“Young” Platelets Regulate Thrombus Formation by Expression of Increased Adhesive Properties and Procoagulant Activity.

Abstract

Previous studies from our lab and others have demonstrated that thrombin mediated activation of platelets results in the formation of a distinct procoagulant subpopulation capable of assembling the components of both the prothrombinase complex and intrinsic tenase complex at its surface. A similar subpopulation has been identified following activation of platelets with the combination of thrombin and convulxin, a glycoprotein VI agonist, and has been found to be enriched in young platelets. In addition, young zebrafish thrombocytes were shown to have a higher density of adhesive receptors and greater functional activity than their more mature counterparts. Since the membrane properties that distinguish the platelet's ability to assemble these procoagulant complexes are unknown, the purpose of this study was to elucidate these important differences. Following their isolation from whole, anticoagulated blood, human platelets were activated with 50 nM thrombin (2 min, ambient temperature) followed by the addition of hirudin (75 nM) to quench the reaction. Equimolar concentrations of factors Va and Xa were added (5 nM, 10 min) to saturate the functional Prothrombinase binding sites determined previously using kinetics of prothrombin activation, prior to platelet fixation with 2% v/v paraformaldehyde. Those platelets capable of binding factors Va and Xa were identified by flow cytometry using specific, non-inhibitory fluorophore-conjugated MoAbs directed against the required cofactor and serine protease, respectively. Analyses of the membrane density of well-known and characterized platelet receptors including glycoprotein IIb/IIIa and glycoprotein Ib-IX-V, were also assessed using specific MoAbs conjugated with the appropriate fluorophore to determine whether expression of any or all of these adhesive receptors defined the platelet subpopulation capable of assembling a competent Prothrombinase. Thiazole orange (TO), a dye frequently used to stain nucleic acid, and previously shown to identify the youngest platelets in circulation, was used to evaluate the effect of platelet age on these various parameters. Using this approach, we demonstrated a 7–8-fold increase in both CD62P (P-selectin) and CD61 (glycoprotein β3) expression in TO-positive platelets, while expression of the platelet marker CD42b (glycoprotein 1bα) increased as much as 14-fold. The subpopulation capable of assembling Prothrombinase likewise demonstrated a 3–4-fold increase in CD62P, CD61, and CD41 (glycoprotein IIb) expression, and a ~7-fold increase in CD42b expression. Dual-labeling studies demonstrated that ~60% of TO-positive platelets are able to bind factor Xa while ~85% of platelets able to bind Xa are TO-positive. Similar results were seen when platelets were evaluated for their ability to bind high levels of factor Va. These data suggest that it is predominantly the younger platelets with increased density of adhesive receptors that are capable of assembling Prothrombinase on their activated surface. Since platelet adhesion is the initiating step in both normal hemostasis and pathological thrombus formation, the observed increase in adhesive receptor density places this platelet subpopulation in a unique position of significance in the regulation of the hemostatic response, and indicates a potential role for these platelets, in the initiation of thrombus formation.