AN ANTIFIBRINOLYTIC MECHANISM DESCRIBING THE PROTHROMBOTIC EFFECT ASSOCIATED WITH FACTOR V-LEIDEN

Factor Va is the essential cofactor in prothrombinase-dependent activa tion of prothrombin. Resistance of Factor Va(Leiden) to inactivation b y activated protein C (APC) contributes to thrombotic tendencies in su bjects with the variant due, in part, to the inability to terminate th rombin production which increases both fibrin accretion and the freque ncy of thrombus formation, A reduced ability to inhibit thrombin gener ation, however, may lead to the stabilization of a clot through the ac tivation of thrombin activatable fibrinolysis inhibitor (TAFI). This h ypothesis was tested by determining the profibrinolytic effect of APC on lysis time using clots formed with plasma from either homozygous no rmal (n = 4) or homozygous factor V-Leiden (n = 4) subjects, Clots wer e formed in the presence of tissue-type plasminogen activator, thrombi n, 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-Leid en compared to normal factor V. This effect was specific to the form o f factor V present in plasma since identical results were obtained in an appropriately reconstituted purified system, which included both TA FI and either form of factor V purified from pooled plasma, In the abs ence of TAFI, APC did not affect clot lysis in experiments with either normal factor V or factor V-Leiden. During the various lysis assays p erformed with purified components, clots were solubilized and the prot eolytic alterations in factor V/Va were assessed by Western blotting u sing a specific factor Va heavy chain monoclonal antibody, The heavy c hain of factor Va(Leiden) persisted for as long as 60 min, in the pres ence of 6.3 nM APC indicating sustained activity of factor Va(Leiden) during the lysis assay, In contrast, no factor Va heavy chain was pres ent 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(L eiden) specifically attenuates the profibrinolytic effect of APC, Thus , an impaired TAFI-dependent profibrinolytic response to APC in APC-re sistant individuals appears to be an additional factor contributing to the prothrombotic tendencies in subjects with factor V-Leiden.