Role of platelets in the procoagulant environment in blood
thesisposted on 15.12.2014, 10:34 by Jackie A. Appleby
The project investigated the mechanisms whereby platelets expose a procoagulant surface and induce tissue factor (TF) in monocytes. A whole blood flow cytometric assay to measure phosphatidylserine (PS) exposure on platelets was established and validated. Using this assay, it was demonstrated that collagen is the primary agonist in eliciting platelet PS exposure, individuals' platelets vary in their response to collagen, and not all express PS despite maximal activation. Collagen-induced PS exposure was independent of other aspects of platelet activation. ADP enhanced the procoagulant effect of collagen, but could not induce PS exposure on its own. The potentiating effect of ADP was mediated mainly through P2Y12 and was required for the formation of microparticles. Procoagulant microparticles were enriched in CD42b and P-selectin, but had reduced ability to bind fibrinogen. Thrombin could instigate PS exposure only where there was platelet-platelet contact. These data suggest that at a wound site, platelet PS exposure is stimulated by subendothelial collagen at the same time as coagulation is initiated by TF. It is not dependent on initial thrombin generation but thrombin and ADP perpetuate thrombin generation by promoting PS exposure where there is platelet-platelet contact within a thrombus. Activated platelets caused upregulation of gene expression of monocyte TF within 2 hours, and, at a later stage, of its inhibitor, tissue factor pathway inhibitor. These effects were primarily mediated via soluble factors. Analysis of TF antigen on monocytes and monocyte-platelet aggregates by flow cytometry suggested that TF might be platelet-associated.