Abstract:
The invention relates to the adhesion of platelet GpIbα to strand β3 of domain A1 of von Willebrand factor (vWF), the strand β3 comprising amino acid residues at amino acid position 560-566 and/or a functional part or equivalent thereof, the platelet GpIbα, the GpIbα region comprising an amino acid sequence corresponding to a beta-switch loop of platelet GpIbα, comprising amino acid residues at amino acid position 227-242 and/or a functional part or equivalent thereof. The invention provides a method of interfering with adhesion of blood platelets to vWF that includes modulating adhesion. The invention further provides proteinaceous compounds, antibodies, medicaments and pharmaceutical compositions to that end. The invention also provides means and methods to increase platelet adhesion by topical application of a compound increasing platelet adhesion.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation of PCT International Patent Application No. PCT/NL2003/000564, filed on Aug. 6, 2003, designating the United States of America, and published, in English, as PCT International Publication No. WO 2004/015425 A1 on Feb. 19, 2004, the contents of the entirety of which is incorporated by this reference, which application claims priority to European Patent Application Serial No. 02078277.7 filed Aug. 7, 2002. 
    
    
     TECHNICAL FIELD 
     The invention relates to the field of medicine, more specifically to the field of hematology, and even more specifically to platelet adhesion to von Willebrand Factor and the treatment of diseases in which platelet adhesion and blood clotting is involved. 
     BACKGROUND 
     Acute Coronary Syndromes (ACS) are a major cause of mortality in Western countries. The key event in an overwhelming number of these cases is platelet thrombus formation. Consequently, there is strong interest in the development of safe and effective anti-thrombotic agents. Numerous agents have been developed that target the platelet activation cascade or prevent fibrinogen-mediated platelet-platelet aggregation (GPIIb/IIIa inhibitors). These approaches have produced some encouraging results. However, direct inhibition of the very first step in thrombus formation, namely, formation of the initial platelet monolayer, has received comparatively little attention to date. Platelet rolling over exposed subendothelium at sites of vascular injury is a crucial initiating step in hemostasis and thrombosis. This process depends critically on the interaction of platelet-receptor glycoprotein Ibα (GpIbα) and plasma-protein von Willebrand Factor (vWF). GpIbα not only plays a role in the adhesion to vWF, but also triggers the platelet into an activated state. 
     DISCLOSURE OF THE INVENTION 
     This application has identified and characterized that a part of (GpIbα) forms a flexible and surface-exposed loop aa227-242, in this application designated as a “Beta-switch.” This peptide, formerly thought to be an a chain, surprisingly, has been found to control platelet binding to vWf (e.g., vWF precursor protein of GenBank Accession Number NP 000543, which is incorporated herein by reference; and mature vWF shown in SEQ ID NO:1) by controlling the binding of platelet GpIbα (e.g., GpIbα precursor protein of GenBank Accession Number NP — 000164, which is incorporated herein by reference; and mature GpIbα shown in SEQ ID NO:2), aa227 to 242 region (e.g., SEQ ID NO:2) to the Beta3 strand and its preceding turn region of the A1 region of vWF protein (aa560-566, e.g., SEQ ID NO:1) to form a continuous Beta-sheet shared between the two molecules. In this application, the Beta3 strand, comprising aa562-566 and its preceding turn region of the A1 region of vWF protein comprising aa560-562, together will be named the Beta3 strand. Binding to vWF is essential for subsequent platelet adhesion to the subendothelial layer or to damaged endothelial cells and the following platelet activation. Thus, the flexible and surface-exposed loop aa227-242, in this application designated as a “Beta-switch” and/or a functional derivative thereof, is an efficient controller of platelet adhesion and platelet activation and thrombus formation in vivo. 
     This application shows structures of the human GpIbα N-terminal domain and its complex with human vWF domain A1. The structure of GpIbα, containing eight leucine-rich repeats flanked by capping sequences characteristic of extra-cellular proteins with these repeats, wraps around one side of vWF-A1 providing a large contact area. Perturbing this in vivo-modulated interaction explains four types of congenital bleeding disorders caused by mutations in either GpIbα or vWF-A1. 
     These findings provide a structural basis for understanding this critical interaction in platelet rolling, which is relevant for the development of novel anti-thrombotics. The ability to inhibit primary platelet adhesion potentially presents significant advantages over alternative thrombotic therapies because the former targets the very first step of platelet aggregation and thrombus formation. 
     Identification of the function of the peptide loop aa227-242 of GpIbα as a Beta-switch and adhesion site to strand β3 of domain A1 of vWF enables a person skilled in the art to develop a method to interfere with platelet adhesion comprising modulating the adhesion of amino acid residues at amino acid position 560-566 of strand β3 of domain A1 of von Willebrand factor (vWF) to amino acid residues at amino acid position 227-242 of a region of platelet GpIbα, the GpIbα region corresponding to a beta-switch loop of platelet GpIbα. In a preferred embodiment of the invention, the GpIbα region comprises amino acid residues at amino acid position 227-242. 
     In a more preferred embodiment, the GpIbα region comprises amino acid residues at amino acid position 200-300. 
     This interfering can be both a decrease or an increase of platelet adhesion. Decreasing the platelet adhesion will result in a decreased tendency of platelets to stick to vessel walls and damaged endothelium, a decreased thrombus formation, and dissolving of an existing thrombus. These features are very helpful for preventing and curing acute coronary syndromes, as well as other diseases wherein platelet adhesion and thrombus formation play a role. 
     Increasing the platelet adhesion will result in more effective stopping of bleeding from wounds or cuts and can result in a treatment for certain bleeding disorders. 
     Now that the specific site of adhesion between vWF and platelet GpIbα is known, it is clear to any person skilled in the art to select or make and test compounds that block this adhesion. 
     In one embodiment of the invention, the compound that interferes with the adhesion of strand β3 of vWF to a region of platelet GpIbα may comprise a chemical or proteinaceous compound capable of interfering with the binding of the platelet GpIbα peptide to the region of vWF. The compound also comprises a fusion protein, at least containing part of amino acids 560-566 of strand β3 of domain A1 of vWF, or of amino acids 227-242 of platelet GpIbα, or both. 
     In a preferred embodiment of the invention, the compound comprises an amino acid sequence corresponding to strand β3 of domain A1 of vWF comprising amino acid residues at amino acid position 560-566 and/or a functional part or equivalent thereof. 
     In another preferred embodiment of the invention, the compound comprises an amino acid sequence corresponding to a beta-switch loop of platelet GpIbα comprising amino acid residues at amino acid position 227-242 and/or a functional part or equivalent thereof. A “functional part of a protein” is defined as a part that has the same properties in kind, not necessarily in amount. By “properties of amino acid position 227-242 of platelet GpIbα” is meant the flexible and surface-exposed loop called the Beta-switch and the adhesion site of platelet GpIbα to strand β3 of domain A1 of vWF. A “functional equivalent of a protein” is defined as a protein that has been altered such that the properties of the molecule are essentially the same in kind, not necessarily in amount. An equivalent can be provided in many ways, for instance, through conservative amino acid substitution. 
     A person skilled in the art is well able to generate analogous compounds or derivatives of a protein. This can, for instance, be done through screening of a peptide library. Such an analogue has essentially the same properties of the protein in kind, not necessarily in amount. In this application, an equivalent also includes analogous compounds. Derivatives may include chemical modifications of biological molecules or equivalents thereof as defined above. Such chemical modifications may be introduced, for instance, to provide an (additional) group for coupling vWF and/or platelet GpIbα or to prolong the circulation time in the body. A proteinaceous molecule comprises at least two amino acids linked to each other with a peptide bond. It preferably further comprises additional amino acids linked with peptide bonds. A proteinaceous molecule may comprise a modification such as typically added in post-translational modification reactions of proteins. Non-limiting examples are glycosylation and/or myristillization. One or more of the amino acids may also comprise a chemical modification. 
     The adhesion of platelet GpIbα to vWF can also be interfered by antibodies specifically recognizing either the strand β3 of domain A1 of vWF comprising amino acid residues at amino acid position 560-566 or the beta-switch loop of platelet GpIbα comprising amino acid residues at amino acid position 227-242. In a preferred embodiment of the invention, the compound comprises an antibody and/or a functional part or equivalent thereof, specifically recognizing the region of platelet GpIbα and/or the region of strand β3 of domain A1 of vWF. Above-mentioned compounds can be administered to individuals for interfering with platelet adhesion and/or thrombus formation. It may be favorable to couple the compound to another compound, which is capable of prolonging the circulation time. Therefore, this application teaches the use of the compound either directly and/or linked to another chemical and/or a proteinaceous compound, wherein the second compound modulates the circulation time of the first compound. The second compound comprises any chemical or proteinaceous substance that is known in the art to extend the circulation time of the first compound. 
     In another embodiment, the compound is used for the preparation of a medicament for the treatment or prevention of a disease in which platelet adhesion and/or thrombus formation is involved. The medicament can, for example, be administered by parenteral inoculation or by oral administration in the form of tablets or pills or as a potion. This medicament may be combined with other substances to optimize the action, for example by combination with a slow release compound, and the shelf-life of the medicament, for example, by adding stabilizing agents, and to facilitate the administration. 
     In another embodiment, this application teaches a pharmaceutical composition for inhibiting platelet adhesion and/or thrombus formation comprising the compound further comprising a suitable carrier or solvent. 
     In yet another embodiment of the invention, the medicament or pharmaceutical composition is used to modulate platelet adhesion and/or thrombus formation. 
     Because this application teaches the specific adhesion sites of strand β3 of domain A1 of vWF comprising amino acid residues at amino acid position 560-566 and of a beta-switch loop of platelet GpIbα comprising amino acid residues at amino acid position 227-242, antibodies can be produced against these sites that interfere with platelet adhesion. Therefore, this application teaches an antibody and/or a functional part or equivalent thereof, specifically recognizing a region of platelet GpIbα according to claim  1 , capable of interfering with platelet adhesion. And, in another embodiment, this application teaches an antibody and/or a functional part or equivalent thereof, specifically recognizing strand β3 of domain A1 of vWF capable of interfering with platelet adhesion. In yet another embodiment, this application teaches the use of antibodies and/or a functional part or equivalent thereof, for the preparation of a medicament for the treatment of a disease in which platelet adhesion and/or blood clotting is involved. 
     The amino acid sequence of the beta-switch loop of GpIbα and the amino acid sequence of strand β3 of domain A1 of vWF can be built into a vector using standard techniques. Therefore, this application teaches a vector which is capable of effecting the expression of a peptide with an amino acid sequence corresponding to a beta-switch loop of platelet GpIbα comprising amino acid residues at amino acid position 227-242 and/or a functional part or equivalent thereof, in an appropriate environment. And, in another embodiment, this application teaches a vector which is capable of effecting the expression of a peptide with an amino acid sequence corresponding to strand β3 of domain A1 of vWF comprising amino acid residues at amino acid position 560-566 and/or a functional part or equivalent thereof, in an appropriate environment. Because this application teaches how to produce a medicament, a pharmaceutical composition, antibodies, and/or vectors as mentioned above, it also teaches a method of treatment or prevention of a disease condition in which platelet adhesion is involved comprising administering the medicament, the pharmaceutical composition, the antibody, and/or the vector. 
     As has been mentioned above, a peptide or a proteinaceous compound with an amino acid sequence corresponding to strand β3 of domain A1 of vWF comprising amino acid residues at amino acid position 560-566 and/or a functional part or equivalent thereof, capable of binding platelet GpIbα, can be used to locally increase the adhesion of platelets and the formation of a thrombus. Therefore, this application also teaches a method to enhance platelet adhesion comprising local application of a peptide or a proteinaceous compound with an amino acid sequence corresponding to strand β3 of domain A1 of vWF comprising amino acid residues at amino acid position 560-566 and/or a functional part or equivalent thereof, capable of binding platelet GpIbα. The compound may be bound to a matrix like, for example, a wound dressing or a bandage, or may be combined with an ointment or a glue or another suitable carrier or solvent. In another embodiment of this invention, the peptide or a proteinaceous compound with an amino acid, sequence corresponding to strand β3 of domain A1 of vWF comprising amino acid residues at amino acid position 560-566 and/or a functional part or equivalent thereof, can be used for the manufacturing of a topical medicament for local enhancement of platelet adhesion and/or thrombus formation and for the manufacturing of a pharmaceutical composition for local enhancement of platelet adhesion and/or thrombus formation comprising a peptide or a proteinaceous compound with an amino acid sequence corresponding to strand β3 of domain A1 of vWF comprising amino acid residues at amino acid position 560-566 and/or a functional part or equivalent thereof, and a suitable carrier or solvent. Therefore, this application also teaches such a pharmaceutical composition for local enhancement of platelet adhesion and/or thrombus formation comprising a peptide or a proteinaceous compound with an amino acid sequence corresponding to strand β3 of domain A1 of vWF comprising amino acid residues at amino acid position 560-566 and/or a functional part or equivalent thereof, and a suitable carrier or solvent. This application also teaches providing the medicament and/or the pharmaceutical composition to an individual, for example, suffering from a bleeding disorder, such as, but not limited to, von Willebrand disease type 2M, as a method of treatment or prevention of a disease condition in which platelet adhesion is decreased. 
    
    
     
       BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS 
         FIG. 1  depicts structures of the vWF-binding domain of GpIbα and the complex of GpIbα with the A1 domain of vWF. Panel A is a ribbon representation of GpIbα. The N-terminal β-hairpin, called “β-finger,” is colored blue, the eight leucine-rich repeats are green, and the C-terminal flanking region is red. Disulphide bridges are indicated in yellow ball-and-stick representation. The C-terminal flank contains an exposed loop (residues 227-242) called “β-switch,” which is disordered in the uncomplexed structure. Panel B is a stereo view of a ribbon representation of the complex GpIbα-A1. GpIbα is shown in green and A1 in pale blue with mutations GpIbα M239V and A1 R543Q shown in red ball-and-stick representation. The structure reveals an extended site of interactions in which the leucine-rich repeat curve folds around one side of the A1 domain. In the complex, the β-switch of GpIbα adopts a β3-hairpin structure that aligns with the central β-sheet of A1. Panel C is a representation of residues at the A1-GpIbα interface. Residues involved in inter-molecular contacts shorter than 4.0 Å are shown in stick representation. Residues of GpIbα from leucine-rich repeats 4 to 8 (Val104, Glu128, Lys152, Asp175, Thr176, Pro198 and Phe199, for example, see SEQ ID NO:2) and the C-terminal flank (Glu225, Asn226, Tyr228 and Ser241, for example, see SEQ ID NO:2) form a continuous surface that interacts with A1 residues Glu596, Lys599, Phe603, Gln604 and Ser607 (for example, see SEQ ID NO:1) located in helix α3 and loop α3β4. Residues Lys8, Ser11, His12, Glu14 and Asn16 of the β-finger and His37 (e.g., SEQ ID NO:2) of Irr-1 of GpIbα interact with residues Trp550, Arg571 and Glu613 (e.g., SEQ ID NO:1) located in loops α1β2, β3α2, and α3α4 in A1. Leucine-rich repeats 1 to 4 of GpIbα shows no, or few, short contacts with A1. Panel D is a close-up of the β-switch and its interaction with strand β3 of A1. Main-chain hydrogen bonds are shown by dotted lines. Amino acids known to have gain-of-function mutations related to platelet-type von Willebrand disease have red labels and likely induce β-hairpin formation in the β-switch of GpIbα. 
         FIG. 2  shows loss- and gain-of-function mutations in A1 and GpIbα. Mutations shown have been identified by site-directed mutagenesis or were detected in patients suffering from four different bleeding disorders. Panel a shows loss-of-function mutations. Mutations in GpIbα causing Bernard-Soulier syndrome are shown in purple. Mutations in A1 related to type 2M von Willebrand disease are shown in red ball-and-stick representation. Panel b depicts gain-of-function mutations. Mutations in GpIbα causing a platelet-type von Willebrand disease phenotype are shown in purple. Mutations in A1 related to type 2B von Willebrand disease are shown in red ball-and-stick representation. The A1-domain and GpIbα are shown in blue and green ribbon representation, respectively. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The invention is further explained in the following example. 
     EXAMPLE 
     Insight into Platelet Rolling by Crystal Structures of Glycoprotein Ibα and von Willebrand Factor A1 Domain 
     The interaction of platelet-receptor glycoprotein Ibα (GpIbα) and immobilized von Willebrand Factor (vWF) at sites of vascular damage mediates rolling of platelets 1 . Transient interactions of the platelet with vWF greatly reduces platelet velocity and prolongs the contact time with reactive components of the cell matrix necessary for platelet-activation and integrin-mediated firm attachment. In rapidly flowing blood, this vWF-mediated rolling is essential for platelet adhesion. 2    
     GpIbα is the central component of a receptor complex consisting of glycoproteins Ibα, Ibβ, IX and V. It anchors the complex to the cytoskeleton and contains the vWF-binding function in its ˜290 N-terminal residues. The vWF-binding site is exposed well above the platelet surface, being connected to a ˜45 nm long highly O-glycosylated stalk. 3  The ˜250-kDa vWF protein forms large disulfide-bonded multimers with molecular weights of up to 10 MDa. It is found in plasma and the subendothelial cell matrix and is released from storage granules when platelets and endothelial cells are activated. A vWF multimer acts as bridging ligand between platelets and the cell matrix through collagen binding by its A3 domain and binding to GpIbα by its A1 domain. 4    
     and platelet GpIbα coexist in the circulation and interact with each other when vWF has been bound to a surface. A disturbed balance with deleterious consequences is apparent in a number of congenital bleeding disorders. 5,6  In von Willebrand disease type 2M and the Bernard-Soulier syndrome, loss-of-function mutations in the vWF-A1 domain and GpIbα, respectively, lead to reduced affinity and cause a bleeding tendency. In von Willebrand disease type 2B and platelet-type pseudo-von Willebrand disease, gain-of-function mutations in vWF and GpIbα, respectively, cause an increased affinity sufficient to support interaction between soluble vWF and platelets, leading to intravascular platelet clumping, reduced platelet counts, and a resulting bleeding tendency. Shear stress modulates the GpIbα-vWF affinity; 7  vigorous stirring can induce vWF-mediated platelet aggregation. In thrombosis, shear stress-induced interaction of GpIbα and vWF occurs in arteries occluded by atherosclerotic plaque. The precise interactions between GpIbα and A1 of vWF, the molecular basis of the bleeding disorders, and the mechanism of activation are poorly understood. Crystal structures of the N-terminal domain of GpIbα (residues 1-290) and its complex with the vWF-A1 domain (residues 498-705), revealing key interactions in these processes, is presented herein. 
     Structure of GpIbα 
     The von Willebrand factor-binding domain of GpIbα displays an elongated curved shape ( FIG. 1 , Panel A) that is typical for proteins containing leucine-rich repeats. The structure shows, for the first time, the flanking regions conserved among numerous extracellular proteins, including the other members of the GpIb-IX-V complex. The central region of the molecule consists of eight short leucine-rich repeats of which seven were predicted based on the amino acid sequence. At the N-terminal flank, the leucine-rich repeats are preceded by a 14-residue β-hairpin delimited by a conserved disulphide bond between Cys4 and Cys17. The tip of the β-hairpin, which we refer to as β-finger, protrudes from the protein surface and is disordered in one of the two molecules in the asymmetric unit. At the C-terminal flank, up to residue 267, the leucine-rich repeats are followed by a region containing a 9-residue α-helix and four short 3 10 -helices. The subsequent anionic region of GpIbα was invisible in the electron density and has not been modeled. Conserved cysteine residues in the C-terminal flank at positions 209 and 211 form disulphide bonds to Cys248 and Cys264, respectively, stabilizing the observed irregular fold. Residues 227 to 242 project from the concave face of the molecule forming a highly flexible loop that shows disorder in both molecules in the asymmetric unit. Sequence alignment of C-terminal flanking regions shows that the protruding loop is not a conserved feature in this domain family. Remarkably, it is this flexible protruding loop that contains the gain-of-function mutations causing platelet-type pseudo-von Willebrand disease. 
     Structure of the GpIbα-A1 Complex 
     The structure of GpIbα in complex with vWF-A1 using mutant proteins, related to platelet-type pseudo- and type 2B von Willebrand diseases, which increase the affinity of complex formation, was solved. Individually, mutations GpIbα M239V and vWF-A1 R543Q increase the affinity by a factor of 3.0 and 2.5, respectively. Together, these mutations increase the affinity 5.7-fold, yielding a Kd of 5.8 nM (Biacore binding data are given in Table 2). 
     In the complex, the A1 domain fits into the concave curve of GpIbα ( FIG. 1 , Panel B), burying a solvent-accessible surface of ˜1,900 Å. 2  The N-terminal β-hairpin, the leucine-rich repeat region, and the C-terminal flank of GpIbα all interact with A1 and define an extended but discontinuous binding site that involves residues close to the top face and on the bottom face of the A1 domain. As for the native structure of GpIbα, electron density for the anionic region of GpIbα in the complex was not observed. This is surprising in view of the role ascribed to the anionic region in vWF-binding, 8  but is consistent with our observation that the anionic region has no significant effect on Kds (Table 2). 
     The flexible and surface-exposed loop 227-242 in GpIbα, which contains mutation M239V, undergoes a remarkable conformational change upon complex formation. In the complex this loop, that is called β-switch, forms a 16-residue β-hairpin that extends from residues 227 to 242. It aligns with strand β3 of A1 (residues 562-566), thus forming a continuous β-sheet shared between the two molecules ( FIG. 1 , Panel D). Interactions between the β-switch and strand β3 of A1 are predominantly backbone-backbone in nature. The mutated residue Val239 of GpIbα is located in the β-switch strand that directly hydrogen bonds to β3 of A1. Its side chain has hydrophobic contacts with residues Phe199 and Phe201 of GpIbα and Tyr600 of helix α3 in A1. 
     Residues of the GpIbα C-terminal flank and leucine-rich repeats Irr-8 to Irr-6 form a continuous surface that interacts tightly with A1 helix α3 and loop α3β4 ( FIG. 1 , Panel C). Glu596 and Lys599 of helix α3 of A1 are indispensable for GpIbα binding 9,10  and are engaged in extensive interactions. From Irr-5 to Irr-1, the interactions between the molecules peter out. In fact, A1 interacts only with a rim of the leucine-rich repeat concave face made up of residues that are located in the first position of the β-strand or immediately N-terminal to the P-strand in Irr-4 to Irr-8. At the bottom, the N-terminal β-finger of GpIbα regains contact with A1 near its N- and C-terminus and contacts residues in loops α1β2, β3α2, and α3β4. Compared to the free structure of GpIbα, the β-finger has made a small relative shift of 2 Å away from the A1 domain. Remarkably, residues Lys549, Trp550 and Arg571 are disordered at this interaction site showing reduced electron density for side chains. The mutation R543Q in A1 is located 20 Å away from the interaction region with GpIbα. It is part of loop α1β2 that in other structures of A1 interacts with the N-terminal region of the A1 domain. 11    
     Molecular Basis of Congenital Bleeding Disorders 
       FIG. 2 , Panel A, shows point mutations associated with the Bernard-Soulier syndrome and von Willebrand type 2M diseases, which strongly reduce the interaction between GpIbα and A1, yielding a loss of function. Mutations in GpIbα, L57F, C65R, L129P and a deletion of Leu179, occur at buried sites critical for the structural integrity of the leucine-rich repeats. Mutation C209S prevents the formation of a conserved disulphide bond in the C-terminal flanking region. Mutation A156V occurs in Irr-6 at the concave face of the protein. Replacement by valine at this site may not be tolerated because of tight packing interactions with neighboring residues (Ala156 is not solvent-accessible) and could affect the conformation of nearby residue Lys159 and the β-switch that interact with A1. 
     Loss-of-function mutations in A1, detected in von Willebrand disease type 2M patients or identified by site-directed mutagenesis, 9,10,12-14  are numerous and scattered throughout the A1 domain. Interpretation of the site-directed mutagenesis data is complicated by differences in experimental procedures, i.e., the use of single-domain A1 vs. full-length vWF and the use of the artificial activators ristocetin and botrocetin. Sixteen loss-of-function mutations involve residues in or directly next to the GpIbα binding site, whereas ten occur at buried positions inside the A1 domain and are likely to disrupt the A1 structure or induce conformational changes to A1 incompatible with binding to GpIbα. The remaining 14 mutations are at the surface but outside the observed GpIbα binding site. 
     In platelet-type pseudo-von Willebrand disease, mutations in GpIbα enhance the interaction with A1 leading to a gain-of-function phenotype. The five known gain-of-function mutations, G233V and M239V detected in patients, 15,16  and V234G, D235V and K237V identified by site-directed mutagenesis, 17,18  occur in the β-switch loop (see  FIG. 1 , Panel D). Four of these mutations are known to stabilize β-hairpin structures 19  by introducing a Cβ-branched residue in the strands (G233V, M239V and K237V) or a glycine residue in the tight turn (V234G). Kinetic analysis shows that mutation M239V yields a three-fold increase in the association rate and hardly affects the dissociation rate, which is consistent with stabilization of the β-hairpin priming the mutant GpIbα for A1 binding. Unclear at this stage is how D235V, at the second position in the tight turn, induces β-hairpin formation and enhancement of the binding affinity. However, all five mutations support an indirect mechanism by influencing the conformation, because the side chains have either no or few direct contacts to A1 in the complex (see  FIG. 1 , Panel D). Other valine substitutions in this region, K231V, Q232V, A238V and T240V reduce binding affinity. 18  These may be explained in part by steric hindrance (Q232V and A238V) and loss of a hydrogen bond with Asp560 of A1 (T240V). Altogether, it may be concluded that the conformational equilibrium of the β-switch of GpIbα is a critical factor in the precisely balanced affinity of the interacting partners. 
     The von Willebrand disease type 2B gain-of-function mutations in A1 cluster on one side of the central β-sheet at the bottom face of the domain (see  FIG. 2 , Panel b). Type 2B mutation R543Q that was used in the structure determination of the complex causes a 2.5-fold decrease in Kd and affects both the association and dissociation rate. The wild-type A1 domain has a Kd of 33 nM and binds GpIbα much stronger than multimeric vWF, for which no binding up to a concentration of 150 nM was detected (data not shown). This indicates that important structural elements required for keeping A1 in a low-affinity state are not present in the isolated domain used for crystallization. Some of the type 2B mutations (K549A, W550C) are close to the interaction site with the β-finger of GpIbα, but most are far from the interaction site and appear involved in interactions with the N- and C-terminal flanking peptides of the A1 domain. Comparison of A1 (R543Q) of the GpIbα A1 complex with wt-A1 structures 11,20  shows several differences in the region of type 2B mutations. However, the interpretation of these differences is complicated by extensive crystal contacts in this area. Differences involve N-terminal residues 505-511 and C-terminal residues 694-696 and 700-703, including disulphide bond 509-695 that links the N- and C-terminal regions and shifts by about 2.5 Å. The conformation of the main chain of residues 544-551 is largely unchanged, but this loop appears more flexible in the complex with poorly resolved electron density for the side chains. The conformational differences observed could represent activation of the A1 domain towards GpIbα binding. 
     Implications for Rolling Interactions 
     Tethering and rolling in platelet adhesion to sites of vascular damage is achieved by extended interactions between one side of the globular A1-domain of vWF and the concave face of the GpIbα N-terminal domain. Two contact sites appear critical in achieving a fine-tuned balance between premature vWF-mediated platelet aggregation in the circulation and lack of binding to immobilized vWF blocking efficient platelet adhesion. The β-switch of GpIbα forms a β-hairpin upon complex formation that aligns with the central β-sheet of vWF-A1. Mutations in the β-switch that increase the β-sheet propensity disturb the balance in the direction of enhanced binding, causing platelet-type pseudo-von Willebrand disease. The N-terminal β-finger of GpIbα contacts A1 on the lower face of the domain, where the gain-of-function mutations related to type 2B von Willebrand disease are clustered, which likely influence the interactions between the A1 domain and its N- and C-terminal flanking peptides. It is possible that shear stress affects this site of A1 in immobilized vWF causing activation in the platelet adhesion process. For treating or preventing arterial thrombosis, the interactions between A1 and GpIbα must be destabilized. The data indicates molecular regions that can be targeted for development of intervening molecules. 
     Methods 
     Protein Expression and Purification 
     GpIbα residues 1 to 269 and 1 to 290 preceded by the signal peptide and fused to a C-terminal (His) 6  or Arg-(His) 6  sequence, respectively, were cloned into expression vector pCDNA3.1. The QuickChange™ kit from Stratagene was used to introduce mutations N21Q and N159Q, removing two N-glycosylation sites and mutation M239V, a platelet-type von Willebrand disease mutation. Proteins were expressed in stable BHK cell lines. BHK cells were cultured in Dulbecco&#39;s MEM/F-12 Ham medium containing 5% fetal calf serum. During protein production, serum was replaced by 1% Ultroser G (Gibco). GpIbα fragments were purified from expression medium by Ni 2+ /NTA chromatography, followed by anion exchange (MonoQ) and gel filtration (Superdex 200). The protein was concentrated to ˜7 mg/ml in the gel filtration buffer (50 mM NaCl, 20 mM Tris/HCl pH 8.0). Anion exchange of wild-type and mutant GpIbα (1-290) proteins yielded four base-line separated peaks. Electro-spray mass spectroscopic analysis showed that these peaks contain GpIbα modified by sulfation at 0, 1, 2 or 3 sites, likely tyrosine residues in the anionic region (data not shown). Fully sulphated GpIbα was used for crystallization experiments. 
     Wild-type vWF-A1 domain residues 498 to 705 and mutant A1 R543Q were cloned in expression vector pPIC9 and over-expressed in Pichia pastoris strain GS115, according to the Invitrogen manual. After three days of induction, expression medium was collected and dialyzed against standard buffer (25 mM Tris, 100 mM NaCl, pH 7.8). The protein was purified on heparin Sepharose, followed by gel filtration (Superdex 200). It was dialyzed against standard buffer and concentrated to ˜4 mg ml −1 . 
     Crystallization 
     Crystals were grown using the hanging-drop vapor diffusion technique. GpIbα (N21Q, N159Q) crystals were obtained at 28° C. by mixing 1 μl of protein (7 mg ml −1 ) and 1 μl reservoir solution (1.8 M ammonium sulphate, 0.2 M lithium sulphate and 100 mM CAPS pH 8.2). Before flash freezing, crystals were transferred to a cryo-protective solution (25% (w/v) PEG 3000, 200 mM NaCl, 100 mM Tris pH 8.2 and 15% (v/v) glycerol). GpIbα crystals have space group C2 with cell constants: a=121.5 Å, b=54.5 Å, c=101.8 Å, β=103.7°, and contain two molecules per asymmetric unit. 
     Crystals of a complex of GpIbα mutant (N21Q, N159Q, M239V) and A1 mutant (R543Q) were obtained at 4° C. by mixing 1 μl protein solution (7 mg ml −1 ) containing a 1:1 molar ratio of A1 and GpIb and 1 μl precipitant solution (10% (w/v) PEG 3000, 200 mM NaCl and 100 mM MES pH 5.5). Before flash freezing, crystals were transferred to precipitant solution containing 20% (v/v) glycerol. Crystals have space group P6 1  with cell constants: a=b=89.8 Å, and c=124.6 Å, and contain one complex per asymmetric unit. 
     Structure Determination and Refinement 
     Diffraction data of the GpIbα-A1 complex at the X11 beam line of the EMBL outstation at the DESY synchrotron in Hamburg and data of GpIbα at beam line ID 14-2 of the ESRF, Grenoble (Table 1) was collected. Data were processed with DENZO and SCALEPACK software. Structures of GpIbα and the GpIbα-A1 complex were solved in conjunction. Molecular replacement with CNS placed A1 in the unit cell of the complex. After solvent flattening with CNS, β-strands of GpIbα leucine-rich repeats were clearly resolved. A mask was constructed around the putative GpIbα molecule. Electron density inside the mask was used for molecular replacement with AMORE. This identified two GpIbα molecules in the asymmetric unit of the GpIbα crystal. After refinement of the non-crystallographic symmetry operator, electron density was improved by two-fold averaging, solvent flattening and phase extension to 2.5 Å. A model was built in the 2.5 Å map with O and later refined at 1.8 Å resolution using CNS. The refined model of GpIbα together with A1 was then used as the starting point for refinement of the GpIbα-A1 complex to a resolution of 3.1 Å. 
     Biacore Analysis 
     Binding studies were performed on a Biacore 2000 (Biacore AB, Uppsala Sweden). GpIbα monoclonal antibody 2D4 was immobilized on CM5-sensor chips by amine coupling as instructed by the supplier. A control channel was activated and blocked by using the amine-coupling reagents in the absence of protein. Proteins were dialyzed to standard Biacore buffer (150 mM NaCl, 0.005% (v/v) Tween-20 and 25 mM HEPES pH 7.4) and analyzed at 25° C. GpIbα (150 nM) was injected for one minute, followed by a two-minute association phase of A1 (5-100 nM) and a five-minute dissociation phase, during which standard buffer was injected. The sensor chip was regenerated by first injecting 50 mM triethylamine, then 10 mM sodium formate pH 2.0 and 150 mM NaCl, and finally another injection of 50 mM triethylamine. Each run was performed in triplicate. Data evaluation was performed with Bia evaluation software (Biacore AB) using a 1:1 Langmuir model with baseline drift to compensate for the slow release of GpIbα from 2D4. Control experiments included immobilization of GpIbα via His-tag antibody 3D5 (Novagen) which excluded artifacts caused by antibody 2D4 and a comparison of glycosylated and non-glycosylated mutant GpIbα to confirm that glycosylation has no effect on binding. 
     
       
         
               
             
               
               
               
             
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Data collection and refinement statistics 
               
             
          
           
               
                   
                 Crystal 
                   
               
             
          
           
               
                   
                 GpIbα 
                 GpIbα A1 
               
               
                   
                   
               
             
          
           
               
                   
                 Resolution (Å) 
                    1.85/1.9-1.85 
                    3.1/3.2-3.1 
               
               
                   
                 Completeness (%) 
                 97.8/80.8 
                 99.9/99.9 
               
               
                   
                 Mosaicity (°) 
                 0.4 
                 0.2 
               
               
                   
                 Redundancy 
                 3.6/2.4 
                 5.8/5.4 
               
               
                   
                 R merge  (%) 
                  7.3/33.7 
                  8.7/48.0 
               
               
                   
                 I/ σI   
                 16.3/2.9  
                 19.3/3.6  
               
               
                   
                 R factor (%) 
                 19.0 
                 25.1 
               
               
                   
                 R free (%) 
                 22.0 
                 29.7 
               
               
                   
                 No. of protein atoms 
                 4113 
                 3667 
               
               
                   
                   
                 (dimer) 
               
               
                   
                 No. of waters 
                 638 
                 0 
               
               
                   
                 r.m.s.d. bonds (Å) 
                 0.006 
                 0.009 
               
               
                   
                 r.m.s.d. angles (°) 
                 1.4 
                 1.7 
               
               
                   
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
             
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Biacore data 
               
             
          
           
               
                   
                 Dissociation constant (standard deviation) 
               
               
                   
                 (nM) 
               
             
          
           
               
                   
                 GpIbα 1-290 
                 GpIbα 1-269 
               
               
                   
                   
               
             
          
           
               
                 GpIbα (wt) + A1(wt) 
                 33.3 
                 (14.0) 
                 38.4 
                 (14.4) 
               
               
                 GpIbα (wt) + A1(mt) 
                 13.6 
                 (3.5) 
                 16.5 
                 (3.14) 
               
               
                 GpIbα (mt) + A1(wt) 
                 11.1 
                 (2.1) 
                 12.6 
                 (2.7) 
               
               
                 GpIbα (mt) + A1(mt) 
                 5.8 
                 (1.6) 
                 7.5 
                 (2.2) 
               
               
                   
               
             
          
         
       
     
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