The coagulation protein Factor Va forms the receptor for the serine protease Factor Xa at the platelet surface. This membrane-bound complex of Factor Va and Factor Xa plus calcium constitutes the enzymatic complex prothrombinase, which effects the conversion of prothrombin to the clotting enzyme, thrombin. Studies were undertaken to investigate the proteolytic events accompanying the inactivation of platelet-bound Factor Va by activated protein C as well as the ability of Factor Xa to protect Factor Va from activated protein C inactivation. During the course of these studies, observations were made which indicated that Factor Va was also cleaved by both a platelet-associated protease, as well as Factor Xa. When Factor Va was incubated with washed platelets, electrophoresis and autoradiography of solubilized platelet pellets indicated that three Factor Va peptides were associated with the platelet: component D (Mr = 94,000), component E (Mr = 74,000), and a 90,000-dalton peptide (component D') which appeared with time as the result of a platelet-associated protease cleavage of component D. The Factor Va peptides bound to platelets were proteolytically inactivated by activated protein C, resulting in five peptide products, all of which remained associated with the platelet-membrane surface. Factor Va was protected from activated protein C proteolysis by complex formation with Factor Xa or active site-blocked Factor Xa. However, active Factor Xa cleaved platelet-bound Factor Va to peptide products which also remained associated with the platelet. Whereas activated protein C rapidly cleaved components D and D' with secondary cleavages occurring in component E, Factor Xa rapidly cleaved component E with secondary cleavages occurring in components D and D'. The Factor Xa-cleaved Factor Va is catalytically functional. To determine whether cleavage was necessary for function, prothrombin conversion reaction mixtures were monitored for thrombin formation and Factor Va cleavage with time in a defined phospholipid vesicle model system. The results indicated that Factor Xa cleavage of Factor Va is not essential for Factor Va activity but may promote its ability to function in the prothrombinase complex.