PROTEOLYSIS OF FACTOR-V BY CATHEPSIN-G AND ELASTASE INDICATES THAT CLEAVAGE AT ARG(1545) OPTIMIZES COFACTOR FUNCTION BY FACILITATING FACTORXA 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 prot eases to generate a cofactor species with various levels of cofactor f unction. Having demonstrated previously that monocyte-bound forms of c athepsin G and elastase cleave and activate factor V, studies were ini tiated here using purified proteins to probe factor V structure/functi on. Electrophoretic, Western blotting, and amino-terminal sequence ana lyses revealed that cathepsin G cleaves factor V at several sites (Phe (1031), Leu(1447), Tyr(1518), and potentially Tyr(696)), ultimately ge nerating an amino-terminal 103 kDa heavy chain and a carboxy-terminal 80 kDa light chain (FVa(CG)), Elastase also cleaves factor V at severa l sites (Ile(708), Ile(819), Ile(1484), and potentially Thr(678)), gen erating a cofactor species, FVa(HNE), With an amino-terminal 102 kDa h eavy chain and a carboxy-terminal 90 kDa light chain. Incubation of FV a(IIa), with either cathepsin G or elastase resulted in cleavage withi n the heavy chain, releasing peptides of similar to 2000 and similar t o 3000 Da, respectively, generating FVa(IIa/CG) and FVa(IIa/HNE). The functional activity of each cofactor species was assessed either by cl otting assay or by employing a purified prothrombinase assay using sat urating amounts of factor Xa, Significant differences in cofactor func tion were observed between the two assay systems, Whereas FVa(IIa), FV a(CG), FVa(IIa/CG), FVa(HNE), and FVa(IIa/HNE) all had similar cofacto r 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 inter actions of these cofactors with the various prothrombinase components. Kinetic analyses indicated that FVa(IIa) (K-d(app) = 0.096 nM), FVa(I Ia/CG) (K-d(app) = 0.244 nM), and FVa(IIa/HNE) (K-d(app) = 0.137 nM) b ound 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, st udies of the activated protein C (APC)-catalyzed inactivation of each of the factor V(a) species indicated that they were all equivalent sub strates for APC with no differences observed in the rate of inactivati on or the cleavage mechanism, suggesting that APC interacts with the l ight chain at a site distinct from factor Xa. The K-m values for proth rombin, as well as the k(cat) values for each of the FV(a) species, we re 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 ve sicles (similar to 2-3-fold), the remaining FV(a) species assembled eq ually well on this surface, Collectively, these data indicate that FVa (CG) and FVa(HNE) have a diminished capacity to support factor Xa bind ing; however, cleavage at Arg(1545) and removal of the extended B-doma in in these cofactors restore near-total factor Xa binding. Thus, clea vage at Arg(1545) optimizes cofactor function within prothrombinase by facilitating factor Xa binding to membrane-bound FVa.