Factor V-short and protein S as synergistic tissue factor pathway inhibitor (TFPIα) cofactors
Björn Dahlbäck1, Li Jun Guo1, Ruzica Livaja-Koshiar1
1Department of Translational Medicine Lund University Skåne University Hospital Malmö Sweden.
Background:
FV-Short is a normal splice variant of Factor V (FV) having a short B domain, which exposes a high affinity-binding site for tissue factor pathway inhibitor α (TFPIα). FV-Short and TFPIα circulate in complex in plasma.
Objectives:
The aim was to elucidate whether FV-Short affects TFPIα as inhibitor of coagulation FXa and to test whether the TFPIα-cofactor activity of protein S is influenced by FV-Short.
Methods:
Recombinant FV, wild-type FV-Short and a FV-Short thrombin-cleavage resistant variant were expressed and purified. The influence of FV and FV-Short variants and/or protein S on the FXa inhibitory activity of TFPIα was monitored both in a purified system and in a plasma-based thrombin generation assay.
Results:
FV-Short had intrinsically weak TFPIα-cofactor activity but with protein S present, FV-Short yielded efficient inactivation of FXa. Protein S alone did not promote full TFPIα-activity. Intact FV was inefficient at low protein S concentrations and had 10-fold lower activity compared to FV-Short at physiological protein S levels. Activation of FV-Short by thrombin resulted in the loss of the TFPIα-cofactor activity. The synergistic TFPIα-cofactor activity of FV-Short and protein S was also demonstrated in plasma using a thrombin generation assay.
Conclusions:
FV-Short and protein S are highly efficient, synergistic cofactors to TFPIα in the regulation of FXa activity, whereas full length FV has lower activity. Our results suggest the formation of an efficient FXa-inhibitory complex between FV-Short, TFPIα and protein S on the surface of negatively charged phospholipids.
Insights
Factor V-Short (FV-Short) and protein S act as efficient cofactors for tissue factor pathway inhibitor α (TFPIα), enhancing the inhibition of Factor Xa (FXa) coagulation. This synergistic activity is crucial for regulating blood clotting.
Area of Science:
- Biochemistry
- Hematology
- Molecular Biology
Background:
- Factor V-Short (FV-Short) is a splice variant of Factor V (FV) with a truncated B domain.
- FV-Short exposes a high-affinity binding site for tissue factor pathway inhibitor α (TFPIα).
- FV-Short and TFPIα form a complex in plasma, suggesting a role in coagulation regulation.
Purpose of the Study:
- To investigate FV-Short's effect on TFPIα's inhibition of coagulation Factor Xa (FXa).
- To determine if protein S influences the TFPIα-cofactor activity of FV-Short.
- To compare the cofactor activity of FV-Short with intact Factor V (FV).
Main Methods:
- Recombinant FV, FV-Short, and a thrombin-cleavage resistant FV-Short variant were expressed and purified.
- TFPIα's FXa inhibitory activity was assessed in the presence of FV, FV-Short, and/or protein S.
- Assays included purified systems and plasma-based thrombin generation tests.
Main Results:
- FV-Short exhibited weak intrinsic TFPIα-cofactor activity, but became efficient with protein S.
- Protein S alone did not fully support TFPIα activity; intact FV showed lower activity than FV-Short.
- Thrombin activation of FV-Short abolished its TFPIα-cofactor activity, while synergistic activity was confirmed in plasma.
Conclusions:
- FV-Short and protein S are potent synergistic cofactors for TFPIα in regulating FXa activity.
- FV-Short, TFPIα, and protein S form an efficient FXa inhibitory complex on phospholipid surfaces.
- This complex plays a significant role in the physiological regulation of coagulation.
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