Two subpopulations of thrombin-activated platelets differ in their binding of the components of the intrinsic factor X-activating complex

Binding of fluorescein-labeled coagulation factors IXa, VIII, X and allophycocyanin-labeled annexin V to thrombin-activated platelets was studied using flow cytometry. Upon activation, two platelet subpopulation; were detected, which differed by 1-2 orders Of magnitude in the binding of the coagulation factors and by 2-3 orders of magnitude in the binding of annexin V. The percentage of the high-binding platelets increased dose dependently or thrombin concentration. At 100 nM of thrombin, platelets with elevated binding capability Constituted similar to 4% of total platelets and were responsible for the binding of similar to 50% of the total bound factor. Binding of factors to the high-binding subpopulation was calcium-dependent and specific as evidenced by experiments in the presence of excess unlabeled factor. The percentage of the high-binding platelets was not affected by echistatin, a potent aggregation inhibitor, confirming that the high-binding platelets were not platelet aggregates. Despite the difference in the coagulation factors binding, the subpopulations were indistinguishable by the expression of general platelet marker CD42b and activation markers PAC1 (an epitope of glycoprotein IIb/IIIa) and CD62P (P-selectin). Dual-labeling, binding studies involving Coagulation factors (IXa, VIII, Or X) and annexin V demonstrated that the high-binding platelet subpopulation Was identical for all Coagulation factors and for annexin v. The high-binding subpopulation had lower rilean forward and side scatters compared with the low-binding subpopulation (-80% and -60%, respectively). In its turn, the high-binding subpopulation was not homogeneous and included two subpopulation with different scatter values. We Conclude that activation by thrombin induces the formation of two distinct subpopulations of platelets different in their binding of the components of the intrinsic fX-activating complex, which may have certain physiological or pathological significance.