THE MECHANISM OF INACTIVATION OF HUMAN FACTOR-V AND HUMAN FACTOR VA BY ACTIVATED PROTEIN-C

The cleavage of human factor V and human factor Va by human activated protein C (APC) was analyzed in the absence and presence of phospholip id vesicles containing 75% phosphatidylcholine (PC) and 25% phosphatid ylserine (PS). Membrane-bound human factor V (250 nM) is cleaved by AP C (2.5 nM) to give M(r) = 200,000, 70,000, 45,000, and 30,000 fragment s and an M(r) = 22/20,000 doublet. These fragments are released after four sequential cleavages of the membrane bound procofactor at Arg(308 ), Arg(506), Arg(679) and Lys994. No cofactor activity is observed fol lowing thrombin treatment of the membrane-bound APC-cleaved procofacto r. In the absence of a membrane surface, no cleavage of factor V by AP C is observed, and following thrombin activation factor Va retains ful l cofactor activity, Membrane-bound human factor Va (600 nM) loses mor e than 90% of its initial cofactor activity after 10 min of incubation with APC (10.9 nM), and virtually no cofactor activity is observed af ter 1 h of incubation. Under similar conditions but in the absence of PCPS vesicles, factor Va is cleaved but retains approximately 80% of i ts initial cofactor activity after 2 h of incubation with APC, In the presence of PCPS vesicles, the APC related loss of activity is correla ted with cleavage of the heavy chain and appearance of fragments of M( r) = 45,000, 30,000, and of 28/26,000, and 22/20,000 doublets, These p roducts correspond to three cleavages of the heavy chain (at Arg(306), Arg(506) and Arg(679)). Cleavage at Arg(506) of factor Va precedes an d appears to be required for cleavage at Arg(306) and Arg(679). In the absence of membrane, proteolysis at Arg(506) produces an M(r) = 75,00 0 fragment which corresponds to the NH2-terminal portion of the human factor Va heavy chain (residues 1-506), and a carboxyI-terminal double t of M(r) = 28/26,000 (residues 507-709) which is cleaved by APC at Ar g(679) to generate an M(r) = 22/20,000 doublet and an M(r) = 6,000 pep tide. No cleavage of the light chain of the human cofactor is observed in the presence or absence of PCPS vesicles following 2 h of incubati on with APC, Our data demonstrate that inactivation of human factor V and human factor Va only occurs in the presence of a membrane surface after cleavage at Arg(306). However, while this cleavage site is expos ed an membrane bound human factor V, cleavage at Arg(506) on the heavy chain of factor Va appears necessary for complete exposure of the cle avage site at Arg(306).