Phosphatidylserine-induced factor Xa dimerization and binding to factor Va are competing processes in solution.

Area of Science:

• Biochemistry
• Molecular Biology
• Hematology

Background:

• Phosphatidylserine (PS) plays a crucial role in coagulation factor assembly.
• Soluble short-chain PS, like 1,2-dicaproyl-sn-glycero-3-phospho-l-serine (C6PS), can modulate coagulation factor activity.
• FXa, FVa, and prothrombin are key components of the prothrombinase complex.

Purpose of the Study:

• To investigate the competitive nature of FXa dimerization and FVa binding in the presence of C6PS.
• To determine the specific domains of FXa involved in C6PS-mediated dimerization and FVa interaction.
• To elucidate the thermodynamic linkage between FXa dimerization and FVa binding sites.

Main Methods:

• Enzyme kinetics assays to measure FXa activity and binding constants.
• Titration experiments with varying concentrations of FXa and FVa in the presence of C6PS and calcium.
• Site-directed mutagenesis of FXa (R165A) to assess the impact on dimerization and FVa binding.
• Native gel electrophoresis to analyze protein complex formation.

Main Results:

• C6PS promotes FXa dimerization with a K(d) of ~14 nM and enhances FXa catalytic and FVa cofactor activities.
• FXa dimerization and FVa binding sites are both located within the catalytic domain of FXa.
• The binding sites for FXa dimerization and FVa are thermodynamically linked, influencing each other's affinity.
• A mutant FXa (R165A) exhibited weakened dimerization and FVa binding, supporting the role of the catalytic domain.

Conclusions:

• FXa dimerization and FVa binding are not competitive but occur at linked sites within the catalytic domain.
• C6PS binding to FXa alters its conformation, facilitating both homodimerization and interaction with FVa.
• Understanding these interactions is crucial for comprehending prothrombinase complex assembly and regulation.