Factor V (FV) is recently shown to express anticoagulant activity. It functions as a synergistic cofactor with protein S to activated protein C (APC) in the degradation of factor VIIIa (FVIIIa). FV is composed of multiple domains, A1-A2-B-A3-C1-C2. Thrombin cleaves FV at Arg-709, Arg-1018, and Arg-1545 that leads to the generation of a procoagulant FV species which functions as a cofactor to factor Xa (FXa) in the activation of prothrombin to thrombin. During the activation process, the B-domain is released from the heavy (A1-A2) and light chains (A3-C1-C2) which constitute the active FV (FVa). To elucidate which effect the different thrombin cleavages in FV have on the ability of FV to express APC-cofactor activity, seven recombinant FV mutants containing all possible combinations of mutated and native thrombin cleavage sites were tested in a FVIIIa degradation assay. Thrombin cleavage at Arg-709 and/or Arg-1018 yielded FV molecules that were still able to function as APC cofactors, whereas cleavage at Arg-1545 led to a complete loss in APC-cofactor function. This suggests that the APC-cofactor function of FV depends on the B-domain remaining attached to the A3 domain. The importance of the FV B-domain for expression of APC-cofactor activity was further investigated using two B-domain deleted FV molecules, FV des-709-1545 (with the whole B-domain deleted) and FV des-709-1476 (with amino acids 710-1476 of the B-domain being removed). FV des-709-1476 expressed APC-cofactor activity, whereas the FV des-709-1545 was completely devoid of such activity. Thus, the C-terminal part of the B-domain (residues 1477-1545) was crucial for the APC-cofactor function. FV and factor VIII (FVIII) are homologous proteins having similar domain organization. A FV/FVIII chimera, harboring the B-domain from FVIII (FVBVIII) instead of the FV B-domain did not work as an APC cofactor, further illustrating the importance of the FV B-domain for the APC-cofactor function.