A Role for von Willebrand Factor Proline Residues 702-704 in Ristocetin-Mediated Binding to Platelet Glycoprotein Ib

Summary

Mutant domains of von Willebrand factor (vWF) were constructed to determine the effects of altering net charge, and presumably conformation, within a peptide sequence (residues 694-708) previously shown to be involved in the platelet receptor glycoprotein (GP) Ib binding function of vWF. Non-conservative substitutions replaced a triplet of proline residues (proline⁷⁰²–⁷⁰⁴) with either a triplet of arginine (positively-charged) or aspartic acid (negatively-charged) residues. After establishing stable CHO cell transformants, we observed the secretion of covalently-linked dimeric molecules analogous to a domain with native sequence. Functional assays using immunopurified molecules revealed that the ristocetin-dependent binding to GP Ib was abolished with both charge mutants. However, in the absence of disulfide-bond dependent conformation both mutant molecules and the molecule with native sequence interacted with GP Ib. The results demonstrate that vWF proline⁷⁰²–⁷⁰⁴ are important for the ristocetin-mediated interaction between vWF and GP Ib, but are not essential residues of the GP Ib binding site within vWF.

• References

• 1 Baumgartner HR, Tsehopp TB, Weiss HJ. Defective adhesion of platelets to subendothelium in von Willebrand’s disease and Bernard Soulier Syndrome. Thromb Haemostas 1978; 39: 782-783
• 2 Stel HV, Sakariassen KS, de Groot PG, van Mourik JA, Sixma JJ. Von Willebrand factor in the vessel wall mediates platelet adherence. Blood 1985; 65: 85-90
• 3 Weiss HJ, Turitto VT, Baumgartner HR. Platelet adhesion and thrombus formation on subendothelium in platelets deficient in glycoproteins IIb-IIIa, lb, and storage granules. Blood 1986; 67: 322-330
• 4 Sakariassen KS, Bolhuis PA, Sixma JJ. Human blood platelet adhesion to artery subendothelium is mediated by factor VIII/von Willebrand factor bound to the subendothelium. Nature 1979; 279: 636-638
• 5 Ikeda Y, Handa M, Kawano K, Kamata T, Murata M, Araki Y, Anbo H, Kawai Y, Watanabe K, Itagaki I, Sakai K, Ruggeri ZM. The role of von Willebrand factor and fibrinogen in platelet aggregation under varying shear stress. J Clin Invest 1991; 87: 1234-1240
• 6 Ware J, Dent JA, Azuma H, Sugimoto M, Kyrie PA, Yoshioka A, Ruggeri ZM. Identification of a point mutation in Type IIB von Willebrand disease illustrating the regulation of von Willebrand factor affinity for the platelet GP Ib-IX receptor. Proc Natl Acad Sci USA 1991; 88: 2946-2950
• 7 Howard MA, Firkin BG. Ristocetin - A new tool in the investigation of platelet aggregation. Thromb Haemostas 1971; 26: 362-369
• 8 Kao KJ, Pizzo SV, McKee PA. Platelet receptors for human Factor VIII/von Willebrand protein: functional correlation of receptor occupancy and ristocetin-induced platelet aggregation. Proc Natl Acad Sci USA 1979; 76: 5317-5320
• 9 Read MS, Shermer RW, Brinkhous KM. Venom coagglutinin: an activator of platelet aggregation dependent on von Willebrand factor. Proc Natl Acad Sci USA 1978; 75: 4514-4518
• 10 Read MS, Smith SV, Lamb MA, Brinkhous KM. Role of botrocetin in platelet agglutination: formation of an activated complex of botrocetin and von Willebrand factor. Blood 1989; 74: 1031-1035
• 11 Sugimoto M, Mohri H, McClintock RA, Ruggeri ZM. Identification of discontinuous von Willebrand factor sequences involved in complex formation with Botrocetin: a model for the regulation of von Willebrand factor binding to platelet glycoprotein Ib. J Biol Chem 1991; 266: 18172-18178
• 12 Fujimura Y, Titani K, Holland LZ, Russell SR, Roberts JR, Elder JH, Ruggeri ZM, Zimmerman TS. Von Willebrand factor.A reduced and alkylated 52/48 kDa fragment beginning at amino acid residue 449 contains the domain interacting with platelet glycoprotein lb. J Biol Chem 1986; 261: 381-5
• 13 Mohri H, Fujimura Y, Shima M, Yoshioka A, Houghten RA, Ruggeri ZM, Zimmerman TS. Structure of the von Willebrand factor domain interacting with glycoprotein lb. J Biol Chem 1988; 263: 17901-17904
• 14 Azuma H, Dent JA, Sugimoto M, Ruggeri ZM, Ware J. Independent assembly and secretion of a dimeric adhesive domain of von Willebrand factor containing the glycoprotein Ib-binding site. J Biol Chem 1991; 226: 12342-12347
• 15 Seed B. An LFA-3 cDNA encodes a phospholipid-linked membrane protein homologous to its receptor CD2. Nature 1987; 329: 840-842
• 16 Kunkel TA, Roberts JD, Zakour RA. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol 1987; 154: 367-383
• 17 Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 1977; 74: 5463-5467
• 18 Chen C, Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Molec Cell Biol 1987; 7: 2745-2752
• 19 Fujimura Y, Usami Y, Titani K, Niinomi K, Nishio K, Takase T, Yoshioka A, Fukui H. Studies on anti-von Willebrand factor (vWF) monoclonal antibody NMC-4, which inhibits both ristocetin- and botrocetin-induced vWF binding to platelet glycoprotein lb. Blood 1991; 77: 113-120
• 20 Walsh PN, Mills DCB, White JG. Metabolism and function of human platelets washed by albumin density gradient separation. Br J Haematol 1977; 36: 281-298
• 21 Handa M, Titani K, Holland LZ, Roberts JR, Ruggeri ZM. The von Willebrand factor-binding domain of platelet membrane glycoprotein Ib.Characterization by monoclonal antibodies and partial amino acid sequence analysis of proteolytic fragments. J Biol Chem 1986; 261: 12579-12585
• 22 Sugimoto M, Ricca G, Hrinda ME, Schreiber AB, Searfoss GH, Bottini E, Ruggeri ZM. Functional modulation of the isolated glycoprotein Ib-binding domain of von Willebrand factor expressed in Escherichia coli . Biochemistry 1991; 30: 5202-5209
• 23 Fraker PJ, Speck JC. Protein and cell membrane iodinations with a sparingly soluble chloramide, l,3,4,6-tetrachloro-3a, 6a-diphenylgly-coluril. Biochem Biophys Res Commun 1978; 80: 849-857
• 24 Scott JP, Montgomery RR, Retzinger GS. Dimeric ristocetin flocculates proteins, binds to platelets, and mediates von Willebrand factor-dependent agglutination of platelets. J Biol Chem 1991; 266: 8149-8155
• 25 Gooley AA, Classon BJ, Marschalek R, Williams KL. Glycosylation sites identified by detection of glycosylated amino acids released from Edman degradation: the identification of Xaa-Pro-Xaa-Xaa as a motif for thr-O-glycosylation. Biochem Biophys Res Comm 1991; 178: 1194-1201
• 26 Marti T, Roesselet S, Titani K, Walsh KA. Identification of disulfidebridged substructures within human von Willebrand factor. Biochemistry 1987; 26: 8099-8109
• 27 Andrews RK, Gorman JJ, Booth WJ, Corino GL, Castaldi PA, Berndt MC. Cross-linking of a monomeric 39/34-kDa dispase fragment of von Willebrand factor (Leu⁴⁸⁰/Val⁴⁸¹-Gly⁷¹⁸) to the N-terminal region of the α-chain of membrane glycoprotein Ib on intact platelets with lis(sulfosuccinimidyl) suberate. Biochemistry 1989; 28: 8326-8336