The kinetic parameters of the conversion of prothrombin into thrombin by activated clotting factor X (factor Xa) have been determined in the absence and presence of Ca2+, phospholipid (phosphatidyl serine/phosphatidylcholine vesicles) and activated blood clotting factor V (factor Va). In free solution the Km for prothrombin is 298 μM which is well above its plasma concentration of 4μM. Under these conditions the Vmax of thrombin formation is 1.25 Moles min-1 Mole Xa -1. When phospholipid is present the km for prothrombin drops to 0.1μM while the Vmax is only slightly affected (3 Moles min-1 Mo Le Xa -1). For the complete prothrombin activating complex consisting of factor Xa, factor Va, Ca2+ and phospholipids the kinetic constants greatly favour thrombin formation. A for prothrombin of 0.26μM and a Vmax of 2130 Moles min-1 Mole xa -1 are measured under these conditions. These results help to elucidate the role of phospholipid and factor Va in prothrombin activation. The earlier observed rate enhancements caused by phospholipid and factor Va are explained as effects on the Km for prothrombin and the Vmax of thrombin formation, respectively. The changes of the kinetic parameters for prothrombinase complexes of various composition will be considered with respect to the function of the accessory components in the mechanism of prothrombin activation. Implications of these data for in vivo blood coagulation will be discussed.

Objective-Platelets play a dual role in thrombosis by forming aggregates and stimulating coagulation. We investigated the commitment of platelets to these separate functions during collagen-induced thrombus formation in vitro and in vivo. Methods and Results-High-resolution 2-photon fluorescence microscopy revealed that in thrombus formation under flow, fibrin(ogen)-binding platelets assembled into separate aggregates, whereas distinct patches of nonaggregated platelets exposed phosphatidylserine. The latter platelet population had inactivated ␣IIb␤3 integrins and displayed increased binding of coagulation factors. Coated platelets, expressing serotonin binding sites, were not identified as a separate population. Thrombin generation and coagulation favored the transformation to phosphatidylserine-exposing platelets with inactivated integrins and reduced adhesion. Prolonged tyrosine phosphorylation in vitro resulted in secondary downregulation of active ␣IIb␤3. Conclusions-These results lead to a new spatial model of thrombus formation, in which aggregated platelets ensure thrombus stability, whereas distinct patches of nonaggregated platelets effectuate procoagulant activity and generate thrombin and fibrin. Herein, the hemostatic activity of a developing thrombus is determined by the balance in formation of proaggregatory and procoagulant platelets. This balance is influenced by antiplatelet and anticoagulant medication. Key Words: aggregation Ⅲ coagulation Ⅲ microdomains Ⅲ platelets Ⅲ integrin activation A ctivated platelets have a dual role in hemostasis and thrombosis. They form the building blocks of a thrombus and provide the membrane surface for coagulation factor activation, which results in thrombin and fibrin formation. 1 Once formed, thrombin greatly enhances platelet activation and aggregation. Given the strong interdependency of thrombin generation and platelet activation, it is intuitively assumed that those platelets that participate in aggregate formation are also involved in the coagulation process, but this has not been investigated.The mechanism(s) by which platelets contribute to thrombin generation and coagulation have been investigated for decades. Kinetic evidence shows that collagen/thrombin-activated platelets expose phosphatidylserine (PS) at their outer surface and then bind Gla domain-containing coagulation factors, mediating factor Xa and thrombin generation. 2 However, even after activation with strong agonists, only a subpopulation of the platelets tends to expose PS. 3 Conversely, it has been argued that PS exposure alone is insufficient to explain the procoagulant contribution of platelets. 4,5 There is also evidence that subfractions of activated platelets have different roles in the coagulation process. Several reports indicate an imperfect relation of PS exposure and the binding of coagulation factors Va, VIIIa, IXa, and Xa to the platelet surface. 6 -8 Another report characterizes a subfraction of activated platelets according to their so-called SCIP morphology (for susta...

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Meizothrombin-Thrombin-Prothrombin activation lntroduction The factor Xu-catalyzed conversion of the zymogen prothrombin into the active serine protease thrombin is one of the crucial steps of blood coagulation (1). Thrombin is the enzyme responsible for the conversion of fibrinogen into fibrin , a reaction that is followed by fibrin polymerization and clot formation. Thrombin also activates the transglutaminase factor XIII to factor XIII., an enzyme which stabilizes the fibrin clot. Apart from its role in the formation and stabilization of the fibrin clot, thrombin also has an important function in the regulation of the overall hemostatic process. Thus, thrombin accelerates its own rate of formation by activating the blood coagulation factors V and VIII and by stimulating blood platelets. Potential negative feedback control is exerted through the thrombin-dependent activation of protein C, producing an enzyme with anticoagulant properties that inactivates factors % and VIII. and that may also stimulate the fibrinolytic pathway. Considering the key role of thrombin in hemostatic plug formation, it is not surprising that the activation of prothrombin is one of the most intensively studied coagulation reactions (2) and that the product of this reaction, thrombin, is one of the best charact erized coagulation enzymes (3). Thrombin is not the only catalytically active product that is formed during prothrombin activation. In recent years it has been shown that during factor Xu-catalyzed prothrombin activation in addition to thrombin substantial amounts of another enzymatically active product i.e. meizothrombin can be formed (4,5). Meizothrombin differs significantly from thrombin in many of its properties and therefore, it may have different functions during the hemostatic process. In view of the pivotal role of thrombin in blood coagulation it will not be surprising that further studies of another enzymatically active prothrombin derivative with different catalytic properties may add to our understanding of the mechanisms of the reactions leading to thrombus formation. This The nomenclature used for proteolytically derived products of prothrombin is that recommended by the International Committee on Thrombosis and Haemostasis (Jackson C M. Thromb Haemostas 1977;