Functional properties of recombinant factor V mutated in a potential calcium-binding site

Area of Science:

• Biochemistry
• Molecular Biology
• Hematology

Background:

• Activated coagulation factor V (FVa) is essential for prothrombin activation, acting as a cofactor for activated factor X (FXa).
• FVa comprises a light chain (LC) and a heavy chain (HC) noncovalently associated in a calcium-dependent manner.

Purpose of the Study:

• To investigate the role of specific calcium-binding residues in FVa activity.
• To create a recombinant FV mutant (rFV-NN) to abolish calcium binding and assess its functional consequences.

Main Methods:

• Construction of a recombinant FV mutant (rFV-NN) with Asp111Asn/Asp112Asn substitutions.
• Assessing FVa activity after thrombin activation of rFV-wt and rFV-NN.
• Chromatographic analysis (SP-Sepharose) to evaluate HC-LC subunit association.
• Investigating the effect of FXa, inhibited FXa, and prothrombin on rFVa-NN stability.

Main Results:

• Thrombin-activated rFV-wt exhibited stable FVa activity.
• rFV-NN showed transient FVa activity that was rapidly lost upon prolonged incubation.
• Chromatography revealed dissociation of HC and LC in rFVa-NN, unlike the stable association in rFVa-wt.
• FXa and prothrombin partially prevented rFVa-NN activity loss in a dose-dependent manner.

Conclusions:

• The Asp111Asn/Asp112Asn substitutions disrupt the high-affinity calcium-dependent interaction between FVa's HC and LC.
• These mutations likely impair the calcium-binding site in FV, leading to unstable FVa post-activation.
• The rapid loss of activity in rFVa-NN is attributed to HC-LC dissociation.