Abstract
BackgroundThe 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.MethodsC5b-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.ResultsC5b-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 (−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 Mg2+-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.ConclusionsC5b-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.
Highlights
There is a growing body of evidence supporting a bridging role of the complement system (CS) between inflammation and thrombosis through the activation of platelets
Aggregations of Platelet-rich plasma (PRP) adjusted to 250.103 platelets/mL were performed for 30 seconds, 5 and 10 min at 37uC with a Chrono-log aggregometer 570 (Chrono-log Corporation, Havertown, PA, USA) following activation with either 5 mM adenosine diphosphate (ADP), 10 mM Thrombin Receptor Agonist Peptide (TRAP) (Sigma Chemical Co, St-Louis, MO.), 2.5 mg/mL collagen or 50 mM calcium ionophore A23187
The correlation between C5b-9 formation and platelet aggregation was greatest with TRAP (R2 = 0.65 versus = 0.51 and 0.37 for ADP and collagen, respectively), whereas it was noticeably weak with A23187 (R2 = 0.20), which induced a heterogeneous aggregatory response (Figure 1)
Summary
There is a growing body of evidence supporting a bridging role of the complement system (CS) between inflammation and thrombosis through the activation of platelets. The insertion of C5b-9 in the platelet membrane induces reversible membrane depolarisation [4], phosphatidylserine exposure [5], microparticle formation and catalysis of prothrombinase activation in a calcium- and protein kinase-dependent manner [6,7]. Several groups have recently demonstrated that platelet activation induces complement activation via different mechanisms [8]. Platelet P-selectin can bind C3b thereby initiating the activation of the complement alternative pathway (AP) [9], while the classical pathway (CP) is activated on the platelet surface via a C1q and gC1qR/p33-dependent activation of C4 [10]. Chondroitin sulfate secreted by thrombin-activated platelet was found to trigger the fluid phase activation of the classical pathway in a C1q-dependent manner [11]. Objectives: We sought to explore the formation of C5b-9 and anaphylatoxins binding to collagen-activated platelets
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