Determinants of Prothrombinase Activity and Modification of Prothrombin Conversion by Thrombin‐Treated Factor V

摘要

Summary.The development of an intrinsic two‐stage assay for factor V using highly purified protein components is described. Phospholipid, activated factor X, calcium ion and factor V are incubated together to form a prothrombinase. After addition of prothrombin, sequential aliquots are removed and tested for thrombin activity using fibrinogen as a substrate. The rate of thrombin generation is linear for at least 10 min and increases with increasing concentrations of factor V or activated factor X. At a fixed concentration of activated factor X, a linear relationship is observed between the logarithm of the initial rate of thrombin generation and the logarithm of the factor V concentration. Oligomeric forms of factor V generate parallel but non‐identical concentration curves indicating differing abilities to form prothrombinase.The rate of thrombin generation eventually decreases and a maximum thrombin concentration is attained. This phenomenon is not attributable to substrate exhaustion or to decay of prothrombinase activity. Evidence for the formation of an inhibitor during prothrombin conversion, which is directed against both prothrombinase and the reaction of thrombin with fibrinogen, is offered.Activation of factor V by thrombin is demonstrable in the two‐stage intrinsic system and in a modified extrinsic sequential technique. In both systems the time course and extent of activation are similar to those observed using a one‐stage extrinsic assay.Thrombin formation began immediately with or without prior exposure of factor V to thrombin. The ‘lag time’ observed by some previous workers is shown to be an artifact due to inappropriate extrapolation of the standard thr