Role of the N-Terminal Amino Acid Region of Factor Va Light Chain In Prothrombinase Assembly and Function.
Authors: Vaughn John L. , Hirbawi Jamila , Bukys Michael A. , Kalafatis Michael 
Published: October 2019

Abstract 1127

The timely activation of prothrombin (II) to thrombin (IIa) by the prothrombinase (IIase) complex is required for the maintenance of hemostasis. The IIase complex is composed of the enzyme factor Xa (fXa) and the cofactor factor Va (fVa) assembled on a membrane surface in the presence of divalent ions. While fXa alone is capable of activating II, the incorporation of fVa into the IIase complex results in a 300,000-fold increase in the catalytic efficiency of fXa for IIa formation. FVa is generated following limited proteolysis of factor V (fV) by IIa at Arg709, Arg1018, and Arg1545, releasing a heavy chain (residues 1–709) and light chain (residues 1546–2196) that associate noncovalently through divalent ions. The A3 domain of the light chain of fVa has been implicated in the interaction of the cofactor with fXa. To determine the contribution of the A3 domain of fVa to IIase assembly and function, synthetic peptides representing residues 1546–1612 were screened for their ability to inhibit IIase. The peptide representing residues 1546–1558 strongly inhibited IIase with an IC50 of 50 μM. Additional fluorometric studies with overlapping pentapeptides from that region revealed that amino acids 1553–1558 were responsible for the observed inhibition. To verify the data from the overlapping pentapeptide studies, site-directed mutagenesis was used to generate a fVa mutant molecule with amino acids 1549–1558 deleted (fVaLCD1). FVaLCD1 demonstrated greatly reduced clotting activity in two-stage clotting assays. Gel electrophoresis of the IIase catalyzed activation of II using fVaLCD1 revealed a 50% reduction in the rate of prothrombin consumption. IIase assembled with fVaLCD1 exhibited a 5-fold increase in the apparent disassociation constant (Kdapp) for the enzyme-cofactor interaction and a 50% reduction in the turnover number (kcat) for the enzyme. To investigate the possibility that amino acids 1549–1558 are important for procofactor activation, the fVaLCD1 mutant was incubated with IIa and visualized with gel electrophoresis. The fVaLCD1 mutant exhibited a greatly reduced rate of IIa catalyzed activation. Additional recombinant fVa molecules with overlapping alanine mutations within the amino acid region 1548–1559 revealed that amino acids 1554–1555 were responsible for the observed effects in fVaLCD1. The recombinant fVa molecule with amino acids 1554–1555 mutated to alanines (fVaAA) demonstrated a greatly reduced clotting activity, an 8-fold increase in the Kdapp for the enzyme-cofactor interaction, and a 50% reduction in the kcat for the enzyme. To further investigate the mutational robustness of fVa against mutations in amino acids 1554–1555, a recombinant fVa molecule with both tyrosine residues in that region mutated conservatively to phenylalanines (fVaFF) was created. The more conservative fVaFF mutant, which differed from fVaWT only in the absence of side-chain hydroxyl moieties, exhibited normal clotting activity, kinetics, and procofactor activation. Altogether, the data demonstrate that amino acids 1554–1555 from the N-terminal region of the fVa light chain are important for IIase assembly and function because they constitute an interactive site for IIa and fXa.

Disclosures:

No relevant conflicts of interest to declare.