# Patent

## Title
Methods and kits for measuring von Willebrand factor

## Abstract

Methods and kits for measuring levels of von Willebrand factor function in a sample without using a platelet aggregation agonist, such as ristocetin, comprising recombinant glycoprotein Ibα having at least two of a G233V, D235Y and M239V mutations and an agent to detect a complex between the recombinant glycoprotein Ibα and von Willebrand factor.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No. 13/153,105, filed Jun. 3, 2011, which is a continuation of U.S. application Ser. No. 12/197,057, filed Aug. 22, 2008, which claims the benefit of U.S. Provisional Patent Application No. 60/957,604, filed Aug. 23, 2007, each incorporated herein by reference as if set forth in its entirety.


STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
This invention was made with United States government support under grant numbers RO1-HL033721-19 and RO1-HL081588-03, awarded by the National Institutes of Health. The United States government has certain rights in this invention.


BACKGROUND
The invention relates generally to methods and kits for measuring von Willebrand factor (VWF), and more particularly to methods and kits for measuring VWF that do not require a platelet agglutination agonist, such as ristocetin.
VWF is a multimeric glycoprotein synthesized by megakaryocytes and endothelial cells, which is subsequently secreted into blood plasma as a spectrum of multimers. VWF binds other proteins, especially proteins involved in hemostasis, such as Factor VIII (an essential clotting factor that participates in the intrinsic pathway of blood coagulation) and platelet glycoprotein Ib (GPIb; a component of a platelet adhesion receptor complex). VWF is deficient or defective in von Willebrand disease (VWD) and is involved in a large number of other diseases, including thrombotic thrombocytopenic purpura, Heyde's syndrome and possibly hemolytic-uremic syndrome. See, Sadler J, “Biochemistry and genetics of von Willebrand factor”. Annu Rev. Biochem. 67:395-424 (1998). VWF levels can be affected by many factors including ABO blood group and ethnicity.
VWD is a common bleeding disorder characterized by either qualitative or quantitative defects in tests for VWF. Symptoms of VWD include easy bruising, menorrhagia and epistaxis. Currently, many types of hereditary VWD are known (e.g., type 1; type 2A, 2B, 2M, 2N and type 3, as well as platelet-type, pseudo VWD, which results from a defect in platelet GPIb); however, acquired forms of VWD are also known, but are less frequently observed. Of particular interest herein is platelet-type, pseudo VWD. In contrast to the other forms of VWD, the genetic defect in platelet-type, pseudo VWD is in platelets rather than VWF and is characterized by abnormally high binding affinity of an individual's platelets to VWF, leading to a characteristic platelet hyper-responsiveness in vitro to a low concentration of ristocetin.
Additional screening tests for VWD include those that measure Factor VIII activity, VWF antigen (VWF:Ag), VWF binding to collagen (VWF:CB) and VWF ristocetin cofactor activity (VWF:RCo). Of particular interest herein is VWF:RCo, which is presently the standard for measurement of VWF function. VWF:RCo utilizes an ability of VWF to bind platelet GPIb following activation by ristocetin, which results in a VWF-dependent agglutination of platelets that can be measured quantitatively by platelet aggregometry or turbidometry. See, Macfarlane D, et al., “A method for assaying von Willebrand factor (ristocetin cofactor),” Thromb. Diath. Haemorrh. 34:306-308 (1975). In fact, an international reference standard for VWF:RCo was assigned a biologic activity in international units by the World Health Organization (WHO) and the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis (ISTH).
Unfortunately, VWF:RCo, has several shortcomings. For one, VWF:RCo has high intra- and inter-assay imprecision because of its dependence on ristocetin. See, e.g., Chng W, et al., “Differential effect of the ABO blood group on von Willebrand factor collagen binding activity and ristocetin cofactor assay,” Blood Coagul. Fibrinolysis 16:75-78 (2005); Favaloro E, “An update on the von Willebrand factor collagen binding assay: 21 years of age and beyond adolescence but not yet a mature adult,” Semin. Thromb. Hemost. 33:727-744 (2007); and Riddel A, et al., “Use of the collagen-binding assay for von Willebrand factor in the analysis of type 2M von Willebrand disease: a comparison with the ristocetin cofactor assay,” Br. J. Haematol. 116:187-192 (2002). Federici et at recently described an alternative assay with improved reproducibility that used recombinant GPIb in an enzyme-linked immunosorbant assay of VWF binding; however, it is ristocetin dependent. See, Federici A, et al., “A sensitive ristocetin co-factor activity assay with recombinant glycoprotein Ib for diagnosis of patients with low von Willebrand factor levels,” Haematologica 89:77-85 (2004).
In addition, VWF:RCo does not always reflect the true in vivo function of VWF when mutations or polymorphisms are in the ristocetin-binding region of VWF. For example, some individuals have VWF mutations that show a reduced interaction with ristocetin such that VWF:RCo is markedly reduced (e.g., <0.12 IU/dL), although they have no bleeding symptoms even with a major surgical challenge. See, Flood V, et al., “Common VWF haplotypes in normal African-Americans and Caucasians recruited into the ZPMCB-VWD and their impact on VWF laboratory testing,” Blood 10:Abstract 714 (2007); Mackie I, et al., “Ristocetin-induced platelet agglutination in Afro-Caribbean and Caucasian people,” Br. J. Haematol. 50:171-173 (1982); and Miller C, et al., “Measurement of von Willebrand factor activity: relative effects of ABO blood type and race,” J. Thromb. Haemost. 1:2191-2197 (2003). These individuals, who appear to have a polymorphism in the ristocetin-binding region, do not have an abnormality in the binding of VWF to platelet GPIb.
Furthermore, VWF:RCo is affected by high-affinity VWF/platelet disorders. For example, individuals with platelet-type, pseudo VWD have GPIb mutations that cause spontaneous binding of their platelets to VWF. See, Franchini M, et al., “Clinical, laboratory and therapeutic aspects of platelet-type von Willebrand disease,” Int. J. Lab. Hematol. 30:91-94 (2008); Miller J & Castella A, “Platelet-type von Willebrand's disease: characterization of a new bleeding disorder,” Blood 60:790-794 (1982); and Miller J, “Platelet-type von Willebrand's Disease,” Thromb. Haemost. 75:865-869 (1996). Likewise, individuals with type 2B VWD have VWF mutations that cause spontaneous binding to platelets. See, Weiss H, “Type 2B von Willebrand disease and related disorders of patients with increased ristocetin-induced platelet aggregation: what they tell us about the role of von Willebrand factor in hemostasis,” J. Thromb. Haemost. 2:2055-2056 (2004).
Because of the wide variability and reproducibility of VWF:RCo, the art desires a VWF function assay that does not require a platelet aggregation agonist, such as ristocetin (i.e., ristocetinless).
BRIEF SUMMARY
The invention relates generally to methods and kits for measuring VWF without requiring a platelet agglutination agonist by utilizing recombinant platelet GPIb gain-of-function mutations. As used herein, a “platelet agglutination agonist” means an agent that facilitates adhesion between VWF and GPIb in platelet agglutination tests. Examples of platelet agglutination agonist include, but are not limited to, ristocetin and botrocetin.
In a first aspect, the present invention is summarized as a method of measuring VWF without requiring a platelet agglutination agonist by providing a surface with immobilized recombinant platelet GPIbα having at least two mutations selected from G233V, D235Y and M239V relative to SEQ ID NO:2 or a functional fragment thereof. The method also includes contacting a sample having or suspected of having VWF with the immobilized GPIbα or functional fragment thereof without using the platelet agglutination agonist. The method also includes detecting a complex, if any, of VWF and the immobilized GPIbα or functional fragment thereof.
In some embodiments of the first aspect, the surface can be a cell surface such that the method is a flow cytometry (FC) or fluorescence-activated cell sorting (FACS) assay. Suitable host cells can be a Xenopus oocyte, CHO-K1 cell, L929 cell, HEK-293T cell, COS-7 cell or S2 cell engineered to comprise a polynucleotide encoding platelet GPIbα having the at least two mutations selected from the group consisting of G233V, D235Y and M239V relative to SEQ ID NO:2 or a functional fragment thereof. The host cell also can be engineered to further comprise a polynucleotide encoding platelet glycoprotein Ibβ (GPIbβ; SEQ ID NO:4) and/or optionally platelet glycoprotein IX (GP-IX; SEQ ID NO:8) or functional fragments thereof.
In some embodiments of the first aspect, the surface can be a solid-phase surface such that the method is an enzyme-linked immunosorbant assay (ELISA). The solid-phase surface can be agarose, glass, latex or plastic.
In some embodiments of the first aspect, the complex can be detected with a labeled anti-VWF antibody or functional fragment thereof, such as a fluorescently labeled antibody or fluorescently labeled functional fragment thereof. Alternatively, the complex can be detected by surface plasmon resonance or quasi-elastic light scattering.
In some embodiments of the first aspect, the sample can be a biological sample from an individual having or suspected of having VWD, such as plasma.
In some embodiments of the first aspect, the at least two mutations can be D235Y/G233V, D235Y/M239V or G233V/M239V. In other embodiments of the first aspect, the at least two mutation can be a triple mutation, such as D235Y/G233V/M239V.
In a second aspect, the present invention is summarized as a kit for measuring VWF that includes recombinant platelet GPIbα having at least two mutations selected from G233V, D235Y and M239V relative to SEQ ID NO:2 or a functional fragment thereof. The kit also includes a reagent to detect a complex of VWF and GPIbα.
In some embodiments of the second aspect, the reagent can be a labeled anti-VWF antibody or labeled functional fragment thereof, such as a fluorescently labeled antibody or fluorescently labeled functional fragment thereof.
In some embodiments of the second aspect, the kit further includes a negative or positive control or both. If included, the negative control can be VWF-depleted plasma. If included, the positive control can be pooled plasma from individuals that do not have VWD or can be a commercially available standard, such as those available from WHO and ISTH. In other embodiments of the second aspect, the kit further includes an abnormal control. If included, the abnormal control can be pooled plasma from individuals with variant forms of VWD, such as type-2A, 2B or 2M VWD, as well as pooled plasma from individuals with true loss of in vivo VWF function or pooled plasma individuals that are not appropriately assayed using VWF:RCo (i.e., plasma from individuals having any gain-of-function mutation in VWF).
In some embodiments of the second aspect, the at least two mutations can be selected from D235Y/G233V, D235Y/M239V or G233V/M239V. In other embodiments of the second aspect, the at least two mutations can be a triple mutation, such as D235Y/G233V/M239V.
In some embodiments of the second aspect, the recombinant platelet GPIbα having at least two mutations selected from G233V, D235Y and M239V relative to SEQ ID NO:2 or functional fragment thereof can be immobilized to a surface. In certain embodiments, the surface can be a host cell surface of a host cell that does not natively express platelet GPIbα, as described above. In certain other embodiments, the surface can be a solid-phase surface, as described above.
These and other features, objects and advantages of the present invention will become better understood from the description that follows. In the description, reference is made to the accompanying drawings, which form a part hereof and in which there is shown by way of illustration, not limitation, embodiments of the invention. The description of preferred embodiments is not intended to limit the invention to cover all modifications, equivalents and alternatives. Reference should therefore be made to the claims recited herein for interpreting the scope of the invention.



BRIEF DESCRIPTION OF THE DRAWINGS
The present invention will be better understood and features, aspects and advantages other than those set forth above will become apparent when consideration is given to the following detailed description thereof. Such detailed description makes reference to the following drawings, wherein:
FIG. 1 is a schematic illustration of the platelet adhesion receptor, which shows the components of the receptor, including GPIbα, GPIbβ, GP-V and GP-IX;
FIGS. 2A-C show the effect GPIbα mutations (single, double or triple; FIG. 2A, y-axis is mean fluorescence and x-axis is log ristocetin concentration in mg/ml), ristocetin (FIG. 2B, y-axis is mean fluorescence and x-axis is log ristocetin concentration in mg/ml) and botrocetin (FIG. 2C, y-axis is mean fluorescence and x-axis is log botrocetin concentration in mg/ml) during FACS. Mock is a HEK-293T cells transfected with an empty expression vector.
FIG. 3 shows an FACS assay with GPIbα having two platelet-type, pseudo VWD mutations using samples from control individuals and with type 3 VWD, which has low to undetectable VWF (y-axis is mean fluorescence and x-axis is platelet poor plasma (PPP) dilutions).
FIG. 4 shows an FACS assay with additional samples from individuals having type 2B VWD, which has gain-of-function VWF mutations (y-axis is mean fluorescence and x-axis is platelet poor plasma (PPP) dilutions).
FIG. 5 shows a FACS assay with additional samples from individuals having type 2M VWD, which has low GPIb binding, and apparent type 2M VWD, which has low VWF:RCo/VWF:Ag, yet normal levels of VWF (y-axis is mean fluorescence and x-axis is platelet poor plasma (PPP) dilutions).
FIG. 6 shows the effect of charge on the solid-phase surface during an ELISA with immobilized GPIbα having two platelet-type, pseudo VWD mutations (y-axis is mean fluorescence and x-axis is ISTH (a standard) concentration in U/dL).



While the present invention is susceptible to various modifications and alternative forms, exemplary embodiments thereof are shown by way of example in the drawings and are herein described in detail. It should be understood, however, that the description of preferred embodiments is not intended to limit the invention to the particular forms disclosed, but on the contrary, the intention is to cover all modifications, equivalents and alternatives falling within the spirit and scope of the invention as defined by the appended claims.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The present invention stems from the inventor's observation that some individuals with VWD have VWF mutations that lower VWF:RCo (i.e., <10 IU/dL), even though their in vivo VWF function is normal (i.e., VWF still binds to the platelet adhesion receptor component GPIb). See, Friedman K, et al., “Factitious diagnosis of type 2M von Willebrand disease (VWD) with a mutation in von Willebrand factor (VWF) that affects the ristocetin cofactor assay but does not significantly affect VWF function in vitro,” Blood 98:536a (2001).
In contrast, other individuals with VWD (i.e., type 2B and platelet-type VWD) have VWF or GPIbα mutations that lower the concentration of ristocetin required for platelet aggregation in an assay for VWF function. This paradox results from gain-of-function mutations that cause VWF multimers and the GPIb receptors on platelets to bind more tightly to one another. The inventor hypothesized that recombinant gain-of-function GPIbα mutations could be useful in assays for VWF function, thereby avoiding ristocetin (i.e., ristocetinless). As used herein, “ristocetinless” or “agonistless” means that ristocetin or other platelet agglutination agonists (i.e., botrocetin) are not required in a VWF assay.
The present invention therefore broadly relates to novel methods and kits for VWF utilizing gain-of-function GPIbα mutations, especially GPIbα mutations identified in individuals having platelet-type, pseudo VWD, to measure VWF (herein called “VWF:IbCo”). The methods and kits are useful in a variety of applications. For example, the methods and kits disclosed herein may be used for diagnosing VWD in an individual suspected of having VWD, classifying VWD in an individual diagnosed with VWD and monitoring treatment in an individual having VWD.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein.
As used herein, “about” means within 5% of a stated concentration range, purity range, temperature range or stated time frame.
As used herein, a “coding sequence” means a sequence that encodes a particular polypeptide, such as GPIbα, and is a nucleic acid sequence that is transcribed (in the case of DNA) and translated (in the case of mRNA) into that polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at a 5′ (amino) terminus and a translation stop codon at a 3′ (carboxy) terminus. A coding sequence can include, but is not limited to, viral nucleic acid sequences, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence will usually be located 3′ to the coding sequence.
As used herein, an “expression sequence” means a control sequence operably linked to a coding sequence.
As used herein, “control sequences” means promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites (“IRES”), enhancers, and the like, which collectively provide for replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control sequences need always be present so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell.
As used herein, a “promoter” means a nucleotide region comprising a nucleic acid (i.e., DNA) regulatory sequence, wherein the regulatory sequence is derived from a gene that is capable of binding RNA polymerase and initiating transcription of a downstream (3′-direction) coding sequence. Transcription promoters can include “inducible promoters” (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), “repressible promoters” (where expression of a polynucleotide sequence operably linked to the promoter is repressed by an analyte, cofactor, regulatory protein, etc.) and “constitutive promoters” (where expression of a polynucleotide sequence operably linked to the promoter is unregulated and therefore continuous).
As used herein, a “nucleic acid” sequence means a DNA or RNA sequence. The term encompasses sequences that include, but are not limited to, any of the known base analogues of DNA and RNA such as 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, -uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine and 2,6-diaminopurine.
As used herein, “operably linked” means that elements of an expression sequence are configured so as to perform their usual function. Thus, control sequences (i.e., promoters) operably linked to a coding sequence are capable of effecting expression of the coding sequence. The control sequences need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. For example, intervening untranslated, yet transcribed, sequences can be present between a promoter and a coding sequence, and the promoter sequence can still be considered “operably linked” to the coding sequence.
As used herein, “operable interaction” means that subunits of a polypeptide (e.g., the components of the platelet adhesion receptor, such as GPIbβ and/or GP-IX), and any other accessory proteins, that are heterologously expressed in a host cell assemble into a functioning platelet adhesion receptor (i.e., capable of binding with VWF or functional fragments thereof capable of binding VWF).
As used herein, a “vector” means a replicon, such as a plasmid, phage or cosmid, to which another nucleic acid segment may be attached so as to bring about the replication of the attached segment. A vector is capable of transferring gene sequences to target cells (e.g., bacterial plasmid vectors, particulate carriers and liposomes).
Typically, the terms “vector construct,” “expression vector,” “gene expression vector,” “gene delivery vector,” “gene transfer vector” and “expression cassette” all refer to an assembly that is capable of directing the expression of a coding sequence or gene of interest. Thus, the terms include cloning and expression vehicles.
As used herein, an “isolated polynucleotide” or “isolated polypeptide” means a polynucleotide or polypeptide isolated from its natural environment or prepared using synthetic methods such as those known to one of ordinary skill in the art. Complete purification is not required in either case. The polynucleotides and polypeptides described herein can be isolated and purified from normally associated material in conventional ways, such that in the purified preparation the polynucleotide or polypeptide is the predominant species in the preparation. At the very least, the degree of purification is such that extraneous material in the preparation does not interfere with use of the polynucleotide or polypeptide in the manner disclosed herein. The polynucleotide or polypeptide is at least about 85% pure; alternatively, at least about 95% pure; and alternatively, at least about 99% pure.
Further, an isolated polynucleotide has a structure that is not identical to that of any naturally occurring nucleic acid molecule or to that of any fragment of a naturally occurring genomic nucleic acid spanning more than one gene. An isolated polynucleotide also includes, without limitation, (a) a nucleic acid having a sequence of a naturally occurring genomic or extrachromosomal nucleic acid molecule, but which is not flanked by the coding sequences that flank the sequence in its natural position; (b) a nucleic acid incorporated into a vector or into a prokaryote or eukaryote host cell's genome such that the resulting polynucleotide is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR) or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene (i.e., a gene encoding a fusion protein). Specifically excluded from this definition are nucleic acids present in mixtures of clones, e.g., as these occur in a DNA library such as a cDNA or genomic DNA library. An isolated polynucleotide can be modified or unmodified DNA or RNA, whether fully or partially single-stranded or double-stranded or even triple-stranded. In addition, an isolated polynucleotide can be chemically or enzymatically modified and can include so-called non-standard bases such as inosine.
As used herein, “homologous” means those polynucleotides or polypeptides sharing at least about 90% or at least about 95% sequence identity to, e.g., SEQ ID NOS:1-6, respectively, that result in functional polypeptides that bind VWF. For example, a polypeptide that is at least about 90% or at least about 95% identical to the GPIbα mutations discussed herein is expected to be a constituent of the platelet adhesion receptor. One of ordinary skill in the art understands that modifications to either the polynucleotide or the polypeptide includes substitutions, insertions (e.g., adding no more than about ten nucleotides or amino acids) and deletions (e.g., deleting no more than about ten nucleotides or amino acids). These modifications can be introduced into the polynucleotide or polypeptide described below without abolishing structure and ultimately, function. Polynucleotides and/or polypeptides containing such modifications can be used in the methods of the present invention. Such polypeptides can be identified by using the screening methods described below.
An isolated nucleic acid containing a polynucleotide (or its complement) that can hybridize to any of the uninterrupted nucleic acid sequences described above, under either stringent or moderately stringent hybridization conditions, is also within the scope of the present invention. Stringent hybridization conditions are defined as hybridizing at 68° C. in 5×SSC/5×Denhardt's solution/1.0% SDS, and washing in 0.2×SSC/0.1% SDS+/−100 μg/ml denatured salmon sperm DNA at room temperature, and moderately stringent hybridization conditions are defined as washing in the same buffer at 42° C. Additional guidance regarding such conditions is readily available in the art, e.g., in Sambrook J, et al. (eds.), “Molecular cloning: a laboratory manual,” (3rd ed. Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001); and Ausubel F, et al. (eds.), “Current Protocols in Molecular Biology,” (John Wiley & Sons, N.Y. 1995), each of which is incorporated herein by reference as if set forth in its entirety.
It is well known in the art that amino acids within the same conservative group can typically substitute for one another without substantially affecting the function of a protein. For the purpose of the present invention, such conservative groups are set forth in Table 1 and are based on shared properties.







TABLE 1







Amino Acid Conservative Substitutions.










Original Residue
Conservative Substitution







Ala (A)
Val, Leu, Ile



Arg (R)
Lys, Gln, Asn



Asn (N)
Gln, His, Lys, Arg



Asp (D)
Glu



Cys (C)
Ser



Gln (Q)
Asn



Glu (E)
Asp



His (H)
Asn, Gln, Lys, Arg



Ile (I)
Leu, Val, Met, Ala, Phe



Leu (L)
Ile, Val, Met, Ala, Phe



Lys (K)
Arg, Gln, Asn



Met (M)
Leu, Phe, Ile



Phe (F)
Leu, Val, Ile, Ala



Pro (P)
Gly



Ser (S)
Thr



Thr (T)
Ser



Trp (W)
Tyr, Phe



Tyr (Y)
Trp, Phe, Thr, Ser



Val (V)
Ile, Leu, Met, Phe, Ala










As used herein, an “antibody” means a monoclonal and polyclonal antibody and can belong to any antibody class (i.e., IgG, IgM, IgA, etc.). One of ordinary skill in the art is familiar with methods for making monoclonal antibodies (Mab). For example, one of ordinary skill in the art can make monoclonal antibodies by isolating lymphocytes and fusing them with myeloma cells, thereby producing hybridomas. See, e.g., Milstein C, “Handbook of experimental immunology,” (Blackwell Scientific Pub., 1986); and Goding J, “Monoclonal antibodies: principles and practice,” (Academic Press, 1983), each of which is incorporated herein by reference as if set forth in its entirety. The cloned hybridomas are then screened for production of, e.g., “anti-GPIbα” (i.e., antibodies that bind preferentially to GPIbα or fragments thereof) or “anti-VWF” antibodies (i.e., antibodies that bind preferentially to VWF or fragments thereof). Monoclonal antibodies are thus not limited by the manner in which the antibodies are produced, whether such production is in situ or not. Alternatively, antibodies can be produced by recombinant DNA technology including, but not limited, to expression in bacteria, yeast, insect cell lines or mammalian cell lines.
Likewise, one of ordinary skill in the art is familiar with methods of making polyclonal antibodies. For example, one of ordinary skill in the art can make polyclonal antibodies by immunizing a suitable host animal, e.g., such as a rabbit, with an immunogen and using properly diluted serum or isolating immunoglobulins from the serum. The animal may therefore be inoculated with the immunogen, with blood subsequently being removed from the animal and an IgG fraction purified. Other suitable host animals include a chicken, goat, sheep, guinea pig, rat or mouse. If desired, the immunogen may be administered as a conjugate in which the immunogen is coupled, e.g., via a side chain of one of its amino acid residues, to a suitable carrier. The carrier molecule is typically a physiologically acceptable carrier. The antibody obtained may be purified to a purity of up to about 70%, up to about 80%, up to about 90%, up to about 95%, up to about 99% or up to about 100%.
Antibody also encompasses functional fragments, like Fab and F(ab′)2, of anti-GPIbα or anti-VWF antibodies. Treatment of antibodies with proteolytic enzymes, such as papain and pepsin, generates these antibody fragments, especially anti-GPIbα fragments.
Antibodies are typically conjugated to a detectable label for easy visualization. Examples of suitable labels for the methods and kits described herein include, but are not limited to, radiolabels, biotin (which may be detected by avidin or streptavidin conjugated to peroxidase), lanthanides, alkaline phosphatase and fluorescent labels (e.g., fluorescein, rhodamine, especially the Alexa Fluor® family of fluorescent dyes available from Invitrogen/Molecular Probes). Labelling of the antibody can be carried out by, e.g. labeling free amine groups (covalently or non-covalently). Some labels can be detected by using a labeled counter suitable for the detection of the label in question.
Commercially available anti-GPIbα antibodies and anti-VWF antibodies are suitable for use with the methods and kits described herein, and can be obtained from Blood Research Institute (Milwaukee, Wis.) and Dako (Carpinteria, Calif.), respectively.
As shown in FIG. 1, the platelet adhesion receptor is comprised of a combination of four proteins, including GPIb, which is a heterodimer of an alpha chain (GPIbα; GenBank Accession No. NM—000173.4; SEQ ID NOS:1-2) and a beta chain (GPIbβ; GenBank Accession No. NM—000407.4; SEQ ID NOS:3-4) linked by disulfide bonds. Other components of the receptor include GP-V (GenBank Accession No. NM—004488.2; SEQ ID NOS:5-6) and GP-IX (GenBank Accession No. NM—000174.2; SEQ ID NOS:7-8). The platelet adhesion receptor binds to VWF (GenBank Accession No. NM—000552.3; SEQ ID NOS:9-10) to regulate hemostasis and thrombosis.
Of particular interest herein is human GPIbα modified so that a platelet aggregation agonist is not required in assays of VWF function. For example, GPIbα can be modified to include the gain-of-function mutations that cause platelet-type, pseudo VWD including, but not limited to, G233V (see, Miller J, et al., “Mutation in the gene encoding the alpha chain of platelet glycoprotein Ib in platelet-type von Willebrand disease,” Proc. Natl. Acad. Sci. USA 88:4761-4765 (1991), incorporated herein by reference as if set forth in its entirety); D235V (see, Dong J, et al., “Novel gain-of-function mutations of platelet glycoprotein IBα by valine mutagenesis in the Cys209-Cys248 disulfide loop, which interacts with the A1 domain of VWF. Functional analysis under static and dynamic conditions,” J. Biol. Chem. 275:27663-27670 (2000), incorporated herein by reference as if set forth in its entirety); M239V (see, Russell S & Roth G, “Pseudo-von Willebrand disease: a mutation in the platelet glycoprotein Ib alpha gene associated with a hyperactive surface receptor,” Blood 81:1787-1791 (1993), incorporated herein by reference as if set forth in its entirety); G233S (Matsubara Y, et al., “Identification of a novel point mutation in platelet glycoprotein Ib, Gly to Ser at residue 233, in a Japanese family with platelet-type von Willebrand disease,” J. Thromb. Haemost. 1:2198-2205 (2003)); and K237V (see, Dong et al., supra). Advantageously, the mutation(s) can be in the Cys209-Cys248 disulfide loop of GPIbα that compromise hemostasis by increasing the affinity of GPIb for VWF. For example, and as shown below, the inventor found that D235Y is another gain-of-function mutation suitable for use with the methods and kits described herein.
As used herein, a “functional fragment” means a fragment of a component of a platelet adhesion receptor, such as a fragment of GPIbα, having at least two of the previously mentioned mutation, yet retaining its ability to interact with VWF or other substrates. For example, the amino terminus of GPIbα retains its ability to interact with VWF. As shown below, fragments of GPIbα as small as 290 amino acids and having two mutations retained an ability to interact with VWF, although smaller fragments are contemplated. With respect to VWF, a functional fragment may comprise at least the A1 domain, which is the GPIb binding domain. With respect to antibodies, functional fragments are those portions of an antibody that bind to a particular epitope, such as the domains indicated above.
As used herein, a “sample” means a biological sample, such as amniotic fluid, aqueous humor, cerebrospinal fluid, interstitial fluid, lymph, plasma, pleural fluid, saliva, serum, sputum, synovial fluid, sweat, tears, urine, breast milk or tissue that has or is suspected of having VWF. With respect to measuring VWF, plasma is a suitable sample.
As used herein, a “surface” means, e.g., a cell surface or solid-phase surface, such as an unsoluble polymer material, which can be an organic polymer, such as polyamide or a vinyl polymer (e.g., poly(meth)acrylate, polystyrene and polyvinyl alcohol or derivates thereof), a natural polymer, such as cellulose, dextrane, agarose, chitin and polyamino acids, or an inorganic polymer, such as glass or plastic. The solid-phase surface can be in the form of a bead, microcarrier, particle, membrane, strip, paper, film, pearl or plate, particularly a microtiter plate.
One aspect of the present invention includes a diagnostic assay for measuring VWF. The underlying methodology of the assay can be FC, FACS or ELISA, each of which is well known to one of ordinary skill in the art. See, e.g., Alice Giva, “Flow cytometry: first principles,” (2nd ed. Wiley-Liss, New York, 2001); Howard Shapiro, “Practical flow cytometry,” (4th Ed. Wiley-Liss, New York, 2003); Larry Sklar, “Flow cytometry for biotechnology,” (Oxford University Press, New York, 2005); J. Paul Robinson, et al., “Handbook of flow cytometry,” (Wiley-Liss, New York, 1993); “Flow cytometry in clinical diagnosis,” (3rd ed., Carey, McCoy and Keren, eds., ASCP Press 2001); Lequin R, “Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA),” Clin. Chem. 51:2415-2418 (2005); Wide L & Porath J, “Radioimmunoassay of proteins with the use of Sephadex-coupled antibodies,” Biochem. Biophys. Acta. 30:257-260 (1966); Engvall E & Perlman P, “Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G,” Immunochemistry 8:871-874 (1971); and Van Weemen B & Schuurs A, “Immunoassay using antigen-enzyme conjugates,” FEBS Letters 15:232-236 (1971), each of which is incorporated herein by reference as if set forth in its entirety.
As noted above, the surface for the methods and kits described herein can be a host cell surface expressing at least platelet GPIbα for use in FACS. For example, one can heterologously express (either transiently or stably) mutant GPIbα or other components of platelet adhesion receptor (i.e., GPIbβ and/or GP-IX) in host cells. Methods of expressing polynucleotides and their encoded platelet glycoprotein receptor polypeptides in heterologous host cells are known to one of ordinary skill in the art. See, e.g., Tait A, et al., “Phenotype changes resulting in high-affinity binding of von Willebrand factor to recombinant glycoprotein Ib-IX: analysis of the platelet-type von Willebrand disease mutations,” Blood 98:1812-1818 (2001), incorporated herein by reference as if set forth in its entirety; and Dong et al., supra.
Cells suitable for use herein preferably do not natively display GPIbα or the other components of the platelet adhesion receptor complex. One such cell type is HEK-293T cells (American Type Culture Collection (ATCC); Manassas, Va.; Catalog No. CRL-11268). See also, Graham F, et al., “Characteristics of a human cell line transformed by DNA from human adenovirus type 5,” J. Gen. Virol. 36:59-74 (1977), incorporated herein by reference as if set forth in its entirety. HEK-293 cells are easy to reproduce and to maintain, are amenable to transfection using a wide variety of methods, have a high efficiency of transfection and protein production, have faithful translation and processing of proteins and have a small cell size with minimal processes appropriate for electrophysiological experiments.
Another suitable cell type is COS-7 cells (ATCC; Catalog No. CRL-1651). See also, Gluzman Y, “SV40-transformed simian cells support the replication of early SV40 mutants,” Cell 23:175-182 (1981), incorporated herein by reference as if set forth in its entirety. Like HEK-293 cells, COS-7 cells are easy to reproduce and maintain and are amenable to transfection using a wide variety of methods.
Yet another suitable cell type is Xenopus oocytes. Xenopus oocytes are commonly used for heterologous gene expression because of their large size (−1.0 mm), which makes their handling and manipulation easy. Xenopus oocytes are readily amenable to injection, and thus express functional proteins when injected with cRNA for an desired protein.
Yet another suitable cell type is S2 Drosophila melanogaster cells. S2 cells are ideal for difficult-to-express proteins, and a S2 expression system is commercially available from Invitrogen (Carlsbad, Calif.). The S2 expression system can be engineered to preferably lack the Bip secretion sequence so that the encoded proteins are expressed on the cell surface. Expression of platelet adhesion receptor components in S2 cells was previously shown by Celikel et al. See, Celikel R, et al., “Modulation of alpha-thrombin function by distinct interactions with platelet glycoprotein Ibα,” Science 301:218-221 (2003), incorporated herein by reference as if set forth in its entirety.
Any of the contemplated polynucleotides for the platelet adhesion receptor can be cloned into an expression vector (or plurality of expression vectors) engineered to support expression from the polynucleotides. Suitable expression vectors comprise a transcriptional promoter active in a recipient host cell upstream of, e.g., a GPIbα polynucleotide engineered to have the previously mentioned mutations or additional polynucleotides and can optionally comprise a polyA-addition sequence downstream of the polynucleotide.
The vector(s) can be introduced (or co-introduced) by, for example, transfection or lipofection, into recipient host cells competent to receive and express mutant GPIbα and optionally other components of the platelet adhesion receptor. A commercially available lipofection kit, such as a kit available from Mirus Corporation (Madison, Wis.) can be employed. Preferably, the recipient host cells do not natively contain GPIbα, so that the presence of it is completely attributable to expression from the introduced expression vector. Suitable recipient host cells are described above and can express the polypeptides on their surface or secrete them.
Alternatively, the surface for the methods and kits described herein can be a solid-phase surface having platelet GPIbα immobilized thereupon by, e.g., covalent attachment or antibodies. Suitable solid-phase surfaces include the solid-phase surfaces described above. One of ordinary skill in the art is familiar with methods for attaching anti-GPIbα antibodies or functional fragments thereof to solid-phase surfaces. For example, the antibody or functional fragment thereof can be immobilized on the surface directly by covalent coupling or via a carrier such as a linker molecule or an antibody immobilized on the solid-phase surface. The antibody can be a polyclonal or monoclonal antibody, such as anti-GPIbα or a functional fragment thereof. Alternatively, the antibody can be an anti-epitope antibody that recognizes an epitope-tag (e.g., biotin, digoxigenin, GST, hexahistidine, hemagglutinin, FLAGTM, c-Myc, VSV-G, V5 and HSV) complexed with GPIbα. Commercially available epitope tags and their respective antibodies are suitable for use with the methods and kits described herein, and can be obtained from Sigma Aldrich (St. Louis, Mo.) and Abcam, Inc. (Cambridge, Mass.).
The methods and kits described herein are thus sensitive to the measurement of the more functional, large VWF multimers, correlates with VWF:Ag in individuals with reduced VWF function, and remains unaffected by mutations that affect VWF binding of ristocetin but do not have a bleeding phenotype.
The invention will be more fully understood upon consideration of the following non-limiting Examples.
EXAMPLES
Example 1
Cells Heterologously Expressing Mutant GPIbα Spontaneous Binding in the Absence Ristocetin
Methods: A heterologous platelet adhesion receptor expression system was constructed by transiently transfecting HEK-293T cells (ATCC) with a full-length GPIbα construct encoding a single mutation (i.e., G233V, D235Y or M239V), a double mutation (i.e., G233V/M239V, G233V/D235Y or D235Y/M239V) a triple mutation (i.e., G233V/D235Y/M239V) relative to SEQ ID NO:2 or wild-type GPIbα (SEQ ID NO:2). Some HEK-293T cells also were transiently transfected with GPIbβ and GP-IX constructs encoding SEQ ID NOS:4 and 8, respectively. A mock group of HEK-293T cells were treated similarly, but were transfected with an expression vector lacking the above constructs, thereby serving as controls.
The constructs were cloned in to a pCI-neo vector (Promega; Madison, Wis.) and expressed in HEK-293T cells as described below. In some instances, separate constructs were made for each GPIbα mutation; however, in other instances, a single construct was made having multiple GPIbα mutations.
Briefly, HEK-293T cells were first seeded until they were 50-80% confluent (i.e., 3.5-4×106/100 mm dish). Typically, the cells were seeded the day before transfection.
For transfection, Hanks Balanced Salt Solution (HBSS) and OptiMEM (Invitrogen) were warmed to 37° C. 800 μl of OptiMEM was added to 17×100 polystyrene tubes (2 tubes/plate to be transfected). The following was added to one set of tubes: 4.5 μg of DNA (1.5 μg of each construct) and 20 μl PLUS Reagent (Invitrogen). The following was added to another set of tubes: 30 μl Lipofectamine (Invitrogen). Each set was allowed to incubate at room temperature for 15 minutes. The DNA mixture was then added to the Lipofectamine mixture and incubated at room temperature for 15 minutes. During incubation, the cells were washed twice with 5 ml HBSS. 3.4 ml of OptiMEM was added to the DNA/Lipofectamine mixture, and then added to the HEK-293T cells (total volume=5 ml). The cells were then incubated at 37° C. with 5% CO2 for 3-3.5 hours.
Following transfection, the transfection medium was removed and 8 ml of fresh complete medium was added to the cells. The cells were then incubated at 37° C. with 5% CO2 for about 60 hours. Cells were then harvested for use in a standard FACS assay using ristocetin.
For FACS, about 50 μl of a 1:10 dilution of platelet poor plasma (PPP; source of VWF) in assay buffer was added to the plate and serially diluted 1:2 to final dilution of 1:80. ISTH Lot #3 (GTI; Milwaukee, Wis.) was used as a standard and diluted 1:10 in assay buffer and serially diluted 1:2 to a final dilution of 1:320. The plate was then incubated for one hour at room temperature. After the one-hour incubation, the plate was centrifuged again at 1200 rpm for 5 minutes and the supernatant was discarded.
In some experiments, the PPP was diluted in PBS containing 1% BSA and either 1 mg/ml Ristocetin A (American Biochemical & Pharmaceuticals, Ltd.; Marlton, N.J.) or 1 mg/ml Botrocetin (Sigma Aldrich).
Fluorescently labeled antibodies (anti-GPIbα; Blood Research Institute) were diluted to a final concentration of 5 μg/ml in assay buffer. Fluorescently labeled anti-VWF polyclonal was also was diluted to a final concentration of 5 μg/ml in assay buffer and added to transfected cells incubated in PPP. Normal rabbit IgG (NRIgG; Pierce) and AP-1 were added at a concentration of 5 μg/ml to transfected cells as negative and positive controls, respectively. The plate was then incubated in the dark for one hour at room temperature. Assay buffer was added to each well to bring the final volume to 150 μl, and FACS was performed using a BD LSRII System (Becton Dickinson). Results are shown in VWF:IbCo units.
Results: As shown in FIG. 2A, mock transfected HEK-293T cells did not show any binding in the presence of ristocetin, while cells expressing wild-type GPIbα showed a concentration-dependent decrease in ristocetin binding after 1.2 mg/ml. HEK-293T cells expressing only one of the GPIbα mutations showed increased sensitivity even at low concentrations of ristocetin, which suggests that the binding is independent of ristocetin. Cells expressing two GPIbα mutations showed an extreme sensitivity to ristocetin or alternatively, an increased spontaneous binding that was independent of ristocetin. HEK-293T cells expressing the triple GPIbα mutation (i.e., G233V/D235Y/M239V), however, did not show increased sensitivity/spontaneous binding relative to the double mutants. As shown in FIG. 2B, each of the double mutants (i.e., G233V/M239V, G233V/D235Y or D235Y/M239V) showed comparable spontaneous binding relative to one another that was not significantly affected by ristocetin (i.e., ristocetinless). As expected the, wild-type control showed concentration-dependent increases in VWF:IbCo to ristocetin. As shown in FIG. 2C, VWF:IbCo is not affected by the type of platelet aggregation agonist, as none of the double mutants was significantly affected by botrocetin (i.e., botrocetinless). As expected, wild-type control showed concentration-dependent increases in VWF:IbCo to botrocetin.
Example 2
VWF Function in Patient Samples Using Mutant GPIbα in FACS
Methods: HEK293T cells were transiently transfected with a wild-type GPIbα construct or GPIbα encoding one of the double mutants, as described above. The cells were additionally transfected with the GPIbβ and GP-IX constructs. A group of HEK-293T cells were mock transfected, as describe above.
After forty-eight hours, the transfected cells were lifted from the plate with 3 mM EDTA, resuspended in assay buffer (i.e., 1×PBS containing 2% BSA) and counted. Trypsin was not used, as it potentially can cleave GPIbα from the cell surface. After counting, 1.75×105 cells were plated 96-well plate (Becton Dickinson; Franklin Lakes, N.J.) as a way of standardizing GPIbα on the plate surface, and the plate was then centrifuged at 2000 rpm for 5 minutes to pellet the cells. The supernatant was discarded.
HEK-293T cells expressing the GPIbα mutations were used in flow cytometry assays to test VWF binding, which was measured with a fluorescently labeled anti-VWF polyclonal antibody from Dako. A normal curve was developed using serial dilutions of reference plasma previously standardized against both the ISTH and WHO VWF standards based on the VWF:Ag international standard that is also standardized for VWF:RCo.
In one set of experiments, normal patient samples were used to determine whether the HEK-293T cells required all components of the platelet adhesion receptor or simply GPIbα. Normal patient samples were used. In another set of experiments, patient samples from normal individuals and individuals having VWD were used in the FACS assay as described above in Example 2.
Samples included 41 normals, 16 type-2M VWD, 5 type-2B VWD and 5 type-2A VWD plasma, Included therein were individuals with apparent type-2M VWD, but without clinical symptoms, and African Americans with a reduced VWF:RCo/VWF:Ag (RCo/Ag) ratio. Of the 16 type-2M VWD samples, 7 had markedly reduced VWF:IbCo (consistent with the VWF:RCo assay), and 9 had normal VWF:IbCo. African Americans with SNPs associated with reduced RCo/Ag ratios had VWF:IbCo assays that correlated with their VWF:Ag in contrast to the abnormal RCo/Ag ratios identified by standard assays. Type-2A individuals exhibited reduced VWF:IbCo assays and multimer size seemed to correlate with VWF:IbCo activity. Thus, measurement of VWF function using the VWF:IbCo assay more directly correlates with VWF function and avoids some of the pitfalls and functional variability of VWF:RCo assays.
Results: As shown in Table 2, GPIbβ and GP-IX are not required for surface expression of the mutant GPIbα, as FACS results from HEK-293T cells expressing multiple components of the plate adhesion receptor were not significantly different from cells expressing only GPIbα.







TABLE 2







Effect of GPIbα Having a Double Mutation With or Without the Other


Platelet Adhesion Receptor Components in a FACS.















GPIbα


% Diff. btw





(G233V/


GPIbα,



Known
M239V),


GPIbβ and
% Diff. btw



VWF:RCo
GPIbβ

% Diff btw
IX &
GPIbα &


Sample
(IU/dL)
and IX
GPIbα
Transfections
Known
Known
















ISTH 2
71
70.4
70.3
0.1
0.4
0.5


ISTH 3
86
86.9
91.4
2.5
0.5
3.0


CCNRP
82 to 103
89.0
74.6
8.8
4.1
4.7


Cntrl 3
65
64.4
52.1
10.5
0.5
11.0


Cntrl 4
24.6
26.7
22.7
8.0
4.0
4.1


JS
0
0
0
0
0
0


XX-01
200
136.4
119.5
6.6
18.9
25.2





ISTH = reference sample






As shown below in Table 3, the FACS assay resulted in VWF measurements comparable to a method used in clinical laboratories. Samples were normal individuals and individuals having VWD. Table 4 is similar to Table 3, except that the samples were from normal individuals and individuals having Type 2 VWD. Table 5 is also similar to Table 3, except that the samples were from individuals having Type 2M VWD.







TABLE 3





Summary of VWF:IbCo by FACS in Plasma Samples from African


Americans and Caucasians with and without VWF Single Nucleotide Polymorphisms (SNPs).
















1 = DT method (a clinical laboratory method)


2 = BRI method (Blood Research Institute method)


Shaded area = <0.81













TABLE 4







VWF:IbCo by FACS in Plasma from African Americans and Caucasians With/Without Type 2 VWD and Repeats.



















VWD
VWF









Sample
Race
Phenotype
Mutation
VWF:Ag
VWF:RC0
RC0/Ag
FACS1
FACS1/Ag
FACS2
FACS2/Ag




















DB
AA
“2M”
3 AA snps
86
47
0.547
78
0.910
78
0.905


MK0055
AA
“2M”
P1467S
257
36
0.140
214
0.833
184
0.718


LJ
C
“2M”
3 AA snps
66
40
0.606
180
2.734
48
0.735


IN0061

2M
R1374C
22
11
0.500
4
0.204
10
0.432


RH

2B
R1308S
43
37
0.860
67
1.558
67
1.557


LB

2B
V1316M
91
62
0.681
159
1.751
106
1.162


SB

2B
V1316M
27
12
0.444
36
1.347
25
0.914


AJ

2B
H1268D
21
17
0.810
31
1.484
41
1.959


PB0068

2B
R1306W
23
13
0.565
—
—
25
1.065


YG

2A
L1503P
26
13
0.500
—
—
19
0.714


AV

2A
G1579R
46
16
0.348
—
—
1
0.028


AT0021

2A
M7401?
31
12
0.387
—
—
18
0.574


AT0032

2A
I1628T
120
32
0.267
—
—
103
0.586


IA0001

2A
R1597W
33
<10
—
—
—
8
0.247


AT0017
AA
NL
3 AA snps
225
95
0.422
144
0.641
156
0.695


XX0027
AA
NL
3 AA snps
195
130
0.677
147
0.753
116
0.595


XX0004
C
NL
—
96
109
1.135
128
1.334
114
1.183


XX0013
C
NL
—
124
169
1.363
186
1.503
105
0.843


PB0014
AA
NL
—
234
211
0.902
197
0.843
213
0.909


AT0042
AA
NL
—
86
69
0.802
64
0.739
91
1.056





AA = African American


C = Caucasian


NL = normal


“2M” = apparent type 2M













TABLE 5







VWF Function in Plasma from African Americans and Caucasians With/Without Type 2 M VWD.

















VWD
VWF







Sample
Race
Phenotype
Mutation
VWF:Ag 1
VWF:RC0 1
RC0/Ag 1
FACS2
FACS2/Ag


















TB
C
“2M”
—
127
87
0.69
87
0.69


DB
AA
“2M”
3 AA snps
86
47
0.55
78
0.91


AC
C
2M
G13242S
95
13
0.14
<1.1
—


BF
—
2M
I1416T (new)
89
31
0.35
36
0.41


MG
H
2M
I1425F
45
16
0.36
>1.1
—


LG
C
2M
E1359K
67
37
0.55
27
0.41


GI
—
2M
D1283H (new)
16
4
0.25
<1.1
—


KJ
C
2M
—
12
3
0.25
<1.1
—


LJ
AA
“2M”
3 AA snps
66
40
0.61
180
2.73


BM
C
2M
I1426T
156
43
0.28
93
0.60


AR
—
2M
R1374L
48
10
0.21
<1.1
—


DR
AA
“2M”
R1342C;
38
12
0.32
37
0.97





I1343V; 1301-3103





del; and





R2185Q


MK0038
C
2M
R1392-Q1402
47
11
0.23
<1.1
—





del


IN0061
C
2M
R1374C
22
11
0.50
4
0.20


MK0055
AA
“2M”
P1467S
257
36
0.14
214
0.83


MK0058
AA
“2M”
P1467S
265
68
0.14
194
0.73





AA = African American


C = Caucasian


H = Hispanic






Results: As shown in FIG. 4, individuals with normal VWF showed a typical increase in mean fluorescence with lower dilutions of their plasma. As expected, individuals with Type 3 VWD showed change in mean fluorescence because their plasma has low or no VWF.
As shown in FIG. 5, individuals with Type 2B VWD showed a much earlier increase in mean fluorescence when compared to normals, starting at very high dilutions of their plasma (i.e., >1/100). Type 2B VWD is characterized as having gain-of-function mutations. Again, individuals with Type 3 VWD showed no reaction in the assay.
As shown in FIG. 6, individuals with Type 2M VWD showed no increase in mean fluorescence when compared to normals. Type 2M VWD is characterized by defective VWF that does not interact with GPIbα. Individuals with apparent Type 2M (“2M”) showed a much earlier increase in mean fluorescence when compared to normals, starting at very high dilutions of their plasma (i.e., >1/100). Apparent Type 2M is characterized by low VWF:RCo/VWF:Ag, yet normal levels of VWF. Again, individuals with Type 3 VWD showed no reaction in the assay.
Example 3
Mutant GPIbα Function in ELISA
S2 cells (Invitrogen) were stably transfected with a mutant GPIbα construct, a wild-type GPIbα construct and a GP-IX construct. In some experiments, S2 cells were transfected with GPIbα constructs having a C65A mutation and ΔTM290 mutation. The C65A mutation removed a cysteine that could potential allow dimerization of GPIbα; and the ΔTM290 mutation removed the transmembrane region so that the expressed protein was excreted.
Briefly, the constructs were cloned into a pMT/Bip/V5-His:GPIbα C65A, D235Y, M239V ΔTM290 or pMT/Bip/V5-His:GPIbα C65A ΔTM290 secretion vector (Invitrogen). On day 1, S2 cells were counted and seeded into a 35 mm dish or a well of a 6 well plate at 3×106 cells in 3 ml of complete medium (Ex-Cell 420+10% FBS+7 mM L-Glutamine). The cells were allowed to grow 6-8 hours at 28° C. The following was added to one set of tubes: Solution A, which contained 36 μl of 2M CaCl2, 19 μg of plasmid DNA (purified with Qiagen Maxi Kit; Qiagen; Valencia, Calif.), 1 μg pCoBlast (selection vector) and ddH20 up to 300 μl. The following was added to another set of tubes: Solution B, which contained 300 μl of 2×HEPES buffered saline. Solution A was slowly added dropwise to solution B while gently vortexing. The combined solutions then were incubated at room temperature for 30-40 minutes until a fine precipitate formed.
The mixed solution was added dropwise to the plated cells while gently swirling the plate. The cells were then incubated overnight at 28° C. (about 16-24 hours).
The next day, the transfection solution was removed and replaced with 3 ml of fresh complete medium and incubated at 28° C. without CO2. On day 5, the cells were resuspended cells and transferred to a 15 cc conical tube, centrifuged at 2400 rpm for 2 minutes. The medium was decanted, and the cells were resuspended in 3 ml of stable medium (complete medium+25 μg/ml Blastidin-S) and plated in a new dish or well.
Selection began on week 2. As done on Day 5, the selection medium was replaced every 3-4 days with 3 ml fresh selection medium. Selection and expansion continued through week 3. During this time, the cells were resuspended, transferred to 15 cc conical tubes, and centrifuged at 2400 rpm for 2 minutes. The media as decanted, and the cells were resuspended in 5 ml of selector media and plated in new T25 flask. After 4 days, the cells were expanded from 1 T25 to 2 T25 flasks.
Expansion and freezing stocks began on week 4. Cells were expanded from the T25 flasks to T75 flasks (3×106 cells/ml medium). T75 flasks received 15 ml medium, which was about 45×106 cells. The remaining cells (about 2×107 cells/vial) were frozen and stored in liquid nitrogen.
Induction of the cells in the T75 flasks began on week 5. Cells were resuspended, transferred to a 15 cc conical tube for counting and centrifuged. 45×106 cells were resuspended 15 ml induction medium (stable medium+500 μM CuSO4) and transferred to T75 flasks. The cells were then incubated 4 days at 28° C., the supernatant having secreted GPIbα was harvested.
As shown Table 6 and FIG. 3, various solid-phase surfaces were first tested for the ELISA assays. Table 6 shows that the surface density of GPIbα was affected by the surface charge of the solid-phase surface; whereas FIG. 3 shows that different solid-phase surfaces coated with GPIbα having a double mutation affected VWF binding. Solid-phase surface charge appeared to affect GPIbα/VWF binding, suggesting that any solid-phase surface should first be tested for it ability (1) to provide a uniform density of GPIbα and (2) to permit VWF to bind to the GPIbα. After considering both Table 6, and FIG. 3, Immulon® 4 HBX Plates worked best and were used thereafter.







TABLE 6







Effect of Various Solid-Phase Surfaces on Concentration of GPIbα


Double Mutation (G233V/M239V) (same samples on different plates).











Calculated GPIbα


Solid-Phase Surface
Characteristic of the Surface
concentration





Immulon 1
Hydrophobic
635.1


Immulon 2
Hydrophobic
370.8


Immulon 4
Maximum
383.7


Polysorp
Hydrophobic
321.7


Corning Medium
Hydrophobic
576.8


Corning High
Ionic and/or Hydrophobic
414.7


Multisorb
Polar Molecules
No binding


Maxisorb
Hydrophobic/Hydrophilic
408.5









An Immulon® 4 HBX Plate (Thermo Scientific; Waltham, Mass.) was coated with anti-GPIbα monoclonal antibody 142.16 (Blood Research Institute) at a concentration of 5 μg/ml, which was then incubated overnight at 4° C. The plate was blocked with PBS containing 1% BSA for 1 hour at room temperature. Nickel-purified S2-expressed proteins—GPIbα C65A, D235Y, M239V and ΔTM290-were diluted in PBS containing 1% BSA and incubated on the anti-GPIbα antibody-coated plate for 1 hour at 37° C. See, Celikel et al., supra.
PPP from controls or individuals having VWD was diluted 1:50 in PBS containing 1% BSA and serially diluted 1:2 to a final dilution of 1:100. Diluted PPP was added to the plate and incubated for 1 hour at 37° C. ISTH Lot#3 was again used as a standard, with curve dilutions starting at 1:25 in substrate buffer, which was then serially diluted 1:2 to a final dilution of 1:1600. 2 μg/ml biotinylated AVW-1 and AVW-15 (Blood Research Institute) were added to the plate and incubated for 30 minutes at 37° C. Finally, streptavidin-conjugated alkaline phosphatase (Jackson ImmunoResearch Laboratories, Ltd.; West Grove, Pa.), diluted 1:5000 in substrate buffer, was added to the plate and incubated for 30 minutes at 37° C. p-Nitrophenyl Phosphate (PNPP; Invitrogen), an alkaline phosphate substrate, was diluted 1:100 in substrate buffer and added to the plate. The plate was read at 405/650 nm on a plate reader. The plate was washed three times between each step with PBS containing 0.05% Tween-20.
Results: As shown in Tables 7 and 8, individuals with normal VWF showed similar ELISA results whether ristocetin was added to the assay or not. In addition, the ELISA assay resulted in VWF measurements comparable to a method used in clinical laboratories







TABLE 7







Summary of VWF:IbCo by ELISA in Plasma Samples from African Americans and Caucasians with and


without VWF Single Nucleotide Polymorphisms (SNPs).











IbCo
Ristocetin
Clinical















Subject
VWF:Ag
ELISA
ELISA
IbCo/VWF:Ag
Ris/VWF:Ag
VWF:Ag
VWF:RCo
VWF:RCo/VWF:Ag


















ISTH 3 A
121.25
109.4
127.3
0.90
1.05
106
86
0.81


ctrl 5 (70%)
75.62
68.97
69.68
0.91
0.92
742
60.2
0.81


ctrl 6 (35%)
32.28
31.12
28.76
0.96
0.89
37.1
30.1
0.81


CCNRP 7122 A
94.56
84.6
73.34
0.89
0.78
114
71
0.62


ISTH 3 B
96.71
99.71
104.05
1.03
1.08
106
86
0.81


MK0038
33.44
1.41
14.16
0.04
0.42
47
11
0.23


XX0017
139.5
157.35
169.5
1.13
1.22
206
200
0.97


JS
0
0.5
0.99
0.00
0.00
<1
<10
0.00


ctrl 8 (30%)
23.84
26.96
23.58
1.13
0.99
31.8
25.8
0.81


ISTH 3 C
85.14
94.42
90.48
1.11
1.06
106
86
0.81


CCNRP 7122 B
59.61
60.64
55.44
1.02
0.93
114
71
0.62


AT0068
70.4
59.18
31.47
0.84
0.45
99
57
0.58
















TABLE 8







Summary of VWF:IbCo by ELISA in Plasma Samples from African Americans and Caucasians with and


without VWF Single Nucleotide Polymorphisms (SNPs).

















IbCo



Clinical
Clinical
Ristocetin
Clinical
ELISA/BRI














Subject
VWF:Ag
BRI VWF:Ag
VWF/RCo
IbCo ELISA
ELISA
VWF:RCo/VWF:Ag
VWF:Ag

















AA w/ 1380 +









1435 + 1472


HN
334
228
165
165
—
0.494
0.725


XX
278
228
225
235
222
0.809
1.029


AT
257
309
248
234
220
0.965
0.759


AT
225
159
198
149
152
0.880
0.932


AT
225
172
95
67
106
0.422
0.393


IN
215
179
104
77
73
0.484
0.429


XX
193
200
140
123
154
0.725
0.616


NO
179
178
180
171
—
1.006
0.960


AT
103
83
69
58
64
0.670
0.701


XX
85
67
74
73
57
0.871
1.095


AT
71
65
72
70
53
1.014
1.077


HN
67
77
54
54
—
0.806
0.704


AA w/ 1472


alone


NO
259
209
224
151
213
0.865
0.723


XX
195
129
130
118
132
0.667
0.910


XX
185
143
154
143
183
0.832
1.001


XX
167
172
170
123
198
1.018
0.714


NO
166
151
175
155
—
1.054
1.025


IN
153
123
146
120
55
0.954
0.970


NO
144
141
85
92
—
0.590
0.652


DT
141
—
121
—
—
0.858
—


HN
139
—
98
—
—
0.705
—


XX
137
112
123
90
151
0.898
0.801


HN
136
136
113
106
—
0.831
0.784


XX
122
89
85
75
—
0.697
0.839


XX
116
103
89
82
81
0.767
0.793


XX
110
104
91
100
62
0.827
0.967


IN
108
107
101
86
94
0.935
0.800


AT
99
91
57
50
25
0.576
0.550


DT
98
89
85
79
85
0.867
0.885


AT
84
96
79
82
63
0.940
0.856


AA w/ no SNPs


NO
243
237
252
217
—
1.037
0.917


PB
234
192
211
110
83
0.902
0.576


DT
224
185
167
190
—
0.746
1.025


AT
199
178
193
177
—
0.970
0.993


NO
195
179
220
207
233
1.128
1.160


AT
164
132
151
98
96
0.921
0.743


AT
154
139
176
159
135
1.143
1.143


IN
122
76
85
68
74
0.697
0.897


PB
109
91
93
76
63
0.853
0.832


PB
86
63
88
64
52
1.023
1.025


AT
86
107
97
68
63
1.128
0.633


AT
86
57
69
60
56
0.802
1.055


XX
85
79
92
65
44
1.082
0.817


AT
82
93
94
70
49
1.146
0.750


C w/ 1380 +


1435 + 1472


PB
180
144
149
122
115
0.828
0.842


IN
94
91
84
68
79
0.894
0.747


C w/ 1472 alone


XX
206
254
200
266
251
0.971
1.050


IN
192
137
144
133
126
0.750
0.973


DT
174
148
137
165
127
0.787
1.119


IN
171
106
122
94
127
0.713
0.888


PB
129
102
85
76
66
0.659
0.751


IN
111
88
99
76
78
0.892
0.861


XX
97
67
89
82
80
0.918
1.224


HN
94
103
82
82
—
0.872
0.791


IN
91
65
88
58
51
0.967
0.902


C w/ no SNPs


PB
289
313
256
309
292
0.886
0.988


IN
237
171
255
154
275
1.076
0.901


IN
187
165
138
124
144
0.738
0.753


XX
129
121
149
90
112
1.155
0.745


XX
124
128
169
137
127
1.363
1.073


IN
103
82
92
67
78
0.893
0.815


IN
100
71
91
68
72
0.910
0.957


XX
96
93
109
132
103
1.135
1.425


IN
96
86
110
94
87
1.146
1.086


XX
94
77
101
74
83
1.074
0.972


XX
94
100
86
88
93
0.915
0.875


DT
88
90
107
91
83
1.216
1.008


PB
88
79
78
50
54
0.886
0.628


IN
85
61
77
58
57
0.906
0.961


IN
85
74
82
65
56
0.965
0.872


PB
83
80
88
60
51
1.060
0.742


DT
82
73
79
65
63
0.963
0.891


PB
74
52
69
53
34
0.932
1.017


DT
68
57
71
55
56
1.044
0.956


XX
58
54
61
52
49
1.052
0.958





AA = African American


C = Caucasian






Thus, measurement of VWF function using a VWF:IbCo FACS or ELISA assay more directly correlates with VWF function and avoids some of the pitfalls and functional variability observed with VWF:RCo assays.
The invention has been described in connection with what are presently considered to be the most practical and preferred embodiments. However, the present invention has been presented by way of illustration and is not intended to be limited to the disclosed embodiments. Accordingly, those skilled in the art will realize that the invention is intended to encompass all modifications and alternative arrangements within the spirit and scope of the invention as set forth in the appended claims.

## Claims

1. A method of measuring von Willebrand factor (VWF) without using a platelet agglutination agonist, the method comprising the steps of: providing a surface comprising immobilized platelet glycoprotein Ibα (GPibα) or a functional fragment thereof, wherein the immobilized GPibα or functional fragment thereof comprises a combination of mutations selected from G233V, D235Y and M239V relative to SEQ ID NO:2, wherein one of the mutations is D235Y; contacting a patient sample having or suspected of having VWF with the surface, wherein the contacting is done without a platelet aggregation agonist; and detecting a complex of VWF and GPibα.

2. The method of claim 1, wherein the surface is a host cell surface, and wherein the host cell does not natively express GPibα.

3. The method of claim 2, wherein the host cell for the host cell surface is selected from the group consisting of a Xenopus oocyte, a CHO-K1 cell, a L929 cell, a HEK-293T cell, a COS-7 cell and a S2 cell, and wherein the host cell is engineered to comprise a polynucleotide encoding the GPibα or functional fragment thereof.

4. The method of claim 2, wherein the host cell surface also comprises glycoprotein Ibβ (GPibβ) and optionally glycoprotein IX (GP-IX), wherein GPib β comprises SEQ ID NO:4 and GP-IX comprises SEQ ID NO:8.

5. The method of claim 1, wherein the surface is a solid-phase surface selected from the group consisting of agarose, glass, latex and plastic.

6. The method of claim 5, wherein the solid-phase surface comprises an anti-GPibα antibody that binds the GPibα or functional fragment thereof.

7. The method of claim 1, wherein the sample is plasma.

8. The method of claim 1, wherein the combination of mutations is D235Y/G233V or D235Y/M239V.

9. The method of claim 1, wherein the combination of mutations is D235Y/G233V/M239V.

10. The method of claim 1, wherein a labeled anti-VWF antibody is used to detect the complex of VWF and GPibα.

11. The method of claim 1, wherein the patient has or is suspected of having von Willebrand disease.