New Assay for Measuring Binding of Platelet Glycoprotein IIb/IIIa to Unpurified von Willebrand Factor

Summary

Among the numerous variants of vWD, no patient with an abnormal vWF binding to GPIIb/IIIa has been described to date. To search for such potential variants, we developed a two-site assay for measuring the binding of purified GPIIb/IIIa to vWF in biological fluids and we used it to study a large series of plasmas from various types of von Willebrand disease (vWD) and recombinant vWF (rvWF).

vWF in plasma or rvWF in culture medium was immobilized onto anti-vWF monoclonal antibodies (MoAb)-coated wells of microtiter plates. After incubation with either unlabeled GPIIb/IIIa and a ¹²⁵I-anti-GPIIb/IIIa MoAb or ¹²⁵I-GPIIb/IIIa, binding curves and binding isotherms were respectively established. Normal pool plasma and wild-type rvWF were used as reference samples. We tested plasmas from 85 normal subjects, 115 patients with different types of vWD (64 type 1, 2 type 3, 9 type 2A, 4 type 2M, 16 type 2B, 15 type 2N, 3 type IID and 2 acquired forms) and 50 patients with various bleeding disorders. Four mutated rvWF with 2A (Glu875Lys and Pro885Ser) or 2B (Dupl.Met540 and Val551Phe) substitutions and one rvWF mutated in the RGD domain of the C-terminal part of vWF-subunit (Asp1746Gly) were also studied.

Among the various samples tested, only rvWF Asp1746Gly had no affinity for GPIIb/IIIa. In contrast, GPIIb/IIIa similarly bound to the other vWF, independently of the proteic environment, the factor VIII level, the degree of multimerization or the mutation of vWF. Our results indicate that subjects with an abnormal vWF binding to GPIIb/IIIa are probably rare and difficult to target for a specific screening.

• References

• 1 Meyer D, Girma JP. von Willebrand factor: structure and function. Thromb Haemost 1993; 70: 99-104.
• 2 Sadler JE. A revised classification of von Willebrand disease. Thromb Haemost 1994; 71: 520-5.
• 3 Lankhof H, Wu YP, Vink T, Schiphorst ME, Zerwes HG, de Groot PG, Sixma JJ. Role of the glycoprotein Ib-binding A1 repeat and the RGD sequence in platelet adhesion to human recombinant von Willebrand factor. Blood 1995; 86: 1035-42.
• 4 Beacham DA, Wise RJ, Turci SM, Handin I R. Selective inactivation of the Arg-Gly-Asp-Ser (RGDS) binding site in von Willebrand factor by site-directed mutagenesis. J Biol Chem 1992; 79: 2039-47.
• 5 Harverstick DM, Cowan JF, Yamada KM, Santoro SA. Inhibition of platelet adhesion to fibronectin, fibrinogen and von Willebrand factor substrates by a synthetic tetrapeptide derived from the cell-binding domain of fibronectin. Blood 1985; 66: 946-50.
• 6 Steiner B, Cousot D, Trzeciak A, Gillessen D, Hadvary P. Ca²⁺ - dependent binding of a synthetic Arg-Gly-Asp (RGD) peptide to a single site on the purified platelet glycoprotein IIb-IIIa complex. J Biol Chem 1989; 264: 13102-8.
• 7 Fraker PJ, Speck JC. Protein and cell membrane iodinations with a sparingly soluble chloroamide 1, 3, 4, 6 - tetrachloro-3a,6a-diphenylglycoluril. Biochem Biophys Res Commun 1978; 80: 849-57.
• 8 Chopek MW, Girma JP, Fujikawa K, Davie EW, Titani K. Human von Willebrand factor: a multivalent protein composed of identical subunits. Biochemistry 1986; 25: 3146-55.
• 9 Meyer D, Zimmerman TS, Obert B, Edgington TS. Hybridoma antibodies to human von Willebrand factor. Characterization of seven clones. Br J Haematol 1984; 57: 597-608.
• 10 Steinbuch M, Audran R. The isolation og IgG from mammalian sera with the aid of caprilyc acid. Arch Biochem Biophys 1969; 134: 279-84.
• 11 Pietu G, Ribba AS, Chérel G, Siguret V, Obert B, Rouault C, Ginsburg D, Meyer D. Epitope mapping of inhibitory monoclonal antibodies to human von Willebrand factor by using recombinant cDNA libraries. Thromb Haemost 1994; 71: 788-92.
• 12 Nokes TJC, Mahmoud NA, Savidge GF, Goodall AH, Meyer D, Edgington TS, Hardisty RM. von Willebrand factor has more than one binding site for platelets. Thromb Res 1984; 34: 361-6.
• 13 Ardaillou N, Girma JP, Meyer D, Lavergne JM, Shoa’i I, Larrieu MJ. “Variants” of von Willebrand’s disease. Demonstration of a decreased antigenic reactivity by immunoradiometric assay. Thromb Res 1978; 12: 817-30.
• 14 Ribba AS, Voorberg J, Meyer D, Pannekoek H, Pietu G. Characterization of recombinant von Willebrand factors corresponding to mutations in type IIA and IIB von Willebrand disease. J Biol Chem 1992; 267: 23209-15.
• 15 Ribba AS, Christophe O, Derlon A, Cherel G, Siguret V, Lavergne JM, Girma JP, Meyer D, Pietu G. Discrepancy between IIA phenotype and IIB genotype in a patient with a variant of von Willebrand disease. Blood 1994; 83: 833-41.
• 16 Wagner DD, Mayadas T, Marder VJ. Initial glycosylation and acidic pH in the Golgi apparatus are required for multimerization of von Willebrand factor. J Cell Biol 1986; 102: 1320-4.
• 17 Chang DC. Design of protocols for electroporation and electrofusion: selection of electrical parameters. In: Guide to electroporation and electro-fusion Chang DC, Cassy BM, Saunders JA, Sowers AE. (eds) Academic Press: San Diego; 1992: 429-55.
• 18 Laemmli UK. Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 1970; 227: 680-5.
• 19 Towbin H, Stachelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamid gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979; 76: 4350-4.
• 20 Ruggeri ZM, Zimmerman TS. The complex multimeric composition of factor VIII/von Willebrand factor. Blood 1981; 57: 1140-1.
• 21 Federici AB, Bader R, Pagani S, Colibretti ML, DeMarco 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.
• 22 DeMarco L, Mazzucato M, De Roia D, Casanato A, Federici AB, Girolami A, Ruggeri ZM. Distinct abnormalities in the interaction of purified types IIA and IIB von Willebrand factor with the two platelet binding sites, glycoprotein complexes Ib-IX and IIb-IIIa. J Clin Invest 1990; 86: 785-92.
• 23 Christophe O, Ribba AS, Baruch D, Obert B, Rouault C, Niinomi K, Pietu G, Meyer D, Girma JP. Influence of mutations and size of multimers in type II von Willebrand disease upon the function of von Willebrand factor. Blood 1994; 83: 3553-61.
• 24 Fressinaud E, Meyer D. International survey of patients with von Wille-brand disease and angiodysplasia. Thromb Haemost 1993; 70: 546