Cleavage at both Arg306 and Arg506 is required and sufficient for timely and efficient inactivation of factor Va by...

Area of Science:

• Biochemistry
• Hematology
• Molecular Biology

Background:

• Activated protein C (APC) is a key regulator of blood coagulation.
• APC inactivates factor Va, a critical cofactor in the prothrombinase complex, through specific proteolytic cleavages.
• Understanding the contribution of each cleavage site to factor Va inactivation is essential for comprehending coagulation regulation.

Purpose of the Study:

• To determine the relative importance of individual APC cleavage sites (Arg, Arg, Arg) in the inactivation of factor Va.
• To investigate the functional consequences of mutations at these specific cleavage sites on factor Va activity and prothrombinase complex function.

Main Methods:

• Construction and expression of recombinant factor V variants with specific Arg-to-Gln mutations.
• Purification of recombinant factor V molecules.
• Assays for factor Va activity and prothrombinase complex function, including clotting assays and kinetic analyses (Km, kcat) before and after APC treatment.

Main Results:

• Mutations at Arg and Arg rendered factor Va resistant to inactivation by APC, retaining approximately 70% activity in clotting assays.
• Prothrombinase complex assembled with factor Va (R→Q) showed a 20-fold reduction in kcat, while factor Va (R→Q) showed a 2-fold reduction.
• Factor Va variants lacking cleavage sites at Arg and Arg were significantly resistant to APC-mediated inactivation, especially in the presence of a membrane surface.

Conclusions:

• The cleavage sites at Arg and Arg are critical for the efficient inactivation of factor Va by APC.
• The functional impact of APC-mediated cleavage depends on the specific site and the assay employed.
• In the absence of Arg and Arg cleavage sites, factor Va remains largely active and resistant to inactivation by APC on membrane surfaces.