Activity of Human Blood Platelets in Prothrombin and in Factor X Activation Induced by Ionophore A23187

Publisher Website
Google Scholar
Abstract
Two reactions of the blood coagulation cascade are highly stimulated by membranes containing negatively charged phospholipids. These are the activation of prothrombin to thrombin by factor Xa with factor Va as cofactor, and the conversion of factor X into Xa by factor IXa with factor VIIIa as cofactor. It is thought that under physiological conditions these reactions take place with the clotting factors adsorbed at the surface of blood platelets.The role of blood platelets in the two reactions was studied using two assay systems consisting of highly purified clotting factors. The rates of formation of thrombin or factor Xa, measured with specific chromogenic substrates, were dependent on the quantity of negatively charged phospholipid present in the reaction mixture. Phospholipid vesicles with platelet phospholipid composition were highly active in the two reaction systems. When the purified lipids were substituted by disrupted platelets, containing the same quantity of lipids, the same high activities were found. With intact human blood platelets a low but significant activity was observed. Incubation of the platelets with the calcium ionophore A23187, resulted in a 50–60‐fold activity increase in formation of thrombin and factor Xa. Platelet disruption increases procoagulant activity, since the negatively charged phospholipids, originally situated in the inner leaflet of the lipid bilayer of the platelet membrane become exposed upon platelet disruption. Ionophore activation of platelets was however not due to lysis since discharge of platelet lactate dehydrogenase was less then 3%. The activity of A23187‐activated platelets in formation of thrombin and factor Xa was completely abolished after incubation with phospholipase A2, which is to be expected if negatively charged lipids are present at the platelet outer surface. In contrast to platelets, erythrocytes could not be activated by the ionophore.As found by other workers, A23187 induces release of ATP and factor V(a) and results in platelet aggregation. The appearance of procoagulant activity, ATP and factor V(a) release were all dependent on the A23187 concentration and were too rapid to study the time course. All platelet activities were induced between 0.2 μM and 0.7 μM A23187, suggesting a relationship between the release processes and the appearance of procoagulant activity.