Structure and function of the von Willebrand factor A1 domain: analysis with monoclonal antibodies reveals distinct binding sites involved in recognition of the platelet membrane glycoprotein Ib-IX-V complex and ristocetin-dependent activation.
Binding of the adhesive glycoprotein, von Willebrand factor (vWf), to the platelet membrane glycoprotein (GP) Ib-IX-V complex initiates platelet adhesion and aggregation at high shear stress in hemostasis and thrombosis. In this study, the GP Ib-IX-V binding site within the vWf A1 domain was analyzed using a panel of murine monoclonal antibodies raised against a 39/34-kd vWf fragment (Leu-480/Val-481-Gly-718) encompassing the A1 domain. One antibody, 6G1, strongly inhibited ristocetin-dependent vWf binding to platelets, but had no effect on botrocetin- or jaracetin-dependent binding, or asialo-vWf-dependent platelet aggregation. The 6G1 epitope was mapped to Glu-700-Asp-709, confirming the importance of this region for modulation of vWf by ristocetin. Like ristocetin, 6G1 activated the vWf A1 domain, because it enhanced binding of the 39/34-kd fragment to platelets. In contrast, 5D2 and CR1 completely inhibited asialo-vWf-induced platelet aggregation and ristocetin-induced vWf binding to GP Ib-IX-V. However, only 5D2 blocked botrocetin- and jaracetin-induced vWf binding to platelets and binding of vWf to botrocetin- and jaracetin-coated beads. Epitopes for 5D2 and CR1 were conformationally dependent, but not congruent. Other antibodies mapped to epitopes within the A1 domain (CR2 and CR15, Leu-494-Leu-512; CR2, Phe-536-Ala-554; CR3, Arg-578-Glu-596; CR11 and CR15, Ala-564-Ser-582) were not functional, identifying regions of the vWf A1 domain not directly involved in vWf-GP Ib-IX-V interaction. The combined results provide evidence that the proline-rich sequence Glu-700-Asp-709 constitutes a regulatory site for ristocetin, and that ristocetin and botrocetin induce, at least in part, separate receptor-recognition sites on vWf. (Blood. 2000;95:164-172)
De Luca
M
M
Hazel and Pip Appel Vascular Biology Laboratory, Baker Medical Research Institute, Melbourne, VIC, Australia.
Facey
D A
DA
Favaloro
E J
EJ
Hertzberg
M S
MS
Whisstock
J C
JC
McNally
T
T
Andrews
R K
RK
Berndt
M C
MC
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D001666:Binding Sites, Antibody; D003435:Crotalid Venoms / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry; D006388:Hemagglutinins / Q000378:metabolism; D006801:Humans; D008666:Metalloendopeptidases / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010974:Platelet Aggregation; D010975:Platelet Aggregation Inhibitors / Q000378:metabolism; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012310:Ristocetin / Q000494:pharmacology*; D014841:von Willebrand Factor / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D000097146:Bothrops jararaca Venom
D000911:Antibodies, Monoclonal; D003435:Crotalid Venoms; D000939:Epitopes; D006388:Hemagglutinins; D008666:Metalloendopeptidases; D010446:Peptide Fragments; D010975:Platelet Aggregation Inhibitors; D019038:Platelet Glycoprotein GPIb-IX Complex; D012310:Ristocetin; D014841:von Willebrand Factor; D000097146:Bothrops jararaca Venom; C030961:botrocetin