requirements for membrane attack complex formation and anaphylatoxins binding to collagen-activated platelets要求膜攻击复杂地层和过敏毒素绑定collagen-activated血小板.pdfVIP

Requirements for Membrane Attack Complex Formation and Anaphylatoxins Binding to Collagen-Activated Platelets 1 1 1 ¨ 1 1 ´ 1 Catherine Martel , Sylvie Cointe , Pascal Maurice , Saıd Matar , Marta Ghitescu , Pierre Theroux , Arnaud Bonnefoy1,2* 1 Department of Medicine, Montreal Heart Institute and University of Montreal, Montreal, Quebec, Canada, 2 INSERM U743, University of Montreal, Montreal, Quebec, Canada Abstract Background: The activation of complement during platelet activation is incompletely understood. Objectives: We sought to explore the formation of C5b-9 and anaphylatoxins binding to collagen-activated platelets. Methods: C5b-9, anaphylatoxins C3a, C4a and C5a, and anaphylatoxin receptors C3aR1 and C5aR were measured by flow cytometry and/or confocal microscopy. Platelet microparticles were quantified by flow cytometry, and their C5b-9 content was determined by western blot analyses. In all experiments, sodium citrate was used for blood anticoagulation. Results: C5b-9 rapidly formed on the platelet surface following activation with collagen, TRAP, ADP or A23187, but was surprisingly restricted to a subset of platelets (1 to 15%) independently of P-selectin or phosphatidylserine exposure. Following collagen activation, C5b-9-positive platelets in thrombi were found associated with collagen fibres. C5b-9 formation was obliterated by Mg2+-EGTA and significantly reduced by the thrombin inhibitor hirudin ( 237%, p,0.05), but was unaffected by chondroitinase, compstatin, SCH79797 (PAR-1 inhibitor), or in the PRP of a MBL-deficient donor. Compstatin and Mg2+-EGTA, but not hirudin, SCH79797 or chondroitinase, inhibited the formation of collagen-induced