Proteolysis of factor V by cathepsin G and elastase indicates that cleavage at Arg1545 optimizes cofactor function by facilitating factor Xa binding

The single-chain procofactor factor V is cleaved by thrombin (FVa(IIa)) at Arg(709), Arg(1018), and Arg(1545) and by a variety of other proteases to generate a cofactor species with various levels of cofactor function. Having demonstrated previously that monocyte-bound forms of cathepsin G and elastase cleave and activate factor V, studies were initiated here using purified proteins to probe factor V structure/function. Electrophoretic, Western blotting, and amino-terminal sequence analyses revealed that cathepsin G cleaves factor V at several sites (Phe(1031), Leu(1447), Tyr(1518), and potentially Tyr(696)), ultimately generating an amino-terminal 103 kDa heavy chain and a carboxy-terminal 80 kDa light chain (FVa(CG)), Elastase also cleaves factor V at several sites (Ile(708), Ile(819), Ile(1484), and potentially Thr(678)), generating a cofactor species, FVa(HNE), With an amino-terminal 102 kDa heavy chain and a carboxy-terminal 90 kDa light chain. Incubation of FVa(IIa), with either cathepsin G or elastase resulted in cleavage within the heavy chain, releasing peptides of similar to 2000 and similar to 3000 Da, respectively, generating FVa(IIa/CG) and FVa(IIa/HNE). The functional activity of each cofactor species was assessed either by clotting assay or by employing a purified prothrombinase assay using saturating amounts of factor Xa, Significant differences in cofactor function were observed between the two assay systems, Whereas FVa(IIa), FVa(CG), FVa(IIa/CG), FVa(HNE), and FVa(IIa/HNE) all had similar cofactor activities in the purified prothrombinase assay, FVa(CG) and FVa(HNE) had no cofactor activity in the clotting-based assay, and FVa(IIa/CG) and FVa(IIa/HNE) had similar to 30-35% clotting activity relative to FVa(IIa). These disparate results led us to examine the binding interactions of these cofactors with the various prothrombinase components. Kinetic analyses indicated that FVa(IIa) (K-d(app) = 0.096 nM), FVa(IIa/CG) (K-d(app) = 0.244 nM), and FVa(IIa/HNE) (K-d(app) = 0.137 nM) bound to membrane-bound factor Xa much more effectively than FVa(CG) (K-d(app) = 1.46 nM) and FVa(HNE) (K-d(app) = 0.818 nM). In contrast, studies of the activated protein C (APC)-catalyzed inactivation of each of the factor V(a) species indicated that they were all equivalent substrates for APC with no differences observed in the rate of inactivation or the cleavage mechanism, suggesting that APC interacts with the light chain at a site distinct from factor Xa. The K-m values for prothrombin, as well as the k(cat) values for each of the FV(a) species, were all similar (similar to 0.25 mu M and similar to 1900 min(-1)). In addition, kinetic analyses indicated that whereas FVa(CG) and FVa(HNE) exhibited a slightly reduced ability to interact with phospholipid vesicles (similar to 2-3-fold), the remaining FV(a) species assembled equally well on this surface, Collectively, these data indicate that FVa(CG) and FVa(HNE) have a diminished capacity to support factor Xa binding; however, cleavage at Arg(1545) and removal of the extended B-domain in these cofactors restore near-total factor Xa binding. Thus,cleavage at Arg(1545) optimizes cofactor function within prothrombinase by facilitating factor Xa binding to membrane-bound FVa.