Agonist Costimulation Increases Recruitment of Platelet Subpopulations to a Procoagulant Phenotype.

Previously we determined that platelets stimulated with PAR 1 agonists, even at levels eliciting maximal responses for support of factor Xa generation, showed only a subpopulation (~10%) that exposed binding sites for factor IXa. Now we show that the same subpopulation of platelets supported binding of both factor Xa and factor IXa. While stimulation of platelets with GPVI agonists collagen or convulxin also caused exposure of factor Xa binding sites on a small subpopulation (~10%) of platelets, simultaneous stimulation with PAR 1 and GPVI agonists resulted in synergistic recruitment of platelets to expose factor Xa binding sites (30–60%). In kinetic studies of platelet-supported thrombin generation, platelets preactivated with thrombin or collagen showed roughly equivalent initial rates of prothrombinase activity (5nM/min, 10nM/min, and 12nM/min for low, medium and high agonist concentrations) that increased with reaction time up to 20 minutes reflecting platelet stimulation by the thrombin generated. Platelets preactivated with both collagen and thrombin, even at high concentrations (collagen 50μg/ml and thrombin 2U/ml), supported additive rates of prothrombinase in contrast to the synergy achieved in exposure of factor Xa binding sites. These data indicate that although single strong agonists recruit only a small subpopulation of activated platelets to undergo procoagulant surface changes, co-stimulation acts synergistically to recruit a larger population. It was previously shown that treatment of platelets with 100μM of the protein kinase C inhibitor RO318220 permitted an increased (three-fold) PAR 1-stimulated platelet support of prothrombinase and an almost 20-fold increase in the size of the population exposing annexin V binding sites. We now show that agonist-stimulated platelets pretreated with 100μM RO318220, whether stimulated with PAR 1 or GPVI agonists separately or combined, recruited ~80% of platelets to bind factor Xa. These data suggest that exposure of a procoagulant phenotype is negatively regulated by a pathway containing a target of high concentrations of RO318220, and that both PAR 1 and GPVI pathways normally converge in relief of that regulation.