Modulating the Factor V Procofactor to Cofactor Transition Using Recombinant B-Domain Fragments

Abstract

Abstract 376Activation of clotting factors from their inactive precursor states by limited and discrete proteolysis is a hallmark of coagulation. Prothrombinase, the physiological activator of prothrombin, is comprised of the serine protease factor Xa (FXa) and its cofactor factor Va (FVa). The precursor of FVa, FV, is an inactive procofactor and cannot participate to any significant degree in prothrombinase. It is well established that the large central B domain of FV (domain organization of A1-A2-B-A3-C1-C2) stabilizes the inactive procofactor state, however the mechanism by which this occurs is poorly defined. Since FVa has such a profound impact on thrombin generation and clot formation, defining the mechanism by which FV is kept inactive may reveal a unique way to target the cofactor for therapeutic gain. We previously demonstrated that deletion of a conserved region of the B domain enriched in basic residues converts FV to an active cofactor (JBC 2007, 282: 15033). Here we provide new insights into the mechanism by which the basic region of the B-domain contributes to maintaining FV as an inactive procofactor. Using a bacterial expression system, we generated FV B-domain fragments and assessed their ability to disrupt the procofactor to cofactor transition. The FV variant FV-810 (residues 811–1491 deleted), which lacks over 80% of the B-domain including the basic region, possesses activity comparable to FVa in clotting assays, thrombin generation assays, and in vitro prothrombin activation reactions. Addition of a FV B-domain fragment containing the basic region (residues 951–1008; ‘basic peptide’) inhibited FV-810 activity in all three assays. As a control, no inhibitory effect was observed with a non-homologous peptide from the FVIII B-domain. Surprisingly, the basic peptide did not inhibit FVa in these assays, nor did it inhibit FV-810 or other FV variants that had been pre-incubated with thrombin. These results indicate that additional B-domain sequences present in FV-810 must somehow contribute to the inhibitory effect of the basic region. Analysis of the B-domain sequences within FV-810 and other cofactor-like FV variants revealed a concentration of acidic residues between Thr1492 and Asn1538 that may help to stabilize FV in an inactive state most likely through an electrostatic interaction with the basic region. In support of this model, FV BR152, a variant that possesses the basic region but lacks the acidic region, has FVa-like activity in clotting assays and prothrombin activation assays. Unlike the basic peptide, a peptide derived from the acidic region (residues 1492–1538) did not inhibit the activity of FV BR152, indicating that the acidic region must be located proximal to the A3 domain in order to inhibit FV. To further probe the mechanism by which the basic peptide functions, we directly monitored FXa binding to FV-810 on phospholipid vesicles using fluorescence anisotropy. In the absence of the basic peptide, labeled FXa bound to membrane-bound FV-810 with a Kd of ∼1 nM in a 1:1 stoichiometry. Addition of the FV basic peptide significantly reduced FXa binding to FV-810 in a dose-dependent manner, indicating competition between FXa and the basic peptide for FV-810. Consistent with the results of the functional assays, the basic peptide had no effect on FXa binding to membrane-bound FVa. Taken together our data support a model in which the basic B-domain region of FV inhibits the cofactor activity of FV through a functional interaction with an acidic region proximal to the A3 domain, thereby masking a FXa binding site that is exposed following thrombin cleavage of FV at Arg1545. These findings offer important new insights into how FV is kept as an inactive procofactor and provide key mechanistic clues into how the B-domain participates in this process. Moreover, these results demonstrate a novel strategy to regulate FV cofactor activity by using functional elements of the B-domain to shift an active cofactor back to an inactive procofactor. Disclosures:No relevant conflicts of interest to declare.