Requirements for Membrane Attack Complex Formation and Anaphylatoxins Binding to Collagen-Activated Platelets 英文参考文献.docVIP

RequirementsforMembraneAttackComplexFormation andAnaphylatoxinsBindingtoCollagen-Activated Platelets CatherineMartel1,SylvieCointe1,PascalMaurice1,Sa?¨dMatar1,MartaGhitescu1,PierreThe′roux1, ArnaudBonnefoy1,2 * 1DepartmentofMedicine,MontrealHeartInstituteandUniversityofMontreal,Montreal,Quebec,Canada,2INSERMU743,UniversityofMontreal,Montreal,Quebec, Canada Abstract Background:Theactivationofcomplementduringplateletactivationisincompletelyunderstood.Objectives:Wesoughtto exploretheformationofC5b-9andanaphylatoxinsbindingtocollagen-activatedplatelets. Methods:C5b-9,anaphylatoxinsC3a,C4aandC5a,andanaphylatoxinreceptorsC3aR1andC5aRweremeasuredbyflow cytometryand/orconfocalmicroscopy.Plateletmicroparticleswerequantifiedbyflowcytometry,andtheirC5b-9content wasdeterminedbywesternblotanalyses.Inallexperiments,sodiumcitratewasusedforbloodanticoagulation. 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 2+ formationwasobliteratedbyMg -EGTAandsignificantlyreducedbythethrombininhibitorhirudin(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 Mg -EGTA, but not hirudin, SCH79797 or chondroitinase, inhibited the formation of collagen-induced 2+ microparticles (271% and 244%, respectively, p,0.04). These microparticles contained greater amounts of C5b-9 compared with the other agonists. Platelet activation by collagen or convulxin resulted in the strong binding of anaphylatoxinsandtheexposureofreceptorsC3aR1andC5aR(CD88)ontheirsurface. Conclusions:C5b-9formationoncollagen-activatedplateletsisi)partiallycontrolledbythrombin,ii)restrictedtoasubset of platelets, and iii) can occur with