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 (FVaIIa) at Arg709, Arg1018, and Arg1545 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 (Phe1031, Leu1447, Tyr1518, and potentially Tyr696), ultimately generating an amino-terminal 103 kDa heavy chain and a carboxy-terminal 80 kDa light chain (FVaCG). Elastase also cleaves factor V at several sites (Ile708, Ile819, Ile1484, and potentially Thr678), generating a cofactor species, FVaHNE, with an amino-terminal 102 kDa heavy chain and a carboxy-terminal 90 kDa light chain. Incubation of FVaIIa with either cathepsin G or elastase resulted in cleavage within the heavy chain, releasing peptides of approximately 2000 and approximately 3000 Da, respectively, generating FVaIIa/CG and FVaIIa/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 FVaIIa, FVaCG, FVaIIa/CG, FVaHNE, and FVaIIa/HNE all had similar cofactor activities in the purified prothrombinase assay, FVaCG and FVaHNE had no cofactor activity in the clotting-based assay, and FVaIIa/CG and FVaIIa/HNE had approximately 30-35% clotting activity relative to FVaIIa. These disparate results led us to examine the binding interactions of these cofactors with the various prothrombinase components. Kinetic analyses indicated that FVaIIa (Kd(app) = 0.096 nM), FVaIIa/CG (Kd(app) = 0.244 nM), and FVaIIa/HNE (Kd(app) = 0.137 nM) bound to membrane-bound factor Xa much more effectively than FVaCG (Kd(app) = 1.46 nM) and FVaHNE (Kd(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 Km values for prothrombin, as well as the kcat values for each of the FV(a) species, were all similar (approximately 0.25 microM and approximately 1900 min-1). In addition, kinetic analyses indicated that whereas FVaCG and FVaHNE exhibited a slightly reduced ability to interact with phospholipid vesicles (approximately 2-3-fold), the remaining FV(a) species assembled equally well on this surface. Collectively, these data indicate that FVaCG and FVaHNE have a diminished capacity to support factor Xa binding; however, cleavage at Arg1545 and removal of the extended B-domain in these cofactors restore near-total factor Xa binding. Thus, cleavage at Arg1545 optimizes cofactor function within prothrombinase by facilitating factor Xa binding to membrane-bound FVa.
Department of Biochemistry, University of Vermont, College of Medicine, Burlington, Vermont 05405, USA.
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Camire
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Kalafatis
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Tracy
D001120:Arginine / Q000378:metabolism*; D001665:Binding Sites; D001780:Blood Coagulation Tests / Q000379:methods; D002384:Catalysis; D056649:Cathepsin G; D002403:Cathepsins / Q000378:metabolism* / Q000502:physiology; D004789:Enzyme Activation; D005165:Factor V / Q000737:chemistry / Q000378:metabolism*; D015943:Factor Va / Q000378:metabolism; D015951:Factor Xa / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008567:Membranes, Artificial; D010196:Pancreatic Elastase / Q000378:metabolism* / Q000502:physiology; D010713:Phosphatidylcholines / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D011485:Protein Binding; D011486:Protein C / Q000378:metabolism; D011516:Prothrombin / Q000378:metabolism; D012697:Serine Endopeptidases
D008567:Membranes, Artificial; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D011486:Protein C; D015943:Factor Va; D005165:Factor V; D011516:Prothrombin; D001120:Arginine; D002403:Cathepsins; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G; D010196:Pancreatic Elastase; D015951:Factor Xa