The C-terminus of tissue factor pathway inhibitor-alpha inhibits factor V activation by protecting the Arg(1545) cleavage site

Background Factor V (FV) is a carrier and a cofactor of the anticoagulant protein tissue factor pathway inhibitor- (TFPI), whose basic C-terminus binds to an acidic region in the B-domain of FV. Proteolysis of FV at Arg(709), Arg(1018) and Arg(1545) by activated FX (FXa) or thrombin removes the B-domain, and converts FV into a procoagulant cofactor (activated FV [FVa]) of FXa in the prothrombinase complex. However, retention of the acidic region in partially activated FV makes prothrombinase activity susceptible to inhibition by TFPI.

Objective/Methods To investigate the effect of the TFPI C-terminal peptide (TFPI C-term) on thrombin generation in plasma and on FV activation in model systems.

Results TFPI C-term inhibited tissue factor-initiated and FXa-initiated thrombin generation in a dose-dependent manner. Failure to inhibit thrombin generation in FV-depleted plasma reconstituted with FVa indicated that the peptide effect was mediated by the acidic region of FV, and was localized at the level of FV activation and/or prothrombinase. In model systems, TFPI C-term inhibited both FV activation and prothrombinase activity. Western blot analysis showed that the peptide impaired cleavage at Arg(1545) by both thrombin and FXa. The inhibition was stronger for FV-short, which binds TFPI with higher affinity. Similar results were obtained with full-length TFPI.

Conclusions Cleavage of FV at Arg(1545), which abolishes the anticoagulant properties of FV and commits FV to the procoagulant pathway, is inhibited by binding of the TFPI C-terminus to the FV acidic region. Possible targets of this new anticoagulant function of TFPI are low-abundance FV(a) species retaining the acidic region.