A Reliable and Reproducible ELISA Method to Measure Ristocetin Cofactor Activity of von Willebrand Factor

Summary

The ristocetin induced binding of vWF to GPIb, which is routinely tested in a platelet agglutination assay, can be reproducibly studied in an ELISA where plasma vWF binds to a captured rGPIbα-fragment (His1-Val289) in the presence of ristocetin. This binding is specific since the vWF-GPIb interaction could (i) be blocked by inhibitory antiGPIb or anti-(vWF A1 domain) monoclonal antibodies (mAbs) and (ii) be enhanced by an anti-vWF mAb that also facilitates ristocetin induced platelet agglutination. Further studies were undertaken to determine whether the test could be used to differentiate vWF from patients with different types of von Willebrand’s disease. The median vWF:RiCof activity in controls (n = 24) was 0.75 U/ml, in type 1 vWD patients (n = 17) 0.28 U/ml, in type 2A (n = 18) 0.055 U/ml, in type 2B (n = 4) 0.094 U/ml and in type 3 (n = 3) <0.0005 U/ml. Moreover, the values correlated well with those obtained from the vWF:RiCof-agglutination assay (r = 0.873). The vWF:RiCof-ELISA has several advantages: the use of a recombinant fragment instead of donor platelets results in a more reproducible test with a low inter-and intra-assay variability (<14% CV), the test can further be readily automated and for a single determination, only minimal amounts of patient plasma are required (8 µl).

Keywords

Recombinant GpIbα fragment - vWF:RiCof - von Willebrand’s disease - diagnosis

^* These authors contributed equally to this work

• References

• 1 Sadler JE. Biochemistry and genetics of von Willebrand factor. Annu Rev Biochem 1998; 67: 395-424.
• 2 Rodeghiero F, Castaman G, Dini E. Epidemiological investigation of the prevalence of von Willebrand’s disease. Blood 1987; 69: 454-9.
• 3 Sadler JE. A revised classification of von Willebrand disease. For the Subcommittee on von Willebrand Factor of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis. Thromb Haemost 1994; 71: 520-5.
• 4 Ewenstein BM. Von Willebrand’s disease. Annu Rev Med 1997; 48: 525-42.
• 5 Veyradier A, Fressinaud E, Meyer D. Laboratory diagnosis of von Willebrand disease. Int J Clin Lab Res 1998; 28: 201-10.
• 6 Brown JE, Bosak JO. An ELISA test for the binding of von Willebrand antigen to collagen. Thromb Res 1986; 43: 303-11.
• 7 Favaloro EJ, Grispo L, Exner T, Koutts J. Development of a simple collagen based ELISA assay aids in the diagnosis of, and permits sensitive discrimination between type I and type II, von Willebrand’s disease. Blood Coagul Fibrinolysis 1991; 02: 285-91.
• 8 Mazurier C, Parquet-Gernez A, Goudemand M. Enzyme-linked immunoabsorbent assay of factor VIII-related antigen. Interest in study of Von Willebrand’s disease. Pathol Biol (Paris) 1977; 25 Suppl 18-24.
• 9 Howard MA, Firkin BG. Ristocetin – a new tool in the investigation of platelet aggregation. Thromb Diath Haemorrh 1971; 26: 362-9.
• 10 Hoylaerts MF, Nuyts K, Peerlinck K, Deckmyn H, Vermylen J. Promotion of binding of von Willebrand factor to platelet glycoprotein Ib by dimers of ristocetin. Biochem J 1995; 306: 453-63.
• 11 Fischer BE, Kramer G, Mitterer A, Grillberger L, Reiter M, Mundt W, Dorner F, Eibl J. Effect of multimerization of human and recombinant von Willebrand factor on platelet aggregation, binding to collagen and binding of coagulation factor VIII. Thromb Res 1996; 84: 55-66.
• 12 Federici AB, Bader R, Pagani S, Colibretti ML, De Marco L, Mannucci PM. Binding of von Willebrand factor to glycoproteins Ib and IIb/IIIa complex: affinity is related to multimeric size. Br J Haematol 1989; 73: 93-9.
• 13 Vicente V, Houghten RA, Ruggeri ZM. Identification of a site in the alpha chain of platelet glycoprotein Ib that participates in von Willebrand factor binding. J Biol Chem 1990; 265: 274-80.
• 14 Macfarlane DE, Stibbe J, Kirby EP, Zucker MB, Grant RA, McPherson J. Letter: A method for assaying von Willebrand factor (ristocetin cofactor). Thromb Diath Haemorrh 1975; 34: 306-8.
• 15 Brinkhous KM, Graham JE, Cooper HA, Allain JP, Wagner RH. Assay of von Willebrand factor in von Willebrand’s disease and hemophilia: use of a macroscopic platelet aggregation test. Thromb Res 1975; 06 (03) 267-72.
• 16 Favaloro EJ, Koutts J. Laboratory assays for von Willebrand factor: relative contribution to the diagnosis of von Willebrand’s disease. Pathology 1997; 29: 385-91.
• 17 Aihara M, Kimura A, Chiba Y, Yoshida Y. Plasma collagen cofactor correlates with von Willebrand factor antigen and ristocetin cofactor but not with bleeding time. Thromb Haemost 1988; 59: 485-90.
• 18 Schumpp-Vonach B, Kresbach G, Schlaeger EJ, Steiner B. Stable expression in Chinese hamster ovary cells of a homogeneous recombinant active fragment of human platelet glycoprotein Ib alpha. Cytotechnology 1995; 17: 133-41.
• 19 Tornai I, Arnout J, Deckmyn H, Peerlinck K, Vermylen J. A monoclonal antibody recognizes a von Willebrand factor domain within the amino-terminal portion of the subunit that modulates the function of the glycoprotein IB-and IIB/IIIA-binding domains. J Clin Invest 1993; 91: 273-82.
• 20 Obert B, Houllier A, Meyer D, Girma JP. Conformational changes in the A3 domain of von Willebrand factor modulate the interaction of the A1 domain with platelet glycoprotein Ib. Blood 1999; 93: 1959-68.
• 21 Hoylaerts MF, Yamamoto H, Nuyts K, Vreys I, Deckmyn H, Vermylen J. von Willebrand factor binds to native collagen VI primarily via its A1 domain. Biochem J 1997; 324: 185-91.
• 22 Falkenberg FW, Weichert H, Krane M, Bartels I, Palme M, Nagels HO, Fiebig H. In vitro production of monoclonal antibodies in high concentration in a new and easy to handle modular minifermenter. J Immunol Methods 1995; 179: 13-29.
• 23 Murdock PJ, Woodhams BJ, Matthews KB, Pasi KJ, Goodall AH. von Willebrand factor activity detected in a monoclonal antibody-based ELISA: an alternative to the ristocetin cofactor platelet agglutination assay for diagnostic use. Thromb Haemost 1997; 78: 1272-7.
• 24 Preston FE. Assays for von Willebrand factor functional activity: a UK NEQAS survey. National External Quality Assessment Scheme [letter]. Thromb Haemost 1998; 80: 863.
• 25 Fischer BE, Thomas KB, Dorner F. von Willebrand factor: measuring its antigen or function? Correlation between the level of antigen, activity, and multimer size using various detection systems. Thromb Res 1998; 91: 39-43.
• 26 Favaloro EJ, Grispo L, Dinale A, Berndt M, Koutts J. von Willebrand’s disease: laboratory investigation using an improved functional assay for von Willebrand factor. Pathology 1993; 25: 152-8.
• 27 Lankhof H, van Hoeij M, Schiphorst ME, Bracke M, Wu YP, Ijsseldijk MJ, Vink T, de Groot PG, Sixma JJ. A3 domain is essential for interaction of von Willebrand factor with collagen type III. Thromb Haemost 1996; 75: 950-8.
• 28 Sixma JJ, Schiphorst ME, Verweij CL, Pannekoek H. Effect of deletion of the A1 domain of von Willebrand factor on its binding to heparin, collagen and platelets in the presence of ristocetin. Eur J Biochem 1991; 196: 369-75.
• 29 Hillery CA, Mancuso DJ, Evan JSadler, Ponder JW, Jozwiak MA, Christopherson PA, Cox JGill, Paul JScott, Montgomery RR. Type 2M von Willebrand disease: F606I and I662F mutations in the glycoprotein Ib binding domain selectively impair ristocetin-but not botrocetin-mediated binding of von Willebrand factor to platelets. Blood 1998; Mar 1; 91 (05) 1572-81.