Importance of individual activated protein C cleavage site regions in coagulation Factor V for Factor Va inactivation and for Factor Xa activation

Activated protein C (APC) cleavage of Factor Va (FVa) at residues R-506 and R-306 correlates With its inactivation. APC resistance and increased thrombotic risk are due to the mutation R506Q in Factor V (FV). To study the effects of individual cleavages in FVa by APC and the importance of regions near the cleavage sites, the following recombinant (r) human FVs were prepared and purified: wild-type, Q(306)-rFV, Q(506)-rFV, and Q(306)Q(506)-rFV. All had similar time courses for thrombin activation. Q(506)-rFVa was cleaved by APC at R-306 and was moderately resistant to APC in plasma-clotting assays and in prothrombinase assays measuring FVa residual activity, in agreement with studies of purified plasma-derived Q(506)-FVa. Q(506)-rFVa was cleaved by APC at R-306 and gave a low APC-resistance ratio similar to Q(506)-rFVa in clotting assays, whereas unactivated Q(306)-rFV gave a near-normal APC-resistance ratio. When FVa residual activity was measured after lone exposure to APC, Q(306)-rFVa was inactivated by only less than or equal to 40% under conditions where Q(506)-rFVa was inactivated > 90%, supporting the hypothesis that efficient inactivation of normal FVa by APC requires cleavage at R-306. In addition, the heavy chain of Q(306)-rFVa was cleaved at R-506 much more rapidly than activity was lost, suggesting that FVa cleaved at only R-506 is partially active. Under the same conditions, Q(306)Q(506)-rFVa lost no activity and was not cleaved by APC. Therefore, cleavage at either R-506 or R-306 appears essential for significant inactivation of FVa by APC. Modest loss of activity, probably due to cleavage at R-679, was observed for the single site rFVa mutants, as evidenced by a second phase of inactivation. Q(306)Q(506)-rFVa had a low activity-to-antigen ratio of 0.50-0.77, possibly due to abnormal Factor Xa (FXa) binding. Furthermore, Q(306)Q(506)-rFV was very resistant to cleavage and activation by FXa. Q(306)Q(506)-rFV appeared to bind FXa and inhibit FXa's ability to activate normal FV. Thus, APC may downregulate FV/Va partly by impairing FXa-binding sites upon cleavage at R-306 and R-506. This study shows that R-306 is th, most important cleavage site for normal efficient inactivation of FVa by APC and supports Ether studies suggesting that regions near R-306 and R-506 provide FXa-binding sites and that FVa cleaved at only R-506 retains partial activity.