Impaired factor V-related anticoagulant mechanisms and deep vein thrombosis associated with A2086D and W1920R mutations.

Background; Factor (F)V plays pivotal roles in both procoagulant and anticoagulant mechanisms. Genetic mutations, FV-W1920R (FVNara) and FV-A2086D (FVBesançon), in the C1 and C2 domains of FV light chain, respectively, appear to be associated with deep vein thrombosis. The detailed mechanism(s) between these mutations and thrombophilia remains to be fully explored, however. Aim; To clarify thrombotic mechanism(s) in the presence of these FV abnormalities. Methods and Results; Full-length wild-type (WT) and mutated FV were prepared using stable, human cell lines (HEK293T) with the piggyBac transposon system. The susceptibility of FVa-A2086D to activated protein C (APC) was reduced, resulting in significant inhibition of APC-catalyzed inactivation with little cleavage at Arg306 and delayed cleavage at Arg506. Furthermore, APC cofactor activity of FV-A2086D in APC-catalyzed inactivation of FVIIIa through cleavage at Arg336 was impaired. Surface plasmon resonance-based assays demonstrated that FV-A2086D bound to Glu-Gly-Arg-chloromethylketone active site-blocked APC and PS with similar affinities to FV-WT. However, deteriorated interaction between FVa-A2086D to phospholipid membranes was evident, by the prothrombinase assay. Moreover, the addition of FVa-A2086D to plasma failed to inhibit tissue factor (TF)-induced thrombin generation and prothrombin times. This inhibitory effect was independent from protein (P)C, PS, and antithrombin. The coagulant and anticoagulant characteristics of FV(a)-W1920R were similar to those of FV(a)-A2086D. Conclusions; FV-A2086D presented defects in the APC mechanisms associated with FVa inactivation and FV cofactor activity, similar to FV-W1920R. Moreover, both FV proteins that were mutated in the light chain impaired inhibition of TF-induced coagulation reactions. These defects were consistent with congenital thrombophilia.