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Importance of individual activated protein C cleavage site regions in coagulation factor V for factor Va inactivation
M J Heeb1, A Rehemtulla, M Moussalli
1Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, CA 92037, USA. heeb@scripps.edu
Insights
Activated protein C (APC) inactivates Factor Va (FVa) by cleaving it at specific sites. Cleavage at R306 is crucial for efficient FVa inactivation by APC, while cleavage at R506 alone results in partial activity.
Area of Science:
- Biochemistry
- Molecular Biology
- Hematology
Background:
- Activated protein C (APC) is a key regulator of blood coagulation.
- Factor Va (FVa) is a critical cofactor in the prothrombinase complex.
- APC resistance, often due to Factor V (FV) mutation R506Q, is linked to thrombotic risk.
Purpose of the Study:
- To investigate the role of individual APC cleavage sites (R506, R306) in FVa inactivation.
- To determine the importance of regions near these cleavage sites for APC function.
- To elucidate the impact of specific FV mutations on APC resistance and FVa activity.
Main Methods:
- Preparation and purification of recombinant human FV variants (wild-type, Q306-rFV, Q506-rFV, Q306Q506-rFV).
- Assessment of thrombin activation kinetics.
- Evaluation of APC cleavage and inactivation using plasma-clotting assays and prothrombinase assays.
- Measurement of residual FVa activity and Factor Xa (FXa) binding.
Main Results:
- Cleavage at R306 is essential for efficient FVa inactivation by APC.
- FVa cleaved only at R506 retains partial activity, indicating the importance of R306 for complete inactivation.
- The Q306Q506-rFV mutant exhibited significant resistance to APC and FXa, with impaired FXa binding.
- APC-mediated cleavage at R306 and R506 may downregulate FV/Va by affecting FXa-binding sites.
Conclusions:
- Cleavage at either R506 or R306 is necessary for significant FVa inactivation by APC.
- R306 is the primary cleavage site for efficient FVa inactivation by APC.
- Regions near R306 and R506 are involved in FXa binding, and their impairment by APC cleavage affects FV/Va regulation.
Abstract:
Activated protein C (APC) cleavage of Factor Va (FVa) at residues R506 and R306 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, Q306-rFV, Q506-rFV, and Q306Q506-rFV. All had similar time courses for thrombin activation. Q506-rFVa was cleaved by APC at R306 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 Q506-FVa. Q306-rFVa was cleaved by APC at R506 and gave a low APC-resistance ratio similar to Q506-rFVa in clotting assays, whereas unactivated Q306-rFV gave a near-normal APC-resistance ratio. When FVa residual activity was measured after long exposure to APC, Q306-rFVa was inactivated by only < or = 40% under conditions where Q506-rFVa was inactivated > 90%, supporting the hypothesis that efficient inactivation of normal FVa by APC requires cleavage at R306. In addition, the heavy chain of Q306-rFVa was cleaved at R506 much more rapidly than activity was lost, suggesting that FVa cleaved at only R506 is partially active. Under the same conditions, Q306Q506-rFVa lost no activity and was not cleaved by APC. Therefore, cleavage at either R506 or R306 appears essential for significant inactivation of FVa by APC. Modest loss of activity, probably due to cleavage at R679, was observed for the single site rFVa mutants, as evidenced by a second phase of inactivation. Q306Q506-rFVa had a low activity-to-antigen ratio of 0.50-0.77, possibly due to abnormal Factor Xa (FXa) binding. Furthermore, Q306Q506-rFV was very resistant to cleavage and activation by FXa. Q306Q506-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 R306 and R506. This study shows that R306 is the most important cleavage site for normal efficient inactivation of FVa by APC and supports other studies suggesting that regions near R306 and R506 provide FXa-binding sites and that FVa cleaved at only R506 retains partial activity.