An antifibrinolytic mechanism describing the prothrombotic effect associated with factor V-Leiden

Factor Va is the essential cofactor in prothrombinase-dependent activation of prothrombin. Resistance of Factor Va(Leiden) to inactivation by activated protein C (APC) contributes to thrombotic tendencies in subjects with the variant due, in part, to the inability to terminate thrombin production which increases both fibrin accretion and the frequency of thrombus formation, A reduced ability to inhibit thrombin generation, however, may lead to the stabilization of a clot through the activation of thrombin activatable fibrinolysis inhibitor (TAFI). This hypothesis was tested by determining the profibrinolytic effect of APC on lysis time using clots formed with plasma from either homozygous normal (n = 4) or homozygous factor V-Leiden (n = 4) subjects, Clots were formed in the presence of tissue-type plasminogen activator, thrombin, phosphatidylcholine/phosphatidylserine vesicles, Ca2+, and various concentrations of APC, Approximately 10-fold more APC was required to reduce lysis time from 140 to 50 min in clots containing factor V-Leiden compared to normal factor V. This effect was specific to the form of factor V present in plasma since identical results were obtained in an appropriately reconstituted purified system, which included both TAFI and either form of factor V purified from pooled plasma, In the absence of TAFI, APC did not affect clot lysis in experiments with either normal factor V or factor V-Leiden. During the various lysis assays performed with purified components, clots were solubilized and the proteolytic alterations in factor V/Va were assessed by Western blotting using a specific factor Va heavy chain monoclonal antibody, The heavy chain of factor Va(Leiden) persisted for as long as 60 min, in the presence of 6.3 nM APC indicating sustained activity of factor Va(Leiden) during the lysis assay, In contrast, no factor Va heavy chain was present after the first 5.0 min in clots formed in the presence of normal factor V and 6.3 nM APC. These combined data indicate that factor Va(Leiden) specifically attenuates the profibrinolytic effect of APC, Thus, an impaired TAFI-dependent profibrinolytic response to APC in APC-resistant individuals appears to be an additional factor contributing to the prothrombotic tendencies in subjects with factor V-Leiden.