Definition of a factor Va binding site in factor Xa

Area of Science:

• Biochemistry
• Molecular Biology
• Hemostasis

Background:

• Activated factor X (fXa) is a key enzyme in the coagulation cascade.
• Cofactor binding, specifically to factor Va (fVa), is essential for fXa's catalytic efficiency.
• Previous studies identified residue 347 as important for fVa binding.

Purpose of the Study:

• To identify additional residues involved in factor Va (fVa) binding to activated factor X (fXa).
• To characterize the functional consequences of mutations in these residues on fXa activity and cofactor interaction.

Main Methods:

• Site-directed mutagenesis was used to generate five mutant fX species.
• Enzyme kinetics, prothrombin activation assays, and competition binding assays were employed.
• Functional characterization was performed in the absence and presence of phospholipid vesicles and activated platelets.

Main Results:

• Mutations at Arg-347, Lys-351, and Lys-414 significantly reduced the apparent affinity for fVa.
• Mutations at Arg-306 and Glu-310 had less impact on fVa binding but affected prothrombin activation.
• All mutant enzymes showed reduced inhibition rates by antithrombin III in the presence of heparin.
• Enzyme function was preserved on activated platelets, indicating the importance of the surface-bound fVa.

Conclusions:

• Arg-347, Lys-351, and Lys-414 constitute a core binding site for fVa on fXa.
• Arg-306 and Glu-310 play a secondary role in fVa interaction or substrate binding.
• These findings elucidate the structural basis of fXa-fVa complex formation and its regulation.