The mechanism of inactivation of bovine factor Va by plasmin was studied in the presence and absence of phospholipid vesicles (PCPS vesicles). Following 60-min incubation with plasmin (4 nM) membrane-bound factor Va (400 nM) is completely inactive, whereas in the absence of phospholipid vesicles following a 1-h incubation period, the cofactor retains 90% of its initial cofactor activity. Amino acid sequencing of the fragments deriving from cleavage of factor Va by plasmin demonstrated that while both chains of factor Va are cleaved by plasmin, only cleavage of the heavy chain correlates with inactivation of the cofactor. In the presence of a membrane surface the heavy chain of the bovine cofactor is first cleaved at Arg 348 to generate a fragment of M r 47,000 containing the NH 2 -terminal part of the cofactor (amino acid residues 1-348) and a M r 42,000 fragment (amino acid residues 349 -713). This cleavage is associated with minimal loss in cofactor activity. Complete loss of activity of the membrane-bound cofactor coincides with three cleavages at the COOH-terminal portion of the M r 47,000 fragment: Lys 309 , Lys 310 , and Arg 313 . These cleavages result in the release of the COOH terminus of the molecule and the production of a M r 40,000 fragment containing the NH 2 -terminal portion of the factor Va molecule. Factor Va was treated with plasmin in the absence of phospholipid vesicles followed by the addition of PCPS vesicles and activated protein C (APC). A rapid inactivation of the cofactor was observed as a result of cleavage of the M r 47,000 fragment at Arg 306 by APC and appearance of a M r 39,000 fragment. These data suggest a critical role of the amino acid sequence 307-348 of factor Va. A 42-amino acid peptide encompassing the region 307-348 of human factor Va (N42R) was found to be a good inhibitor of factor Va clotting activity with an IC 50 of ϳ1.3 M. These data suggest that plasmin is a potent inactivator of factor Va and that region 307-348 of the cofactor plays a critical role in cofactor function and may be responsible for the interaction of the cofactor with factor Xa and/or prothrombin.Coagulation involves a multitude of proteins that respond to a vascular injury by forming the procoagulant enzyme ␣-thrombin. Prothrombin is activated to ␣-thrombin by the prothrombinase complex, which is composed of the serine protease factor Xa, and the protein cofactor factor Va assembled on a membrane surface in the presence of Ca 2ϩ ions (1). Formation of the prothrombinase complex increases the catalytic efficiency of prothrombin activation by 5 orders of magnitude as compared with factor Xa alone (2).Factor V circulates in plasma as a single chain procofactor (M r 330,000). The cDNA sequence for bovine factor V and the deduced amino acid sequence has been determined previously (3).1 The active form of the protein, factor Va, contains a heavy chain (M r 94,000), which is derived from the NH 2 -terminal part of the procofactor (residues 1-713), and a light chain (M r 74,000), which corresponds to the C...