title: Requirements for membrane attack complex formation and anaphylatoxins binding to collagen-activated platelets.
abstract: BACKGROUND
  The activation of complement during platelet activation is incompletely understood.
  
  
  OBJECTIVE
  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 Mg(2+)-EGTA and significantly reduced by the thrombin inhibitor hirudin (-37%, 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(2+)-EGTA, but not hirudin, SCH79797 or chondroitinase, inhibited the formation of collagen-induced microparticles (-71% and -44%, 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 anaphylatoxins and the exposure of receptors C3aR1 and C5aR (CD88) on their surface.
  
  
  CONCLUSIONS
  C5b-9 formation on collagen-activated platelets is i) partially controlled by thrombin, ii) restricted to a subset of platelets, and iii) can occur without P-selectin expression or phosphatidylserine exposure. Activated platelets bind anaphylatoxins on their surface and express C3a and C5a receptors, which may contribute to the localization of inflammatory processes during thrombosis.
authors.affiliation: Department of Medicine, Montreal Heart Institute and University of Montreal, Montreal, Quebec, Canada.
authors.forename: Catherine
authors.initials: C
authors.lastname: Martel
authors.forename: Sylvie
authors.initials: S
authors.lastname: Cointe
authors.forename: Pascal
authors.initials: P
authors.lastname: Maurice
authors.forename: Saïd
authors.initials: S
authors.lastname: Matar
authors.forename: Marta
authors.initials: M
authors.lastname: Ghitescu
authors.forename: Pierre
authors.initials: P
authors.lastname: Théroux
authors.forename: Arnaud
authors.initials: A
authors.lastname: Bonnefoy
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology; D000706:Anaphylatoxins / Q000378:metabolism*; D001792:Blood Platelets / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D000001:Calcimycin / Q000494:pharmacology; D003094:Collagen / Q000494:pharmacology*; D015938:Complement Membrane Attack Complex / Q000378:metabolism*; D003435:Crotalid Venoms / Q000494:pharmacology; D006629:Hirudins / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D037181:Lectins, C-Type; D019007:P-Selectin / Q000378:metabolism; D015539:Platelet Activation / Q000187:drug effects*; D010974:Platelet Aggregation / Q000187:drug effects; D053657:Platelet-Rich Plasma / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D044087:Receptor, Anaphylatoxin C5a / Q000378:metabolism; D011951:Receptors, Complement / Q000378:metabolism; D018179:Receptors, Thrombin / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D012995:Solubility / Q000187:drug effects
chemical_list: D000706:Anaphylatoxins; D015938:Complement Membrane Attack Complex; D003435:Crotalid Venoms; D006629:Hirudins; D037181:Lectins, C-Type; D019007:P-Selectin; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; D018179:Receptors, Thrombin; C085567:complement C3a receptor; C028890:convulxin; D000001:Calcimycin; D000244:Adenosine Diphosphate; D003094:Collagen