A phosphatidylserine binding site in factor Va C1 domain regulates both assembly and activity of the prothrombinase...

Area of Science:

• Biochemistry
• Hematology
• Molecular Biology

Background:

• Factor V(a) (FVa) and factor X(a) (FXa) form the prothrombinase complex essential for blood coagulation.
• This complex assembles on phosphatidylserine (PS)-rich platelet membranes.
• Previous work showed soluble PS (C6PS) can trigger complex assembly in solution.

Purpose of the Study:

• To investigate the role of FVa domains in binding soluble PS and regulating prothrombinase complex assembly.
• To determine how mutations in FVa's C1 and C2 domains affect FXa binding and prothrombin activation.

Main Methods:

• Expression of human factor V(a) (rFVa) with specific mutations in C1 and C2 domains.
• Assays to measure prothrombin activation rates by FXa in the presence of C6PS.
• Determination of dissociation constants (K(d')) for FXa binding to rFVa variants.
• Equilibrium dialysis to quantify C6PS binding to different rFVa mutants.

Main Results:

• Mutations in the C1 and C1-C2 domains of rFVa significantly reduced prothrombin activation rates (14,000-15,000-fold).
• FXa binding affinity to rFVa was drastically decreased by C1 domain mutations (K(d') increased from 3-4 nM to 564-624 nM).
• C6PS binding experiments indicated that both C1 and C2 domains bind one molecule of C6PS each.

Conclusions:

• The C1 and C2 domains of FVa both bind soluble phosphatidylserine.
• Binding of C6PS to the FVa C1 domain is critical for regulating prothrombinase complex assembly and function.
• These findings elucidate a key mechanism in the regulation of blood coagulation.