MLS Lund University, Dept Translational Medicine Malmö, Skane Lan, Sweden
Background: The factor V (FV) splice isoform FV-Short functions in synergy with protein S as TFPIα-cofactor in inhibition of FXa. TFPIα binds to an exposed acid region (preAR2/AR2; 1481-1537) in the B domain. In intact FV, this region is hidden by an intra-B-domain interaction with the basic region (BR; 963-1008). During FV activation, thrombin or FXa cleaves at Arg709, Arg1018, and Arg1545. As Arg709 and Arg1018 are preferred cleavage sites, an activation intermediate (FVai) is generated with part of its B domain (1019-1545) being retained. As BR dissociates from FVai, the preAR2/AR2 region is exposed and available for TFPIα binding. Partially proteolyzed FV similar to FVai is present in platelets.
Aims: To investigate whether the FVai is able to function as synergistic TFPIα-cofactor with protein S in inhibition of FXa.
Methods: FV variants FVRRQ (Arg1545Gln) and FVQIR (Arg709Gln/Arg1018/Ile) were created and tested in the FXa-inhibition assay with TFPIα and protein S before and after thrombin (1 Unit/ml) cleavage. Thrombin-cleaved FVRRQ (cleaved at positions 709 and 1018 but not at 1545) is a model for FVai. FV and FV-Short were used as controls. To test natural FVai, plasma-derived FV was incubated with low levels of thrombin (titrated down to ≈ 1 mUnit/ml).
Results: After cleavage by thrombin, FVRRQ turns into an efficient synergistic TFPIα-cofactor with protein S being equally active as FV-Short. In contrast, thrombin-treated FVQIR (cleaved at Arg1545) had no synergistic TFPIα-cofactor activity. FVai generated from plasma-derived FV by low-dose thrombin (≈5 mUnits/ml), cleaving at positions 709 and 1018, demonstrated synergistic TFPIα-cofactor activity with protein S.
Conclusion(s): FVai, cleaved at Arg709 and Arg1018 but not at Arg1545, is demonstrated to be a synergistic TFPIα-cofactor activity with protein S. It is equally active as FV-Short. It may be particularly important after platelet activation as FVai and TFPIα are released from platelets.
Learning Objectives:
Upon completion, the participants will be able to describe that circulating coagulation factor V (FV) is a high molecular weight protein having the potential to be converted into either a pro- or an anticoagulant cofactor. They will be able to discuss how FV is activated in a stepwise fashion by thrombin or FXa. Three peptide bonds are cleaved, at Arg709, Arg1018 and Arg1545. The cleavage sites at Arg709 and Arg1018 are more sensitive that that at Arg1545 and thus an activation intermediate (FVai) is formed which is only cleaved at Arg709 and Arg1018. This intermediate is expressing partial activity as a FXa cofactor but is fully active as anticoagulant. The cleavage at Arg1545 generates fully active FVa which can participate in the prothrombinase complex (FXa, FVa and negatively charged phospholipids). The Arg1545 cleavage also results in loss of anticoagulant activity.
Upon completion, the participants will be able to describe that the FVai is able to function as an anticoagulant cofactor to activated protein C (APC) in the degradation of FVIIIa which it is part of the tenase complex (FIXa, FVIIIa and negatively charged phospholipids). The anticoagulant activity is lost when Arg1545 is also cleaved which results in the generation of FVa and full activity as a FXa cofactor in the prothrombinase complex.
Upon completion, the participants will be able to describe that FVai is functioning as an anticoagulant synergistic TFPIa cofactor with protein S. TFPIa binds to a high affinity binding site in the C-terminus of the B domain of FV which is exposed upon cleavage at Arg709 and Arg1018. Full activation of FV resulting from the cleavage at Arg1545 results in the loss of the B domain and the anticoagulant TFPIa cofactor activity.
