated with 25 μg of linearized targeting construct and selected in both G418 (26 μmol/L) and gancyclovir (0.2 μmol/L). Genomic DNA from resistant clones were digested with EcoR1 or KpnI, and analyzed by Southern blot hybridization with probe “a” or “b”, respectively, to determine if the construct was appropriately targeted (FIG. 20B). Targeting both the type 2B (Ile1309Val or 1309I>V) mutation and the Arg1326His (1326R>H) mutant constructs, have been successful. In a second step, embryonic stem cell clones that had undergone homologous recombination were transfected with 25 μg of Cre-recombinase-expressing plasmid and selected for G418. Clones in which the neo-cassette was deleted were identified by PCR and injected into C57BL/6 blastocysts (The Siteman Cancer Center Core Facility, Washington University). Male chimeric mice were bred to C57BL/6 Cre-recombinase (+) females to obtain heterozygous animals. Heterozygous mice lacking the neocassette, but containing the 1326R>H mutation, were interbred to obtain wild-type, heterozygous, and homozygous animals. Animals were identified by both Southern analysis (FIG. 21) and by PCR of the A1 domain (FIGS. 22A-22C; red boxed area denotes the conversion of Arg to His).
Determination of the Multimeric Composition of Murine VWF.
For platelet counts, whole blood will be collected into heparinized tubes and 100 μl volumes will be analyzed on a Hemavet (CBC Tech, Oxford, Conn., USA) Coulter Counter. The multimeric structure of murine VWF will be assayed by using the Pharmacia Phast Gel System (Pharmacia LKB Biotechnology). Briefly, samples diluted in 10 mmol/L Tris/HCl, 1 mmol/L EDTA, 2% SDS, 8 mol/L urea, and 0.05% bromophenol blue, pH 8.0, will be applied to a 1.7% agarose gel (LE, Seakem, FMC Bioproducts) in 0.5 mol/L Tris/HCl, pH 8.8, and 0.1% SDS with a stacking gel consisting of 0.8% agarose (HGT, Seakem) in 0.125 mol/L Tris/HCl, pH 6.8, and 0.1% SDS. After electrophoresis the protein will be transferred to a polyvinylidene fluoride membrane (Immobilon P, Millipore) by diffusion blotting for 1 hour at 60° C. The membrane will be blocked with 5% nonfat dry milk protein solution for 1 hour at room temperature. After washing with PBS/T, pH 7.4, the blot will be incubated with a polyclonal antibody raised in rabbits against murine VWF at a dilution of 1:500, washed, and incubated with a goat anti-rabbit horseradish peroxidase (Sigma) diluted 1:2000 in PBS/T. After three washes with PBS/T, the membrane will be incubated with the substrate solution (25 mg 3,3′-diaminobenzidine tetrahydrochloride (Sigma) in 50 mL PBS with 10 μL 30% H2O2). The enzyme reaction will be stopped by washing the membrane with distilled water.
To demonstrate that conversion of Arg to His in the mouse A1 domain at position 1326 did not alter plasma protein levels of VWF nor its ability to form multimers, we performed an ELISA to detect mouse VWF in plasma obtained from WT and homozygous animals (FIG. 23). As shown in FIG. 23, plasma levels of VWF from homozygous mice (KI) were comparable to WT at all dilutions tested. Moreover, multimer gel analysis of plasma VWF revealed an identical banding pattern between mouse and human VWF. Incorporation of His at position 1326 in the mouse A1 domain had no effect on multimerization of VWF (FIG. 24). Thus, we are the first to successfully introduce a point mutation into mouse VWF A1 domain.
Bleeding Time for Human Platelet-Induced Hemostasis:
This assay provides an indirect measure of the ability of platelets and VWF to support hemostasis by interacting with the injured vessel wall. It also indirectly determines the function of multiple receptors and ligands on platelets that are required to form a hemostatic plug. That said, it provides direct evidence that the bleeding defect in our animals can be corrected by the administration of human platelets (FIG. 12). It is performed by immersing the severed tip (10 mm) of the animal's tail in isotonic saline at 37° C. and monitoring the length of time required for bleeding to cease. Homozygous mutant mice will be infused with an equal volume of either saline or purified human platelets. Platelet specific antibodies or drugs will be administered as described above and their ability to prolong bleeding time evaluated. All experiments will be stopped at 10 min by cauterizing the tail (51).
Although homozygous mice bearing the 1326R>H mutation are viable, they demonstrate a bleeding phenotype similar to that of animals deficient in VWF (KO) only when 10 mm of the tail is cut (FIG. 25 and FIG. 35B). Over 90% of these mice bled for a minimum of 8 minutes (end point) in contrast to 3.5 minutes for WT animals as measured by severing 1 cm of their tail. Moreover, thrombus formation induced by perfusion of whole blood from mutant mice over surface-immobilized collagen in vitro was reduced by  ̃80% as compared to WT controls (FIG. 26). To further characterize the bleeding phenotype depicted in FIG. 35B, standard techniques known in the art were employed, which involves removal of a minimal amount of the animal's tail ( ̃5 mm). A slight, but statistically significant (P<0.01), increase in bleeding was observed in the homozygous 1326R>H mutant mice as compared to WT littermates, but not to the extent of the VWF knockout mouse (a mouse model that bleeds profusely due to complete lack of this plasma glycoprotein). In the homozygous 1326R>H mutant mouse, a larger cut ( ̃10 mm) is needed in order to see a bleeding phenotype comparable to the VWF deficient mouse. Since these mice continue to bleed, the experiment is stopped at 8 min in order to prevent death. This phenotype observed in the 1326R>H mutant mouse is similar to the bleeding observed in human patients who have type 2M VWF disease. As not all type 2M mutations result in a complete loss of interaction between GPIb alpha and VWF-A1, but resemble the adhesion defects outlined for the 1326R>H mutant mouse, this genetically modified mouse model will be useful for directing therapies aimed at patients that have a partial but not complete defect in binding between GPIb alpha and VWF-A1.
Platelet Adhesion Studies in Mice Expressing a Mutant VWF-A1 Domain
One goal of the work is to generate mice with mutant A1 domains that alter the kinetics of its interactions with GPIb alpha on mouse platelets. The first mutation we chose to introduce was the substitution of histidine for arginine at position 1326. This mutation was chosen based on our crystal structure analysis of the mouse and human A1 domains, which suggested that the location of this amino acid is central to GPIb alpha binding. Mice bearing this mutation are viable and demonstrate a bleeding phenotype, albeit not as severe as those lacking VWF (VWF KO) (FIGS. 35A and 35B). This was not unexpected as VWF is still present, but has a reduced ability to interact with platelets at high shear rates (>1600 s−1). FIG. 27 demonstrates reduced thrombus formation that occurs when whole blood from these knock-in animals is perfused over collagen-coated cover slips at a shear rate of 1600 s−1. Results thus far indicate a 70% reduction in thrombi formed on collagen as compared to WT controls.
To demonstrate that the 1326R>H mutant in the A1 domain of mouse VWF (wherein the mutant VWF-A1 domain comprises SEQ ID NO: 5) is far superior to promoting interactions with human platelets under physiologic flow conditions, anticoagulated human blood was infused over surface-immobilized WT or mutant mouse plasma VWF at 1600 s−1 (FIGS. 28A and 28B). Results indicate that the mutant form of mouse VWF can support human platelet attachment to levels observed for its human counterpart, thus making it an ideal system to evaluate human platelet behavior and the impact of novel anti-thrombotic drugs in an animal model. Moreover, this observation was not limited to ex vivo studies, as homozygous mutant mice infused with human (FIG. 29A) but not mouse platelets (FIG. 29B) were able to generate an arterial thrombus that occludes the vessel lumen in response to laser-induced vascular injury as depicted by intravital microscopy (transmitted light). Preliminary results indicate that mouse and human A1 domains are structurally similar and serve an identical functional role in the initiation of thrombus formation. Moreover, the ability of the “humanized” mouse A1 domain to support human platelet adhesion to the same degree as its human VWF-A1 counterpart ex-vivo, as well as its preferential binding of human platelets in vivo (FIG. 29A), suggests that our animal model will be an ideal system for preclinical screening of therapies directed at limiting the interactions between GPIb alpha and the VWF-A1 domain. Moreover, as both hemostasis and thrombosis also rely other key adhesion receptors on human platelets, such as those that interact with collagen OR fibrinogen FIG. 1B), this model can also be used for testing therapies directed against other human platelet receptors and ligands critical for these processes.
To assess the biological significance of this finding in terms of its effect on hemostasis, homozygous mutant animals received an infusion of blood-banked human platelets and bleeding time was subsequently measured by severing 1 cm of their tail. Average bleeding time for mice that received human platelets was  ̃3 minutes vs. 10 minutes (end point) for animals given an intravenous infusion of a physiological buffered saline solution (FIG. 30). Results indicate that not only can the mutant form of mouse VWF support human platelet adhesion both ex-vivo and in vivo assays, but it can also perform its biological function; supporting hemostasis in the context of vascular injury.
Evaluation of Platelet-VWF Behavior in Flow.
Blood will be collected by cardiac puncture from anesthetized mice and thrombin-mediated activation prevented by the addition of hirudin (160 U/ml, Sigma) (68). Platelet adhesion to a glass cover slip coated with 100 μg/ml of equine tendon collagen (Helena Laboratories, Beaumont, Tex.) will be assessed in a parallel-plate flow chamber apparatus. Whole blood will be infused through the chamber at a wall shear rate of 1600 s−1 for 3 minutes. As platelet adhesion under these homodynamic conditions requires VWF deposition and subsequent interactions between its A1 domain and GPIb alpha, the extent of platelet coverage should provide a gross estimate of the degree in impairment between this receptor-ligand pair. In addition, plasma VWF will be purified from these animals to evaluate platelet attachment to this immobilized substrate in flow. The surface area covered by adherent platelets at the end of each experiment will be determined (Image Pro Plus software) and expressed as a percentage of platelet coverage using blood from WT littermates. To better isolate GPIb alpha-VWF A1 interactions, identical experiments can be performed using platelets isolated from alphaIIb beta 3 deficient animals and reconstituting them in platelet poor plasma from our mutant A1 knock-in mice.
Evaluation of Platelet-VWF Behavior In Vivo.
In addition to the proposed in vitro work, platelet-VWF interactions in vivo will also be studied using intravital microscopy (Falati et al. (2002) Nature Medicine 8(10): 1175-80). This is accomplished by using a murine model of thrombosis that involves laser-induced injury to micro-vessels contained within the mouse cremaster muscle. The surgical preparation of animals, insertion of lines for administration of cells and anesthesia, will be performed as previously described (69). Human platelets will be collected and prepared, fluorescently labeled, perfused into a mouse model (such as the transgenic mouse of the current invention) via an intravenous injection (Pozgajova et al., (2006) Blood 108(2):510-4).
Surgical Preparation of Animals: Insertion of Lines for Administration of Cells and Anesthesia.
Briefly, the skin covering the scrotum will be incised and the intact cremaster muscle dissected free from the connections to the subcutis. The mouse will be placed on a custom-built plexiglass board, and the exposed muscle positioned on a heated circular glass coverslip (25 mm) for viewing. The muscle will be slit along the ventral surface (using a thermal cautery), the testis excised, and the muscle spread across the coverslip with attached sutures (6/0 silk) (FIG. 31). The cremaster muscle will be kept continuously moistened by superfusion throughout the experiment with sterile, bicarbonate-buffered (pH 7.4), saline solution (37° C.) that is pregased with a 5% CO2, 95% N2 mixture for O2 depletion. All parts of the setup in contact with the superfusion buffer will be presoaked with 1% Etoxaclean (Sigma Chemical Co., St. Louis, Mo.) overnight followed by extensive rinsing in 70% ethanol and endotoxin-free distilled water. The number of mice used for these experiments will be kept to the minimum necessary to establish statistically significant observations. Anesthetized animals will be euthanized after each experiment by CO2 inhalation.
Vascular Trauma Will be Generated as Follows:
The segment of an arteriole will be visualized and recorded as “pre-injury”. Subsequently, endothelial damage will be induced via a pulsed nitrogen dye laser at 440 nm applied through the microscope objective using the Micropoint laser system (Photonics Instruments, St. Charles, Ill.). The duration of exposure of the endothelium to the laser light will be varied to produce either a mild injury that supports the formation of a platelet monolayer or significant injury resulting in thrombus formation. The region of interest will then be videotaped and analyzed as described below.
For example, vascular damage can subsequently be induced in arterioles contained within the cremaster muscle of mice by either 1) pulsed nitrogen dye laser applied through the objective of an intravital microscope (FIG. 32) or 2) standard application of a ferric chloride solution (Furie et al. J. Clin. Invest. 2005; 115:3355). The latter method has the advantage of exposing significant more subendothelial collagen, which will be beneficial for testing the role of the collagen receptors α2β1 in thrombus formation.
For studies analyzing the dynamic interactions between individual platelets and the injured vessel wall (attachment, translocation, and sticking), cells purified from genetically altered mice will be labeled ex-vivo with a derivative of carboxyfluorescein (BCECF, Molecular Probes) (Diacovo et al. Science. 1996). A human thrombus generated in the mutant mouse can also be visualized by this technique, thus allowing to distinguish human platelets from endogenous circulating mouse platelets upon illumination with an appropriate laser light source (see FIG. 40). Cells (1×107/g of BWT) will be subsequently injected intravenously into mice bearing WT mouse (control) or the “humanized” A1 domains and their behavior visualized in the microcirculation using an intravital microscope (Zeiss, Axiotech Vario; IV500, Mikron Instruments, San Diego, Calif.; and the like) equipped with an iXON EM camera or a silicon-intensified camera (VE1000SIT; Dage mti, Michigan City, Ind.), a Yokogawa CSU22confocal head, and a 488 nm laser line (Andor Technology, Revolution series). A Xenon arc stroboscope (Chadwick Helmuth, El Monte, Calif.) will serve as the light source and fluorescent cells will be viewed through 60× or 100× water immersion objectives (Acroplan, Carl Zeiss Inc.). A tethered platelet will be defined as a cell establishing initial contact with the vessel wall (FIG. 33A, panel 2-3; FIG. 33B). The translocating fraction will be defined as number of tethered platelets that move at a velocity significantly lower than the centerline velocity for >1 s. The sticking fraction will be defined as the number of translocating cells that become stationary for >30 s post-tethering. Second order arterioles (up to 50 μm in diameter) will be evaluated for platelet interactions before and after the injury. Evaluation of platelet circulation in larger arterioles may be less accurate secondary to hemoglobin-mediated quenching of fluorescence emitted from platelets traveling in an area of the blood stream distal to the focal plane of the objective. Epi-illumination will only be used during video recordings to minimize possible phototoxic effects on tissue.
A role for GPIb alpha as well as the collagen (α2β1) and the fibrinogen (αIIb (33) receptors can be evaluated by using function-blocking antibodies to these proteins. Moreover, FDA approved anti-thrombotics (such as clopidogrel and tirofiban) can be examined as to whether the drugs inhibit human platelets from forming a thrombus in vivo, validating the mouse model for use in pre-clinical screening. The effect that antibodies and drugs have on altering the interaction between GPIb alpha-VWF-A1 interaction is determined by evaluating whether thrombus formation in the proposed mice is reduced or augmented upon arteriolar injury (FIGS. 34A and 34B).
For all experiments, the centerline erythrocyte velocity (Vrbc) is measured using an optical doppler velocimeter (Microcirculation Research Institute, Texas A&M College of Medicine, College Station, Tex.) prior to and after inducing the injury. Shear rate (SR) is then calculated based on Poiseulle's law for a Newtonian fluid: SR=8(Vmean/Dv), where Dv is the diameter of the vessel and Vmean is estimated from the measured Vrbc (Vmean=Vrbc/1.6).
Characterization of Thrombus Formation:
Thrombus formation can be characterized as follows: (1) Early individual platelet interactions with the damaged vessel wall (number of fluorescently labeled human platelets that attach during the first minute post-injury); (2) time required for thrombus generation of >20 μm diameter; (3) the ability of thrombi to remain at the initial site of vascular injury and not break free (measure of stability); (4) time until vessel occlusion; and (5) site of vessel occlusion, that is, at the site of injury or downstream from it. Platelet-vessel wall interactions can be viewed through 40× or 60× water immersion objectives. To standardize in vivo conditions, the velocity of flowing blood (shear rate) pre-injury is determined by measuring the centerline erythrocyte velocity (Vrbc) using an optical doppler velocimeter. Shear rate (SR) can then be calculated based on Poiseulle's law for a Newtonian fluid: SR=8×(Vmean/Dv), where Dv is the diameter of the vessel and Vmean is estimated from the measured Vrbc (Vmean=Vrbc/1.6). Vessel and thrombus diameters are measured using imaging software (ImagePro Plus).
Administration of Antibodies:
Function-blocking monoclonal antibodies 6D1 (anti-human GPIb alpha), 6F1 (anti-human α2β1) and 7E3 (anti-human αIIbβ3) have been generously provided by Dr. Barry Coller (Rockefeller University, NY). All antibodies are converted to F(ab′)2 fragments to limit Fc receptor interactions in vivo. An intravenous dose of 10 μg/g body weight is given approximately 10 minutes after the injection of human platelets but 30 minutes prior to inducing vascular injury. Non-function blocking antibodies to these receptors is used as negative controls and administered under identical conditions. To ensure optimal ligand availability for the collagen and fibrinogen receptors on human platelet, mice possessing the A1 domain mutation have been bred with animals genetically deficient in α2β1 or αIIbβ3. Thus, endogenous platelets in these animals not only have a reduced ability to interact with the VWF-A1 domain, but also are incapable of binding to collagen or fibrinogen, respectively. Although human platelets have been shown to circulate in mice for a maximum of 24 hours, we can ensure that an equivalent percentage of human platelets are present at the time of vascular injury under each experimental condition (Xu et al. J. Clin. Invest. 2006; 116:769). Thus, 50 μl is obtained from an inserted venous catheter and flow cytometric analysis will be performed to determine the percentage of circulating fluorescently-labeled human platelets.
Administration of Drugs:
In comparison to aspirin, clopidogrel (Plavix) is the second most commonly used anti-thrombotic drug that targets one of the ADP receptors (P2Y12) on platelets, causing irreversible inhibition (Hankey et al. Med. J. Aust. 2003; 178:568). ADP is a potent mediator of platelet activation and aggregate formation, and thus considerable effort and funds have been devoted to inhibiting this activation pathway in platelets. Clopidogrel was approved by the FDA in 1997 for clinical use and was found to be of benefit in the secondary prevention of major vascular events in patients with a history of cerebrovascular and coronary artery diseases and major cardiac events post coronary artery stent placement (Gachet et al. Semin. Thromb. Hemost. 2005; 31:162). Disadvantages of this drug are: 1) It must be metabolized in the liver to generate an active metabolite, thus limiting its effectiveness in acute settings, and 2) irreversible inhibition that results in a marked prolongation of bleeding time.
Clopidogrel has been shown to reduce thrombus size and delay its formation in mice with a maximal effective dose of 50 mg/kg given the day before and 2 hours prior to experimentation (Lenain et al. J. Throm. Haemost. 2003; 1:1133). This drug will be obtained from the hospital pharmacy and tablets will be dissolved in sterile water for oral administration. Control animals will receive water in lieu of drug. The effectiveness of this treatment regime will be confirmed by first measuring the responsiveness of platelets isolated from drug-treated WT animals to ADP-induced aggregation using an optical aggregometer (Chrono-Log Corp.) as previously described (Leon et al. J. Clin. Invest. 1999; 104:1731). As our mutant VWF-A1 domain mice also have a defect in platelet aggregation, these animals cannot be used for the purpose of testing to ADP-induced aggregation ex-vivo. However, this additional phenotype will be advantageous for us as it limits potential competition between human and mouse platelets for binding to ligands exposed at sites of vascular injury. Human platelets will be administered 30 minutes prior to vascular injury and 50 μl of blood drawn to determine the percentage of circulating cells as described above. Platelet rich plasma will also be purified from control and drug treated animals that receive human platelets to evaluate the effectiveness of clopidogrel on preventing ADP-induced aggregation of these cells ex-vivo.
Tirofiban (Aggrastat) is a non-peptide inhibitor of the fibrinogen receptor αIIbβ3 that limits the ability of platelets to form aggregates, an event required for thrombus progression. It has a plasma half-life of approximately 2 hours but only remains bound to platelets for seconds, thus necessitating continuous intravenous administration. It is currently approved for short-term treatment of patients with acute coronary syndrome that require interventional catheterization. Thus, the animals will be dosed based on that given for interventional procedures such as angioplasty, which consists of a 25 μg/kg bolus over 3 minutes followed by a continuous maintenance infusion of 0.15 μg/kg/min until the completion of the experiment (Valgimigli et al. JAMA. 2005; 293:2109). Human platelets will be administered 30 minutes prior to vascular injury and 50 μl of blood drawn to determine the percentage of circulating cells as described above.
Platelet Donors.
Mice are used as platelet donors. A means to evaluate murine platelet interactions with wild type and mutant VWF-A1 proteins is via in vitro flow chamber assays. Blood from  ̃10 mice are required to purify adequate numbers of platelets per assay. Blood from donor animals is obtained from the retro-orbital plexus using a heparinized glass pipette. Mice will be anesthetized with Ketamine and Xylazine prior to the procedure and are euthanized by CO2 inhalation upon completion.
Bleeding Time for Human Platelet Induced Hemostasis.
This assay is carried out as described above.
Solution-Phase Binding Assay.
For type 2B mutant VWF, its capacity to bind to platelet GPIb alpha in solution can be determined. Plasma is harvested from these mice and VWF purified. Various concentrations of the plasma glycoprotein will be indirectly labeled using a non-function blocking, 125I-labeled mAb to its A1 domain as previously described (67). After a 30 min. incubation, a quantity of this mixture will be incubated with platelets purified from beta 3 deficient mice so to prevent integrin-mediated binding to VWF. After incubation period of 1 hour, an aliquot of this mixture will be added to a sucrose gradient and centrifuged to pellet the platelets. Radioactivity associated with the pellet vs. supernatant will be determined in a γ-scintillation counter, and the binding estimated as the percent of total radioactivity.
Example 4
Defining the In Vivo Role of the Von Willebrand Factor A1 Domain by Modifying a Species-Divergent Bond
Proteins containing von Willebrand Factor (VWF) A domains contribute to human health and disease by promoting adhesive interactions between cells (Whittaker, C. A., & Hynes, R O. Mol. Biol. Cell. 13, 3369-3387 (2002)). The VWF-A1 domain is thought to play a critical role in hemostasis by initiating the rapid deposition of platelets at sites of vascular damage by binding to the platelet receptor glycoprotein Ib alpha (GPIbα) at high shear rates (Roth, G. J. Blood 77, 5-19 (1991); Cruz, M. A., et al., J. Biol. Chem. 268, 21238-21245 (1993); Sugimoto, M. et al., Biochemistry 30, 5202-5209 (1991); Pietu, G. et al., Biochem. Biophys. Res. Commun. 164, 1339-1347 (1989)). Although congenital absence of VWF in humans has established a role for this plasma glycoprotein in hemostasis (Sadler, J. E. et al. J. Thromb. Haemost. 4, 2103-2114 (2006)), the contribution of its A1 domain in clot formation has been questioned in a mouse model of vascular injury (Denis, C. et al. Proc. Natl. Acad. Sci. USA 95, 9524-9529 (1998)).
In this example, murine plasma VWF or its A1 domain fails to support significant interactions with human platelets (and likewise human VWF with murine platelets) under flow conditions. Atomic models of GPIbα-VWF-A1 complexes suggest that the structural basis for this behavior arises primarily from an electrostatic “hot-spot” at the binding interface. Introduction of a single point mutation within this region of murine VWF-A1 is sufficient to switch its binding specificity from murine to human platelets. In addition, introduction of a single point mutation within the electrostatic “hot-spot” region of human VWF-A1 is sufficient to switch its binding specificity from human to murine platelets. Moreover, mice possessing the 1326R>H mutation in their VWF have a bleeding phenotype distinct from VWF-deficient animals, and can be corrected by the administration of human platelets. Mechanistically, mutant animals can generate but not maintain thrombi at sites of vascular injury, whereas those infused with human platelets can form stable thrombi, a process that relies on GPIbα-VWF-A1 interaction. Thus, interspecies differences at protein interfaces can provide insight into the biological importance of a receptor-ligand bond, and aid in the development of an animal model to study human platelet behavior and drug therapies.
Methods
Generation of VWF1326R>H Mice.
The VWF1326R>H targeting vector (FIG. 38A) was prepared from a 129/SvJ mouse genomic library. The clone was identified by PCR using primers specific for exon 28 of the mouse VWF gene and sequence fidelity of the region to be targeted validated by comparison to published sequence for chromosome 6 (GenBank accession number NW_001030811). The targeting vector is identical to the corresponding region in the mouse genome, except the 1326R>H mutation was created in exon 28 and the Neo cassette flanked by loxP sites was inserted into intron 28. This resulted in the loss of an EcoRV site and the introduction of a new EcoR1 and two new XhoI sites. The construct was electroporated into an embryonic stem (ES) cell line, and potential clones identified by continued growth of cells in G418 and Gancyclovir supplemented media. DNA was isolated from surviving colonies, digested with EcoRI, and screened by Southern analysis using a 1.5 kb probe (A) corresponding to a DNA sequence downstream of the targeting construct. Chimeric mice generated from vwF1326R>H targeted ES cell lines were subsequently bred to a Cre transgenic mouse (C57BL/6 background) and animals containing the 1326R>H mutation, but without the Neo cassette, subsequently identified by both PCR and Southern analysis. WT and homozygous animals were the product of matings between heterozygous mice.
Analysis of VWF Transcripts, Antigen Levels, Multimers, and Collagen Binding.
Detection of transcripts from the A1-A2-A3 domains of murine VWF was performed by RT-PCR. Briefly, mRNA was isolated from lung tissue harvested from either homozygous VWF-A11326R>H mice or aged-mated WT littermate controls (Oligotex®, Qiagen). Generation of cDNA and PCR-amplification of desired transcripts was performed using SuperScriptTM One-Step RT-PCR (Invitrogen) and oligos specific for the A domains of VWF.
Functional factor VIII levels were determined by a mechanical clot detection method using the STA automated coagulation analyzer (Diagnostica Stago, Parsippany, N.J.). A log-log calibration curve was established by measuring the activated partial Thromboplastin time (aPTT) of varying dilutions of reference plasma. The aPTT of a 1:10 dilution of sample plasma mixed with factor VIII deficient plasma was determined, compared to the calibration curve, and the activity expressed as a percent of normal.
Evaluation of VWF antigen levels was performed as previously described (Denis, C. et al. Proc. Natl. Acad. Sci. USA 95, 9524-9529 (1998)). For multimer analysis, plasma from sodium citrate treated whole blood was diluted 1:5 in electrophoresis sample buffer (final concentration 10 mM Tris-HCl pH 8.0, 2% SDS, 1 mM EDTA) and heated at 56° C. for 30 minutes. Electrophoresis was carried out overnight (64 volts, 15° C.) through a horizontal SDS-agarose gel in 1.2% agarose (Ruggeri, Z. M. & Zimmerman, T. S. Blood 57, 1140-1143 (1981)). The gel was then electrophoretically transferred (150 mA, 90 minutes) to Immobilon (Millipore) followed by blocking (2 h) with 5% powdered milk in TBST (Tris HCl pH 8.0, 0.15M NaCl, 0.05% Tween-20). The membrane was incubated with a 1:500 dilution of rabbit anti-human VWF antiserum (DAKO) for 1 h, washed in TBST, and then incubated with a 1:10,000 dilution of HRP-conjugated mouse anti-rabbit IgG (Calbiochem). Bands were subsequently detected by chemiluminescence system (GE Healthcare). For comparison, a sample containing pooled human plasma from normals or patients with type 2B VWD was also loaded on the gel. Binding of VWF to surface-immobilized collagen was performed as previously described (Smith, C. et al. (2000) J. Biol. Chem. 275, 4205-4209). Briefly, 100 μg/ml of acid soluble type I collagen from human placenta (Sigma) was added to a 96 well microtiter plate and allowed to incubate overnight (4° C.). After washing and blocking with TBS containing 3% BSA and 0.05% Tween 20, varying concentration of platelet poor plasma harvested and pooled from WT, homozygous vwF1326R>H, and VWF deficient mice was added to the wells and incubated for 1 h (37° C.). Wells were then washed and bound VWF detected by an ELISA as described above.
Ex Vivo Platelet Adhesion Studies.
Experiments were performed in a parallel-plate flow chamber as previously described (Offermanns, S. (2006) Circ. Res. 99, 1293-1304). For studies involving plasma VWF, a polyclonal anti-VWF antibody (Dako) was absorbed overnight (4° C.) to a six well tissue culture plate. Subsequently, the plate was washed and non-specific interactions blocked by the addition of TBS containing 3% BSA, pH 7.4 (1 h, 37° C.). Human or murine plasma obtained from heparinized whole blood was added and the plates placed at 37° C. for an additional 2 h. Generation, purification, and surface-immobilization of recombinant VWF-A1 proteins was performed as previously described (Doggett, T. A. et al. Biophys. J. 83, 194-205 (2002)). Both human and murine VWF-A1 constructs consist of amino acid residues 1238 to 1471, with a single intra-disulfide bond formed between residues 1272 and 1458 and were generated in bacteria. Citrated whole blood (150 μl) collected via cardiac puncture from anesthetized homozygous VWF1326R>H or WT mice or from venopuncture from human volunteers was perfused over the immobilized substrates at a wall shear rate of 1600 s−1 for 4 min, followed by washing with Tyrode's buffer under the identical flow conditions. The number of platelets attached per unit area (0.07 mm2) and translocation velocities were determined by off-line analysis (Image-Pro Plus, Media Cybernetics). For GPIbα inhibition studies, the function-blocking mAb 6D1 (20 μg/ml) or mAb SZ2 (20 μg/ml; Beckman Coulter) was added to anticoagulated human blood for 30 min prior to use. Experiments were performed in triplicate on two separate days. An ELISA was used to ensure equivalent coating concentration of plasma and recombinant proteins (Denis, C. et al. (1998) Proc. Natl. Acad. Sci. USA 95, 9524-9529).
In Vivo Thrombus Formation.
Administration of anesthesia, insertion of venous and arterial catheters, fluorescent labeling and administration of human platelets (5×108/ml), and surgical preparation of the cremaster muscle in mice have been previously described (Doggett, T. A. et al. Biophys. J. 83, 194-205 (2002); Diacovo, T. G., et al., Science 273, 252-255 (1996)). Injury to the vessel wall of arterioles ( ̃40-65 μm diameter) was performed using a pulsed nitrogen dye laser (440 nm, Photonic Instruments) applied through a 20× water-immersion Olympus objective (LUMPlanFI, 0.5 NA) of a Zeiss Axiotech vario microscope. Mouse platelet- and human platelet-vessel wall interactions were visualized using either bright field or fluorescence microscopy. The latter utilized a fluorescent microscope system equipped with a Yokogawa CSU-22 spinning disk confocal scanner and 488 nm laser line (Revolution XD, AndorTM Technology). The extent of thrombus formation was assessed for 2 min post injury and the area (μm2) of coverage determined (Image IQ, AndorTM Technology). For GPIbα or αIIbβ3 inhibition studies, the function-blocking mAb 6D1 or 7E3 (20 μg/ml), respectively (from B. Coller, Rockefeller University), was added to purified human platelets for 30 min prior to administration.
Tail Bleeding Assay.
Bleeding times were measured in 7-week old mice after amputating 1 cm of the tail tip as previously described (Denis, C. et al. (1998) Proc. Natl. Acad. Sci. USA 95, 9524-9529). In studies involving human platelets, platelet concentrates were obtained from Columbia Presbyterian Hospital Blood Bank, washed and resuspended in normal saline (1.5×109/300 μl) before administering through a catheter inserted into the right internal jugular vein. Tail cuts were performed 5 min after completion of the infusion of platelets. PLAVIX and ReoPro® were obtained from the research pharmacy at CUMC. For studies involving PLAVIX, animals received a 50 mg/kg oral dose of the drug the day before and 2 h prior to the administration of human platelets. ReoPro® was given initially as an intravenous bolus (0.25 mg/kg) 5 min after the administration of human platelets, followed by a continuous infusion (0.125 μg/kg/min) as per the manufacturer's recommendations.
Structural Modeling.
There are three crystal structures of the GPIbα-VWF-A1 complex: two are WT except for mutated N-glycosylation sites in GPIbα(Fukuda, K. et al., (2005) Nat. Struct. Mol. Biol. 12, 152-159; Dumas, J. J. et al. (2004) J. Biol. Chem. 279, 23327-23334), and one is a gain-of-function mutant (Huizinga, E. G. et al. (2002) Science 297, 1176-1179). The structures have only small differences that are not the result of the presence of mutations or botrocetin binding (Fukuda, K., et al., (2005) Nat. Struct. Mol. Biol. 12, 152-159). Both N-glycosylation sites in human GPIbα lie on the well-ordered upper ridge of the LRR, 18 Å and 27 Å (Cα-Cα) from the nearest VWF-A1 residue, so their absence is unlikely to affect the structure of the complex. Murine GPIbα has no predicted N-glycosylation sites.
Human GPIbα contains sulfated tyrosines implicated in binding VWF within an acidic loop just C-terminal to the sequence included in the crystal structures. Murine GPIbα has a predicted sulfation site in the same loop, so that the differential binding of human vs. murine GPIbα to VWF-A1 is also likely to be small. The interfacial regions are otherwise highly conserved between species, with the exception of three salt bridges (See FIGS. 37C-G). The conformation of the β-switch region is highly constrained, as noted in the main text. The only change to a buried interfacial residue is M239T (human to mouse), which lies in an invariant pocket. Notably, the crystal structure of the human “gain-of-function” mutant, M239V, shows no perturbations in this region, and given that valine is isosteric with threonine, this species difference is unlikely to affect either the complex structure or interspecies binding.
We used a consensus model of the human complex to build the murine model. We first overlaid murine A1 onto human A1 by fitting the central β-sheets (RMSD 0.3 Å; within experimental error); the only notable difference is the location of helix α4, which is shifted by 2-3 Å away from the GPIbα interface in the mouse owing to a larger residue on the buried face of this helix. For the GPIbα model, only the side-chains were altered, since the human and murine LRRs have identical lengths. Consensus rotamers with minimal steric clashes were chosen, followed by manual adjustment where necessary to create sensible van der waals interactions and H-bonding, using TURBOFRODO (Bio-Graphics, Marseille, France). Molecular overlays were optimized using LSQKAB (Collaborative Computational Project, No. 4. Acta Crystallogr. D50, 760-763 (1994)); molecular figures were created using MOLSCRIPT (Esnouf, R. M. J. Mol. Graph. Model. 15, 132-136 (1997)) and OPENGL (http://www.rush3d.com/reference/opengl-bluebook-1.0)
Statistics.
An unpaired, two-tailed Student t test was used for multiple comparisons.
Results and Discussion
As the interaction between GPIbα and VWF-A1 is a prerequisite for effective thrombus formation in the arterial circulation, we first tested the ex vivo ability of surface-bound murine plasma VWF or its recombinant A1 domain (rVWF-A1) to support human platelet adhesion under physiologically relevant flow conditions: that is at a shear rate exceeding 1000 s−1 using a parallel-plate flow chamber (Ruggeri, Z. M. et al., (2006) Blood. 108, 1903-1910). The adhesive properties of VWF are tightly regulated such that it preferentially binds to platelets only when immobilized to sites of vascular injury and under hydrodynamic conditions encountered on the arterial side of the circulation (Sakariassen et al., (1979) Nature 279, 636-638; Ruggeri, Z. M. et al., (2006) Blood. 108, 1903-1910). Perfusion of human whole blood over murine plasma VWF or rVWF-A1 resulted in limited platelet deposition (10 to 25%, respectively) as compared with same-species controls (FIGS. 36A and 36B). Similarly, human VWF proteins had a diminished capacity to support murine platelet accumulation under identical conditions (FIG. FIG. 36C-D). This interspecies incompatibility would seem to preclude the study of human platelet behavior in a mouse model of arterial thrombosis.
In order to gain insight into the structural origins of this species incompatibility, we built models of murine-murine and human-murine GPIbα-VWF-A1 complexes based on the crystal structures of the human complex (Fukuda, K., et al., Nat. Struct. Mol. Biol. 12, 152-159 (2005); Dumas, J. J. et al., J. Biol. Chem. 279, 23327-23334 (2004); Huizinga, E. G. et al., Science 297, 1176-1179 (2002)) and human and murine VWF-A1 (Fukuda, K., et al., Nat. Struct. Mol. Biol. 12, 152-159 (2005)) (FIGS. 37A through 37E; see Methods).
The A1 domain comprises a Rossmann-like fold with a central, mostly parallel β-sheet flanked on both sides by α-helices (Fukuda, K., et al., Nat. Struct. Mol. Biol. 12, 152-159 (2005)). Human and murine VWF-A1 share considerable sequence (86% identity) and structure homology; in fact, the β-sheets of both species are identical within experimental error (a root mean square difference of 0.33 Å for Cα-atoms). The only major difference is the location of helix α4 (nomenclature as previously described in Dumas, J. J. et al., J. Biol. Chem. 279, 23327-23334 (2004)), which is shifted 2-3 Å away from the GPIbα binding site in the mouse, owing to a difference in a buried hydrophobic residue (FIGS. 37A-37B). Although neither the structure of murine GPIbα nor its complex with the VWF-A1 domain are known, the high sequence similarity of the murine and human proteins (including the complex interface), as well as the rigid architecture of the leucine-rich repeats (LRR) of GPIbα, provide high confidence that their 3D structures will be highly homologous.
In the complexes, the major contact region involves the “β-switch” region (residues 227 to 241 in the C-terminal flank of GPIb alpha), which forms a β-hairpin that augments the β-sheet of the VWF-A1 domain. On its other side, this region of GPIbα packs tightly against the concave face of the LRR, which highly constrains it movement. Residues in mouse and human are mostly invariant on both sides of this interface. Notable exceptions are at position 1326 in VWF-A1, which is histidine (H) in humans versus an arginine (R) in mouse, and at position 238 in GPIb alpha, which is alanine (A) in humans versus an aspartic acid (D) in mouse (FIGS. 37C and 37D). A model of the murine complex suggests that these changes are complementary, since D238 can form an intermolecular salt-bridge with R1326; D238 in murine GPIb alpha also shields the positively charged flanking lysine (K) at position 231 (a conserved residue in both species) from unfavorable interactions with R1326 in murine VWF-A1. This salt-bridge cannot form in the human complex due to the presence of a histidine at 1326. However, an intermolecular salt-bridge can occur between R1395 and E225 located at the top of the human complex, which may compensate for this loss (FIG. 37D). No such interaction can occur in the murine complex (FIG. 37C).
In the human GPIb alpha-murine VWF-A1 interspecies complex, we predict that the two positively charged residues (GPIb alpha K231 and VWF-A1 R1326) create an electrostatic clash that impedes binding, owing to the lack of a negatively charged group at position 238 (FIG. 37E). In the murine GPIb alpha-human VWF-A1 interspecies complex, however, no such electrostatic clash occurs despite the absence of the salt-bridge. There is, however, an overall change in net charge in the binding interface compared with the murine GPIb alpha-murine VWF-A1 complex (FIG. 37F). This, together with the loss of critical salt-bridges, most likely accounts for the reduced interaction between mouse platelets and human VWF (See Tables 5 and 6).







TABLE 5







Predicted effect of species differences in residues on the human GPIb 


alpha-murine VWF-A1 interspecies complex.











hGPIb-alpha
hGPIb alpha-



mVWF-A1
partner
mVWF-A1
Reason





R1326
A238
(−)
Permits electrostatic clash of 





R1326 with K231 in GPIb alpha


E1330
K237
(+)
New salt-bridge E1330-K237


G1370
none
0
No interactions


R1395
E225
(−)
Loss of salt-bridge


(shifts 





position)





(+) = net positive,


(−) = net negative,


0 = minimal effect compared with syngeneic complexes.













TABLE 6







Predicted effect of species differences in residues on the murine 


GPIb alpha-human VWF-A1 interspecies complex.











mGPIb-alpha
hGPIb-alpha-



hVWF-A1
partner
mVWF-A1
Reason





H1326
D238
(−)
Loss of R1326-D238 





salt-bridge


G1330
K237
(−)
Loss of E1330-K237 





salt-bridge


S1370
none
0
No interactions


R1395
N225
(+)
New polar interactions 


(shifts 


with R1395


position)





(+) = net positive,


(−) = net negative,


0 = minimal effect compared with syngeneic complexes.






To explore the importance of the electrostatic mismatches in destabilizing the interspecies complexes, we substituted human residues into murine rVWF-A1 at positions 1326 (R>H), 1330 (E>G), and 1370 (S>G), and analyzed the ability of the mutant proteins to support human platelet accumulation under flow. As expected, amino acid substitutions at positions 1330 (predicted to remove a salt-bridge) and 1370 (predicted to have no effect) failed to promote the interaction between murine rVWF-A1 and human GPIbα. However, the 1326R>H mutation, which eliminates the electrostatic clash with K231, rendered murine A1 capable of supporting interactions at a level comparable to its wild-type (WT) human counterpart (FIG. 37G). Similarly, conversion of 1326H>R in the human rVWF-A1 protein promoted the binding of mouse platelets, while the reverse substitution in its murine counterpart reduced adhesion by  ̃75%. That a single residue change is sufficient for shifting the binding preferences across species supports the notion that this contact region is a “hot-spot” in the protein interface (Bogan, A. A. & Thorn, K. S. J. Mol. Biol. 280, 1-9 (1998)).
In order to determine the ability of full-length murine VWF containing the 1326R>H mutation (VWF1326R>H) to support human platelet interactions and ultimately thrombus formation in vivo, we genetically modified mice to express VWF1326R>H (FIGS. 38B-38C). Both homozygous and heterozygous animals were viable, fertile, born at the expected Mendelian ratio, and had platelet counts comparable to WT littermate controls. Moreover, reverse transcription-PCR (RT-PCR) of lung tissue from mutant mice with primers specific for the A1, A2, and/or A3 domains of VWF amplified cDNAs of the correct size and of similar intensity as compared to WT littermate controls (FIG. 39A). VWF antigen levels, Factor VIII function, as well as VWF multimer pattern in homozygous mutant plasma were found to be equivalent to WT controls (FIGS. 39B-39C). These results indicate that VWF gene translation, transcription, and posttranslational modifications were not perturbed by our targeting strategy. The ability of plasma VWF to bind to collagen was also not affected by the introduction of the point mutation.
Hemostasis relies on platelet adhesion and activation at sites of vascular injury, which ultimately results in the formation of a hemostatic plug. To demonstrate the importance of VWF-A1 in this process, we measured bleeding times for mice possessing the 1326R>H mutation by removing 1 cm of distal tail (FIG. 41A). In contrast to their WT counterparts, the vast majority of homozygous VWF1326R>H mice were incapable of forming an effective hemostatic plug, as they continued to bleed profusely throughout the duration of the experiment (10 min). Moreover, a smaller but statistically significant increase in bleeding time was noted for heterozygous animals (1.9-fold compared with WT; P=0.0055). Thus, disruption of a single salt bridge between murine VWF-A1 and GPIbα is sufficient to impair hemostasis.
To gain insight into how the 1326R>H mutation alterations hemostasis, we evaluated murine platelet adhesion at sites of vascular damage in vivo. A laser-induced vascular injury model was utilized to initiate platelet deposition in arterioles located in the microcirculation of the cremaster muscle of WT and homozygous mutant mice (Furie, B. & Furie., B. C. (2005) J. Clin. Invest. 115, 3355-3362). Although VWF1326R>H animals can initially form thrombi that fill the vessel lumen, they rapidly dissipated under the prevailing hydrodynamic conditions (FIG. 40). By contrast, thrombi in WT mice continue to enlarge and eventually occlude blood flow under identical conditions.
To demonstrate that the removal of the electrostatic clash between residues 1326 and 231 in murine VWF-A1 and human GPIb alpha, respectively, promotes substantial interactions between this chimeric receptor-ligand pair, we perfused human whole blood over surface-immobilized plasma VWF obtained from mice homozygous for VWF1326R>H. Remarkably, mutant murine VWF bound human platelets at levels comparable with its human counterpart (FIG. 41B). Moreover, a function-blocking antibody 6D1, which binds exclusively to human GPIbα at the fourth LRR (Shen, Y. et al. (2000) Blood. 95, 903-910), inhibited platelet adhesion, demonstrating a key role for the platelet receptor in adhesion to VWF1326R>H.
By contrast, the antibody SZ2 that recognizes the anionic-sulfated tyrosine sequence of GPIbα (residues 276 to 282) had a minimal affect on platelet accumulation, results consistent with a previous report (Fredrickson et al., (1998) Blood. 92, 3684-3693). The A1 domain also possesses the ability to support the movement of attached platelets in the direction of the prevailing hydrodynamic force owing to rapid and reversible interactions with GPIbα (Savage et al., (1996) Cell 84, 289-297; Doggett, T. A. et al. (2002) Biophys. J. 83, 194-205). We therefore compared translocation velocities of human platelets on either human or mutant murine VWF. Translocation velocities of human platelets on either homozygous mutant murine or native human plasma VWF were also similar (3.5±0.1 μm/sec vs. 3.2±0.1 μm/sec, respectively; mean±s.e.m, n=4), demonstrating that VWF1326R−>H functions in a manner indistinguishable from its human counterpart.
We next tested the ability of murine VWF1326R>H to support human platelet adhesion in vivo. The ability of human platelets to preferentially support thrombus formation was monitored simultaneously by labeling purified human cells with BCECF ex-vivo, and mouse platelets with rhodamine 6G by intravenous administration. Fluorescently-labeled human platelets were infused continuously via a catheter inserted into the femoral artery, resulting in a high local concentration of these cells within the microcirculation of the cremaster muscle. Their behavior in response to laser-induced vascular injury was monitored in real-time using confocal intravital microscopy (Furie, B. & Furie., B. C. (2005) J. Clin. Invest. 115, 3355-3362). Upon induction of laser damage to the vessel wall of arterioles in mice homozygous for VWF1326R>H, human platelets rapidly adhered to the site of injury, forming large thrombi composed mainly of human cells (91.7±1.2%; mean±s.e.m) (FIGS. 41C-41D); average thrombus size was 8,950±1,620 μm2 (mean±s.e.m.). In WT mice, by contrast, human platelets had only a limited capacity to bind to the damaged vessel wall, accounting for only 5.7±0.4% of total thrombus area (mean±s.e.m.) (FIGS. 41C-41D).
Consistent with the critical role of platelet GPIb alpha in mediating interactions with VWF-A1, pre-treatment of human platelets with mAb 6D1 greatly reduced thrombus size in the vasculature of VWF1326R>H mice (265±125 μm2; mean±s.e.m.) (FIG. 41E). This was also validated by our observations that human platelet deposition at sites of arterial injury is limited in VWF-deficient mice (185±35 μm2; mean±s.e.m.), demonstrating that the A1 domain of this plasma protein serves as the major ligand for GPIbα in our humanized animal model of thrombosis.
Although GPIbα initiates platelet deposition at arterial shear rates, it is ultimately the platelet integrin αIIbβ3 that supports thrombus growth by promoting platelet-platelet interactions. The contribution of human αIIbβ3 in this process is demonstrated by the ability of the function blocking antibody 7E3 to also limit thrombus size (529±150 μm2) (FIG. 41E). Confirmation that the interaction formed between human platelets and murine VWF1326R>H is sufficient to promote effective hemostasis is provided by the ability of infused human cells to restore bleeding times in mutant mice to a level observed for their WT counterparts (182±14.5 s vs. 131.5±11.2 s, respectively; mean±s.e.m.) (FIG. 41F). Importantly, human platelet-induced hemostatic clot formation can be completely disrupted in these animals by the preadministration of either clopidogrel (PLAVIX), an inhibitor of ADP-induced platelet activation, or abciximab (ReoPro®), a Fab fragment of the chimeric human-mouse monoclonal antibody 7E3, which blocks the function of αIIbβ3 on human but not murine platelets (Hankey, G. J. & Eikelboom, J. W. Antiplatelet drugs. Med. J. Aust. 178, 568-574; Bennett, J. S. (2001) Annu. Rev. Med. 52, 161-184) (FIG. 41F).
In summary, these studies demonstrate how one can effectively utilize atomic models of interspecies complexes to identify a binding hot spot where a disproportionate amount of the binding free energy is localized, such that a single amino acid substitution significantly affects the interaction (Bogan, A. A. & Thorn, K. S. (1998) J. Mol. Biol. 280, 1-9), and in this case switches species specificity. Moreover, a subtle and localized change of this nature limits the possibility of inducing structural perturbations that impact on the function of other domains contained within VWF. These show that human platelet adhesion to VWF1326R>H is dependent on GPIbα binding to VWF-A1, with other potential ligands for this receptor playing a subservient role in this process (Bergmeier, W. et al. (2006) Proc. Natl. Acad. Sci. USA. 103, 16900-16905). The reliance of human thrombus formation on the integrin αIIbβ3, as well as the ability of the FDA approved drugs PLAVIX and ReoPro® to impair human platelet-mediated hemostasis indicate that downstream adhesive and activation events known to be critical for clot formation and stability are intact in the mutant VWF animals. Thus, we anticipate that the VWF1326R>H knock-in mice will prove useful in the preclinical evaluation of new antithrombotic therapeutics designed ultimately for human use. These results also have implications for advancing both knowledge of human platelet biology and in preclinical testing of antithrombotic therapies in vivo.
Example 5
Use of “Humanized” VWF-A1 Animal for Developing Technologies to Image Sites of Occult Bleeding or Thrombus Formation in Humans
Perfluorocarbon Nanoparticle Based Imaging Platform.
The ability of a VWF-A1 mutant animal, such as our 1326R>H mutant mouse, to generate thrombi composed of human platelets at sites of vascular injury in vivo, provides a means for developing imaging technologies designed to detect sites of occult bleeding or thrombus formation in humans. For example, such technologies may prove useful expediting the discovery of sites of internal bleeding in humans as a result of injuries obtained form a motor vehicle accident. Similarly, it may be useful in detecting injuries obtained in a military battle. Suitable probes include antibodies, small molecules, peptides that recognize molecules expressed on human platelets or the various domains of VWF. However, coupling contrast agents directly to antibodies is cumbersome and insufficient for detection of such complexes in the body by various imaging modalities (i.e. MRI) due to low signal to noise output. Thus, an ideal candidate for detection would not only preserve the specificity associated with monoclonal antibodies, small molecules, or peptides but also have the following properties: 1) high signal-to-noise ratio, 2) long circulating half-life, 3) acceptable toxicity profile, 4) ease of use and production, and 5) compatibility with standard commercially available imaging modalities. Perfluorocarbon Nanoparticle (PNP) may provide the answer. This proposal will take advantage of a novel nanoparticle contrast agent that can be imaged by ultrasound, magnetic resonance, and nuclear imaging (Lanza et al., (2000) Invest Radiol, 35: 227-234; Lanza et al., (1997) Ultrasound Med Biol, 23: 863-870; Yu et al., (2000) Magn Reson Med. 44(6):867-72). This agent is a small ( ̃150-250 nanometer diameter), lipid encapsulated, perfluorocarbon emulsion that can be administered by vein. Importantly, monoclonal antibodies as well as small molecules and peptides that recognize platelets and/or VWF can be covalently coupled to PNPs. Moreover, PNPs can also be potentially used for targeted drug delivery (FIG. 42).
PNPs have been shown to remain stable in the circulation with a half-life of >1 hour, which permits rapid binding and local contrast enhancement sufficient for diagnostic imaging within 30-60 minutes. PNPs are cleared by the liver and spleen, and are similar to “artificial blood” formulations used to enhance oxygen, which have acceptable safety profiles for clinical use at 10 times greater dose than would be required for targeted contrast enhancement. In addition, perfluorocarbon to be used in this study (perfluorooctylbromide) has an extensive tract record for human safety in clinical trials (i.e., Oxygent, Alliance Pharmaceuticals). Thus, this nanoparticle platform provides an ideal opportunity to prove that contrast agents can be targeted specifically to sites of human thrombus formation.
Preparation of Fluorescently-Labeled Antibody Targeted Nanoparticles.
The basic method for formulating perfluorocarbon nanoparticles comprised of perfluorooctyl bromide (40% w/v), a surfactant co-mixture (2.0%, w/v) and glycerin 9(1.7%, w/v) has been well described (Lanza et al., (2000) Invest Radiol, 35: 227-234; Lanza et al., (1997) Ultrasound Med Biol, 23: 863-870).
Briefly, the surfactant co-mixture is dissolved in chloroform/methanol, evaporated under reduced pressure, dried in a 50° C. vacuum oven, and finally dispersed into water by sonication. The suspension is combined with perfluorocarbon and then emulsified at 20,000 PSI. Fluorescent nanoparticles are manufactured by including in the lipid mixture 0.1 mole % Fluorescence-FITC or PE prior to the emulsification step. Coupling of monoclonal antibodies involves the introduction of a sulfhydrl group onto the protein by modification of amines with N-succinimidyl S-acetylthioacetate (SATA), which then is reacted with nanoparticles containing activated maleimide. We coupled an antibody that recognizes the human, but not mouse, platelet receptor alphaIIb beta3 and determined the ability of FITC-labeled PNPs to detect a thrombus composed of human platelets at a site of laser-induced vascular injury in the cremaster muscle of a mouse homozygous for the 1326R>H mutation. These antibody-coupled PNPs rapidly and selectively accumulated at the site of the developing human thrombus (FIG. 43).
Example 6
Identification of Small Molecules that Mitigate Binding Between GPIb Alpha and the VWF-A1 Domain
Small molecules, often with molecular weights of 500 or below, have proven to be extremely important to researchers to explore function at the molecular, cellular, and in vivo level. Such compounds have also been proven to be valuable as drugs to treat diseases, and most medicines marketed today are from this class (i.e., Aggrastat-see above). As the interaction between GPIb alpha and VWF-A1 is essential for the platelet deposition in damaged arterioles, it is a reasonable to assume that disruption of this adhesive event will inhibit or ameliorate thrombus formation. Moreover, we speculate that only partial inhibition is required to achieve this goal based on the phenotype of our mutant A1 domain mice, the inability to form stable thrombi in vivo.
Computational Design Based on the Structure of the Binary Complex.
Traditional approaches to small molecule discovery typically rely on a step-wise synthesis and screening program for large numbers of compounds to optimize activity profiles. Over the past decade, scientists have used computer models to aid in the development of new chemical agonists or antagonists as well as to better define activity profiles and binding affinities of such compounds. In particular, these tools are being successfully used, in conjunction with traditional research techniques, to examine the structural properties of existing compounds in order to predict their ability to alter the function of biologically relevant proteins. For this approach to be successful, one must have high quality crystal structures of the biological molecule(s) in order to generate an accurate 3-dimensional model so that it can then be used to identify binding regions for small molecules.
The structure of the binary complex formed when GPIb alpha binds to the A1 domain of VWF can be determined using such methods. For example, a mechanism by which the snake venom protein botrocetin enhances the interaction between GPIb alpha and the VWF-A1 in order to promote spontaneous platelet aggregation, resulting in death has been elucidated. Botrocetin was known to bind with high affinity to the A1 domain [see Table 7 for the crystallization data summary and Table 8 for atomic coordinate data], but was not thought to interact directly with GPIb alpha. This snake venom has the capacity to form a small, but distinct interface with this platelet receptor so to prevent its release from the A1 domain, thus facilitating platelet aggregation (FIGS. 44A and 44B). In a sense, nature has created a molecule that modifies the behavior of a known biological interaction, suggesting that one may be able to target man-made structures to this domain as well.







TABLE 7







Summary of Crystallization data (from PDB access no. 


1AUQ and J Biol Chem (1998) 273(17):10396-10401)










Structure
Free enzyme (see Table 8)







Space Group
P61



Maximum resolution (Å)
2.3



Resolution range for refinement
10-2.3



Number of reflections
11,849



Completeness (%)
85.4



R factor2 (%)
18.6



Free R factor2 (%)
23.8



rms deviation in bond lengths (Å)
0.011



rms deviation in bond angles (°)
1.43

















TABLE 8







Atomic Coordinates for Residues of a Crystal of murine VWF-A1 


(SEQ ID NO: 10).















CRYST1
86.395
86.395
68.125
90.00
90.00
120.00
P 61
6




















ATOM
1
N
ASP
498
22.142
52.453
−14.520
1.00
83.59
N


ATOM
2
CA
ASP
498
20.770
52.768
−14.026
1.00
83.86
C


ATOM
3
C
ASP
498
20.803
53.068
−12.522
1.00
81.16
C


ATOM
4
O
ASP
498
20.978
52.165
−11.696
1.00
81.93
O


ATOM
5
CB
ASP
498
19.821
51.597
−14.327
1.00
87.23
C


ATOM
6
CG
ASP
498
18.352
51.961
−14.133
1.00
88.94
C


ATOM
7
OD1
ASP
498
18.022
53.171
−14.084
1.00
88.91
O


ATOM
8
OD2
ASP
498
17.521
51.027
−14.033
1.00
90.11
O


ATOM
9
N
ILE
499
20.620
54.343
−12.190
1.00
76.43
N


ATOM
10
CA
ILE
499
20.649
54.829
−10.809
1.00
70.82
C


ATOM
11
C
ILE
499
19.237
55.126
−10.255
1.00
64.40
C


ATOM
12
O
ILE
499
18.888
56.265
−9.941
1.00
62.53
O


ATOM
13
CB
ILE
499
21.596
56.076
−10.724
1.00
75.17
C


ATOM
14
CG1
ILE
499
21.872
56.476
−9.278
1.00
77.18
C


ATOM
15
CG2
ILE
499
21.030
57.256
−11.527
1.00
77.54
C


ATOM
16
CD1
ILE
499
23.010
57.485
−9.141
1.00
79.34
C


ATOM
17
N
SER
500
18.445
54.071
−10.111
1.00
58.81
N


ATOM
18
CA
SER
500
17.073
54.170
−9.616
1.00
52.67
C


ATOM
19
C
SER
500
16.771
52.914
−8.797
1.00
46.03
C


ATOM
20
O
SER
500
17.477
51.915
−8.928
1.00
46.10
O


ATOM
21
CB
SER
500
16.085
54.306
−10.779
1.00
55.03
C


ATOM
22
OG
SER
500
14.814
54.747
−10.320
1.00
55.43
O


ATOM
23
N
GLU
501
15.690
52.946
−8.017
1.00
39.21
N


ATOM
24
CA
GLU
501
15.341
51.839
−7.124
1.00
33.62
C


ATOM
25
C
GLU
501
13.826
51.632
−6.967
1.00
33.73
C


ATOM
26
O
GLU
501
13.055
52.575
−7.148
1.00
35.42
O


ATOM
27
CB
GLU
501
16.025
52.088
−5.772
1.00
27.09
C


ATOM
28
CG
GLU
501
15.928
53.517
−5.328
1.00
23.81
C


ATOM
29
CD
GLU
501
17.036
53.971
−4.394
1.00
22.26
C


ATOM
30
OE1
GLU
501
18.150
53.442
−4.407
1.00
23.46
O


ATOM
31
OE2
GLU
501
16.790
54.912
−3.645
1.00
19.78
O


ATOM
32
N
PRO
502
13.390
50.408
−6.585
1.00
32.77
N


ATOM
33
CA
PRO
502
11.980
50.038
−6.406
1.00
33.37
C


ATOM
34
C
PRO
502
11.257
50.886
−5.377
1.00
36.76
C


ATOM
35
O
PRO
502
11.881
51.657
−4.655
1.00
40.35
O


ATOM
36
CB
PRO
502
12.056
48.588
−5.911
1.00
32.84
C


ATOM
37
CG
PRO
502
13.426
48.154
−6.188
1.00
30.80
C


ATOM
38
CD
PRO
502
14.248
49.372
−5.994
1.00
33.53
C


ATOM
39
N
PRO
503
9.921
50.743
−5.283
1.00
38.50
N


ATOM
40
CA
PRO
503
9.095
51.495
−4.332
1.00
36.00
C


ATOM
41
C
PRO
503
9.217
50.861
−2.962
1.00
33.78
C


ATOM
42
O
PRO
503
9.351
49.642
−2.870
1.00
33.98
O


ATOM
43
CB
PRO
503
7.670
51.270
−4.844
1.00
35.94
C


ATOM
44
CG
PRO
503
7.844
50.708
−6.240
1.00
36.76
C


ATOM
45
CD
PRO
503
9.074
49.889
−6.137
1.00
37.21
C


ATOM
46
N
LEU
504
9.225
51.670
−1.910
1.00
32.48
N


ATOM
47
CA
LEU
504
9.284
51.120
−0.562
1.00
31.29
C


ATOM
48
C
LEU
504
7.869
50.821
−0.120
1.00
31.74
C


ATOM
49
O
LEU
504
7.663
50.104
0.847
1.00
34.42
O


ATOM
50
CB
LEU
504
9.857
52.133
0.427
1.00
27.31
C


ATOM
51
CG
LEU
504
11.346
52.216
0.633
1.00
20.47
C


ATOM
52
CD1
LEU
504
11.608
53.354
1.577
1.00
16.31
C


ATOM
53
CD2
LEU
504
11.865
50.886
1.213
1.00
22.70
C


ATOM
54
N
HIS
505
6.892
51.433
−0.779
1.00
32.33
N


ATOM
55
CA
HIS
505
5.508
51.234
−0.382
1.00
33.59
C


ATOM
56
C
HIS
505
4.574
50.872
−1.506
1.00
37.41
C


ATOM
57
O
HIS
505
4.980
50.817
−2.669
1.00
38.66
O


ATOM
58
CB
HIS
505
4.977
52.504
0.277
1.00
30.39
C


ATOM
59
CG
HIS
505
5.799
52.956
1.432
1.00
24.50
C


ATOM
60
ND1
HIS
505
6.533
54.118
1.413
1.00
26.88
N


ATOM
61
CD2
HIS
505
6.041
52.377
2.628
1.00
22.75
C


ATOM
62
CE1
HIS
505
7.195
54.235
2.543
1.00
22.86
C


ATOM
63
NE2
HIS
505
6.915
53.193
3.297
1.00
23.78
N


ATOM
64
N
ASP
506
3.323
50.612
−1.125
1.00
39.57
N


ATOM
65
CA
ASP
506
2.254
50.291
−2.072
1.00
41.22
C


ATOM
66
C
ASP
506
1.600
51.614
−2.482
1.00
39.47
C


ATOM
67
O
ASP
506
1.306
51.826
−3.651
1.00
42.48
O


ATOM
68
CB
ASP
506
1.231
49.325
−1.443
1.00
44.20
C


ATOM
69
CG
ASP
506
1.831
47.941
−1.106
1.00
47.68
C


ATOM
70
OD1
ASP
506
2.837
47.508
−1.730
1.00
48.34
O


ATOM
71
OD2
ASP
506
1.276
47.272
−0.209
1.00
49.92
O


ATOM
72
N
PHE
507
1.367
52.497
−1.507
1.00
38.50
N


ATOM
73
CA
PHE
507
0.800
53.830
−1.756
1.00
35.03
C


ATOM
74
C
PHE
507
2.016
54.527
−2.406
1.00
36.00
C


ATOM
75
O
PHE
507
3.086
54.637
−1.780
1.00
35.29
O


ATOM
76
CB
PHE
507
0.408
54.471
−0.418
1.00
32.75
C


ATOM
77
CG
PHE
507
−0.252
55.810
−0.552
1.00
32.71
C


ATOM
78
CD1
PHE
507
−1.605
55.915
−0.866
1.00
33.60
C


ATOM
79
CD2
PHE
507
0.471
56.969
−0.343
1.00
29.61
C


ATOM
80
CE1
PHE
507
−2.216
57.157
−0.962
1.00
31.49
C


ATOM
81
CE2
PHE
507
−0.132
58.205
−0.437
1.00
29.32
C


ATOM
82
CZ
PHE
507
−1.477
58.298
−0.750
1.00
33.05
C


ATOM
83
N
TYR
508
1.839
55.032
−3.624
1.00
35.32
N


ATOM
84
CA
TYR
508
2.955
55.578
−4.396
1.00
32.82
C


ATOM
85
C
TYR
508
2.445
56.499
5.515
1.00
33.01
C


ATOM
86
O
TYR
508
1.484
56.154
−6.200
1.00
39.06
O


ATOM
87
CB
TYR
508
3.612
54.364
−5.053
1.00
27.12
C


ATOM
88
CG
TYR
508
5.027
54.464
−5.529
1.00
31.57
C


ATOM
89
CD1
TYR
508
6.041
54.922
−4.680
1.00
35.35
C


ATOM
90
CD2
TYR
508
5.389
53.946
−6.771
1.00
28.70
C


ATOM
91
CE1
TYR
508
7.397
54.848
−5.054
1.00
36.51
C


ATOM
92
CE2
TYR
508
6.730
53.862
−7.154
1.00
33.58
C


ATOM
93
CZ
TYR
508
7.735
54.309
−6.294
1.00
36.83
C


ATOM
94
OH
TYR
508
9.075
54.176
−6.649
1.00
40.00
O


ATOM
95
N
CYS
509
3.046
57.662
−5.717
1.00
28.11
N


ATOM
96
CA
CYS
509
2.593
58.516
−6.822
1.00
27.15
C


ATOM
97
C
CYS
509
3.509
58.310
−8.009
1.00
25.32
C


ATOM
98
O
CYS
509
4.717
58.512
−7.909
1.00
30.67
O


ATOM
99
CB
CYS
509
2.572
59.997
−6.464
1.00
25.77
C


ATOM
100
SG
CYS
509
1.888
61.004
−7.834
1.00
21.50
S


ATOM
101
N
SER
510
2.939
57.837
−9.102
1.00
23.50
N


ATOM
102
CA
SER
510
3.686
57.590
−10.321
1.00
25.91
C


ATOM
103
C
SER
510
3.052
58.414
−11.475
1.00
25.38
C


ATOM
104
O
SER
510
2.384
57.882
−12.364
1.00
32.01
O


ATOM
105
CB
SER
510
3.687
56.087
−10.612
1.00
24.85
C


ATOM
106
OG
SER
510
4.275
55.783
−11.867
1.00
29.60
O


ATOM
107
N
ARG
511
3.236
59.726
−11.434
1.00
19.35
N


ATOM
108
CA
ARG
511
2.689
60.596
−12.451
1.00
16.16
C


ATOM
109
C
ARG
511
3.757
61.589
−12.810
1.00
20.03
C


ATOM
110
O
ARG
511
4.825
61.584
−12.191
1.00
24.48
O


ATOM
111
CB
ARG
511
1.439
61.310
−11.952
1.00
13.51
C


ATOM
112
CG
ARG
511
0.300
60.358
−11.708
1.00
13.74
C


ATOM
113
CD
ARG
511
−0.994
61.084
−11.674
1.00
12.03
C


ATOM
114
NE
ARG
511
−2.111
60.169
−11.505
1.00
12.96
N


ATOM
115
CZ
ARG
511
−3.388
60.547
−11.582
1.00
16.84
C


ATOM
116
NH1
ARG
511
−3.700
61.814
−11.841
1.00
18.71
N


ATOM
117
NH2
ARG
511
−4.361
59.680
−11.342
1.00
18.41
N


ATOM
118
N
LEU
512
3.482
62.427
−13.810
1.00
16.95
N


ATOM
119
CA
LEU
512
4.457
63.400
−14.279
1.00
17.28
C


ATOM
120
C
LEU
512
4.337
64.645
−13.434
1.00
16.82
C


ATOM
121
O
LEU
512
3.544
65.534
−13.715
1.00
18.14
O


ATOM
122
CB
LEU
512
4.220
63.668
−15.756
1.00
18.43
C


ATOM
123
CG
LEU
512
4.357
62.372
−16.556
1.00
17.02
C


ATOM
124
CD1
LEU
512
3.947
62.602
−17.993
1.00
15.13
C


ATOM
125
CD2
LEU
512
5.796
61.865
−16.461
1.00
16.19
C


ATOM
126
N
LEU
513
5.226
64.748
−12.458
1.00
19.32
N


ATOM
127
CA
LEU
513
5.162
65.827
−11.501
1.00
18.08
C


ATOM
128
C
LEU
513
6.506
66.390
−11.089
1.00
15.12
C


ATOM
129
O
LEU
513
7.477
65.655
−10.948
1.00
14.11
O


ATOM
130
CB
LEU
513
4.454
65.273
−10.257
1.00
19.36
C


ATOM
131
CG
LEU
513
4.260
66.123
−9.012
1.00
20.52
C


ATOM
132
CD1
LEU
513
3.338
67.259
−9.353
1.00
18.37
C


ATOM
133
CD2
LEU
513
3.698
65.270
−7.902
1.00
12.16
C


ATOM
134
N
ASP
514
6.534
67.699
−10.853
1.00
16.07
N


ATOM
135
CA
ASP
514
7.735
68.384
−10.383
1.00
16.17
C


ATOM
136
C
ASP
514
7.257
68.946
9.041
1.00
15.61
C


ATOM
137
O
ASP
514
6.480
69.890
9.014
1.00
16.76
O


ATOM
138
CB
ASP
514
8.136
69.530
−11.327
1.00
15.99
C


ATOM
139
CG
ASP
514
9.016
69.076
−12.511
1.00
18.33
C


ATOM
140
OD1
ASP
514
9.255
67.874
−12.736
1.00
17.22
O


ATOM
141
OD2
ASP
514
9.492
69.961
−13.240
1.00
25.18
O


ATOM
142
N
LEU
515
7.660
68.338
−7.929
1.00
15.44
N


ATOM
143
CA
LEU
515
7.205
68.808
−6.623
1.00
14.64
C


ATOM
144
C
LEU
515
8.282
69.524
−5.802
1.00
14.01
C


ATOM
145
O
LEU
515
9.382
68.995
−5.603
1.00
8.40
O


ATOM
146
CB
LEU
515
6.613
67.625
−5.853
1.00
14.99
C


ATOM
147
CG
LEU
515
6.168
67.788
−4.396
1.00
9.59
C


ATOM
148
CD1
LEU
515
5.054
68.775
4.249
1.00
5.58
C


ATOM
149
CD2
LEU
515
5.714
66.455
3.924
1.00
8.85
C


ATOM
150
N
VAL
516
7.975
70.743
−5.355
1.00
10.50
N


ATOM
151
CA
VAL
516
8.927
71.514
−4.566
1.00
13.88
C


ATOM
152
C
VAL
516
8.504
71.601
−3.100
1.00
14.19
C


ATOM
153
O
VAL
516
7.400
72.059
−2.796
1.00
14.17
O


ATOM
154
CB
VAL
516
9.103
72.966
−5.111
1.00
15.97
C


ATOM
155
CG1
VAL
516
10.134
73.711
−4.278
1.00
14.88
C


ATOM
156
CG2
VAL
516
9.580
72.947
−6.570
1.00
14.16
C


ATOM
157
N
PHE
517
9.359
71.127
−2.191
1.00
17.00
N


ATOM
158
CA
PHE
517
9.072
71.205
−0.747
1.00
13.51
C


ATOM
159
C
PHE
517
9.776
72.469
−0.179
1.00
12.08
C


ATOM
160
O
PHE
517
11.009
72.587
−0.233
1.00
10.17
O


ATOM
161
CB
PHE
517
9.602
69.968
−0.024
1.00
15.54
C


ATOM
162
CG
PHE
517
8.940
68.662
−0.426
1.00
11.63
C


ATOM
163
CD1
PHE
517
7.805
68.200
0.230
1.00
13.47
C


ATOM
164
CD2
PHE
517
9.517
67.852
−1.400
1.00
9.53
C


ATOM
165
CE1
PHE
517
7.256
66.952
−0.075
1.00
10.36
C


ATOM
166
CE2
PHE
517
8.989
66.625
−1.703
1.00
8.49
C


ATOM
167
CZ
PHE
517
7.847
66.164
−1.036
1.00
10.76
C


ATOM
168
N
LEU
518
9.005
73.405
0.378
1.00
12.33
N


ATOM
169
CA
LEU
518
9.574
74.643
0.926
1.00
12.65
C


ATOM
170
C
LEU
518
9.420
74.659
2.453
1.00
11.22
C


ATOM
171
O
LEU
518
8.333
74.940
2.959
1.00
15.25
O


ATOM
172
CB
LEU
518
8.884
75.865
0.287
1.00
9.93
C


ATOM
173
CG
LEU
518
8.791
75.956
−1.244
1.00
8.05
C


ATOM
174
CD1
LEU
518
7.840
77.076
−1.640
1.00
3.79
C


ATOM
175
CD2
LEU
518
10.166
76.182
−1.857
1.00
10.27
C


ATOM
176
N
LEU
519
10.514
74.399
3.168
1.00
7.04
N


ATOM
177
CA
LEU
519
10.518
74.319
4.635
1.00
7.57
C


ATOM
178
C
LEU
519
10.795
75.567
5.440
1.00
4.28
C


ATOM
179
O
LEU
519
11.867
76.131
5.339
1.00
10.10
O


ATOM
180
CB
LEU
519
11.544
73.283
5.090
1.00
6.92
C


ATOM
181
CG
LEU
519
11.188
71.821
4.925
1.00
10.92
C


ATOM
182
CD1
LEU
519
11.011
71.550
3.470
1.00
8.66
C


ATOM
183
CD2
LEU
519
12.295
70.951
5.489
1.00
7.00
C


ATOM
184
N
ASP
520
9.891
75.908
6.352
1.00
11.69
N


ATOM
185
CA
ASP
520
10.051
77.081
7.217
1.00
13.58
C


ATOM
186
C
ASP
520
11.330
76.914
8.057
1.00
14.38
C


ATOM
187
O
ASP
520
11.469
75.933
8.775
1.00
18.86
O


ATOM
188
CB
ASP
520
8.813
77.201
8.116
1.00
15.76
C


ATOM
189
CG
ASP
520
8.737
78.528
8.839
1.00
14.91
C


ATOM
190
OD1
ASP
520
9.744
78.970
9.394
1.00
15.53
O


ATOM
191
OD2
ASP
520
7.671
79.143
8.870
1.00
15.00
O


ATOM
192
N
GLY
521
12.272
77.847
7.958
1.00
12.62
N


ATOM
193
CA
GLY
521
13.514
77.720
8.713
1.00
12.88
C


ATOM
194
C
GLY
521
13.617
78.637
9.919
1.00
17.26
C


ATOM
195
O
GLY
521
14.690
78.785
10.508
1.00
16.89
O


ATOM
196
N
SER
522
12.500
79.256
10.291
1.00
16.08
N


ATOM
197
CA
SER
522
12.452
80.168
11.420
1.00
17.98
C


ATOM
198
C
SER
522
12.484
79.424
12.750
1.00
18.81
C


ATOM
199
O
SER
522
12.359
78.197
12.792
1.00
17.50
O


ATOM
200
CB
ER
522
11.158
80.967
11.371
1.00
12.88
C


ATOM
201
OG
SER
522
10.069
80.128
11.708
1.00
12.60
O


ATOM
202
N
SER
523
12.547
80.206
13.628
1.00
17.68
N


ATOM
203
CA
SER
523
12.558
79.701
15.187
1.00
20.71
C


ATOM
204
C
SER
523
11.141
79.389
15.694
1.00
20.97
C


ATOM
205
O
SER
523
10.971
78.999
16.833
1.00
27.46
O


ATOM
206
CB
SER
523
13.253
80.693
16.111
1.00
21.32
C


ATOM
207
OG
SER
523
12.701
81.986
15.933
1.00
26.32
O


ATOM
208
N
ARG
524
10.117
79.590
14.872
1.00
16.19
N


ATOM
209
CA
ARG
524
8.765
79.240
15.278
1.00
19.04
C


ATOM
210
C
ARG
524
8.655
77.734
15.460
1.00
20.87
C


ATOM
211
O
ARG
524
7.724
77.235
16.106
1.00
22.79
O


ATOM
212
CB
ARG
524
7.758
79.709
14 .246
1.00
20.78
C


ATOM
213
CC
ARG
524
7.654
81.183
14.225
1.00
22.74
C


ATOM
214
CD
ARG
524
7.449
81.671
15.627
1.00
23.78
C


ATOM
215
NE
ARG
524
7.265
83.107
15.661
1.00
29.27
N


ATOM
216
CZ
ARG
524
7.116
83.815
16.774
1.00
29.96
C


ATOM
217
NH1
ARG
524
7.140
83.218
17.959
1.00
25.48
N


ATOM
218
NH2
ARG
524
6.899
85.118
16.686
1.00
26.64
N


ATOM
219
N
LEU
525
9.561
77.014
14.804
1.00
19.12
N


ATOM
220
CA
LEU
525
9.645
75.571
14.915
1.00
17.29
C


ATOM
221
C
LEU
525
10.942
75.381
15.674
1.00
22.24
C


ATOM
222
O
LEU
525
11.950
76.041
15.374
1.00
21.49
O


ATOM
223
CB
LEU
525
9.817
74.913
13.546
1.00
19.88
C


ATOM
224
CG
LEU
525
8.695
74.845
12.523
1.00
15.92
C


ATOM
225
CD1
LEU
525
9.236
74.241
11.253
1.00
11.38
C


ATOM
226
CD2
LEU
525
7.556
73.989
13.080
1.00
16.42
C


ATOM
227
N
SER
526
10.912
74.519
16.682
1.00
23.55
N


ATOM
228
CA
SER
526
12.113
74.240
17.460
1.00
24.19
C


ATOM
229
C
SER
526
12.873
73.147
16.725
1.00
25.31
C


ATOM
230
O
SER
526
12.342
72.534
15.797
1.00
26.87
O


ATOM
231
CB
SER
526
11.737
73.746
18.855
1.00
22.27
C


ATOM
232
OG
SER
526
10.926
72.579
18.785
1.00
18.28
O


ATOM
233
N
GLU
527
14.093
72.868
17.160
1.00
26.48
N


ATOM
234
CA
GLU
527
14.900
71.829
16.539
1.00
26.18
C


ATOM
235
C
GLU
527
14.144
70.514
16.449
1.00
25.55
C


ATOM
236
O
GLU
527
14.134
69.859
15.406
1.00
30.55
O


ATOM
237
CB
GLU
527
16.172
71.620
17.349
1.00
28.26
C


ATOM
238
CG
GLU
527
17.025
70.471
16.880
1.00
37.14
C


ATOM
239
CD
GLU
527
18.417
70.909
16.459
1.00
44.14
C


ATOM
240
OE1
GLU
527
19.082
71.637
17.238
1.00
44.73
O


ATOM
241
OE2
GLU
527
18.853
70.506
15.351
1.00
47.92
O


ATOM
242
N
ALA
528
13.458
70.159
17.526
1.00
23.12
N


ATOM
243
CA
ALA
528
12.733
68.901
17.580
1.00
19.11
C


ATOM
244
C
ALA
528
11.547
68.875
16.650
1.00
19.81
C


ATOM
245
O
ALA
528
11.237
67.834
16.078
1.00
24.34
O


ATOM
246
CB
ALA
528
12.301
68.609
18.992
1.00
11.67
C


ATOM
247
N
GLU
529
10.873
70.015
16.518
1.00
19.26
N


ATOM
248
CA
GLU
529
9.697
70.126
15.659
1.00
19.04
C


ATOM
249
C
GLU
529
10.146
70.132
14.212
1.00
21.14
C


ATOM
250
O
GLU
529
9.491
69.543
13.358
1.00
26.65
O


ATOM
251
CB
GLU
529
8.908
71.403
15.974
1.00
17.25
C


ATOM
252
CG
GLU
529
8.144
71.355
17.292
1.00
12.54
C


ATOM
253
CD
GLU
529
7.447
72.663
17.624
1.00
18.73
C


ATOM
254
OE1
GLU
529
8.120
73.725
17.576
1.00
16.59
O


ATOM
255
OE2
GLU
529
6.236
72.623
17.966
1.00
18.64
O


ATOM
256
N
PHE
530
11.292
70.750
13.945
1.00
17.88
N


ATOM
257
CA
PHE
530
11.828
70.807
12.600
1.00
18.23
C


ATOM
258
C
PHE
530
12.139
69.383
12.147
1.00
21.70
C


ATOM
259
O
PHE
530
11.848
69.007
11.003
1.00
28.22
O


ATOM
260
CB
PHE
530
13.083
71.658
12.571
1.00
17.84
C


ATOM
261
CG
PHE
530
13.534
72.028
11.196
1.00
19.34
C


ATOM
262
CD1
PHE
530
12.696
72.758
10.342
1.00
17.07
C


ATOM
263
CD2
PHE
530
14.806
71.685
10.759
1.00
16.08
C


ATOM
264
CE1
PHE
530
13.127
73.139
9.089
1.00
12.46
C


ATOM
265
CE2
PHE
530
15.248
72.066
9.492
1.00
13.83
C


ATOM
266
CZ
PHE
530
14.413
72.789
8.664
1.00
11.80
C


ATOM
267
N
GLU
531
12.650
68.563
13.052
1.00
18.89
N


ATOM
266
CA
GLU
531
12.932
67.182
12.695
1.00
21.46
C


ATOM
269
C
GLU
531
11.660
66.478
12.263
1.00
17.63
C


ATOM
270
O
GLU
531
11.711
65.606
11.397
1.00
22.80
O


ATOM
271
CB
GLU
531
13.556
66.414
13.862
1.00
19.16
C


ATOM
272
CG
GLU
531
14.685
66.955
14.274
1.00
24.82
C


ATOM
273
CD
GLU
531
15.827
67.120
13.105
1.00
28.99
C


ATOM
274
OE1
GLU
531
16.177
66.101
12.469
1.00
24.87
O


ATOM
275
OE2
GLU
531
16.211
68.282
12.831
1.00
35.27
O


ATOM
276
N
VAL
532
10.529
66.840
12.864
1.00
15.71
N


ATOM
277
CA
VAL
532
9.257
66.211
12.509
1.00
15.96
C


ATOM
278
C
VAL
532
8.820
66.691
11.133
1.00
18.38
C


ATOM
279
O
VAL
532
8.234
65.921
10.357
1.00
22.15
O


ATOM
280
CB
VAL
532
8.149
66.493
13.551
1.00
15.96
C


ATOM
281
CG1
VAL
532
6.825
65.847
13.117
1.00
6.01
C


ATOM
282
CG2
VAL
532
8.575
65.950
14.908
1.00
13.74
C


ATOM
283
N
LEU
533
9.150
67.943
10.821
1.00
14.73
N


ATOM
284
CA
LEU
533
8.826
68.530
9.536
1.00
14.97
C


ATOM
285
C
LEU
533
9.602
67.771
8.476
1.00
16.19
C


ATOM
286
O
LEU
533
9.044
67.348
7.458
1.00
18.03
O


ATOM
287
CB
LEU
533
9.243
70.008
9.496
1.00
14.45
C


ATOM
288
CG
LEU
533
8.780
70.717
8.220
1.00
12.15
C


ATOM
289
CD1
LEU
533
7.283
70.472
8.038
1.00
14.73
C


ATOM
290
CD2
LEU
533
9.060
72.183
8.282
1.00
8.85
C


ATOM
291
N
LYS
534
10.899
67.640
8.704
1.00
12.22
N


ATOM
292
CA
LYS
534
11.766
66.936
7.785
1.00
13.88
C


ATOM
293
C
LYS
534
11.337
65.492
7.592
1.00
13.67
C


ATOM
294
O
LYS
534
11.356
64.974
6.478
1.00
19.68
O


ATOM
295
CB
LYS
534
13.210
67.016
8.282
1.00
17.43
C


ATOM
296
CG
LYS
534
13.819
68.393
8.100
1.00
13.24
C


ATOM
297
CD
LYS
534
15.OD2
68.635
9.036
1.00
18.41
C


ATOM
298
CE
LYS
534
15.936
67.455
9.114
1.00
20.81
C


ATOM
299
NZ
LYS
534
17.017
67.696
10.101
1.00
18.52
N


ATOM
300
N
ALA
535
10.871
64.858
8.656
1.00
13.06
N


ATOM
301
CA
ALA
535
10.442
63.472
8.563
1.00
9.85
C


ATOM
302
C
ALA
535
9.164
63.382
7.745
1.00
13.30
C


ATOM
303
O
ALA
535
8.911
62.361
7.082
1.00
15.28
O


ATOM
304
CB
ALA
535
10.254
62.901
9.923
1.00
8.53
C


ATOM
305
N
PHE
536
8.348
64.428
7.797
1.00
13.50
N


ATOM
306
CA
PHE
536
7.117
64.490
6.996
1.00
11.73
C


ATOM
307
C
PHE
536
7.537
64.558
5.521
1.00
14.75
C


ATOM
308
O
PHE
536
6.962
63.887
4.654
1.00
17.83
O


ATOM
309
CB
PHE
536
6.369
65.756
7.350
1.00
6.75
C


ATOM
310
CG
PHE
536
5.252
66.076
6.419
1.00
12.43
C


ATOM
311
CD1
PHE
536
4.113
65.288
6.388
1.00
13.94
C


ATOM
312
CD2
PHE
536
5.314
67.191
5.585
1.00
10.83
C


ATOM
313
CE1
PHE
536
3.040
65.604
5.534
1.00
15.05
C


ATOM
314
CE2
PHE
536
4.260
67.511
4.736
1.00
11.68
C


ATOM
315
CZ
PHE
536
3.114
66.720
4.711
1.00
9.74
C


ATOM
316
N
VAL
537
8.550
65.375
5.250
1.00
13.66
N


ATOM
317
CA
VAL
537
9.068
65.549
3.903
1.00
14.58
C


ATOM
318
C
VAL
537
9.593
64.209
3.418
1.00
16.40
C


ATOM
319
O
VAL
537
9.176
63.735
2.376
1.00
21.04
O


ATOM
320
CB
VAL
537
10.143
66.684
3.858
1.00
12.13
C


ATOM
321
CG1
VAL
537
10.864
66.706
2.527
1.00
7.80
C


ATOM
322
CG2
VAL
537
9.459
68.040
4.070
1.00
2.11
C


ATOM
323
N
VAL
538
10.397
63.541
4.231
1.00
17.90
N


ATOM
324
CA
VAL
538
10.929
62.228
3.867
1.00
15.91
C


ATOM
325
C
VAL
538
9.842
61.187
3.559
1.00
17.55
C


ATOM
326
O
VAL
538
9.946
60.4 91
2.538
1.00
15.35
O


ATOM
327
CB
VAL
538
11.893
61.693
4.948
1.00
17.65
C


ATOM
328
CG1
VAL
538
12.129
60.169
4.791
1.00
9.43
C


ATOM
329
CG2
VAL
538
13.200
62.447
4.849
1.00
9.34
C


ATOM
330
N
ASP
539
6.618
61.040
4.410
1.00
17.17
N


ATOM
331
CA
ASP
539
7.798
60.050
4.068
1.00
21.22
C


ATOM
332
C
ASP
539
6.892
60.418
2.919
1.00
17.63
C


ATOM
333
O
ASP
539
6.313
59.526
2.305
1.00
20.04
O


ATOM
334
CB
ASP
539
7.048
59.412
5.257
1.00
29.14
C


ATOM
335
CG
ASP
539
6.636
60.345
6.384
1.00
35.47
C


ATOM
336
OD1
ASP
539
6.283
61.428
6.128
1.00
44.75
O


ATOM
337
OD2
ASP
539
7.219
59.993
7.523
1.00
36.63
O


ATCM
338
N
MET
540
6.610
61.709
2.601
1.00
16.39
N


ATOM
339
CA
MET
540
6.063
62.172
1.423
1.00
13.96
C


ATOM
340
C
MET
540
6.857
61.593
0.269
1.00
10.86
C


ATOM
341
O
MET
540
6.333
60.862
−0.539
1.00
14.71
O


ATOM
342
CB
MET
540
6.159
63.688
1.294
1.00
22.58
C


ATOM
343
CG
MET
540
5.149
64.497
2.065
1.00
31.44
C


ATOM
344
SD
MET
540
3.503
64.541
1.326
1.00
45.37
S


ATOM
345
CE
MET
540
3.679
65.729
0.060
1.00
31.22
C


ATOM
346
N
MET
541
8.163
61.870
0.261
1.00
13.18
N


ATOM
347
CA
MET
541
9.116
61.398
−0.751
1.00
10.35
C


ATOM
348
C
MET
541
9.107
59.895
−0.969
1.00
16.49
C


ATOM
349
O
MET
541
9.225
59.438
−2.111
1.00
20.40
O


ATOM
350
CB
MET
541
10.537
61.813
−0.370
1.00
9.58
C


ATOM
351
CG
MET
541
10.820
63.276
−0.620
1.00
11.21
C


ATOM
352
SD
MET
541
12.434
63.842
−0.058
1.00
12.89
S


ATOM
353
CE
MET
541
13.392
63.835
−1.500
1.00
15.28
C


ATOM
354
N
GLU
542
8.985
59.119
0.107
1.00
16.56
N


ATOM
355
CA
GLU
542
8.971
57.657
−0.008
1.00
13.92
C


ATOM
356
C
GLU
542
7.759
57.174
−0.818
1.00
14.26
C


ATOM
357
O
GLU
542
7.789
56.104
−1.430
1.00
15.31
O


ATOM
358
CB
GLU
542
8.925
57.018
1.382
1.00
12.65
C


ATOM
359
CG
GLU
542
10.175
57.228
2.230
1.00
15.01
C


ATOM
360
CD
GLU
542
10.155
56.422
3.528
1.00
13.47
C


ATOM
361
OE1
GLU
542
9.181
55.694
3.785
1.00
18.27
O


ATOM
362
OE2
GLU
542
11.109
56.518
4.308
1.00
15.89
O


ATOM
363
N
ARG
543
6.688
57.961
−0.789
1.00
12.61
N


ATOM
364
CA
ARG
543
5.451
57.633
−1.473
1.00
10.82
C


ATOM
365
C
ARG
543
5.388
58.327
−2.827
1.00
11.37
C


ATOM
366
O
ARG
543
4.310
58.574
−3.373
1.00
13.23
O


ATOM
367
CB
ARG
543
4.285
58.069
−0.593
1.00
9.75
C


ATOM
368
CG
ARG
543
4.168
57.203
0.632
1.00
8.93
C


ATOM
369
CD
ARG
543
3.353
57.841
1.737
1.00
9.66
C


ATOM
370
NE
ARG
543
3.241
56.878
2.835
1.00
17.05
N


ATOM
371
CZ
ARG
543
4.196
56.652
3.737
1.00
12.00
C


ATOM
372
NH1
ARG
543
5.341
57.332
3.697
1.00
9.90
N


ATOM
373
NH2
ARG
543
4.044
55.681
4.634
1.00
15.47
N


ATOM
374
N
LEU
544
6.554
58.559
−3.401
1.00
13.34
N


ATOM
375
CA
LEU
544
6.676
59.214
−4.688
1.00
15.24
C


ATOM
376
C
LEU
544
7.606
58.388
−5.574
1.00
16.79
C


ATOM
377
O
LEU
544
8.633
57.871
−5.103
1.00
17.01
O


ATOM
378
CB
LEU
544
7.318
60.578
−4.465
1.00
12.06
C


ATOM
379
CG
LEU
544
6.539
61.886
−4.546
1.00
11.78
C


ATOM
380
CD1
LEU
544
5.138
61.769
−4.046
1.00
11.41
C


ATOM
381
CD2
LEU
544
7.315
62.896
−3.751
1.00
8.97
C


ATOM
382
N
ARG
545
7.283
58.269
−6.855
1.00
15.28
N


ATOM
383
CA
ARG
545
8.187
57.551
−7.740
1.00
15.08
C


ATOM
384
C
ARG
545
9.110
58.627
−8.278
1.00
17.33
C


ATOM
385
O
ARG
545
8.794
59.266
−9.283
1.00
19.41
O


ATOM
386
CB
ARG
545
7.431
56.887
−8.875
1.00
16.99
C


ATOM
387
CG
ARG
545
8.327
56.142
−9.837
1.00
20.50
C


ATOM
388
CD
ARG
545
7.453
55.4 93
10.868
1.00
26.46
C


ATOM
389
NE
ARG
545
8.195
54.796
−11.903
1.00
28.51
N


ATOM
390
CZ
ARG
545
7.644
53.877
−12.690
1.00
33.26
C


ATOM
391
NH1
ARG
545
6.357
53.572
−12.541
1.00
31.05
N


ATOM
392
NH2
ARG
545
8.368
53.259
−13.617
1.00
30.37
N


ATOM
393
N
ILE
546
10.205
58.879
−7.556
1.00
16.27
N


ATOM
394
CA
ILE
546
11.177
59.909
−7.919
1.00
18.03
C


ATOM
395
C
ILE
546
12.039
59.480
−9.084
1.00
19.30
C


ATOM
396
O
ILE
546
12.750
58.477
−9.014
1.00
20.66
O


ATOM
397
CB
ILE
546
12.065
60.290
−6.714
1.00
13.91
C


ATOM
398
CG1
ILE
546
11.234
60.956
−5.622
1.00
10.37
C


ATOM
399
CG2
ILE
546
13.134
61.243
−7.134
1.00
10.93
C


ATOM
400
CD1
ILE
546
11.981
61.088
4.306
1.00
8.23
C


ATOM
401
N
SER
547
11.986
60.270
−10.152
1.00
20.13
N


ATOM
402
CA
SER
547
12.725
59.968
−11.360
1.00
16.38
C


ATOM
403
C
SER
547
12.518
61.108
−12.343
1.00
20.11
C


ATOM
404
O
SER
547
11.443
61.710
−12.407
1.00
19.86
O


ATOM
405
CB
SER
547
12.191
58.664
−11.956
1.00
19.67
C


ATOM
406
OG
SER
547
12.939
58.238
−13.078
1.00
24.56
O


ATOM
407
N
GLN
548
13.542
61.364
−13.156
1.00
21.04
N


ATOM
408
CA
GLN
548
13.527
62.426
−14.159
1.00
22.05
C


ATOM
409
C
GLN
548
12.335
62.253
−15.121
1.00
23.36
C


ATOM
410
O
GLN
548
11.802
63.236
−15.645
1.00
26.68
O


ATOM
411
CB
GLN
548
14.869
62.399
−14.905
1.00
23.09
C


ATOM
412
CG
GLN
548
15.601
63.727
−15.086
1.00
25.87
C


ATOM
413
CD
GLN
548
15.738
64.560
−13.825
1.00
27.98
C


ATOM
414
OE1
GLN
548
15.630
65.783
−13.890
1.00
34.12
O


ATOM
415
NE2
GLN
548
15.977
63.925
−12.684
1.00
25.55
N


ATOM
416
N
LYS
549
11.894
61.009
−15.304
1.00
21.82
N


ATOM
417
CA
LYS
549
10.766
60.701
−16.182
1.00
21.44
C


ATOM
418
C
LYS
549
9.453
60.554
−15.390
1.00
22.62
C


ATOM
419
O
LYS
549
8.374
60.330
−15.971
1.00
18.28
O


ATOM
420
CB
LYS
549
11.041
59.406
−16.955
1.00
24.39
C


ATOM
421
CG
LYS
549
12.431
59.320
−17.575
1.00
34.15
C


ATOM
422
CD
LYS
549
12.762
60.573
−18.390
1.00
44.91
C


ATOM
423
CE
LYS
549
14.154
60.513
−19.044
1.00
51.62
C


ATOM
424
NZ
LYS
549
15.302
60.529
−18.076
1.00
53.07
N


ATOM
425
N
TRP
550
9.535
60.665
−14.064
1.00
19.15
N


ATOM
426
CA
TRP
550
8.350
60.544
−13.241
1.00
18.15
C


ATOM
427
C
TRP
550
8.178
61.740
−12.313
1.00
17.14
C


ATOM
428
O
TRP
550
7.755
62.808
−12.759
1.00
17.54
O


ATOM
429
CB
TRP
550
8.383
59.219
−12.473
1.00
22.31
C


ATOM
430
CG
TRP
550
8.360
58.034
−13.381
1.00
24.07
C


ATOM
431
CD1
TRP
550
9.420
57.250
−13.752
1.00
25.74
C


ATOM
432
CD2
TRP
550
7.228
57.533
−14.096
1.00
23.63
C


ATOM
433
NE1
TRP
550
9.016
56.302
−14.667
1.00
25.59
N


ATOM
434
CE2
TRP
550
7.665
56.461
−14.894
1.00
25.02
C


ATOM
435
CE3
TRP
550
5.875
57.905
−14.136
1.00
23.56
C


ATOM
436
CZ2
TRP
550
6.807
55.743
−15.734
1.00
25.72
C


ATOM
437
CZ3
TRP
550
5.017
57.196
−14.970
1.00
25.40
C


ATOM
438
CH2
TRP
550
5.487
56.125
−15.758
1.00
24.34
C


ATOM
439
N
VAL
551
8.465
61.572
−11.022
1.00
18.80
N


ATOM
440
CA
VAL
551
8.321
62.674
−10.069
1.00
16.63
C


ATOM
441
C
VAL
551
9.695
63.240
−9.739
1.00
14.99
C


ATOM
442
O
VAL
551
10.599
62.501
−9.362
1.00
15.74
O


ATOM
443
CB
VAL
551
7.614
62.214
−8.731
1.00
16.18
C


ATOM
444
CG1
VAL
551
7.477
63.406
−7.761
1.00
10.00
C


ATOM
445
CG2
VAL
551
6.231
61.611
−9.016
1.00
12.35
C


ATOM
446
N
ARG
552
9.876
64.536
−9.950
1.00
14.51
N


ATOM
447
CA
ARG
552
11.142
65.180
−9.612
1.00
14.95
C


ATOM
448
C
ARG
552
10.860
66.010
−8.358
1.00
14.50
C


ATOM
449
O
ARG
552
9.756
66.543
−8.188
1.00
12.82
O


ATOM
450
CB
ARG
552
11.639
66.066
−10.757
1.00
13.60
C


ATOM
451
CG
ARG
552
11.890
65.318
−12.052
1.00
14.38
C


ATOM
452
CD
ARG
552
12.686
66.146
−13.018
1.00
14.42
C


ATOM
453
NE
ARG
552
11.980
67.368
−13.388
1.00
22.75
N


ATOM
454
CZ
ARG
552
12.544
68.383
−14.035
1.00
19.53
C


ATOM
455
NH1
ARG
552
13.824
68.318
−14.381
1.00
24.67
N


ATOM
456
NH2
ARG
552
11.823
69.436
−14.383
1.00
12.20
N


ATOM
457
N
VAL
553
11.844
66.108
−7.469
1.00
18.50
N


ATOM
458
CA
VAL
553
11.654
66.840
−6.209
1.00
19.75
C


ATOM
459
C
VAL
553
12.740
67.861
−5.972
1.00
19.66
C


ATOM
460
O
VAL
553
13.872
67.695
−6.406
1.00
22.27
O


ATOM
461
CB
VAL
553
11.677
65.886
−4.956
1.00
16.81
C


ATOM
462
CG1
VAL
553
10.441
64.952
−4.920
1.00
7.54
C


ATOM
463
CG2
VAL
553
12.966
65.070
4.942
1.00
19.00
C


ATOM
464
N
ALA
554
12.385
68.930
−5.279
1.00
17.64
N


ATOM
465
CA
ALA
554
13.356
69.958
4.924
1.00
16.45
C


ATOM
466
C
ALA
554
13.038
70.208
−3.450
1.00
14.99
C


ATOM
467
O
ALA
554
11.882
70.006
−3.030
1.00
12.08
O


ATOM
468
CB
ALA
554
13.153
71.224
−5.764
1.00
6.96
C


ATOM
469
N
VAL
555
14.063
70.516
−2.651
1.00
13.54
N


ATOM
470
CA
VAL
555
13.879
70.778
−1.220
1.00
14.53
C


ATOM
471
C
VAL
555
14.590
72.068
−0.892
1.00
11.41
C


ATOM
472
O
VAL
555
15.769
72.191
−1.175
1.00
10.57
O


ATOM
473
CB
VAL
555
14.402
69.594
−0.318
1.00
14.50
C


ATOM
474
CG1
VAL
555
14.355
69.980
1.182
1.00
10.37
C


ATOM
475
CG2
VAL
555
13.491
68.358
−0.515
1.00
11.88
C


ATOM
476
N
VAL
556
13.857
73.044
−0.359
1.00
11.91
N


ATOM
477
CA
VAL
556
14.424
74.337
−0.026
1.00
14.48
C


ATOM
478
C
VAL
556
13.996
74.729
1.394
1.00
18.03
C


ATOM
479
O
VAL
556
12.807
74.771
1.717
1.00
18.24
O


ATOM
480
CB
VAL
556
13.929
75.435
−0.999
1.00
16.63
C


ATOM
481
CG1
VAL
556
14.721
76.700
−0.812
1.00
12.12
C


ATOM
482
CG2
VAL
556
14.022
74.948
−2.450
1.00
17.97
C


ATOM
483
N
GLU
557
14.977
75.023
2.228
1.00
15.76
N


ATOM
484
CA
GLU
557
14.750
75.431
3.594
1.00
16.18
C


ATOM
485
C
GLU
557
14.810
76.937
3.456
1.00
19.44
C


ATOM
486
O
GLU
557
15.751
77.448
2.847
1.00
20.78
O


ATOM
487
CB
GLU
557
15.922
74.921
4.451
1.00
21.73
C


ATOM
488
CG
GLU
557
16.042
75.479
5.890
1.00
22.98
C


ATOM
489
CD
GLU
557
17.093
76.581
6.011
1.00
26.66
C


ATOM
490
OE1
GLU
557
18.322
76.271
6.047
1.00
27.04
O


ATOM
491
OE2
GLU
557
16.666
77.755
6.075
1.00
18.13
O


ATOM
492
N
TYR
558
13.823
77.668
3.964
1.00
16.82
N


ATOM
493
CA
TYR
558
13.878
79.117
3.826
1.00
19.30
C


ATOM
494
C
TYR
558
13.894
79.898
5.123
1.00
21.56
C


ATOM
495
O
TYR
558
13.367
79.450
6.146
1.00
18.80
O


ATOM
496
CB
TYR
558
12.769
79.638
2.903
1.00
18.20
C


ATOM
497
CG
TYR
558
11.350
79.457
3.414
1.00
21.51
C


ATOM
498
CD1
TYR
558
10.681
78.250
3.251
1.00
19.46
C


ATOM
499
CD2
TYR
558
10.662
80.511
4.037
1.00
17.80
C


ATOM
500
CE1
TYR
558
9.360
78.101
3.689
1.00
17.32
C


ATOM
501
CE2
TYR
558
9.361
80.361
4.462
1.00
11.71
C


ATOM
502
CZ
TYR
558
8.727
79.158
4.289
1.00
14.15
C


ATOM
503
OH
TYR
558
7.443
78.997
4.727
1.00
21.33
O


ATOM
504
N
HIS
559
14.548
81.054
5.077
1.00
22.82
N


ATOM
505
CA
HIS
559
14.659
81.942
6.228
1.00
22.57
C


ATOM
506
C
HIS
559
14.761
83.343
5.632
1.00
24.28
C


ATOM
507
O
HIS
559
13.803
83.820
5.035
1.00
24.51
O


ATOM
508
CB
HIS
559
15.877
81.580
7.084
1.00
23.05
C


ATOM
509
CG
HIS
559
17.138
81.409
6.295
1.00
31.12
C


ATOM
510
ND1
HIS
559
17.327
80.384
5.400
1.00
35.63
N


ATOM
511
CD2
HIS
559
18.274
82.154
6.254
1.00
35.29
C


ATOM
512
CE1
HIS
559
18.512
80.499
4.841
1.00
34.53
C


ATOM
513
NE2
HIS
559
19.109
81.564
5.342
1.00
33.84
N


ATOM
514
N
ASP
560
15.895
84.013
5.788
1.00
29.85
N


ATOM
515
CA
ASP
560
16.077
85.336
5.194
1.00
37.48
C


ATOM
516
C
ASP
560
16.114
85.132
3.676
1.00
36.28
C


ATOM
517
O
ASP
560
15.522
85.892
2.912
1.00
39.28
O


ATOM
518
CB
ASP
560
17.391
85.957
5.686
1.00
46.15
C


ATOM
519
CG
ASP
560
17.242
87.430
6.059
1.00
55.52
C


ATOM
520
OD1
ASP
560
16.086
87.905
6.195
1.00
60.53
O


ATOM
521
OD2
ASP
560
18.286
88.112
6.218
1.00
59.22
O


ATOM
522
N
GLY
561
16.849
84.101
3.271
1.00
34.51
N


ATOM
523
CA
GLY
561
16.973
83.729
1.879
1.00
29.45
C


ATOM
524
C
GLY
561
16.599
82.261
1.769
1.00
28.57
C


ATOM
525
O
GLY
561
15.991
81.698
2.689
1.00
25.19
O


ATOM
526
N
SER
562
17.044
81.614
0.700
1.00
27.35
N


ATOM
527
CA
SER
562
16.730
80.216
0.502
1.00
26.74
C


ATOM
528
C
SER
562
17.984
79.376
0.408
1.00
28.35
C


ATOM
529
O
SER
562
18.981
79.803
−0.164
1.00
29.74
O


ATOM
530
CB
SER
562
15.903
80.028
−0.770
1.00
25.65
C


ATOM
531
OG
SER
562
14.733
80.834
−0.746
1.00
25.69
O


ATOM
532
N
HIS
563
17.952
78.221
1.058
1.00
26.61
N


ATOM
533
CA
HIS
563
19.052
77.273
1.016
1.00
28.05
C


ATOM
534
C
HIS
563
18.452
76.026
0.354
1.00
25.45
C


ATOM
535
O
HIS
563
17.662
75.317
0.977
1.00
26.54
O


ATOM
536
CB
HIS
563
19.565
76.923
2.425
1.00
33.01
C


ATOM
537
CG
HIS
563
20.592
77.879
2.971
1.00
42.56
C


ATOM
538
ND1
HIS
563
21.368
77.586
4.081
1.00
41.44
N


ATOM
539
CD2
HIS
563
20.950
79.129
2.588
1.00
41.45
C


ATOM
540
CE1
HIS
563
22.148
78.615
4.351
1.00
39.61
C


ATOM
541
NE2
HIS
563
21.919
79.566
3.466
1.00
38.40
N


ATOM
542
N
ALA
564
18.736
75.824
−0.929
1.00
22.09
N


ATOM
543
CA
ALA
564
18.220
74.671
−1.653
1.00
20.28
C


ATOM
544
C
ALA
564
19.183
73.513
−1.505
1.00
18.53
C


ATOM
545
O
ALA
564
20.359
73.650
−1.785
1.00
21.05
O


ATOM
546
CB
ALA
564
18.040
75.005
−3.108
1.00
16.92
C


ATOM
547
N
TYR
565
18.675
72.384
−1.028
1.00
18.56
N


ATOM
548
CA
TYR
565
19.463
71.183
−0.840
1.00
15.69
C


ATOM
549
C
TYR
565
19.236
70.245
−2.021
1.00
16.43
C


ATOM
550
O
TYR
565
20.096
69.461
−2.376
1.00
14.38
O


ATOM
551
CB
TYR
565
19.035
70.473
0.434
1.00
18.28
C


ATOM
552
CG
TYR
565
19.373
71.214
1.691
1.00
20.29
C


ATOM
553
CD1
TYR
565
18.609
72.296
2.105
1.00
19.07
C


ATOM
554
CD2
TYR
565
20.450
70.816
2.485
1.00
24.63
C


ATOM
555
CE1
TYR
565
18.899
72.966
3.277
1.00
23.97
C


ATOM
556
CE2
TYR
565
20.752
71.476
3.670
1.00
25.91
C


ATOM
557
CZ
TYR
565
19.971
72.552
4.061
1.00
24.70
C


ATOM
558
OH
TYR
565
20.247
73.212
5.240
1.00
28.07
O


ATOM
559
N
ILE
566
18.050
70.302
−2.599
1.00
16.43
N


ATOM
560
CA
ILE
566
17.720
69.445
−3.721
1.00
16.83
C


ATOM
561
C
ILE
566
17.121
70.236
−4.876
1.00
14.70
C


ATOM
562
O
ILE
566
16.222
71.044
−4.662
1.00
16.54
O


ATOM
563
CB
ILE
566
16.690
68.368
−3.301
1.00
15.97
C


ATOM
564
CG1
ILE
566
17.187
67.570
−2.097
1.00
18.44
C


ATOM
565
CG2
ILE
566
16.392
67.432
−4.446
1.00
16.90
C


ATOM
566
CD1
ILE
566
18.416
66.752
−2.370
1.00
28.29
C


ATOM
567
N
GLY
567
17.667
70.052
−6.075
1.00
14.70
N


ATOM
568
CA
GLY
567
17.123
70.694
−7.261
1.00
14.69
C


ATOM
569
C
GLY
567
16.372
69.660
−8.106
1.00
15.85
C


ATOM
570
O
GLY
567
16.730
68.469
−8.109
1.00
16.56
O


ATOM
571
N
LEU
568
15.392
70.097
−8.892
1.00
17.74
N


ATOM
572
CA
LEU
568
14.622
69.174
−9.722
1.00
16.77
C


ATOM
573
C
LEU
568
15.471
68.292
−10.628
1.00
18.54
C


ATOM
574
O
LEU
568
15.177
67.117
−10.807
1.00
24.33
O


ATOM
575
CB
LEU
568
13.591
69.935
−10.559
1.00
11.30
C


ATOM
576
CG
LEU
568
12.456
70.603
−9.767
1.00
11.42
C


ATOM
577
CD1
LEU
568
11.744
71.638
−10.640
1.00
6.22
C


ATOM
578
CD2
LEU
568
11.508
69.583
−9.227
1.00
3.91
C


ATOM
579
N
LYS
569
16.512
68.864
−11.212
1.00
23.19
N


ATOM
580
CA
LYS
569
17.393
68.123
−12.124
1.00
23.15
C


ATOM
581
C
LYS
569
18.459
67.248
−11.458
1.00
23.97
C


ATOM
582
O
LYS
569
19.272
66.623
−12.149
1.00
21.55
O


ATOM
583
CB
LYS
569
18.087
69.098
−13.067
1.00
23.83
C


ATOM
584
CG
LYS
569
17.154
69.828
−14.027
1.00
29.42
C


ATOM
585
CD
LYS
569
17.882
71.015
−14.638
1.00
32.89
C


ATOM
586
CE
LYS
569
16.994
71.782
−15.588
1.00
36.32
C


ATOM
587
NZ
LYS
569
17.497
71.604
−16.981
1.00
42.82
N


ATOM
588
N
ASP
570
18.477
67.207
−10.128
1.00
24.14
N


ATOM
589
CA
ASP
570
19.476
66.409
−9.435
1.00
26.34
C


ATOM
590
C
ASP
570
19.300
64.950
−9.749
1.00
31.25
C


ATOM
591
O
ASP
570
18.335
64.336
−9.314
1.00
34.28
O


ATOM
592
CB
ASP
570
19.389
66.624
−7.930
1.00
23.64
C


ATOM
593
CG
ASP
570
20.081
67.890
−7.493
1.00
24.59
C


ATOM
594
OD1
ASP
570
20.678
68.575
−8.352
1.00
27.05
O


ATOM
595
OD2
ASP
570
20.041
68.200
−6.289
1.00
24.49
O


ATOM
596
N
ARG
571
20.223
64.390
−10.523
1.00
34.80
N


ATOM
597
CA
ARG
571
20.126
62.983
−10.869
1.00
35.48
C


ATOM
598
C
ARG
571
20.829
62.155
−9.817
1.00
30.97
C


ATOM
599
O
ARG
571
22.025
61.861
−9.922
1.00
29.82
O


ATOM
600
CB
ARG
571
20.710
62.721
−12.246
1.00
46.16
C


ATOM
601
CG
ARG
571
20.453
61.315
−12.743
1.00
60.41
C


ATOM
602
CD
ARG
571
21.501
60.929
−13.762
1.00
70.06
C


ATOM
603
NE
ARG
571
21.379
59.540
−14.198
1.00
76.92
N


ATOM
604
CZ
ARG
571
22.061
59.032
−15.218
1.00
82.89
C


ATOM
605
NH1
ARG
571
22.910
59.802
−15.892
1.00
86.30
N


ATOM
606
NH2
ARG
571
21.877
57.770
−15.579
1.00
85.49
N


ATOM
607
N
LYS
572
20.093
61.876
−8.750
1.00
23.93
N


ATOM
608
CA
LYS
572
20.588
61.086
−7.634
1.00
20.81
C


ATOM
609
C
LYS
572
19.511
60.067
−7.365
1.00
16.06
C


ATOM
610
O
LYS
572
18.381
60.237
−7.789
1.00
17.31
O


ATOM
611
CB
LYS
572
20.843
61.966
−6.393
1.00
18.06
C


ATOM
612
CG
LYS
572
22.004
62.929
−6.578
1.00
20.31
C


ATOM
613
CD
LYS
572
22.138
63.949
−5.468
1.00
23.14
C


ATOM
614
CE
LYS
572
23.202
64.988
−5.836
1.00
24.18
C


ATOM
615
NZ
LYS
572
23.545
65.977
−4.755
1.00
20.57
N


ATOM
616
N
ARG
573
19.857
58.956
−6.750
1.00
20.74
N


ATOM
617
CA
ARG
573
18.834
57.949
−6.475
1.00
22.56
C


ATOM
618
C
ARG
573
17.883
58.457
−5.379
1.00
20.74
C


ATOM
619
O
ARG
573
18.280
59.311
−4.565
1.00
23.29
O


ATOM
620
CB
ARG
573
19.498
56.617
−6.138
1.00
27.37
C


ATOM
621
CG
ARG
573
20.682
56.741
−5.228
1.00
35.56
C


ATOM
622
CD
ARG
573
21.369
55.399
−5.040
1.00
44.36
C


ATOM
623
NE
ARG
573
22.340
55.095
−6.087
1.00
49.02
N


ATOM
624
CZ
ARG
573
22.261
54.052
−6.916
1.00
54.28
C


ATOM
625
NH1
ARG
573
21.240
53.191
−6.847
1.00
56.03
N


ATOM
626
NH2
ARG
573
23.226
53.851
−7.805
1.00
52.89
N


ATOM
627
N
PRO
574
16.610
57.997
−5.375
1.00
17.46
N


ATOM
628
CA
PRO
574
15.610
58.412
−4.392
1.00
12.01
C


ATOM
629
C
PRO
574
16.092
58.327
−2.953
1.00
14.44
C


ATOM
630
O
PRO
574
15.816
59.220
−2.163
1.00
17.36
O


ATOM
631
CB
PRO
574
14.472
57.442
−4.650
1.00
16.28
C


ATOM
632
CG
PRO
574
14.547
57.219
−6.088
1.00
11.38
C


ATOM
633
CD
PRO
574
16.024
57.OD2
−6.291
1.00
18.26
C


ATOM
634
N
SER
575
16.880
57.305
−2.639
1.00
13.89
N


ATOM
635
CA
SER
575
17.394
57.104
−1.289
1.00
15.54
C


ATOM
636
C
SER
575
18.385
58.170
−0.880
1.00
18.01
C


ATOM
637
O
SER
575
18.394
58.619
0.269
1.00
19.91
O


ATOM
638
CB
SER
575
18.052
55.742
−1.163
1.00
11.08
C


ATOM
639
OG
SER
575
19.038
55.601
−2.157
1.00
15.54
O


ATOM
640
N
GLU
576
19.221
58.599
−1.810
1.00
19.82
N


ATOM
641
CA
GLU
576
20.180
59.616
−1.445
1.00
19.22
C


ATOM
642
C
GLU
576
19.456
60.923
−1.244
1.00
19.69
C


ATOM
643
O
GLU
576
19.754
61.678
−0.322
1.00
21.30
O


ATOM
644
CB
GLU
576
21.287
59.774
−2.483
1.00
24.50
C


ATOM
645
CG
GLU
576
22.414
60.647
−1.930
1.00
34.07
C


ATOM
646
CD
GLU
576
23.375
61.108
−2.979
1.00
44.16
C


ATOM
647
OE1
GLU
576
23.681
60.305
3.893
1.00
48.95
O


ATOM
648
OE2
GLU
576
23.816
62.283
−2.891
1.00
50.40
O


ATOM
649
N
LEU
577
18.495
61.191
−2.106
1.00
18.39
N


ATOM
650
CA
LEU
577
17.734
62.419
−1.993
1.00
18.06
C


ATOM
651
C
LEU
577
17.042
62.469
−0.640
1.00
15.66
C


ATOM
652
O
LEU
577
17.081
63.482
0.056
1.00
16.14
O


ATOM
653
CB
LEU
577
16.715
62.518
−3.141
1.00
14.46
C


ATOM
654
CG
LEU
577
17.362
62.681
−4.522
1.00
13.01
C


ATOM
655
CD1
LEU
577
16.309
62.686
−5.592
1.00
12.16
C


ATOM
656
CD2
LEU
577
18.182
63.963
−4.564
1.00
10.44
C


ATOM
657
N
ARG
578
16.431
61.356
−0.258
1.00
18.03
N


ATOM
658
CA
ARG
578
15.721
61.282
1.013
1.00
16.42
C


ATOM
659
C
ARG
578
16.656
61.533
2.177
1.00
18.59
C


ATOM
660
O
ARG
578
16.276
62.207
3.136
1.00
23.30
O


ATOM
661
CB
ARG
578
15.005
59.939
1.169
1.00
14.49
C


ATOM
662
CG
ARG
578
13.822
59.783
0.220
1.00
9.10
C


ATOM
663
CD
ARG
578
12.924
58.631
0.583
1.00
7.18
C


ATOM
664
NE
ARG
578
13.668
57.391
0.758
1.00
13.10
N


ATOM
665
CZ
ARG
578
13.780
56.455
−0.164
1.00
11.87
C


ATOM
666
NH1
ARG
578
13.191
56.622
−1.340
1.00
9.76
N


ATOM
667
NH2
ARG
578
14.501
55.368
0.083
1.00
14.50
N


ATOM
668
N
ARG
579
17.889
61.046
2.070
1.00
17.36
N


ATOM
669
CA
ARG
579
18.879
61.248
3.119
1.00
17.76
C


ATOM
670
C
ARG
579
19.246
62.719
3.237
1.00
18.99
C


ATOM
671
O
ARG
579
19.372
63.252
4.341
1.00
21.54
O


ATOM
672
CB
ARG
579
20.132
60.414
2.833
1.00
18.77
C


ATOM
673
CG
ARG
579
21.347
60.787
3.684
1.00
24.17
C


ATOM
674
CD
ARG
579
22.532
59.857
3.461
1.00
26.24
C


ATOM
675
NE
ARG
579
22.856
59.678
2.045
1.00
36.84
N


ATOM
676
CZ
ARG
579
22.428
58.654
1.296
1.00
39.67
C


ATOM
677
NH1
ARG
579
21.640
57.703
1.800
1.00
37.99
N


ATOM
678
NH2
ARG
579
22.861
58.521
0.052
1.00
40.49
N


ATOM
679
N
ILE
580
19.433
63.377
2.101
1.00
18.54
N


ATOM
680
CA
ILE
580
19.784
64.790
2.098
1.00
14.36
C


ATOM
681
C
ILE
580
18.681
65.556
2.800
1.00
14.39
C


ATOM
682
O
ILE
580
18.956
66.396
3.643
1.00
15.74
O


ATOM
683
CB
ILE
580
19.969
65.315
0.673
1.00
10.89
C


ATOM
684
CG1
ILE
580
21.209
64.682
0.067
1.00
16.10
C


ATOM
685
CG2
ILE
580
20.087
66.835
0.663
1.00
13.34
C


ATOM
686
CD1
ILE
580
21.457
65.030
−1.408
1.00
15.50
C


ATOM
687
N
ALA
581
17.432
65.208
2.508
1.00
16.77
N


ATOM
688
CA
ALA
581
16.294
65.881
3.121
1.00
16.31
C


ATOM
689
C
ALA
581
16.252
65.723
4.646
1.00
17.47
C


ATOM
690
O
ALA
581
15.942
66.670
5.350
1.00
18.94
O


ATOM
691
CB
ALA
581
14.991
65.386
2.487
1.00
12.38
C


ATOM
692
N
SER
582
16.559
64.539
5.167
1.00
21.47
N


ATOM
693
CA
SER
582
16.516
64.344
6.617
1.00
22.46
C


ATOM
694
C
SER
582
17.700
64.984
7.295
1.00
23.44
C


ATOM
695
O
SER
582
17.673
65.229
8.497
1.00
29.23
O


ATOM
696
CB
SER
582
16.469
62.867
6.974
1.00
21.25
C


ATOM
697
OG
SER
582
17.659
62.228
6.577
1.00
25.04
O


ATOM
698
N
GLN
583
18.728
65.293
6.522
1.00
22.77
N


ATOM
699
CA
GLN
583
19.919
65.910
7.077
1.00
23.36
C


ATOM
700
C
GLN
583
19.874
67.425
6.981
1.00
21.29
C


ATOM
701
O
GLN
583
20.812
68.117
7.416
1.00
20.02
O


ATOM
702
CB
GLN
583
21.177
65.369
6.398
1.00
30.71
C


ATOM
703
CG
GLN
583
21.524
63.939
6.797
1.00
42.85
C


ATOM
704
CD
GLN
583
22.843
63.455
6.192
1.00
55.80
C


ATOM
705
OE1
GLN
583
23.589
62.715
6.839
1.00
62.99
O


ATOM
706
NE2
GLN
583
23.141
63.873
4.950
1.00
59.38
N


ATOM
707
N
VAL
584
18.804
67.940
6.389
1.00
15.51
N


ATOM
708
CA
VAL
584
18.622
69.371
6.255
1.00
21.88
C


ATOM
709
C
VAL
584
18.891
70.018
7.615
1.00
26.42
C


ATOM
710
O
VAL
584
18.367
69.585
8.643
1.00
26.77
O


ATOM
711
CB
VAL
584
17.194
69.706
5.768
1.00
25.45
C


ATOM
712
CG1
VAL
584
16.944
71.199
5.861
1.00
30.17
C


ATOM
713
CG2
VAL
584
17.013
69.269
4.333
1.00
27.33
C


ATOM
714
N
LYS
585
19.690
71.073
7.597
1.00
30.58
N


ATOM
715
CA
LYS
585
20.108
71.788
8.798
1.00
33.89
C


ATOM
716
C
LYS
585
19.091
72.734
9.417
1.00
32.68
C


ATOM
717
O
LYS
585
18.570
73.641
8.747
1.00
33.70
O


ATOM
718
CB
LYS
585
21.398
72.551
8.497
1.00
41.79
C


ATOM
719
CG
LYS
585
22.643
71.977
9.165
1.00
52.09
C


ATOM
720
CD
LYS
585
22.693
72.340
10.657
1.00
63.92
C


ATOM
721
CE
LYS
585
22.857
73.859
10.873
1.00
70.35
C


ATOM
722
NZ
LYS
585
22.735
74.271
12.316
1.00
74.91
N


ATOM
723
N
TYR
586
18.859
72.557
10.718
1.00
30.31
N


ATOM
724
CA
TYR
586
17.928
73.416
11.454
1.00
29.54
C


ATOM
725
C
TYR
586
18.583
74.776
11.564
1.00
29.75
C


ATOM
726
O
TYR
586
19.696
74.885
12.079
1.00
30.67
O


ATOM
727
CB
TYR
586
17.619
72.856
12.861
1.00
27.95
C


ATOM
728
CG
TYR
586
16.772
73.793
13.721
1.00
26.56
C


ATOM
729
CD1
TYR
586
15.389
73.906
13.522
1.00
24.59
C


ATOM
730
CD2
TYR
586
17.361
74.604
14.699
1.00
21.69
C


ATOM
731
CE1
TYR
586
14.616
74.806
14.265
1.00
20.84
C


ATOM
732
CE2
TYR
586
16.590
75.508
15.457
1.00
17.53
C


ATOM
733
CZ
TYR
586
15.222
75.601
15.233
1.00
23.50
C


ATOM
734
OH
TYR
586
14.461
76.482
15.978
1.00
21.84
O


ATOM
735
N
ALA
587
17.932
75.788
10.994
1.00
28.03
N


ATOM
736
CA
ALA
587
18.433
77.150
11.016
1.00
27.73
C


ATOM
737
C
ALA
587
17.945
77.932
12.220
1.00
27.05
C


ATOM
738
O
ALA
587
18.735
78.601
12.870
1.00
26.39
O


ATOM
739
CB
ALA
587
18.056
77.882
9.719
1.00
29.03
C


ATOM
740
N
GLY
588
16.643
77.880
12.490
1.00
25.16
N


ATOM
741
CA
GLY
588
16.093
78.606
13.619
1.00
24.35
C


ATOM
742
C
GLY
588
16.224
80.115
13.484
1.00
26.00
C


ATOM
743
O
GLY
588
16.582
80.806
14.429
1.00
27.67
O


ATOM
744
N
SER
589
15.891
80.626
12.309
1.00
25.60
N


ATOM
745
CA
SER
589
15.985
82.044
12.009
1.00
25.91
C


ATOM
746
C
SER
589
14.916
82.905
12.679
1.00
27.91
C


ATOM
747
O
SER
589
13.849
82.426
13.050
1.00
23.56
O


ATOM
748
CB
SER
589
15.879
82.228
10.495
1.00
26.02
C


ATOM
749
OG
SER
589
16.935
83.028
9.992
1.00
41.28
O


ATOM
750
N
GLN
590
15.212
84.193
12.811
1.00
32.43
N


ATOM
751
CA
GLN
590
14.256
85.130
13.373
1.00
37.48
C


ATOM
752
C
GLN
590
13.253
85.476
12.296
1.00
35.55
C


ATOM
753
O
GLN
590
12.160
85.937
12.588
1.00
38.88
O


ATOM
754
CB
GLN
590
14.955
86.415
13.821
1.00
39.80
C


ATOM
755
CG
GLN
590
15.288
86.435
15.295
1.00
56.15
C


ATOM
756
CD
GLN
590
16.155
87.622
15.686
1.00
65.66
C


ATOM
757
OE1
GLN
590
15.649
88.723
15.932
1.00
71.38
O


ATOM
758
NE2
GLN
590
17.473
87.403
15.742
1.00
67.58
N


ATOM
759
N
VAL
591
13.647
85.246
11.047
1.00
34.29
N


ATOM
760
CA
VAL
591
12.832
85.570
9.880
1.00
30.91
C


ATOM
761
C
VAL
591
12.827
84.471
8.809
1.00
26.09
C


ATOM
762
O
VAL
591
13.885
84.022
8.392
1.00
23.24
O


ATOM
763
CB
VAL
591
13.359
86.907
9.243
1.00
30.16
C


ATOM
764
CG1
VAL
591
13.067
86.983
7.758
1.00
33.78
C


ATOM
765
CG2
VAL
591
12.750
88.102
9.949
1.00
29.76
C


ATOM
766
N
ALA
592
11.636
84.000
8.437
1.00
24.70
N


ATOM
767
CA
ALA
592
11.462
83.007
7.372
1.00
20.42
C


ATOM
758
C
ALA
592
10.529
83.712
6.375
1.00
23.74
C


ATOM
769
O
ALA
592
9.320
83.852
6.629
1.00
19.55
O


ATOM
770
CB
ALA
592
10.829
81.750
7.893
1.00
14.37
C


ATOM
771
N
SER
593
11.104
84.209
5.277
1.00
22.94
N


ATOM
772
CA
SER
593
10.349
84.951
4.270
1.00
23.81
C


ATOM
773
C
SER
593
9.677
84.093
3.224
1.00
22.86
C


ATOM
774
O
SER
593
10.336
83.470
2.401
1.00
22.82
O


ATOM
775
CB
SER
593
11.232
85.974
3.569
1.00
23.57
C


ATOM
776
OG
SER
593
10.496
86.636
2.556
1.00
14.43
O


ATOM
777
N
THR
594
8.354
84.150
3.212
1.00
22.76
N


ATOM
778
CA
THR
594
7.568
83.380
2.263
1.00
22.25
C


ATOM
779
C
THR
594
7.608
84.069
0.898
1.00
21.75
C


ATOM
780
O
THR
594
7.593
83.411
−0.143
1.00
21.45
O


ATOM
781
CB
THR
594
6.122
83.217
2.768
1.00
19.99
C


ATOM
782
OG1
THR
594
5.593
84.505
3.106
1.00
18.50
O


ATOM
783
CG2
THR
594
6.110
82.375
4.021
1.00
15.10
C


ATOM
784
N
SER
595
7.663
85.400
0.901
1.00
21.20
N


ATOM
785
CA
SER
595
7.738
86.164
−0.334
1.00
18.60
C


ATOM
786
C
SER
595
9.032
85.850
−1.040
1.00
19.69
C


ATOM
787
O
SER
595
9.044
85.513
−2.228
1.00
22.57
O


ATOM
788
CB
SER
595
7.661
87.653
−0.037
1.00
11.10
C


ATOM
789
OG
SER
595
6.344
87.985
0.326
1.00
19.37
O


ATOM
790
N
GLU
596
10.122
85.922
−0.294
1.00
21.05
N


ATOM
791
CA
GLU
596
11.418
85.638
−0.863
1.00
23.66
C


ATOM
792
C
GLU
596
11.591
84.184
−1.299
1.00
22.27
C


ATOM
793
O
GLU
596
12.302
83.905
−2.260
1.00
24.61
O


ATOM
794
CB
GLU
596
12.543
86.151
0.043
1.00
30.29
C


ATOM
795
CG
GLU
596
13.058
87.575
−0.366
1.00
39.81
C


ATOM
796
CD
GLU
596
11.948
88.663
−0.449
1.00
45.46
C


ATOM
797
OE1
GLU
596
11.391
89.067
0.602
1.00
52.14
O


ATOM
798
OE2
GLU
596
11.648
89.142
−1.565
1.00
47.05
O


ATOM
799
N
VAL
597
10.886
83.257
−0.661
1.00
19.54
N


ATOM
800
CA
VAL
597
10.993
81.869
−1.079
1.00
14.34
C


ATOM
801
C
VAL
597
10.056
81.566
−2.257
1.00
14.34
C


ATOM
802
O
VAL
597
10.322
80.677
−3.047
1.00
15.50
O


ATOM
803
CB
VAL
597
10.768
80.893
0.077
1.00
10.95
C


ATOM
804
CG1
VAL
597
9.297
80.603
0.266
1.00
9.01
C


ATOM
805
CG2
VAL
597
11.555
79.624
−0.172
1.00
10.66
C


ATOM
806
N
LEU
598
8.951
82.287
−2.372
1.00
12.70
N


ATOM
807
CA
LEU
598
8.067
82.083
−3.502
1.00
12.15
C


ATOM
808
C
LEU
598
8.749
82.749
−4.699
1.00
17.31
C


ATOM
809
O
LEU
598
8.579
82.324
−5.840
1.00
18.32
O


ATOM
810
CB
LEU
598
6.700
82.699
−3.240
1.00
8.81
C


ATOM
811
CG
LEU
598
5.728
81.891
−2.363
1.00
9.60
C


ATOM
812
CD1
LEU
598
4.467
82.716
−2.154
1.00
9.06
C


ATOM
813
CD2
LEU
598
5.370
80.522
−2.975
1.00
7.06
C


ATOM
814
N
LYS
599
9.552
83.777
−4.432
1.00
18.09
N


ATOM
815
CA
LYS
599
10.288
84.442
−5.492
1.00
20.39
C


ATOM
816
C
LYS
599
11.328
83.475
−6.055
1.00
20.58
C


ATOM
817
O
LYS
599
11.552
83.432
−7.260
1.00
23.57
O


ATOM
818
CB
LYS
599
11.OD1
85.693
−4.982
1.00
21.30
C


ATOM
819
CG
LYS
599
11.836
86.366
−6.063
1.00
20.62
C


ATOM
820
CD
LYS
599
12.331
87.732
−5.633
1.00
28.26
C


ATOM
821
CE
LYS
599
13.674
87.677
−4.914
1.00
28.15
C


ATOM
822
NZ
LYS
599
14.463
88.927
−5.268
1.00
36.90
N


ATOM
823
N
TYR
600
11.972
82.712
−5.184
1.00
15.33
N


ATOM
824
CA
TYR
600
12.975
81.739
−5.605
1.00
14.72
C


ATOM
825
C
TYR
600
12.380
80.614
−6.470
1.00
15.26
C


ATOM
826
O
TYR
600
13.031
80.110
−7.394
1.00
14.23
O


ATOM
827
CB
TYR
600
13.659
81.159
−4.369
1.00
10.22
C


ATOM
828
CG
TYR
600
14.763
80.168
−4.658
1.00
13.36
C


ATOM
829
CD1
TYR
600
16.091
80.594
−4.820
1.00
9.42
C


ATOM
830
CD2
TYR
600
14.488
78.791
−4.745
1.00
9.40
C


ATOM
831
CE1
TYR
600
17.123
79.670
−5.066
1.00
8.00
C


ATOM
832
CE2
TYR
600
15.502
77.874
−4.987
1.00
11.12
C


ATOM
833
CZ
TYR
600
16.812
78.320
−5.154
1.00
13.46
C


ATOM
834
OH
TYR
600
17.796
77.412
−5.477
1.00
18.52
O


ATOM
835
N
THR
601
11.144
80.228
−6.167
1.00
15.74
N


ATOM
836
CA
THR
601
10.449
79.163
−6.887
1.00
16.26
C


ATOM
837
C
THR
601
10.062
79.658
−8.291
1.00
18.06
C


ATOM
838
O
THR
601
10.189
78.942
−9.297
1.00
20.51
O


ATOM
839
CB
THR
601
9.184
78.724
−6.074
1.00
13.62
C


ATOM
840
OG1
THR
601
9.583
78.361
−4.745
1.00
14.88
O


ATOM
841
CG2
THR
601
8.493
77.517
−6.690
1.00
8.81
C


ATOM
842
N
LEU
602
9.660
80.914
−8.358
1.00
18.80
N


ATOM
843
CA
LEU
602
9.239
81.517
−9.594
1.00
12.28
C


ATOM
844
C
LEU
602
10.394
81.808
−10.528
1.00
12.80
C


ATOM
845
O
LEU
602
10.365
81.445
−11.686
1.00
15.88
O


ATOM
846
CB
LEU
602
8.497
82.800
−9.259
1.00
7.41
C


ATOM
847
CG
LEU
602
7.971
83.645
−10.410
1.00
4.81
C


ATOM
848
CD1
LEU
602
7.137
82.788
−11.305
1.00
11.16
C


ATOM
849
CD2
LEU
602
7.163
84.783
−9.881
1.00
3.73
C


ATOM
850
N
PHE
603
11.409
82.496
−10.031
1.00
17.52
N


ATOM
851
CA
PHE
603
12.537
82.883
−10.862
1.00
18.59
C


ATOM
852
C
PHE
603
13.743
81.966
−10.844
1.00
21.46
C


ATOM
853
O
PHE
603
14.577
82.022
−11.746
1.00
21.41
O


ATOM
854
CB
PHE
603
12.954
84.322
−10.533
1.00
13.06
C


ATOM
855
CG
PHE
603
11.860
85.333
−10.758
1.00
13.57
C


ATOM
856
CD1
PHE
603
11.257
85.469
−12.006
1.00
13.30
C


ATOM
857
CD2
PHE
603
11.422
86.138
−9.722
1.00
11.61
C


ATOM
858
CE1
PHE
603
10.232
86.385
−12.212
1.00
8.39
C


ATOM
859
CE2
PHE
603
10.403
87.054
−9.916
1.00
14.44
C


ATOM
860
CZ
PHE
603
9.804
87.180
−11.160
1.00
14.78
C


ATOM
861
N
GLN
604
13.828
81.087
−9.856
1.00
24.28
N


ATOM
862
CA
GLN
604
14.983
80.205
−9.787
1.00
27.12
C


ATOM
863
C
GLN
604
14.687
78.739
−10.058
1.00
24.50
C


ATOM
864
O
GLN
604
15.512
78.040
−10.605
1.00
24.04
O


ATOM
865
CB
GLN
604
15.689
80.368
−8.440
1.00
31.32
C


ATOM
866
CG
GLN
604
16.522
81.621
−8.287
1.00
35.80
C


ATOM
867
CD
GLN
604
17.861
81.469
−8.960
1.00
45.50
C


ATOM
868
OE1
GLN
604
18.784
80.859
−8.418
1.00
49.26
O


ATOM
869
NE2
GLN
604
17.973
81.999
−10.166
1.00
54.22
N


ATOM
870
N
ILE
605
13.521
78.260
−9.660
1.00
23.49
N


ATOM
871
CA
ILE
605
13.196
76.859
−9.881
1.00
22.06
C


ATOM
872
C
ILE
605
12.418
76.627
−11.163
1.00
25.79
C


ATOM
873
O
ILE
605
12.635
75.633
−11.861
1.00
28.80
O


ATOM
874
CB
ILE
605
12.425
76.243
−8.678
1.00
18.12
C


ATOM
875
CG1
ILE
605
13.356
76.119
−7.470
1.00
16.09
C


ATOM
876
CG2
ILE
605
11.862
74.874
−9.035
1.00
15.46
C


ATOM
877
CD1
ILE
605
12.661
75.604
−6.225
1.00
12.12
C


ATOM
878
N
PHE
606
11.444
77.484
−11.431
1.00
21.94
N


ATOM
879
CA
PHE
606
10.670
77.333
−12.645
1.00
22.03
C


ATOM
880
C
PHE
606
10.903
78.556
−13.533
1.00
22.92
C


ATOM
881
O
PHE
606
9.965
79.130
−14.083
1.00
19.90
O


ATOM
882
CB
PHE
606
9.188
77.149
−12.310
1.00
19.38
C


ATOM
883
CG
PHE
606
8.899
75.943
−11.442
1.00
24.03
C


ATOM
884
CD1
PHE
606
8.941
74.656
−11.969
1.00
21.11
C


ATOM
885
CD2
PHE
606
8.498
76.107
−10.108
1.00
23.28
C


ATOM
886
CE1
PHE
606
8.582
73.560
−11.178
1.00
22.09
C


ATOM
887
CE2
PHE
606
8.141
75.025
−9.319
1.00
19.77
C


ATOM
888
CZ
PHE
606
8.175
73.749
−9.850
1.00
22.92
C


ATOM
889
N
SER
607
12.165
78.971
−13.641
1.00
27.16
N


ATOM
890
CA
SER
607
12.505
80.127
−14.456
1.00
34.01
C


ATOM
891
C
SER
607
11.927
79.863
−15.832
1.00
41.74
C


ATOM
892
O
SER
607
11.276
80.737
−16.421
1.00
49.40
O


ATOM
893
CB
SER
607
13.997
80.342
−14.543
1.00
29.88
C


ATOM
894
OG
SER
607
14.209
81.653
−15.023
1.00
31.36
O


ATOM
895
N
LYS
608
12.279
78.717
−16.404
1.00
43.35
N


ATOM
896
CA
LYS
608
11.647
78.316
−17.648
1.00
46.17
C


ATOM
897
C
LYS
608
11.480
76.817
−17.716
1.00
42.64
C


ATOM
898
O
LYS
608
12.365
76.029
−17.390
1.00
40.78
O


ATOM
899
CB
LYS
608
12.195
78.939
−18.935
1.00
54.48
C


ATOM
900
CG
LYS
608
11.035
79.221
−19.979
1.00
55.51
C


ATOM
901
CD
LYS
608
9.997
80.303
−19.504
1.00
54.84
C


ATOM
902
CE
LYS
608
8.628
79.732
−19.088
1.00
53.13
C


ATOM
903
NZ
LYS
608
7.777
79.171
−20.196
1.00
50.29
N


ATOM
904
N
ILE
609
10.244
76.475
−18.018
1.00
38.42
N


ATOM
905
CA
ILE
609
9.753
75.134
−18.077
1.00
32.87
C


ATOM
906
C
ILE
609
10.437
74.185
−19.038
1.00
30.65
C


ATOM
907
O
ILE
609
10.108
74.152
−20.208
1.00
34.42
O


ATOM
908
CB
ILE
609
8.235
75.198
−18.302
1.00
31.16
C


ATOM
909
CG1
ILE
609
7.626
76.217
−17.324
1.00
30.77
C


ATOM
910
CG2
ILE
609
7.595
73.836
−18.148
1.00
32.93
C


ATOM
911
CD1
ILE
609
8.177
76.152
−15.893
1.00
27.01
C


ATOM
912
N
ASP
610
11.382
73.407
−18.512
1.00
29.30
N


ATOM
913
CA
ASP
610
12.115
72.397
−19.278
1.00
26.65
C


ATOM
914
C
ASP
610
11.296
71.090
−19.348
1.00
24.52
C


ATOM
915
O
ASP
610
11.667
70.160
−20.061
1.00
22.38
O


ATOM
916
CB
ASP
610
13.472
72.100
−18.610
1.00
31.38
C


ATOM
917
CG
ASP
610
13.326
71.445
−17.208
1.00
40.16
C


ATOM
918
OD1
ASP
610
12.777
72.111
−16.296
1.00
44.05
O


ATOM
919
OD2
ASP
610
13.781
70.280
−17.006
1.00
37.91
O


ATOM
920
N
ARG
611
10.207
71.023
−18.586
1.00
20.53
N


ATOM
921
CA
ARG
611
9.338
69.847
−18.523
1.00
17.10
C


ATOM
922
C
ARG
611
7.882
70.318
−18.565
1.00
18.14
C


ATOM
923
O
ARG
611
7.147
70.151
−17.579
1.00
17.77
O


ATOM
924
CB
ARG
611
9.555
69.117
−17.188
1.00
15.35
C


ATOM
925
CG
ARG
611
10.090
67.715
−17.279
1.00
19.43
C


ATOM
926
CD
ARG
611
9.053
66.659
−16.878
1.00
17.95
C


ATOM
927
NE
ARG
611
9.225
66.220
−15.507
1.00
15.53
N


ATOM
928
CZ
ARG
611
8.787
65.070
−15.009
1.00
15.73
C


ATOM
929
NH1
ARG
611
8.143
64.187
−15.746
1.00
14.15
N


ATOM
930
NH2
ARG
611
8.950
64.830
−13.726
1.00
20.70
N


ATOM
931
N
PRO
612
7.410
70.799
−19.734
1.00
18.33
N


ATOM
932
CA
PRO
612
6.023
71.278
−19.846
1.00
18.14
C


ATOM
933
C
PRO
612
4.985
70.176
−19.746
1.00
16.54
C


ATOM
934
O
PRO
612
3.800
70.458
−19.694
1.00
18.83
O


ATOM
935
CB
PRO
612
6.003
71.928
−21.226
1.00
20.27
C


ATOM
936
CG
PRO
612
6.909
70.998
−22.015
1.00
21.20
C


ATOM
937
CD
PRO
612
8.066
70.757
−21.054
1.00
19.55
C


ATOM
938
N
GLU
613
5.439
68.926
−19.694
1.00
17.56
N


ATOM
939
CA
GLU
613
4.539
67.780
−19.590
1.00
18.04
C


ATOM
940
C
GLU
613
4.265
67.341
−18.137
1.00
20.09
C


ATOM
941
O
GLU
613
3.525
66.376
−17.895
1.00
19.26
O


ATOM
942
CB
GLU
613
5.088
66.593
−20.380
1.00
17.32
C


ATOM
943
CG
GLU
613
6.320
65.939
−19.764
1.00
16.50
C


ATOM
944
CD
GLU
613
7.622
66.575
−20.203
1.00
17.75
C


ATOM
945
OE1
GLU
613
7.609
67.682
−20.775
1.00
21.13
O


ATOM
946
OE2
GLU
613
8.680
65.949
−19.979
1.00
18.68
O


ATOM
947
N
ALA
614
4.896
67.997
−17.172
1.00
17.80
N


ATOM
948
CA
ALA
614
4.645
67.643
−15.782
1.00
17.35
C


ATOM
949
C
ALA
614
3.998
68.805
−15.068
1.00
17.82
C


ATOM
950
O
ALA
614
4.245
69.962
−15.422
1.00
18.03
O


ATOM
951
CB
ALA
614
5.929
67.302
−15.103
1.00
14.29
C


ATOM
952
N
SER
615
3.143
68.502
−14.088
1.00
14.90
N


ATOM
953
CA
SER
615
2.532
69.553
−13.297
1.00
13.08
C


ATOM
954
C
SER
615
3.622
70.036
−12.347
1.00
14.27
C


ATOM
955
O
SER
615
4.577
69.304
−12.056
1.00
17.25
O


ATOM
956
CB
SER
615
1.362
69.029
−12.491
1.00
10.01
C


ATOM
957
OG
SER
615
0.379
68.502
−13.348
1.00
13.32
O


ATOM
958
N
ARG
616
3.505
71.290
−11.929
1.00
13.79
N


ATOM
959
CA
ARG
616
4.441
71.923
−11.021
1.00
10.29
C


ATOM
950
C
ARG
616
3.696
72.218
−9.722
1.00
8.30
C


ATOM
961
O
ARG
616
2.638
72.841
−9.721
1.00
8.31
O


ATOM
962
CB
ARG
616
4.966
73.188
−11.671
1.00
10.60
C


ATOM
963
CG
ARG
616
5.580
72.883
−13.020
1.00
11.29
C


ATOM
964
CD
ARG
616
5.648
74.101
−13.902
1.00
18.79
C


ATOM
965
NE
ARG
616
5.380
73.740
−15.286
1.00
25.65
N


ATOM
956
CZ
ARG
616
4.217
73.955
−15.877
1.00
28.89
C


ATOM
967
NH1
ARG
616
3.238
74.543
−15.223
1.00
36.05
N


ATOM
968
NH2
ARG
616
3.977
73.463
−17.075
1.00
37.22
N


ATOM
959
N
ILE
617
4.187
71.673
−8.616
1.00
10.46
N


ATOM
970
CA
ILE
617
3.532
71.893
−7.341
1.00
8.09
C


ATOM
971
C
ILE
617
4.549
72.311
−6.289
1.00
9.44
C


ATOM
972
O
ILE
617
5.625
71.717
−6.176
1.00
13.23
O


ATOM
973
CB
ILE
617
2.790
70.616
−6.898
1.00
13.19
C


ATOM
974
CG1
ILE
617
1.747
70.212
−7.951
1.00
12.54
C


ATOM
975
CG2
ILE
617
2.102
70.832
−5.549
1.00
17.10
C


ATOM
976
CD1
ILE
617
0.818
69.049
−7.489
1.00
9.40
C


ATOM
977
N
ALA
618
4.241
73.396
−5.587
1.00
11.26
N


ATOM
978
CA
ALA
618
5.092
73.932
−4.523
1.00
9.88
C


ATOM
979
C
ALA
618
4.319
73.708
−3.221
1.00
13.13
C


ATOM
980
O
ALA
618
3.164
74.135
−3.084
1.00
13.87
O


ATOM
981
CB
ALA
618
5.332
75.403
−4.747
1.00
6.93
C


ATOM
982
N
LEU
619
4.920
72.964
−2.304
1.00
11.48
N


ATOM
983
CA
LEU
619
4.286
72.655
−1.029
1.00
14.03
C


ATOM
984
C
LEU
619
4.900
73.622
−0.022
1.00
12.75
C


ATOM
985
O
LEU
619
6.035
73.447
0.395
1.00
9.90
O


ATOM
986
CB
LEU
619
4.604
71.206
−0.675
1.00
14.36
C


ATOM
987
CG
LEU
619
3.800
70.416
0.353
1.00
19.68
C


ATOM
988
CD1
LEU
619
2.311
70.465
0.078
1.00
19.00
C


ATOM
989
CD2
LEU
619
4.319
68.989
0.289
1.00
14.02
C


ATOM
990
N
LEU
620
4.186
74.700
0.281
1.00
11.45
N


ATOM
991
CA
LEU
620
4.706
75.711
1.201
1.00
14.00
C


ATOM
992
C
LEU
620
4.397
75.384
2.663
1.00
13.84
C


ATOM
993
O
LEU
620
3.271
75.581
3.123
1.00
10.40
O


ATOM
994
CB
LEU
620
4.156
77.095
0.823
1.00
11.10
C


ATOM
995
CG
LEU
620
4.779
78.300
1.525
1.00
10.11
C


ATOM
996
CD1
LEU
620
6.276
78.366
1.250
1.00
10.19
C


ATOM
997
CD2
LEU
620
4.089
79.568
1.058
1.00
11.71
C


ATOM
998
N
LEU
621
5.397
74.880
3.380
1.00
12.67
N


ATOM
999
CA
LEU
621
5.226
74.528
4.785
1.00
15.42
C


ATOM
1000
C
LEU
621
5.536
75.770
5.639
1.00
15.14
C


ATOM
1OD1
O
LEU
621
6.697
76.117
5.882
1.00
17.05
O


ATOM
1OD2
CB
LEU
621
6.117
73.327
5.100
1.00
11.95
C


ATOM
1003
CG
LEU
621
5.761
72.185
4.115
1.00
11.75
C


ATOM
1004
CD1
LEU
621
6.957
71.338
3.727
1.00
7.16
C


ATOM
1005
CD2
LEU
621
4.678
71.351
4.702
1.00
7.13
C


ATOM
1006
N
MET
622
4.467
76.390
6.131
1.00
13.49
N


ATOM
1007
CA
MET
622
4.538
77.624
6.899
1.00
17.11
C


ATOM
1008
C
MET
622
4.273
77.505
8.396
1.00
17.35
C


ATOM
1009
O
MET
622
3.211
77.062
8.803
1.00
18.55
O


ATOM
1010
CB
MET
622
3.499
78.593
6.341
1.00
19.32
C


ATOM
1011
CG
MET
622
3.634
78.898
4.878
1.00
19.64
C


ATOM
1012
SD
MET
622
2.174
79.715
4.255
1.00
22.75
S


ATOM
1013
CE
MET
622
2.302
81.305
4.988
1.00
20.32
C


ATOM
1014
N
ALA
623
5.199
78.003
9.204
1.00
16.59
N


ATOM
1015
CA
ALA
623
5.057
78.000
10.654
1.00
14.97
C


ATOM
1016
C
ALA
623
5.115
79.425
11.201
1.00
18.32
C


ATOM
1017
O
ALA
623
4.952
79.648
12.405
1.00
21.62
O


ATOM
1018
CB
ALA
623
6.167
77.173
11.279
1.00
15.96
C


ATOM
1019
N
SER
624
5.300
80.397
10.323
1.00
19.46
N


ATOM
1020
CA
SER
624
5.433
81.772
10.771
1.00
21.55
C


ATOM
1021
C
SER
624
4.869
82.812
9.799
1.00
22.83
C


ATOM
1022
O
SER
624
4.108
82.483
8.855
1.00
18.76
O


ATOM
1023
CB
SER
624
6.921
82.065
11.026
1.00
18.76
C


ATOM
1024
OG
SER
624
7.721
81.753
9.896
1.00
22.42
O


ATOM
1025
N
GLN
625
5.152
84.075
10.122
1.00
22.23
N


ATOM
1026
CA
GLN
625
4.776
85.204
9.282
1.00
27.39
C


ATOM
1027
C
GLN
625
5.948
86.161
9.222
1.00
27.09
C


ATOM
1028
O
GLN
625
6.583
86.445
10.227
1.00
27.21
O


ATOM
1029
CB
GLN
625
3.498
85.908
9.742
1.00
27.20
C


ATOM
1030
CG
GLN
625
3.294
86.097
11.206
1.00
33.26
C


ATOM
1031
CD
GLN
625
1.881
86.577
11.507
1.00
32.60
C


ATOM
1032
OE1
GLN
625
1.168
85.979
12.305
1.00
41.09
O


ATOM
1033
NE2
GLN
625
1.471
87.647
10.854
1.00
32.88
N


ATOM
1034
N
GLU
626
6.326
86.519
8.004
1.00
28.00
N


ATOM
1035
CA
GLU
626
7.434
87.437
7.771
1.00
28.38
C


ATOM
1036
C
GLU
626
7.013
88.885
8.071
1.00
29.02
C


ATOM
1037
O
GLU
626
5.813
89.221
8.041
1.00
25.62
O


ATOM
1038
CB
GLU
626
7.852
87.355
6.304
1.00
25.11
C


ATOM
1039
CG
GLU
626
6.807
87.963
5.378
1.00
23.18
C


ATOM
1040
CD
GLU
626
7.062
87.693
3.915
1.00
24.81
C


ATOM
1041
OE1
GLU
626
8.171
87.208
3.580
1.00
20.13
O


ATOM
1042
OE2
GLU
626
6.129
87.946
3.116
1.00
17.26
O


ATOM
1043
N
PRO
627
7.995
89.762
8.367
1.00
29.70
N


ATOM
1044
CA
PRO
627
7.708
91.173
8.653
1.00
31.82
C


ATOM
1045
C
PRO
627
7.050
91.835
7.441
1.00
33.52
C


ATOM
1046
O
PRO
627
7.505
91.662
6.310
1.00
33.48
O


ATOM
1047
CB
PRO
627
9.096
91.759
8.947
1.00
28.98
C


ATOM
1048
CG
PRO
627
10.037
90.803
8.297
1.00
31.86
C


ATOM
1049
CD
PRO
627
9.420
89.477
8.587
1.00
29.78
C


ATOM
1050
N
GLN
628
6.000
92.609
7.702
1.00
36.73
N


ATOM
1051
CA
GLN
628
5.208
93.302
6.679
1.00
38.04
C


ATOM
1052
C
GLN
628
6.026
93.896
5.542
1.00
35.21
C


ATOM
1053
O
GLN
628
5.629
93.832
4.375
1.00
34.60
O


ATOM
1054
CB
GLN
628
4.353
94.406
7.332
1.00
44.33
C


ATOM
1055
CG
GLN
628
2.922
94.535
6.808
1.00
52.77
C


ATOM
1056
CD
GLN
628
1.885
94.095
7.834
1.00
60.03
C


ATOM
1057
OE1
GLN
628
1.070
94.899
8.302
1.00
61.34
O


ATOM
1058
NE2
GLN
628
1.923
92.814
8.206
1.00
63.65
N


ATOM
1059
N
ARG
629
7.180
94.448
5.884
1.00
32.79
N


ATOM
1060
CA
ARG
629
8.051
95.067
4.898
1.00
34.31
C


ATOM
1061
C
ARG
629
8.588
94.118
3.822
1.00
30.81
C


ATOM
1062
O
ARG
629
8.919
94.556
2.723
1.00
36.74
O


ATOM
1063
CB
ARG
629
9.209
95.784
5.592
1.00
38.79
C


ATOM
1064
CG
ARG
629
10.042
94.861
6.431
1.00
47.03
C


ATOM
1065
CD
ARG
629
11.399
95.422
6.687
1.00
49.94
C


ATOM
1066
NE
ARG
629
12.324
94.336
6.978
1.00
58.97
N


ATOM
1067
CZ
ARG
629
13.603
94.339
6.633
1.00
65.16
C


ATOM
1068
NH1
ARG
629
14.109
95.379
5.985
1.00
70.46
N


ATOM
1069
NH2
ARG
629
14.378
93.314
6.952
1.00
68.49
N


ATOM
1070
N
MET
630
8.655
92.828
4.110
1.00
25.21
N


ATOM
1071
CA
MET
630
9.171
91.879
3.134
1.00
23.72
C


ATOM
1072
C
MET
630
8.061
91.297
2.268
1.00
24.23
C


ATOM
1073
O
MET
630
8.346
90.631
1.270
1.00
25.88
O


ATOM
1074
CB
MET
630
9.905
90.735
3.828
1.00
25.55
C


ATOM
1075
CG
MET
630
11.078
91.153
4.687
1.00
28.67
C


ATOM
1076
SD
MET
630
11.844
89.733
5.470
1.00
30.08
S


ATOM
1077
CE
MET
630
13.015
89.228
4.233
1.00
33.64
C


ATOM
1078
N
SER
631
6.811
91.617
2.606
1.00
23.98
N


ATOM
1079
CA
SER
631
5.634
91.098
1.906
1.00
24.27
C


ATOM
1080
C
SER
631
5.076
91.919
0.751
1.00
26.26
C


ATOM
1081
O
SER
631
4.010
91.584
0.210
1.00
21.34
O


ATOM
1082
CB
SER
631
4.504
90.879
2.911
1.00
22.54
C


ATOM
1083
OG
SER
631
4.923
90.050
3.983
1.00
24.17
O


ATOM
1084
N
ARG
632
5.797
92.967
0.357
1.00
29.47
N


ATOM
1085
CA
ARG
632
5.348
93.865
−0.711
1.00
27.92
C


ATOM
1086
C
ARG
632
4.990
93.255
−2.075
1.00
24.00
C


ATOM
1087
O
ARG
632
4.013
93.653
−2.698
1.00
19.20
O


ATOM
1088
CB
ARG
632
6.350
95.003
−0.869
1.00
32.62
C


ATOM
1089
CG
ARG
632
6.093
96.167
0.064
1.00
38.66
C


ATOM
1090
CD
ARG
632
7.198
97.197
−0.041
1.00
48.62
C


ATOM
1091
NE
ARG
632
8.267
96.958
0.932
1.00
58.66
N


ATOM
1092
CZ
ARG
632
9.535
97.343
0.786
1.00
61.73
C


ATOM
1093
NH1
ARG
632
9.927
97.984
−0.317
1.00
62.36
N


ATOM
1094
NH2
ARG
632
10.396
97.139
1.779
1.00
60.51
N


ATOM
1095
N
ASN
633
5.754
92.267
−2.520
1.00
22.34
N


ATOM
1096
CA
ASN
633
5.501
91.630
−3.812
1.00
23.39
C


ATOM
1097
C
ASN
633
4.823
90.274
−3.673
1.00
22.81
C


ATOM
1098
O
ASN
633
4.613
89.592
−4.665
1.00
20.30
O


ATOM
1099
CB
ASN
633
6.815
91.430
−4.583
1.00
19.08
C


ATOM
1100
CG
ASN
633
7.352
92.708
−5.169
1.00
17.97
C


ATOM
1101
OD1
ASN
633
6.663
93.724
−5.197
1.00
12.49
O


ATOM
1102
NO2
ASN
633
8.602
92.667
−5.644
1.00
17.03
N


ATOM
1103
N
PHE
634
4.494
89.889
−2.445
1.00
22.28
N


ATOM
1104
CA
PHE
634
3.878
88.603
−2.182
1.00
19.74
C


ATOM
1105
C
PHE
634
2.821
88.172
−3.190
1.00
18.80
C


ATOM
1106
O
PHE
634
2.994
87.142
−3.849
1.00
19.49
O


ATOM
1107
CB
PHE
634
3.303
88.536
−0.756
1.00
14.84
C


ATOM
1108
CG
PHE
634
2.736
87.184
−0.414
1.00
11.94
C


ATOM
1109
CD1
PHE
634
3.583
86.149
−0.048
1.00
4.10
C


ATOM
1110
CD2
PHE
634
1.373
86.920
−0.576
1.00
11.23
C


ATOM
1111
CE1
PHE
634
3.095
84.880
0.136
1.00
5.83
C


ATOM
1112
CE2
PHE
634
0.858
85.645
−0.393
1.00
10.88
C


ATOM
1113
CZ
PHE
634
1.719
84.617
−0.040
1.00
9.89
C


ATOM
1114
N
VAL
635
1.751
88.942
−3.365
1.00
18.69
N


ATOM
1115
CA
VAL
635
0.707
88.514
−4.314
1.00
21.28
C


ATOM
1116
C
VAL
635
1.214
88.374
−5.751
1.00
21.28
C


ATOM
1117
O
VAL
635
0.803
87.478
−6.480
1.00
22.82
O


ATOM
1118
CB
VAL
635
−0.500
89.440
−4.280
1.00
20.51
C


ATOM
1119
CG1
VAL
635
−1.510
89.022
−5.316
1.00
25.23
C


ATOM
1120
CG2
VAL
635
−1.129
89.383
−2.922
1.00
23.14
C


ATOM
1121
N
ARG
636
2.160
89.227
−6.111
1.00
18.25
N


ATOM
1122
CA
ARG
636
2.778
89.239
−7.420
1.00
13.92
C


ATOM
1123
C
ARG
636
3.522
87.936
−7.677
1.00
15.53
C


ATOM
1124
O
ARG
636
3.419
87.366
−8.760
1.00
19.16
O


ATOM
1125
CB
ARG
636
3.719
90.446
−7.480
1.00
13.52
C


ATOM
1126
CG
ARG
636
2.923
91.760
−7.492
1.00
21.06
C


ATOM
1127
CD
ARG
636
3.742
92.986
−7.115
1.00
31.89
C


ATOM
1128
NE
ARG
636
3.059
94.231
−7.497
1.00
34.04
N


ATOM
1129
CZ
ARG
636
2.973
95.330
−6.747
1.00
33.28
C


ATOM
1130
NH1
ARG
636
3.523
95.373
−5.545
1.00
32.30
N


ATOM
1131
NH2
ARG
636
2.396
96.421
−7.233
1.00
35.94
N


ATOM
1132
N
TYR
637
4.249
87.439
−6.683
1.00
12.24
N


ATOM
1133
CA
TYR
637
4.969
86.187
−6.837
1.00
13.29
C


ATOM
1134
C
TYR
637
3.984
85.046
−6.970
1.00
15.41
C


ATOM
1135
O
TYR
637
4.176
84.141
−7.789
1.00
17.67
O


ATOM
1136
CB
TYR
637
5.912
85.942
−5.654
1.00
16.02
C


ATOM
1137
CG
TYR
637
7.003
86.987
−5.572
1.00
17.66
C


ATOM
1138
CD1
TYR
637
7.556
87.512
−6.726
1.00
23.47
C


ATOM
1139
CD2
TYR
637
7.438
87.486
−4.356
1.00
23.35
C


ATOM
1140
CE1
TYR
637
8.507
88.512
−6.684
1.00
24.21
C


ATOM
1141
CE2
TYR
637
8.398
88.493
−4.296
1.00
25.27
C


ATOM
1142
CZ
TYR
637
8.929
89.000
−5.471
1.00
27.11
C


ATOM
1143
OH
TYR
637
9.897
89.989
−5.446
1.00
27.67
O


ATOM
1144
N
VAL
638
2.892
85.114
−6.212
1.00
17.90
N


ATOM
1145
CA
VAL
638
1.866
84.073
−6.264
1.00
15.71
C


ATOM
1146
C
VAL
638
1.110
84.107
−7.591
1.00
16.42
C


ATOM
1147
O
VAL
638
0.730
83.057
−8.121
1.00
13.74
O


ATOM
1148
CB
VAL
638
0.879
84.189
−5.070
1.00
14.51
C


ATOM
1149
CG1
VAL
638
−0.224
83.196
−5.201
1.00
10.64
C


ATOM
1150
CG2
VAL
638
1.623
83.907
−3.762
1.00
15.68
C


ATOM
1151
N
GLN
639
0.858
85.306
−8.122
1.00
17.70
N


ATOM
1152
CA
GLN
639
0.173
85.418
−9.401
1.00
19.16
C


ATOM
1153
C
GLN
639
1.132
84.973
−10.494
1.00
20.06
C


ATOM
1154
O
GLN
639
0.705
84.381
−11.500
1.00
21.31
O


ATOM
1155
CB
GLN
639
−0.280
86.842
−9.661
1.00
24.29
C


ATOM
1156
CG
GLN
639
−1.377
87.339
−8.758
1.00
33.20
C


ATOM
1157
CD
GLN
639
−1.509
88.855
−8.824
1.00
42.75
C


ATOM
1158
OE1
GLN
639
−0.534
89.573
−9.095
1.00
47.34
O


ATOM
1159
NE2
GLN
639
−2.709
89.353
−8.572
1.00
47.82
N


ATOM
1160
N
GLY
640
2.428
85.212
−10.272
1.00
19.08
N


ATOM
1161
CA
GLY
640
3.465
84.789
−11.212
1.00
16.51
C


ATOM
1162
C
GLY
640
3.515
83.268
−11.342
1.00
16.94
C


ATOM
1163
O
GLY
640
3.492
82.740
−12.454
1.00
19.48
O


ATOM
1164
N
LEU
641
3.553
82.551
−10.221
1.00
16.74
N


ATOM
1165
CA
LEU
641
3.579
81.088
−10.252
1.00
13.10
C


ATOM
1166
C
LEU
641
2.269
80.555
−10.792
1.00
13.33
C


ATOM
1167
O
LEU
641
2.206
79.465
−11.355
1.00
18.15
O


ATOM
1168
CB
LEU
641
3.802
80.530
−8.852
1.00
14.38
C


ATOM
1169
CG
LEU
641
5.198
80.681
−8.238
1.00
16.65
C


ATOM
1170
CD1
LEU
641
5.139
80.462
−6.733
1.00
18.86
C


ATOM
1171
CD2
LEU
641
6.206
79.737
−8.886
1.00
6.08
C


ATOM
1172
N
LYS
642
1.205
81.311
−10.605
1.00
14.11
N


ATOM
1173
CA
LYS
642
−0.101
80.898
−11.096
1.00
16.06
C


ATOM
1174
C
LYS
642
−0.150
80.870
−12.623
1.00
16.97
C


ATOM
1175
O
LYS
642
−0.809
80.019
−13.210
1.00
19.62
O


ATOM
1176
CB
LYS
642
−1.169
81.847
−10.555
1.00
16.72
C


ATOM
1177
CG
LYS
642
−2.558
81.585
−11.078
1.00
18.86
C


ATOM
1178
CD
LYS
642
−3.085
82.761
−11.881
1.00
14.53
C


ATOM
1179
CE
LYS
642
−4.522
82.498
−12.267
1.00
25.13
C


ATOM
1180
NZ
LYS
642
−5.053
83.581
−13.128
1.00
33.85
N


ATOM
1181
N
LYS
643
0.517
81.829
−13.256
1.00
17.23
N


ATOM
1182
CA
LYS
643
0.554
81.933
−14.714
1.00
15.33
C


ATOM
1183
C
LYS
643
1.409
80.845
−15.322
1.00
14.03
C


ATOM
1184
O
LYS
643
1.183
80.439
−16.464
1.00
13.60
O


ATOM
1185
CB
LYS
643
1.127
83.281
−15.127
1.00
16.90
C


ATOM
1186
CG
LYS
643
0.301
84.453
−14.692
1.00
23.60
C


ATOM
1187
CD
LYS
643
0.990
85.751
−15.080
1.00
29.59
C


ATOM
1188
CE
LYS
643
0.321
86.922
−14.390
1.00
34.30
C


ATOM
1189
NZ
LYS
643
1.216
88.106
−14.326
1.00
35.59
N


ATOM
1190
N
LYS
644
2.442
80.431
−14.598
1.00
15.71
N


ATOM
1191
CA
LYS
644
3.321
79.373
−15.059
1.00
15.56
C


ATOM
1192
C
LYS
644
2.720
78.041
−14.644
1.00
13.62
C


ATOM
1193
O
LYS
644
3.351
77.009
−14.825
1.00
16.26
O


ATOM
1194
CB
LYS
644
4.737
79.517
−14.466
1.00
19.22
C


ATOM
1195
CG
LYS
644
5.710
80.398
−15.264
1.00
22.27
C


ATOM
1196
CD
LYS
644
7.123
80.272
−14.690
1.00
24.71
C


ATOM
1197
CE
LYS
644
8.123
81.299
−15.260
1.00
31.85
C


ATOM
1198
NZ
LYS
644
9.118
81.871
−14.243
1.00
23.70
N


ATOM
1199
N
LYS
645
1.502
78.076
−14.098
1.00
10.95
N


ATOM
1200
CA
LYS
645
0.761
76.876
−13.644
1.00
14.53
C


ATOM
1201
C
LYS
645
1.425
76.068
−12.527
1.00
15.63
C


ATOM
1202
O
LYS
645
1.324
74.833
−12.481
1.00
10.70
O


ATOM
1203
CB
LYS
645
0.397
75.957
−14.803
1.00
14.90
C


ATOM
1204
CG
LYS
645
−1.087
75.949
−15.174
1.00
16.56
C


ATOM
1205
CD
LYS
645
−1.866
75.078
−14.228
1.00
19.52
C


ATOM
1206
CE
LYS
645
−3.242
75.666
−13.949
1.00
28.98
C


ATOM
1207
NZ
LYS
645
−4.386
74.911
−14.522
1.00
29.12
N


ATOM
1208
N
VAL
646
2.095
76.784
−11.630
1.00
13.98
N


ATOM
1209
CA
VAL
646
2.745
76.182
−10.481
1.00
17.17
C


ATOM
1210
C
VAL
646
1.723
76.303
−9.379
1.00
17.08
C


ATOM
1211
O
VAL
646
1.333
77.413
−9.028
1.00
15.83
O


ATOM
1212
CB
VAL
646
4.004
76.952
−10.065
1.00
15.22
C


ATOM
1213
CG1
VAL
646
4.628
76.308
−8.805
1.00
14.08
C


ATOM
1214
CG2
VAL
646
4.980
76.955
−11.204
1.00
10.59
C


ATOM
1215
N
ILE
647
1.247
75.160
−8.895
1.00
17.86
N


ATOM
1216
CA
ILE
647
0.235
75.107
−7.852
1.00
15.74
C


ATOM
1217
C
ILE
647
0.858
75.303
−6.464
1.00
15.68
C


ATOM
1218
O
ILE
647
1.785
74.592
−6.086
1.00
17.61
O


ATOM
1219
CB
ILE
647
−0.514
73.765
−7.947
1.00
14.07
C


ATOM
1220
CG1
ILE
647
−1.094
73.600
−9.364
1.00
17.76
C


ATOM
1221
CG2
ILE
647
−1.620
73.665
−6.896
1.00
12.37
C


ATOM
1222
CD1
ILE
647
−1.913
74.795
−9.865
1.00
11.46
C


ATOM
1223
N
VAL
648
0.400
76.298
−5.722
1.00
16.62
N


ATOM
1224
CA
VAL
648
0.969
76.519
−4.387
1.00
16.18
C


ATOM
1225
C
VAL
648
0.004
75.932
−3.365
1.00
15.96
C


ATOM
1226
O
VAL
648
−1.158
76.338
−3.284
1.00
14.76
O


ATOM
1227
CB
VAL
648
1.258
78.035
−4.079
1.00
12.13
C


ATOM
1228
CG1
VAL
648
2.023
78.179
−2.775
1.00
9.31
C


ATOM
1229
CG2
VAL
648
2.078
78.678
−5.223
1.00
11.58
C


ATOM
1230
N
ILE
649
0.456
74.906
−2.659
1.00
15.66
N


ATOM
1231
CA
ILE
649
−0.369
74.276
−1.644
1.00
16.78
C


ATOM
1232
C
ILE
649
0.222
74.671
−0.306
1.00
17.79
C


ATOM
1233
O
ILE
649
1.223
74.095
0.124
1.00
18.08
O


ATOM
1234
CB
ILE
649
−0.376
72.747
−1.769
1.00
14.88
C


ATOM
1235
CG1
ILE
649
−0.873
72.352
−3.159
1.00
16.15
C


ATOM
1236
CG2
ILE
649
−1.297
72.142
−0.708
1.00
16.62
C


ATOM
1237
CD1
ILE
649
−0.925
70.873
−3.418
1.00
17.42
C


ATOM
1238
N
PRO
650
−0.351
75.709
0.334
1.00
16.08
N


ATOM
1239
CA
PRO
650
0.076
76.238
1.627
1.00
12.67
C


ATOM
1240
C
PRO
650
−0.300
75.297
2.775
1.00
15.98
C


ATOM
1241
O
PRO
650
−1.478
74.921
2.940
1.00
13.96
O


ATOM
1242
CB
PRO
650
−0.737
77.512
1.764
1.00
9.54
C


ATOM
1243
CG
PRO
650
−1.475
77.687
0.465
1.00
13.54
C


ATOM
1244
CD
PRO
650
−1.626
76.326
−0.068
1.00
16.46
C


ATOM
1245
N
VAL
651
0.693
74.893
3.553
1.00
14.32
N


ATOM
1246
CA
VAL
651
0.422
74.040
4.697
1.00
14.36
C


ATOM
1247
C
VAL
651
0.728
74.922
5.911
1.00
12.69
C


ATOM
1248
O
VAL
651
1.871
75.307
6.129
1.00
15.37
O


ATOM
1249
CB
VAL
651
1.293
72.782
4.691
1.00
14.81
C


ATOM
1250
CG1
VAL
651
0.942
71.918
5.884
1.00
15.50
C


ATOM
1251
CG2
VAL
651
1.075
72.004
3.392
1.00
10.07
C


ATOM
1252
N
GLY
652
−0.307
75.355
6.617
1.00
9.84
N


ATOM
1253
CA
GLY
652
−0.102
76.201
7.776
1.00
12.11
C


ATOM
1254
C
GLY
652
0.074
75.324
8.991
1.00
11.33
C


ATOM
1255
O
GLY
652
−0.778
74.503
9.244
1.00
17.62
O


ATOM
1256
N
ILE
653
1.150
75.492
9.742
1.00
13.97
N


ATOM
1257
CA
ILE
653
1.397
74.679
10.931
1.00
18.24
C


ATOM
1258
C
ILE
653
1.481
75.547
12.197
1.00
21.88
C


ATOM
1259
O
ILE
653
2.468
76.273
12.417
1.00
19.90
O


ATOM
1260
CB
ILE
653
2.728
73.937
10.804
1.00
22.12
C


ATOM
1261
CG1
ILE
653
2.805
73.167
9.478
1.00
18.02
C


ATOM
1262
CG2
ILE
653
2.957
73.057
12.034
1.00
18.87
C


ATOM
1263
CD1
ILE
653
4.217
72.868
9.064
1.00
14.43
C


ATOM
1264
N
GLY
654
0.445
75.491
13.023
1.00
17.34
N


ATOM
1265
CA
GLY
654
0.480
76.279
14.245
1.00
18.39
C


ATOM
1266
C
GLY
654
−0.111
77.682
14.187
1.00
20.92
C


ATOM
1267
O
GLY
654
−0.373
78.214
13.102
1.00
24.39
O


ATOM
1268
N
PRO
655
−0.264
78.337
15.363
1.00
20.00
N


ATOM
1269
CA
PRO
655
−0.806
79.684
15.601
1.00
15.28
C


ATOM
1270
C
PRO
655
−0.034
80.794
14.911
1.00
16.41
C


ATOM
1271
O
PRO
655
−0.588
81.825
14.552
1.00
18.28
O


ATOM
1272
CB
PRO
655
−0.642
79.852
17.116
1.00
12.24
C


ATOM
1273
CG
PRO
655
−0.672
78.460
17.630
1.00
13.05
C


ATOM
1274
CD
PRO
655
0.153
77.719
16.635
1.00
14.07
C


ATOM
1275
N
HIS
656
1.272
80.622
14.794
1.00
16.41
N


ATOM
1276
CA
HIS
656
2.082
81.667
14.194
1.00
18.52
C


ATOM
1277
C
HIS
656
2.109
81.703
12.664
1.00
17.35
C


ATOM
1278
O
HIS
656
2.603
82.672
12.084
1.00
17.65
O


ATOM
1279
CB
HIS
656
3.492
81.676
14.815
1.00
16.60
C


ATOM
1280
CG
HIS
656
3.498
82.026
16.283
1.00
18.39
C


ATOM
1281
ND1
HIS
656
3.392
81.077
17.277
1.00
18.56
N


ATOM
1282
CD2
HIS
656
3.554
83.224
16.915
1.00
16.46
C


ATOM
1283
CE1
HIS
656
3.376
81.672
18.451
1.00
13.19
C


ATOM
1284
NE2
HIS
656
3.474
82.972
18.258
1.00
16.12
N


ATOM
1285
N
ALA
657
1.504
80.703
12.027
1.00
17.30
N


ATOM
1286
CA
ALA
657
1.459
80.616
10.564
1.00
18.06
C


ATOM
1287
C
ALA
657
0.729
81.810
9.961
1.00
16.82
C


ATOM
1288
O
ALA
657
−0.300
82.248
10.464
1.00
19.76
O


ATOM
1289
CB
ALA
657
0.808
79.297
10.121
1.00
13.90
C


ATOM
1290
N
ASN
658
1.277
82.340
8.877
1.00
19.53
N


ATOM
1291
CA
ASN
658
0.699
83.502
8.226
1.00
16.37
C


ATOM
1292
C
ASN
658
−0.569
83.144
7.494
1.00
18.85
C


ATOM
1293
O
ASN
658
−0.575
82.964
6.284
1.00
20.50
O


ATOM
1294
CB
ASN
658
1.704
84.142
7.276
1.00
11.24
C


ATOM
1295
CG
ASN
658
1.282
85.534
6.844
1.00
11.66
C


ATOM
1296
OD1
ASN
658
0.098
85.829
6.766
1.00
14.75
O


ATOM
1297
NO2
ASN
658
2.251
86.396
6.563
1.00
16.32
N


ATOM
1298
N
LEU
659
−1.648
83.035
8.251
1.00
20.82
N


ATOM
1299
CA
LEU
659
−2.943
82.704
7.700
1.00
19.30
C


ATOM
1300
C
LEU
659
−3.460
83.699
6.664
1.00
20.85
C


ATOM
1301
O
LEU
659
−4.230
83.317
5.782
1.00
22.69
O


ATOM
1302
CB
LEU
659
−3.950
82.569
8.824
1.00
17.74
C


ATOM
1303
CG
LEU
659
−4.579
81.198
8.897
1.00
21.02
C


ATOM
1304
CD1
LEU
659
−3.505
80.188
9.248
1.00
24.59
C


ATOM
1305
CD2
LEU
659
−5.665
81.212
9.935
1.00
27.06
C


ATOM
1306
N
LYS
660
−3.078
84.968
6.782
1.00
22.50
N


ATOM
1307
CA
LYS
660
−3.520
85.982
5.822
1.00
23.11
C


ATOM
1308
C
LYS
660
−2.945
85.639
4.452
1.00
24.26
C


ATOM
1309
O
LYS
660
−3.663
85.697
3.448
1.00
27.91
O


ATOM
1310
CB
LYS
660
−3.068
87.361
6.251
1.00
21.43
C


ATOM
1311
N
GLN
661
−1.667
85.240
4.420
1.00
21.76
N


ATOM
1312
CA
GLN
661
−0.991
84.844
3.183
1.00
17.85
C


ATOM
1313
C
GLN
661
−1.570
83.531
2.688
1.00
18.55
C


ATOM
1314
O
GLN
661
−1.828
83.372
1.499
1.00
23.06
O


ATOM
1315
CB
GLN
661
0.525
84.730
3.377
1.00
12.86
C


ATOM
1316
CG
GLN
661
1.237
86.102
3.366
1.00
10.63
C


ATOM
1317
CD
GLN
661
2.755
86.013
3.544
1.00
14.73
C


ATOM
1318
OE1
GLN
661
3.287
84.965
3.905
1.00
17.21
O


ATOM
1319
NE2
GLN
661
3.451
87.118
3.305
1.00
15.89
N


ATOM
1320
N
ILE
662
−1.850
82.613
3.607
1.00
17.78
N


ATOM
1321
CA
ILE
662
−2.438
81.336
3.237
1.00
15.00
C


ATOM
1322
C
ILE
662
−3.775
81.610
2.537
1.00
19.04
C


ATOM
1323
O
ILE
662
−4.050
81.031
1.487
1.00
21.90
O


ATOM
1324
CB
ILE
662
−2.580
80.411
4.489
1.00
11.85
C


ATOM
1325
CG1
ILE
662
−1.196
80.040
4.995
1.00
11.79
C


ATOM
1326
CG2
ILE
662
−3.337
79.126
4.196
1.00
7.88
C


ATOM
1327
CD1
ILE
662
−1.191
79.084
6.171
1.00
13.32
C


ATOM
1328
N
ARG
663
−4.558
82.560
3.047
1.00
20.47
N


ATOM
1329
CA
ARG
663
−5.850
82.874
2.436
1.00
20.44
C


ATOM
1330
C
ARG
663
−5.704
83.569
1.078
1.00
19.11
C


ATOM
1331
O
ARG
663
−6.548
83.416
0.195
1.00
19.37
O


ATOM
1332
CB
ARG
663
−6.733
83.701
3.390
1.00
23.96
C


ATOM
1333
CG
ARG
663
−7.123
82.987
4.686
1.00
32.86
C


ATOM
1334
CD
ARG
663
−7.800
81.612
4.456
1.00
41.17
C


ATOM
1335
NE
ARG
663
−7.818
80.785
5.672
1.00
43.59
N


ATOM
1336
CZ
ARG
663
−8.042
79.471
5.708
1.00
44.43
C


ATOM
1337
NH1
ARG
663
−8.287
78.791
4.604
1.00
40.35
N


ATOM
1338
NH2
ARG
663
−7.942
78.818
6.853
1.00
47.33
N


ATOM
1339
N
LEU
664
−4.641
84.338
0.911
1.00
20.67
N


ATOM
1340
CA
LEU
664
4.400
85.016
−0.351
1.00
23.77
C


ATOM
1341
C
LEU
664
−4.006
83.990
−1.425
1.00
24.53
C


ATOM
1342
O
LEU
664
−4.470
84.066
−2.570
1.00
25.26
O


ATOM
1343
CB
LEU
664
−3.287
86.048
−0.200
1.00
22.42
C


ATOM
1344
CG
LEU
664
−3.695
87.458
0.188
1.00
23.55
C


ATOM
1345
CD1
LEU
664
−2.434
88.240
0.463
1.00
22.20
C


ATOM
1346
CD2
LEU
664
−4.517
88.113
−0.910
1.00
21.42
C


ATOM
1347
N
ILE
665
−3.147
83.040
−1.053
1.00
23.53
N


ATOM
1348
CA
ILE
665
−2.697
82.009
−1.970
1.00
21.00
C


ATOM
1349
C
ILE
665
−3.895
81.209
−2.462
1.00
22.09
C


ATOM
1350
O
ILE
665
−4.064
81.029
−3.659
1.00
24.56
O


ATOM
1351
CB
ILE
665
−1.680
81.078
−1.316
1.00
17.66
C


ATOM
1352
CG1
ILE
665
−0.391
81.841
−1.015
1.00
15.92
C


ATOM
1353
CG2
ILE
665
−1.375
79.913
−2.234
1.00
18.28
C


ATOM
1354
CD1
ILE
665
0.629
81.031
−0.194
1.00
6.96
C


ATOM
1355
N
GLU
666
−4.732
80.747
−1.549
1.00
23.35
N


ATOM
1356
CA
GLU
666
−5.917
79.983
−1.918
1.00
19.08
C


ATOM
1357
C
GLU
666
−6.813
80.616
−2.967
1.00
19.61
C


ATOM
1358
O
GLU
666
−7.341
79.920
−3.827
1.00
18.02
O


ATOM
1359
CB
GLU
666
−6.784
79.754
−0.704
1.00
17.71
C


ATOM
1360
CG
GLU
666
−6.367
78.618
0.172
1.00
26.16
C


ATOM
1361
CD
GLU
666
−7.515
78.164
1.049
1.00
28.97
C


ATOM
1362
OE1
GLU
666
−7.910
78.950
1.944
1.00
22.27
O


ATOM
1363
OE2
GLU
666
−8.030
77.036
0.815
1.00
30.15
O


ATOM
1364
N
LYS
667
−7.009
81.928
−2.878
1.00
21.77
N


ATOM
1365
CA
LYS
667
−7.908
82.604
−3.800
1.00
27.64
C


ATOM
1366
C
LYS
667
−7.336
83.042
−5.140
1.00
28.79
C


ATOM
1367
O
LYS
667
−8.067
83.548
−5.999
1.00
33.07
O


ATOM
1368
CB
LYS
667
−8.613
83.773
−3.108
1.00
29.02
C


ATOM
1369
CG
LYS
667
−7.764
84.976
−2.848
1.00
34.25
C


ATOM
1370
CD
LYS
667
−8.600
86.097
−2.236
1.00
45.36
C


ATOM
1371
CE
LYS
667
−7.746
87.344
−1.955
1.00
55.46
C


ATOM
1372
NZ
LYS
667
−8.130
88.070
−0.688
1.00
58.49
N


ATOM
1373
N
GLN
668
−6.042
82.833
−5.329
1.00
29.18
N


ATOM
1374
CA
GLN
668
−5.370
83.189
−6.570
1.00
27.43
C


ATOM
1375
C
GLN
668
−5.720
82.143
−7.648
1.00
27.44
C


ATOM
1376
O
GLN
668
−5.795
82.459
−8.834
1.00
29.48
O


ATOM
1377
CB
GLN
668
−3.856
83.236
−6.324
1.00
24.44
C


ATOM
1378
CG
GLN
668
−3.199
84.560
−6.643
1.00
31.15
C


ATOM
1379
CD
GLN
668
−3.934
85.764
−6.071
1.00
33.50
C


ATOM
1380
OE1
GLN
668
−4.353
86.659
−6.818
1.00
32.99
O


ATOM
1381
NE2
GLN
668
−4.054
85.819
−4.747
1.00
30.05
N


ATOM
1382
N
ALA
669
−5.930
80.895
−7.231
1.00
23.35
N


ATOM
1383
CA
ALA
669
−6.275
79.816
−8.153
1.00
21.93
C


ATOM
1384
C
ALA
669
−6.968
78.683
−7.379
1.00
20.53
C


ATOM
1385
O
ALA
669
−6.590
78.375
−6.267
1.00
22.44
O


ATOM
1386
CB
ALA
669
−5.010
79.303
−8.854
1.00
21.51
C


ATOM
1387
N
PRO
670
−7.983
78.041
−7.977
1.00
22.07
N


ATOM
1388
CA
PRO
670
−8.697
76.959
−7.300
1.00
19.32
C


ATOM
1389
C
PRO
670
−7.916
75.769
−6.744
1.00
24.31
C


ATOM
1390
O
PRO
670
−8.338
75.182
−5.735
1.00
22.85
O


ATOM
1391
CB
PRO
670
−9.740
76.517
−8.344
1.00
19.25
C


ATOM
1392
CG
PRO
670
−9.251
77.058
−9.633
1.00
18.97
C


ATOM
1393
CD
PRO
670
−8.649
78.372
−9.251
1.00
22.64
C


ATOM
1394
N
GLU
671
−6.765
75.444
−7.332
1.00
21.90
N


ATOM
1395
CA
GLU
671
−6.037
74.282
−6.872
1.00
21.53
C


ATOM
1396
C
GLU
671
−5.121
74.573
−5.724
1.00
18.85
C


ATOM
1397
O
GLU
671
−4.526
73.648
−5.152
1.00
17.02
O


ATOM
1398
CB
GLU
671
−5.237
73.622
−7.996
1.00
28.16
C


ATOM
1399
CG
GLU
671
−6.053
73.156
−9.167
1.00
30.85
C


ATOM
1400
CD
GLU
671
−6.052
74.163
−10.295
1.00
40.33
C


ATOM
1401
OE1
GLU
671
−5.922
75.389
−10.033
1.00
37.89
O


ATOM
1402
OE2
GLU
671
−6.181
73.718
−11.456
1.00
49.39
O


ATOM
1403
N
ASN
672
4.915
75.849
−5.440
1.00
16.70
N


ATOM
1404
CA
ASN
672
−4.035
76.229
−4.324
1.00
20.34
C


ATOM
1405
C
ASN
672
−4.755
75.885
−3.007
1.00
18.55
C


ATOM
1406
O
ASN
672
−5.136
76.780
−2.248
1.00
18.65
O


ATOM
1407
CB
ASN
672
−3.751
77.739
−4.359
1.00
19.53
C


ATOM
1408
CG
ASN
672
−2.994
78.186
−5.601
1.00
15.33
C


ATOM
1409
OD1
ASN
672
−2.466
77.384
−6.369
1.00
12.87
O


ATOM
1410
ND2
ASN
672
−2.914
79.494
−5.778
1.00
17.27
N


ATOM
1411
N
LYS
673
−4.986
74.601
−2.761
1.00
17.76
N


ATOM
1412
CA
LYS
673
−5.708
74.178
−1.568
1.00
21.07
C


ATOM
1413
C
LYS
673
−4.820
74.299
−0.335
1.00
20.99
C


ATOM
1414
O
LYS
673
−3.655
73.896
−0.360
1.00
26.08
O


ATOM
1415
CB
LYS
673
−6.222
72.732
−1.710
1.00
19.00
C


ATOM
1416
CG
LYS
673
−7.480
72.469
−0.858
1.00
30.07
C


ATOM
1417
CD
LYS
673
−7.457
71.176
−0.047
1.00
30.38
C


ATOM
1418
CE
LYS
673
−7.751
69.983
−0.913
1.00
36.80
C


ATOM
1419
NZ
LYS
673
−7.645
68.682
−0.181
1.00
45.16
N


ATOM
1420
N
ALA
674
−5.357
74.891
0.727
1.00
18.75
N


ATOM
1421
CA
ALA
674
−4.613
75.043
1.959
1.00
15.72
C


ATOM
1422
C
ALA
674
−4.928
73.914
2.916
1.00
18.41
C


ATOM
1423
O
ALA
674
−6.047
73.370
2.935
1.00
12.37
O


ATOM
1424
CB
ALA
674
−4.938
76.362
2.607
1.00
14.82
C


ATOM
1425
N
PHE
675
−3.916
73.577
3.716
1.00
20.72
N


ATOM
1426
CA
PHE
675
−4.021
72.560
4.751
1.00
17.35
C


ATOM
1427
C
PHE
675
−3.565
73.235
6.032
1.00
13.94
C


ATOM
1428
O
PHE
675
−2.414
73.594
6.162
1.00
16.79
O


ATOM
1429
CB
PHE
675
−3.148
71.357
4.421
1.00
17.30
C


ATOM
1430
CG
PHE
675
−3.667
70.547
3.276
1.00
16.73
C


ATOM
1431
CD1
PHE
675
−3.357
70.890
1.961
1.00
18.39
C


ATOM
1432
CD2
PHE
675
−4.502
69.469
3.501
1.00
11.98
C


ATOM
1433
CE1
PHE
675
−3.880
70.177
0.880
1.00
11.40
C


ATOM
1434
CE2
PHE
675
−5.031
68.746
2.416
1.00
15.53
C


ATOM
1435
CZ
PHE
675
−4.717
69.111
1.108
1.00
13.44
C


ATOM
1436
N
VAL
676
−4.509
73.561
6.899
1.00
17.73
N


ATOM
1437
CA
VAL
676
−4.176
74.211
8.163
1.00
19.36
C


ATOM
1438
C
VAL
676
−4.128
73.145
9.260
1.00
20.91
C


ATOM
1439
O
VAL
676
−5.035
72.324
9.395
1.00
19.66
O


ATOM
1440
CB
VAL
676
−5.175
75.326
8.489
1.00
17.55
C


ATOM
1441
CG1
VAL
676
−4.779
76.031
9.769
1.00
18.65
C


ATOM
1442
CG2
VAL
676
−5.219
76.328
7.325
1.00
19.14
C


ATOM
1443
N
LEU
677
−2.994
73.100
9.952
1.00
23.37
N


ATOM
1444
CA
LEU
677
−2.740
72.122
11.003
1.00
21.69
C


ATOM
1445
C
LEU
677
−2.362
72.830
12.284
1.00
17.44
C


ATOM
1446
O
LEU
677
−1.870
73.965
12.263
1.00
11.94
O


ATOM
1447
CB
LEU
677
−1.597
71.184
10.603
1.00
25.46
C


ATOM
1448
CG
LEU
677
−1.766
70.099
9.540
1.00
25.42
C


ATOM
1449
CD1
LEU
677
−2.269
70.624
8.236
1.00
23.58
C


ATOM
1450
CD2
LEU
677
−0.422
69.495
9.346
1.00
30.67
C


ATOM
1451
N
SER
678
−2.591
72.153
13.403
1.00
15.75
N


ATOM
1452
CA
SER
678
−2.279
72.728
14.704
1.00
18.74
C


ATOM
1453
C
SER
678
−0.805
72.690
15.096
1.00
15.98
C


ATOM
1454
O
SER
678
−0.305
73.594
15.775
1.00
16.52
O


ATOM
1455
CB
SER
678
−3.143
72.085
15.783
1.00
17.24
C


ATOM
1456
OG
SER
678
−4.495
72.476
15.621
1.00
23.02
O


ATOM
1457
N
SER
679
−0.095
71.679
14.621
1.00
15.13
N


ATOM
1458
CA
SER
679
1.305
71.550
14.954
1.00
16.64
C


ATOM
1459
C
SER
679
1.891
7C.526
13.993
1.00
16.28
C


ATOM
1460
O
SER
679
1.152
69.830
13.284
1.00
16.75
O


ATOM
1461
CB
SER
679
1.424
71.025
16.389
1.00
20.41
C


ATOM
1462
OG
SER
679
C.924
69.683
16.472
1.00
15.18
O


ATOM
1463
N
VAL
680
3.212
70.389
14.023
1.00
14.70
N


ATOM
1464
CA
VAL
680
3.881
69.426
13.165
1.00
18.08
C


ATOM
1465
C
VAL
680
3.441
67.999
13.506
1.00
21.23
C


ATOM
1466
O
VAL
680
3.598
67.092
12.692
1.00
22.59
O


ATOM
1467
CB
VAL
680
5.421
69.505
13.291
1.00
15.16
C


ATOM
1468
CG1
VAL
680
5.953
70.852
12.792
1.00
14.14
C


ATOM
1469
CG2
VAL
680
5.844
69.260
14.728
1.00
15.78
C


ATOM
1470
N
ASP
681
2.845
67.803
14.680
1.00
23.61
N


ATOM
1471
CA
ASP
681
2.431
66.465
15.090
1.00
20.29
C


ATOM
1472
C
ASP
681
1.267
65.967
14.292
1.00
17.46
C


ATOM
1473
O
ASP
681
1.021
64 .758
14.254
1.00
19.43
O


ATOM
1474
CB
ASP
681
2.118
66.404
16.586
1.00
22.71
C


ATOM
1475
CG
ASP
681
3.360
66.605
17.455
1.00
27.60
C


ATOM
1476
OD1
ASP
681
4.485
66.251
17.021
1.00
31.79
O


ATOM
1477
OD2
ASP
681
3.205
67.117
18.585
1.00
30.83
O


ATOM
1478
N
GLU
682
0.559
66.891
13.648
1.00
19.92
N


ATOM
1479
CA
GLU
682
−0.593
66.532
12.823
1.00
19.43
C


ATOM
1480
C
GLU
682
−0.246
66.200
11.373
1.00
16.94
C


ATOM
1481
O
GLU
682
−1.031
65.593
10.678
1.00
20.34
O


ATOM
1482
CB
GLU
682
−1.664
67.616
12.904
1.00
19.71
C


ATOM
1483
CG
GLU
682
−2.031
67.903
14.337
1.00
27.72
C


ATOM
1484
CD
GLU
682
−3.385
68.541
14.516
1.00
32.65
C


ATOM
1485
OE1
GLU
682
−3.806
69.338
13.643
1.00
32.50
O


ATOM
1486
OE2
GLU
682
−4.013
68.250
15.567
1.00
36.39
O


ATOM
1487
N
LEU
683
0.968
66.533
10.953
1.00
19.46
N


ATOM
1488
CA
LEU
683
1.430
66.270
9.591
1.00
18.53
C


ATOM
1489
C
LEU
683
1.230
64.820
9.159
1.00
19.54
C


ATOM
1490
O
LEU
683
0.759
64.567
8.050
1.00
21.33
O


ATOM
1491
CB
LEU
683
2.913
66.629
9.458
1.00
9.01
C


ATOM
1492
CG
LEU
683
3.212
68.119
9.485
1.00
11.07
C


ATOM
1493
CD1
LEU
683
4.725
68.380
9.521
1.00
6.20
C


ATOM
1494
CD2
LEU
683
2.581
68.729
8.268
1.00
9.94
C


ATOM
1495
N
GLU
684
1.577
63.879
10.027
1.00
17.82
N


ATOM
1496
CA
GLU
684
1.449
62.470
9.701
1.00
21.58
C


ATOM
1497
C
GLU
684
0.026
62.043
9.404
1.00
20.80
C


ATOM
1498
O
GLU
684
−0.204
61.216
8.521
1.00
21.28
O


ATOM
1499
CB
GLU
684
2.022
61.614
10.826
1.00
27.44
C


ATOM
1500
CG
GLU
684
1.799
60.112
10.601
1.00
42.61
C


ATOM
1501
CD
GLU
684
2.605
59.219
11.539
1.00
48.25
C


ATOM
1502
OE1
GLU
684
2.910
59.655
12.683
1.00
46.17
O


ATOM
1503
OE2
GLU
684
2.915
58.071
11.116
1.00
50.28
O


ATOM
1504
N
GLN
685
−0.914
62.632
10.133
1.00
22.35
N


ATOM
1505
CA
GLN
685
−2.349
62.353
10.005
1.00
25.85
C


ATOM
1506
C
GLN
685
−2.958
62.892
8.709
1.00
22.14
C


ATOM
1507
O
GLN
685
−4.089
62.579
8.381
1.00
25.03
O


ATOM
1508
CB
GLN
685
−3.117
63.008
11.162
1.00
25.71
C


ATOM
1509
CG
GLN
685
−2.414
62.982
12.509
1.00
34.50
C


ATOM
1510
CD
GLN
685
−3.192
63.753
13.571
1.00
41.31
C


ATOM
1511
OE1
GLN
685
−4.338
64.167
13.356
1.00
40.86
O


ATOM
1512
NE2
GLN
685
−2.565
63.957
14.726
1.00
45.33
N


ATOM
1513
N
GLN
686
−2.245
63.789
8.044
1.00
20.96
N


ATOM
1514
CA
GLN
686
−2.715
64.404
6.813
1.00
23.16
C


ATOM
1515
C
GLN
686
−1.882
64.075
5.583
1.00
19.32
C


ATOM
1516
O
GLN
686
−2.328
64.299
4.456
1.00
22.21
O


ATOM
1517
CB
GLN
686
−2.709
65.912
6.982
1.00
26.35
C


ATOM
1518
CG
GLN
686
−3.709
66.416
7.948
1.00
35.22
C


ATOM
1519
CD
GLN
686
−4.816
67.142
7.241
1.00
42.30
C


ATOM
1520
OE1
GLN
686
−4.941
68.365
7.350
1.00
45.54
O


ATOM
1521
NE2
GLN
686
−5.616
66.400
6.478
1.00
44.88
N


ATOM
1522
N
ARG
687
−0.683
63.557
5.803
1.00
14.84
N


ATOM
1523
CA
ARG
687
0.238
63.225
4.731
1.00
17.34
C


ATOM
1524
C
ARG
687
−0.364
62.511
3.524
1.00
17.95
C


ATOM
1525
O
ARG
687
−0.248
62.982
2.402
1.00
18.16
O


ATOM
1526
CB
ARG
687
1.405
62.429
5.303
1.00
12.76
C


ATOM
1527
CG
ARG
687
2.512
62.076
4.320
1.00
12.31
C


ATOM
1528
CD
ARG
687
2.612
60.589
4.398
1.00
15.47
C


ATOM
1529
NE
ARG
687
2.836
60.134
5.786
1.00
27.33
N


ATOM
1530
CZ
ARG
687
2.343
58.985
6.234
1.00
25.36
C


ATOM
1531
NH1
ARG
687
1.783
58.118
5.416
1.00
25.62
N


ATOM
1532
NH2
ARG
687
2.342
58.744
7.535
1.00
34.14
N


ATOM
1533
N
ASP
688
−1.044
61.397
3.749
1.00
22.35
N


ATOM
1534
CA
ASP
688
−1.603
60.649
2.632
1.00
20.59
C


ATOM
1535
C
ASP
688
−2.651
61.418
1.872
1.00
23.19
C


ATOM
1536
O
ASP
688
−2.732
61.326
0.653
1.00
23.82
O


ATOM
1537
CB
ASP
688
−2.128
59.301
3.096
1.00
21.54
C


ATOM
1538
CG
ASP
688
−1.008
58.365
3.518
1.00
24.25
C


ATOM
1539
OD1
ASP
688
0.172
58.697
3.266
1.00
31.52
O


ATOM
1540
OD2
ASP
688
−1.287
57.289
4.083
1.00
26.17
O


ATOM
1541
N
GLU
689
−3.395
62.253
2.575
1.00
23.99
N


ATOM
1542
CA
GLU
689
−4.419
63.045
1.927
1.00
26.49
C


ATOM
1543
C
GLU
689
−3.796
64.142
1.054
1.00
25.62
C


ATOM
1544
O
GLU
689
−4.376
64.536
0.034
1.00
26.10
O


ATOM
1545
CB
GLU
689
−5.334
63.663
2.981
1.00
34.19
C


ATOM
1546
CG
GLU
689
−6.456
64.543
2.429
1.00
36.12
C


ATOM
1547
CD
GLU
689
−7.358
65.061
3.527
1.00
42.19
C


ATOM
1548
OE1
GLU
689
−7.532
64.327
4.532
1.00
50.63
O


ATOM
1549
OE2
GLU
689
−7.895
66.189
3.385
1.00
42.61
O


ATOM
1550
N
ILE
690
−2.645
64.665
1.478
1.00
21.08
N


ATOM
1551
CA
ILE
690
−1.951
65.705
0.726
1.00
19.71
C


ATOM
1552
C
ILE
690
−1.312
65.124
−0.514
1.00
20.60
C


ATOM
1553
O
ILE
690
−1.368
65.722
−1.567
1.00
23.29
O


ATOM
1554
CB
ILE
690
−0.881
66.388
1.572
1.00
20.01
C


ATOM
1555
CG1
ILE
690
−1.550
67.130
2.737
1.00
18.80
C


ATOM
1556
CG2
ILE
690
−0.054
67.322
0.713
1.00
19.33
C


ATOM
1557
CD1
ILE
690
−0.610
67.889
3.623
1.00
13.89
C


ATOM
1558
N
VAL
691
−0.694
63.958
−0.394
1.00
22.47
N


ATOM
1559
CA
VAL
691
−0.083
63.301
−1.543
1.00
21.73
C


ATOM
1560
C
VAL
691
−1.144
62.902
−2.579
1.00
20.40
C


ATOM
1561
O
VAL
691
−0.923
63.076
−3.767
1.00
22.98
O


ATOM
1562
CB
VAL
691
0.750
62.083
−1.110
1.00
22.75
C


ATOM
1563
CG1
VAL
691
1.147
61.263
−2.296
1.00
29.43
C


ATOM
1564
CG2
VAL
691
1.988
62.555
−0.431
1.00
23.51
C


ATOM
1565
N
SER
692
−2.296
62.407
−2.139
1.00
17.46
N


ATOM
1566
CA
SER
692
−3.371
62.031
−3.054
1.00
15.69
C


ATOM
1567
C
SER
692
−3.894
63.216
−3.856
1.00
16.29
C


ATOM
1568
O
SER
692
−4.106
63.117
−5.060
1.00
19.86
O


ATOM
1569
CB
SER
692
−4.532
61.435
−2.283
1.00
15.65
C


ATOM
1570
OG
SER
692
−4.134
60.230
−1.674
1.00
28.15
O


ATOM
1571
N
TYR
693
−4.129
64.319
−3.168
1.00
16.74
N


ATOM
1572
CA
TYR
693
−4.632
65.536
−3.768
1.00
15.99
C


ATOM
1573
C
TYR
693
−3.654
65.960
−4.833
1.00
18.79
C


ATOM
1574
O
TYR
693
−3.983
66.084
−6.006
1.00
21.36
O


ATOM
1575
CB
TYR
693
−4.725
66.619
−2.702
1.00
11.55
C


ATOM
1576
CG
TYR
693
−5.078
67.969
−3.259
1.00
19.55
C


ATOM
1577
CD1
TYR
693
−6.374
68.238
−3.748
1.00
20.06
C


ATOM
1578
CD2
TYR
693
−4.102
68.960
−3.394
1.00
13.67
C


ATOM
1579
CE1
TYR
693
−6.665
69.458
−4.365
1.00
14.61
C


ATOM
1580
CE2
TYR
693
−4.394
70.165
−4.002
1.00
14.06
C


ATOM
1581
CZ
TYR
693
−5.664
70.402
−4.489
1.00
14.63
C


ATOM
1582
OH
TYR
693
−5.911
71.592
−5.123
1.00
17.27
O


ATOM
1583
N
LEU
694
−2.416
66.070
−4.401
1.00
21.64
N


ATOM
1584
CA
LEU
694
−1.287
66.480
−5.216
1.00
22.01
C


ATOM
1585
C
LEU
694
−1.001
65.549
−6.414
1.00
24.72
C


ATOM
1586
O
LEU
694
−0.819
66.007
−7.560
1.00
21.88
O


ATOM
1587
CB
LEU
694
−0.131
66.631
−4.229
1.00
19.20
C


ATOM
1588
CG
LEU
694
1.365
66.561
−4.388
1.00
23.50
C


ATOM
1589
CD1
LEU
694
1.939
67.326
−3.227
1.00
20.23
C


ATOM
1590
CD2
LEU
694
1.840
65.126
−4.387
1.00
21.48
C


ATOM
1591
N
CYS
695
−1.033
64.243
−6.178
1.00
24.01
N


ATOM
1592
CA
CYS
695
−0.805
63.264
−7.230
1.00
23.59
C


ATOM
1593
C
CYS
695
−1.913
63.363
−8.266
1.00
23.92
C


ATOM
1594
O
CYS
695
−1.662
63.443
−9.476
1.00
20.77
O


ATOM
1595
CB
CYS
695
−0.785
61.851
−6.639
1.00
21.84
C


ATOM
1596
SG
CYS
695
−0.122
60.600
−7.770
1.00
25.31
S


ATOM
1597
N
ASP
696
−3.141
63.401
−7.775
1.00
25.04
N


ATOM
1598
CA
ASP
696
−4.312
63.463
−8.634
1.00
30.16
C


ATOM
1599
C
ASP
696
−4.295
64.617
−9.630
1.00
29.12
C


ATOM
1600
O
ASP
696
−4.933
64.557
−10.678
1.00
33.49
O


ATOM
1601
CB
ASP
696
−5.569
63.536
−7.775
1.00
33.29
C


ATOM
1602
CG
ASP
696
−6.827
63.645
−8.598
1.00
40.85
C


ATOM
1603
OD1
ASP
696
−7.215
62.641
−9.244
1.00
44.44
O


ATOM
1604
OD2
ASP
696
−7.416
64.749
−8.613
1.00
45.63
O


ATOM
1605
N
LEU
697
−3.544
65.655
−9.296
1.00
27.97
N


ATOM
1606
CA
LEU
697
−3.421
66.863
−10.108
1.00
23.65
C


ATOM
1607
C
LEU
697
−2.498
66.673
−11.297
1.00
21.19
C


ATOM
1608
O
LEU
697
−2.676
67.307
−12.337
1.00
23.84
O


ATOM
1609
CB
LEU
697
−2.863
67.990
−9.227
1.00
19.78
C


ATOM
1610
CG
LEU
697
−3.679
69.216
−8.812
1.00
18.30
C


ATOM
1611
CD1
LEU
697
−5.186
68.949
−8.767
1.00
12.42
C


ATOM
1612
CD2
LEU
697
−3.128
69.715
−7.478
1.00
11.15
C


ATOM
1613
N
ALA
698
−1.467
65.864
−11.106
1.00
20.21
N


ATOM
1614
CA
ALA
698
−0.472
65.597
−12.128
1.00
18.13
C


ATOM
1615
C
ALA
698
−0.972
64.732
−13.286
1.00
18.60
C


ATOM
1616
O
ALA
698
−1.904
63.935
−13.128
1.00
20.41
O


ATOM
1617
CB
ALA
698
0.746
64.965
−11.481
1.00
16.85
C


ATOM
1618
N
PRO
699
−0.362
64.887
−14.479
1.00
15.84
N


ATOM
1619
CA
PRO
699
−0.802
64.081
−15.622
1.00
14.86
C


ATOM
1620
C
PRO
699
−0.313
62.649
−15.472
1.00
17.66
C


ATOM
1621
O
PRO
699
0.687
62.397
−14.808
1.00
18.33
O


ATOM
1622
CB
PRO
699
−0.131
64.763
−16.817
1.00
10.16
C


ATOM
1623
CG
PRO
699
0.224
66.151
−16.293
1.00
11.52
C


ATOM
1624
CD
PRO
699
0.636
65.888
−14.889
1.00
12.64
C


ATOM
1625
N
GLU
700
−1.029
61.702
−16.058
1.00
18.23
N


ATOM
1626
CA
GLU
700
−0.585
60.329
−15.996
1.00
21.35
C


ATOM
1627
C
GLU
700
0.318
60.141
−17.171
1.00
24.65
C


ATOM
1628
O
GLU
700
0.213
60.873
−18.137
1.00
29.66
O


ATOM
1629
CB
GLU
700
−1.730
59.377
−16.176
1.00
19.18
C


ATOM
1630
CG
GLU
700
−2.640
59.327
−15.036
1.00
27.76
C


ATOM
1631
CD
GLU
700
−3.623
58.221
−15.210
1.00
30.98
C


ATOM
1632
OE1
GLU
700
−3.184
57.061
−15.339
1.00
35.58
O


ATOM
1633
OE2
GLU
700
−4.833
58.511
−15.269
1.00
39.02
O


ATOM
1634
N
ALA
701
1.223
59.173
−17.083
1.00
33.25
N


ATOM
1635
CA
ALA
701
2.109
58.864
−18.203
1.00
34.46
C


ATOM
1636
C
ALA
701
1.251
58.146
−19.254
1.00
36.32
C


ATOM
1637
O
ALA
701
0.277
57.456
−18.916
1.00
30.64
O


ATOM
1638
CB
ALA
701
3.251
57.962
−17.739
1.00
32.72
C


ATOM
1639
N
PRO
702
1.537
58.370
−20.545
1.00
42.71
N


ATOM
1640
CA
PRO
702
0.790
57.732
−21.634
1.00
47.12
C


ATOM
1641
C
PRO
702
1.089
56.237
−21.591
1.00
49.56
C


ATOM
1642
O
PRO
702
2.244
55.851
−21.342
1.00
48.33
O


ATOM
1643
CB
PRO
702
1.402
58.353
−22.886
1.00
45.52
C


ATOM
1644
CG
PRO
702
1.918
59.687
−22.387
1.00
48.78
C


ATOM
1645
CD
PRO
702
2.530
59.322
−21.073
1.00
44.35
C


ATOM
1646
N
PRO
703
0.080
55.383
−21.849
1.00
51.95
N


ATOM
1647
CA
PRO
703
0.189
53.922
−21.841
1.00
54.88
C


ATOM
1648
C
PRO
703
1.480
53.458
−22.521
1.00
59.06
C


ATOM
1649
O
PRO
703
1.819
53.929
−23.616
1.00
56.81
O


ATOM
1650
CB
PRO
703
−1.057
53.477
−22.606
1.00
53.66
C


ATOM
1651
CG
PRO
703
−2.041
54.516
−22.235
1.00
53.29
C


ATOM
1652
CD
PRO
703
−1.240
55.790
−22.379
1.00
53.11
C


ATOM
1653
N
PRO
704
2.271
52.622
−21.816
1.00
64.17
N


ATOM
1654
CA
PRO
704
3.533
52.117
−22.370
1.00
66.20
C


ATOM
1655
C
PRO
704
3.250
51.526
−23.741
1.00
67.33
C


ATOM
1656
O
PRO
704
2.480
50.565
−23.877
1.00
66.63
O


ATOM
1657
CB
PRO
704
3.946
51.050
−21.356
1.00
66.86
C


ATOM
1658
CG
PRO
704
3.457
51.639
−20.067
1.00
65.74
C


ATOM
1659
CD
PRO
704
2.065
52.108
−20.449
1.00
64.51
C


ATOM
1660
N
THR
705
3.885
52.116
−24.741
1.00
67.88
N


ATOM
1661
CA
THR
705
3.712
51.735
−26.130
1.00
68.43
C


ATOM
1662
C
THR
705
5.009
51.114
−26.688
1.00
68.19
C


ATOM
1663
O
THR
705
4.932
50.046
−27.340
1.00
66.67
O


ATOM
1664
CB
THR
705
3.231
52.998
−26.933
1.00
69.60
C


ATOM
1665
OG1
THR
705
2.672
52.619
−28.198
1.00
70.05
O


ATOM
1666
CG2
THR
705
4.357
54.013
−27.124
1.00
68.20
C


ATOM
1667
OXT
THR
705
6.102
51.650
−26.404
1.00
67.96
O









To demonstrate the feasibility of identifying potential small molecule inhibitors in silico, computational modeling software was utilized in conjunction with high-resolution crystal structure results to screen databases for existing compounds that would bind to the A1 domain where it interfaces with botrocetin (exogenous ligand binding site). Several small molecules predicted to bind with sub-micromolar IC50s (concentration of drug required to inhibit the activity by 50%) and that could also severely disrupt binding of this snake venom protein were identified. Thus, potential candidate small molecules can be identified that may interfere with the interaction between GPIb alpha and the A1 domain of VWF.
Screening Small Molecule Library for Inhibitors.
Although the use of computational modeling is a state-of-the-art method for identifying lead compounds, it is not without its limitations. Thus, we will also screen an actual library of 20,000 small molecules manufactured by the Chembridge Corporation (San Diego, Calif.). The library consists of handcrafted drug-like organic molecules with molecular weights in a range of 25-550, which are soluble in DMSO at concentrations ranging from 10-20 mM. The structure and purity (>95%) of these compounds have been validated by NMR. The library is formatted in a 96 well plate for high throughput screening using instrumentation made available through the OCCC (under supervision of the Landry laboratory) and includes a robot plate reader (FLexStation II 384, Molecular Devices, Sunnyvale, Calif.), an 8-tip robotic pipettor (Multiprobe II Plus, Perkin Elmer, Shelton, Conn.), a 96-tip robotic pipettor (Mintrak, Perkin Elmer), and an automated 96 well plate washer (Perkin Elmer).
An ELISA based system will be used to screen for compounds that may inhibit the interaction between GPIb alpha and the VWF-A1 domain. Enzyme-Linked Immunosorbent Assay (ELISA) methods are immunoassay techniques used for detection or quantification of a substance. An example of this assay is demonstrated in FIG. 45A, where an antibody conjugated with horseradish peroxidase (HRP) was used to identify the presence of VWF. Depending on the substrate added, HRP enzyme activity can be detected by either a change in color (chromogenic product) or fluorescence (most sensitive indicator). Schematic representation of the proposed assay system to be used for screening is shown in FIG. 45A.
Assay System:
Recombinant GPIb alpha and VWF-A1 proteins will be generated and purified as described in the attached articles, with the latter containing a 6×His tag. Purified GPIb alpha will be absorbed overnight (4° C.) to PRO-BIND polystyrene 96-well assay plates (Falcon) at 10 μg/ml per well. Plates will be washed and non-specific binding sites blocked by the addition of TENTC buffer (50 mM Tris, 1 mM EDTA, 0.15M NaCl, 0.2% casein, 0.05% Tween 20, pH 8.0) for 1 hour at room temperature. Subsequently, plates will be washed with and resuspended in TBS buffer (50 mM Tris, 150 mM NaCl, pH 8.0) and 1 test compound per well added at a final concentration of 10 μM (final DMSO concentration 0.5%). After 30 min, recombinant His tagged VWF-A1 protein will be added at a 1:1 Molar ratio to that of GPIb alpha and left to incubate for 1 hour before washing with TBS buffer. VWF-A1 bound to surface-immobilized GPIb alpha will be determined by the addition of HRP-conjugated anti-His tag antibody and the A1-antibody conjugate detected by the addition of LumiGlow reagent (KPL, Gaithersburg, Md.). The resulting fluorescence will be quantified by of the number of luminescence emissions per second using a FLexStation II 384 plate reader. A sample will be considered positive when the luminescence (in counts per second) is more than 2 standard deviations above the mean value for negative-controls.
Negative Controls:
Addition of mAb 6D1 to certain wells to prevent VWF-A1 binding to GPIb alpha or no addition of VWF-A1 protein (FIG. 45B). In either case, no significant fluorescence should be detected. Once compounds of interest have been identified, solubility of these molecules will be confirmed to rule out precipitation as the etiology for blocking interactions between GPIb alpha and VWF-A1. In addition, a dose effect curve will also be generated (1 nM to 100 μM) to obtain preliminary information regarding the IC50 of the inhibitor. Lead molecules will then be tested for their ability to limit human platelet interactions with plasma VWF in aggregometry and flow chamber assays as described in preliminary results. Ultimately, the most promising compound will be tested in our humanized mouse model of thrombosis.
Example 7
Effect of Plavix or ReoPro on Human Platelet-Induced Hemostasis in Homozygous VWF1326R>H Mice
To demonstrate the feasibility of our VWF1326R>H mice to identify anti-thrombotic drugs capable of perturbing human platelet function in vivo, we tested the ability of 2 FDA approved drugs, Plavix and ReoPro, to prevent human platelet-induced hemostasis. Plavix is the second most commonly used anti-thrombotic drug that targets one of the ADP receptors (P2Y12) on platelets, causing irreversible inhibition (Hankey et al. Med. J. Aust. 2003; 178:568). ADP is a potent mediator of platelet activation and aggregate formation, and thus considerable effort and funds have been devoted to inhibiting this activation pathway in platelets. Clopidogrel was approved by the FDA in 1997 for clinical use and was found to be of benefit in the secondary prevention of major vascular events in patients with a history of cerebrovascular and coronary artery diseases and major cardiac events post coronary artery stent placement (Gachet et al. Semin. Thromb. Hemost. 2005; 31:162). Disadvantages of this drug are: 1) It must be metabolized in the liver to generate an active metabolite, thus limiting its effectiveness in acute settings, and 2) irreversible inhibition that results in a marked prolongation of bleeding time. Clopidogrel has been shown to reduce thrombus size and delay its formation in mice with a maximal effective dose of 50 mg/kg given the day before and 2 hours prior to experimentation. Homozygous VWF1326R>H mice that received this dosing schema, were unable to produce a hemostatic clot when administered human platelets in contrast to homozygous VWF1326R>H mice that received saline in lieu of drug.
As Plavix can also block the function of the ADP receptor on murine platelets (see FIG. 46A), we also tested the ability of ReoPro to prevent the formation of a hemostatic plug in homozygous VWF1326R>H mice. ReoPro is a Fab fragment of a human-murine chimeric monoclonal antibody that blocks fibrinogen binding to the platelet integrin receptor αIIbβ3, thus limiting thrombus growth (Bennett, J. S. Novel platelet inhibitors. Annu. Rev. Med. 52, 161-184 (2001)). It is currently approved for short-term treatment of patients with acute coronary syndrome that require interventional catheterization. It is administered by intravenously bolus (0.25 mg/kg), followed by an infusion of 0.125 μg/kg/min. This results in >80% αIIbβ3 occupancy, and disrupts platelet function for 24-36 h. It does not bind or disrupt the function of murine αIIbβ3. Administration of ReoPro to homozygous VWF1326R>H mice 5 minutes after the infusion of human platelets, prevented the formation of a hemostatic plug (mean bleeding time 579 sec) (FIG. 46B). By contrast, animals that received a non-function blocking antibody to human αIIbβ3 were able to form a hemostatic plug (mean bleeding 175 sec).
REFERENCES


    1) Jaffe E A, Hoyer L W and Nachman R L Synthesis of antihemophilic factor antigen by cultured human endothelial cells. 1973. J Clin Invest. 52, 2757-2764.
    2) Nachman R L., Levine R, Jaffe E A. Synthesis of factor VIII antigen by cultured guinea-pig megakaryocytes 1977. J Clin Invest. 60, 914-921.
    3) Sporn L A, Chavin S I, Marder V J and Wagner, D D. Biosynthesis of von Willebrand protein by human megakaryocytes. 1985. J Clin Invest. 76, 1102-1106.
    4) Sakariassen K S, Bolhuis P A and Sixma J J. human platelet adhesion to artery subendothelium is mediated by Factor VIII-von Willebrand factor bound to the subendothelium. 1979. Nature 279:636-638.
    5) Meyer D, Baumgartner H R. Role of von Willebrand factor in platelet adhesion to subendothelium. 1983. Br J Haematol. 54:1-9.
    6) Cruz M A, Yuan H, Lee J R, et al. Interaction of von Willebrand factor (vWF) with collagen. 1995. J Biol Chem. 270, 10822-10827.
    7) Handa M, Titani K, Holland L Z, et al. The von Willebrand factor-binding domain of platelet membrane glycoprotein Ib. Characterization by monoclonal antibodies and partial amino acid sequence analysis of proteolytic fragments. 1986. J Biol Chem. 26, 12579-12585.
    8) Murata M, Ware J, and Ruggeri Z M. Site-directed mutagenesis of a soluble recombinant fragment of platelet glycoprotein Ib alpha demonstrating negatively charged residues involved in von Willebrand factor binding. 1991. J Biol Chem. 266, 8149-8155.
    9) Fressinaud E, Baruch, D, Girma J P, et al.: von Willebrand factor-mediate platelet adhesion to collagen involves platelet glycoprotein IIb/IIIa as well as glycoprotein Ib. 1988. J Lab Clin Med 112, 58-67.
    10) Weiss H J, Sussman I I, Hoyer L W. Stabilization of factor VIII in plasma by the von Willebrand factor. 1977. J Clin Invest. 60, 390-404.
    11) Ewenstein B M. von Willebrand's disease. 1997. Ann Rev Med 48, 525-542.
    12) Sadler J E, Matsushita T, Dong Z, Tuley E A, Westfield L A. Molecular mechanism and classification of von Willebrand disease. 1995. Thromb and Haemost 74, 161-166.
    13. Bonthron D T, Handin R I, Kaufman Rj et al. Structure of pre-pro-von Willebrand factor and its expression in heterologous cells. Nature 1986; 324:270-273.
    14. Shelton-Inloes B B, Titani K, Sadler E. cDNA sequences for human von Willebrand factor reveal five types of repeated domains and five possible protein sequence polymorphisms. Biochemistry 1986; 25:3164-3171.
    15. Verweij, C L, Diergaarde P J, Hart, M, et al. Full length von Willebrand factor (vWF) cDNA encodes a highly repetitive protein considerably larger than the mature vWF subunit. EMBO J. 1986; 5:1839-1863.
    16. Mancuso D J, Tuley E A, Westfield L A et al. Structure of the gene for human von Willebrand factor. J Biol Chem. 1989; 264:19514-19527.
    17. Sadler J E, Shelton-Inloes B B, Sorace J M, Harlan J et al. Cloning and characterization of two cDNAs coding for human von Willebrand factor. Proc Natl Acad Sci USA 1985; 82:6394-6398.
    18) CRUZ M A, DIACOVO T G, EMSLEY J, LIDDINGTON R, HANDIN R I. 2000. MAPPING THE GPIB BINDING SITE IN THE VON WILLEBRAND FACTOR A1 DOMAIN. J BIOL CHEM. 275, 19098-19105.
    19) Savage B, Saldivar E, Ruggeri Z M. 1996. Initiation of platelet adhesion by arrest onto fibrinogen or translocation on von Willebrand factor. Cell. 84, 289-297.
    20) Kalafatis M, Takahashi Y, Girma J P, et al. Localization of the collagen-interactive domain of human von Willebrand factor between amino acid residues Gly 911 and Glu 1365. Blood 1987; 70:1577-1583.
    21) Pareti F I, Niiya K, McPherson J M, et al. Isolation and characterization of two domains of human von Willebrand factor that interact with fibillar collagen types I and III. J Biol Chem. 1987; 262:13835-13841.
    22) Roth G J, Titani K, Hoyer L W, et al. Localization of binding sites within human von Willebrand factor for monomeric type III collagen. Biochemistry 1986; 25:8357-8361.
    23) Lankhof H, van Hoeij M, Schiphorst M E, Bracke M et al. A3 domain is essential for interaction of von Willebrand factor with collagen type III. Thromb Haemost. 1996; 75:950-958.
    24) Pareti F I, Fujimura Y, Dent J A, et al. Isolation and characterization of a collagen binding domain in human von Willebrand factor. J Biol Chem. 1986; 261:15310-15315.
    25) Pietu G, Meulien P, Cherel G, et al. Production in Escherichia coli of a biologically active subfragment of von Willebrand factor corresponding to the platelet glycoprotein Ib, collagen, and heparin binding domains. Biochem. Biophys. Res. Comm. 1989; 164:1339-1347.
    26) Meyer D, Fressinaud E, Gaucher C, Lavergne J-M, Hilbert L, Ribba A S et al. Gene defects in 150 unrelated French cases with type 2 von Willebrand disease: from patient to gene. Thromb and Haemost. 1997; 78:451-456.
    27) Ginsburg, D., and J. E. Sadler. 1993. von Willebrand disease: a database of point mutations, insertions, and deletions. For the Consortium on von Willebrand Factor Mutations and Polymorphisms, and the Subcommittee on von Willebrand Factor of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis. Thromb. Haemost. 69:177-184.
    28) Hillery C A, Mancuso D J, Sadler J E, Ponder J W et al. Type 2M von Willebrand disease: F6061 and 1662F mutations in the glycoprotein Ib binding domain selectively impair ristocetin- but not botrocetin-mediated binding of von Willebrand factor to platelets. Blood 1998; 91:1572-15781.
    29) Mancuso D J, Kroner P A, Christopherson P A et al. Type 2M:Milwaukee-1 von Willebrand disease: an in-frame deletion in the Cys509-Cys695 loop of von Willebrand factor A1 domain causes deficient binding of von Willebrand factor to platelets. Blood 1996; 88: 2559-2568.
    30) Ruggeri, Z. M., F. I. Pareti, P. M. Mannucci, N., Ciavarella, and T. S. Zimmerman. 1980.
    Heightened interaction between platelets and factor VIII/von Willebrand factor in a new subtype of von Willebrand's disease. N. Engl. J. Med. 302:1047-1051.
    31) Cooney, K. A., and D. Ginsburg. 1996. Comparative analysis of type 2B von Willebrand disease mutations: implications for the mechanism of von Willebrand factor binding to platelets. Blood 87:2322-2328.
    32) Cruz, M. A., T. G. Diacovo, J. Emsley, R. Liddington, and R. I. Handin. 2000. Mapping the glycoprotein Ib-binding site in the von willebrand factor A1 domain. J Biol Chem. 275:19098-19105.
    33) Huizinga E G, Tsuji S, Romijn R A P, et al. Structures of Glycoprotein Ibα and its complex with von Willebrand Factor A1 domain. Science. 2002; 297:1176-1179.
    34) Federici A B, Bader R, Pagani S, Colibretti M L, De Marco L, and Mannucci P M. Binding of von Willebrand factor to glycoproteins Ib and IIb/IIIa complex: affinity is related to multimeric size. Br J Haematol. 1989; 73:93-99.
    35) Roth G J. Developing relationships: arterial platelet adhesion, glycoprotein Ib, and leucine-rich glycoproteins. Blood. 1991; 77:5-19.
    36) Siedlecki C A, Lestini B J, Kottke-Marchant K K, Eppell S J, Wilson D L, and Marchant R E. Shear-dependent changes in the three-dimensional structure of human von Willebrand factor. Blood 1996; 88:2939-2950.
    37) Ruggeri Z M, and Ware J. The structure and function of von Willebrand factor. Thromb Haemost. 1992; 67:594-599.
    38) Howard M A, and Firkin B G. Ristocetin—a new tool in the investigation of platelet aggregation. Thromb Diath Haemorrh. 1971; 26:362-369.
    39) Read M S, Smith S V, Lamb, M A, and Brinkhous K M. Role of botrocetin in platelet agglutination: formation of an activated complex of botrocetin and von Willebrand factor. Blood 1989; 74:1031-1035
    40) Emsley J, Knight C G, Farndale R W, Barnes M J, and Liddington R C. Structural basis of collagen recognition by integrin alpha2 beta1. Cell 2000; 101:47-56.
    41) CELIKEL R, RUGGERI Z M, AND VARUGHESE K I. VON WILLEBRAND FACTOR CONFORMATION AND ADHESIVE FUNCTION IS MODULATED BY AN INTERNALIZED WATER MOLECULE. NAT STRUCT BIOL. 2000; 7:881-884.
    42) Fukuda K, Doggett T A, Bankston L A, Cruz M A, Diacovo T G, Liddington R C. Structural basis of von Willebrand factor activation by the snake toxin botrocetin. Structure. 2002; 10:943-950.
    43) Sen U, Vasudevan S, Subbarao G, McClintock R A, Celikel R, Ruggeri Z M, Varughese K I. Crystal structure of the von Willebrand factor modulator botrocetin. Biochemistry. 2001; 40:345-352.
    44) Andrews R K, Booth W J, Gorman J J, Castaldi P A, Berndt M C. Purification of botrocetin from Bothrops jararaca venom. Analysis of the botrocetin-mediated interaction between von Willebrand factor and the human platelet membrane glycoprotein Ib-IX complex. Biochemistry. 1989; 28:8317-8326.
    45) Yoshida E, Fujimura Y, Miura S, Sugimoto M, Fukui H, Narita N, Usami Y, Suzuki M, Titani K. Alboaggregin-B and botrocetin, two snake venom proteins with highly homologous amino acid sequences but totally distinct functions on von Willebrand factor binding to platelets. Biochem Biophys Res Commun. 1993; 19:1386-1392.
    46) Italiano J E Jr, Bergmeier W, Tiwari S, Falet H, Hartwig J H, Hoffmeister K M, Andre P, Wagner D D, Shivdasani R A. Mechanisms and implications of platelet discoid shape. Blood. 2003 Jun. 15; 101(12):4789-96. Epub 2003 Feb. 13.
    47) Yoon, B. J., and S. Kim. 1990. A boundary collocation method for the motion of two spheroids in Stokes flow: Hydrodynamic and colloidal interactions. Int. J. Multiphase Flow. 16:639-649.
    48) Chesla S E, Selvaraj P, Zhu C. Measuring two-dimensional receptor-ligand binding kinetics by micropipette. Biophys J. 1998; 75:1553-1572.
    49) Yamamoto H, Vreys I, Stassen J M et al. Antagonism of vWF inhibits both injury induced arterial and venous thrombosis in the hamster. Thromb Haemost 1998: 79:202-210.
    50) Azzam K, Garfinkel L I, Bal dit Sollier C et al. Antithrombotic effect of a recombinant von Willebrand factor, VCL, on nitrogen laser-induced thrombus formation in guinea pig mesenteric arteries. Thromb Haemost 1995: 73:318-323.
    51) Denis C, Methia N, Frenette P S, et al. A mouse model of severe von Willebrand disease:defects in hemostasis and thrombosis. Proc Natl Acad Sci, USA. 1998; 95:9524-9529.
    52) Ware J, Russell S, Ruggeri Z M. Generation and rescue of a murine model of platelet dysfunction: the Bernard-Soulier syndrome. Proc Natl Acad Sci USA. 2000 Mar. 14; 97(6):2803-8.
    53) Miura, S., C. Q. Li, Z. Cao, H. Wang, M. R. Wardell, and J. E. Sadler. 2000. Interaction of von Willebrand factor domain A1 with platelet glycoprotein Ib alpha-(1-289). Slow intrinsic binding kinetics mediate rapid platelet adhesion. J. Biol. Chem. 275:7539-7546.
    54) KOHLER G, MILSTEIN C: CONTINUOUS CULTURES OF FUSED CELLS SECRETING ANTIBODY OF PREDEFINED SPECIFICITY. NATURE 1975; 256:495-497.
    55) Alon R, Chen S, Fuhlbrigge R, Puri K D, Springer T A. The kinetics and shear threshold of transient and rolling interactions of L-selectin with its ligand on leukocytes. Proc Natl Acad Sci USA. 1998; 95:11631-1166.
    56) Gillespie, D. T. 1976. A general method for numerically simulating the stochastic time evolution of coupled chemical reactions. J. Comput. Phys. 22:403-434.
    57) Tees, D. F. J., and H. L. Goldsmith. 1996. Kinetics and locus of failure of receptor-ligand-mediated adhesion between latex spheres. I. Protein-carbohydrate bond. Biophys. J. 71:1102-1114.
    58) Marshall B T, Long M, Piper J W, Yago T, McEver R P, Zhu C. Direct observation of catch bonds involving cell-adhesion molecules. Nature. 2003; 423:190-193.
    59) CHEN S, SPRINGER T A. SELECTIN RECEPTOR-LIGAND BONDS: FORMATION LIMITED BY SHEAR RATE AND DISSOCIATION GOVERNED BY THE BELL MODEL. PROC NATL ACAD SCI USA. 2001; 98:950-955.
    60) Greenberg A W, Brunk D K, Hammer D A. Cell-free rolling mediated by L-selectin and sialyl Lewis (x) reveals the shear threshold effect. Biophys J. 2002; 79:2391-2402.
    61) Chen S and Springer T A. An automatic braking system that stabilizes leukocyte rolling by an increase in selectin bond number with shear. 1999. J Cell Biol. 144:185-200.
    62) Cooney K A, Ginsburg D. Comparative analysis of type 2b von Willebrand disease mutations: implications for the mechanism of von Willebrand factor binding to platelets. Blood. 1996; 87:2322-2328.
    63) Merkel R, Nassoy P, Leung A, Ritchie K, Evans E. Energy landscapes of receptor-ligand bonds explored with dynamic force spectroscopy. Nature. 1999 Jan. 7; 397(6714):50-3.
    64) Evans E, Leung A, Hammer D, Simon S. Chemically distinct transition states govern rapid dissociation of single L-selectin bonds under force. Proc Natl Acad Sci USA. 2001 Mar. 27; 98(7):3784-9. Epub 2001 Mar. 13.
    65) Simson D A, Ziemann F, Strigl M, Merkel R. Micropipet-based pico force transducer: in depth analysis and experimental verification. Biophys J. 1998 April; 74(4):2080-8.
    66) Evans E, Berk D, Leung A. Detachment of agglutinin-bonded red blood cells. I. Forces to rupture molecular-point attachments, Biophys J. 1991 April; 59(4):838-48.
    67) Ribba A S, Voorberg J, Meyer D, Pannekoek H, Pietu G. Related Articles, Links Free Full Text Characterization of recombinant von Willebrand factor corresponding to mutations in type IIA and type IIB von Willebrand disease. J Biol Chem. 1992; 267:23209-23215.
    68) Andre P, Prasad K S, Denis C V, He M, Papalia J M, Hynes R O, Phillips D R, Wagner D D. CD40L stabilizes arterial thrombi by a beta3 integrin-dependent mechanism. Nat Med. 2002 March; 8(3):247-52.
    69) Coxon, A., P. Rieu, F. J. Barkalow, S. Askari, A. H. Sharpe, U. H. von Andrian, M. A. Arnaout, and T. N. Mayadas. 1996. A novel role for the beta 2 integrin CD11b/CD18 in neutrophil apoptosis: a homeostatic mechanism in inflammation. Immunity. 5:653-666.
    70) Ni H, Ramakrishnan V, Ruggeri Z M, Papalia J M, Phillips D R, Wagner D D. Increased thrombogenesis and embolus formation in mice lacking glycoprotein V Blood. 2001 Jul. 15; 98(2):368-73
    71) King M R, Hammer D A. Multiparticle adhesive dynamics: hydrodynamic recruitment of rolling leukocytes. Proc Natl Acad Sci USA. 2001 Dec. 18; 98(26):14919-24.


Example 8
Determining the Efficacy of Anti-Platelet Drugs Administered to Patients by Studying the Ability of Platelets Harvested from Patients on Therapies in the VWF1326R>H Mouse
Intravital microscopic study was carried out to evaluate the ability of the VWF1326R>H mouse to determine the efficacy of anti-platelet therapies given to patients at risk or with active cardiovascular disease. The typical prophylactic dose of aspirin (ASA) of 81 mg did not prevent laser-injury induced human platelet thrombus formation in the genetically modified animal while increasing the daily dose to 162 mg was preventative (FIG. 47). Similarly, platelets administered from a patient on 81 mg of ASA and 75 mg Plavix also prevented thrombus formation.

## Claims

1. A transgenic mouse expressing a von Willebrand Factor A1 protein comprising an amino acid sequence at least 85% identical to SEQ ID NO: 2, wherein said von Willebrand Factor A1 protein comprises an arginine to histidine substitution at amino acid position 67.

2. The transgenic mouse of claim 1, wherein said von Willebrand Factor A1 protein is at least 90% identical to SEQ ID NO: 2 and wherein said von Willebrand Factor A1 protein comprises an arginine to histidine substitution at amino acid position 67.

3. The transgenic mouse of claim 1, wherein said von Willebrand Factor A1 protein is at least 95% identical to SEQ ID NO: 2, and wherein said von Willebrand Factor A1 protein comprises an arginine to histidine substitution at amino acid position 67.

4. The transgenic mouse of claim 1, wherein said von Willebrand Factor A1 protein is at least 99% identical to SEQ ID NO: 2 and wherein said von Willebrand Factor A1 protein comprises an arginine to histidine substitution at amino acid position 67.