Trp2063and Trp2064in the Factor Va C2 Domain Are Required for High-Affinity Binding to Phospholipid Membranes but Not for Assembly of the Prothrombinase Complex

Citations Over TimeTop 17% of 2004 papers

Abstract

Interactions between factor Va and membrane phosphatidylserine (PS) regulate activity of the prothrombinase complex. Two solvent-exposed hydrophobic residues located in the C2 domain, Trp(2063) and Trp(2064), have been proposed to contribute to factor Va membrane interactions by insertion into the hydrophobic membrane bilayer. However, the prothrombinase activity of rHFVa W(2063, 2064)A was found to be significantly impaired only at low concentrations of PS (5 mol %). In this study, we find that 10-fold higher concentrations of mutant factor Va are required for half-maximal prothrombinase activity on membranes containing 25% PS. The ability of the mutant factor Va to interact with factor Xa on a membrane was also impaired since 4-fold higher concentrations of factor Xa were required for half-maximal prothrombinase activity. The interaction of factor Va with 25% PS membranes was also characterized using fluorescence energy transfer and surface plasmon resonance. We found that the affinity of mutant factor Va for membranes containing 25% PS was reduced at least 400-fold with a K(d) > 10(-7) M. The binding of mutant factor Va to 25% PS membranes was markedly enhanced in the presence of factor Xa, indicating stabilization of the factor Va-factor Xa-membrane complex. Our findings indicate that Trp(2063) and Trp(2064) play a critical role in the high-affinity binding of factor Va to PS membranes. It remains to be determined whether occupancy of this PS binding site in factor Va is also required for high-affinity binding to factor Xa.

Related Papers

• → Differentiation-dependent expression of phosphatidylserine in mammalian plasma membranes: quantitative assessment of outer-leaflet lipid by prothrombinase complex formation.(1989)186 cited
• → A phosphatidylserine binding site in factor Va C1 domain regulates both assembly and activity of the prothrombinase complex(2008)57 cited
• → Prothrombin Contributes to the Assembly of the Factor Va-Factor Xa Complex at Phosphatidylserine-containing Phospholipid Membranes(1995)34 cited
• Characterization of tryptophan residues of human coagulation factor V required for binding to phospholipid membranes(2003)
• → Cholesterol and Phosphatidylethanolamine Enhance the Binding of Factor Va and Factor Xa to Phospholipid Vesicles and Promote Assembly of the Prothrombinase Complex on Membranes Containing Low Concentrations of Phosphatidylserine.(2006)