Activated protein C (APC) exerts its anticoagulant activity via proteolytic degradation of the heavy chains of activated factor VIII (FVIIIa) and activated factor V (FVa). So far, three APC cleavage sites have been identified in the heavy chain of FVa: Arg-306, Arg-506, and Arg-679. To obtain more insight in the structural and functional implications of each individual cleavage, recombinant factor V (rFV) mutants were constructed in which two or three of the APC cleavage sites were mutated. After expression in COS-1 cells, rFV mutants were purified, activated with thrombin, and inactivated by APC. During this study we observed that activated rFV-GQA (rFVa-GQA), in which the arginines at positions 306, 506, and 679 were replaced by glycine, glutamine, and alanine, respectively, was still inactivated by APC. Further analysis showed that the inactivation of rFVa-GQA by APC was phospholipid-dependent and sensitive to an inhibitory monoclonal antibody against protein C. Inactivation proceeded via a rapid phase (kx1=5.4 x 10(4) M(-1) s(-1)) and a slow phase (kx2=3.2 x 10(3) M(-1) s(-1)). Analysis of the inactivation curves showed that the rapid phase yielded a reaction intermediate that retained approximately 80% of the original FVa activity, whereas the slow cleavage resulted in formation of a completely inactive reaction product. Inactivation of rFVa-GQA was accelerated by protein S, most likely via stimulation of the slow phase. Immunoblot analysis using a monoclonal antibody recognizing an epitope between Arg-306 and Arg-506 indicated that during the rapid phase of inactivation a fragment of 80 kDa was generated that resulted from cleavage at a residue very close to Arg-506. The slow phase was associated with the formation of fragments resulting from cleavage at a residue 1.5-2 kDa carboxyl-terminal to Arg-306. Our observations may explain the unexpectedly mild APC resistance associated with mutations at Arg-306 (FV HongKong and FV Cambridge) in the heavy chain of FV.