Trp²⁰⁶³ and Trp²⁰⁶⁴ in the Factor Va C2 Domain Are Required for High-Affinity Binding to Phospholipid Membranes but Not for Assembly of the Prothrombinase Complex

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.

Crystal Structure of Lactadherin C2 Domain at 1.7Å Resolution with Mutational and Computational Analyses of Its Membrane-binding Motif

“…Prior mutagenesis studies have confirmed the membrane-interactive role of four hydrophobic amino acids for factor VIII (25) and at least two amino acids for factor V (24,44). The interactive amino acids are localized on spikes 1 and 3, with no demonstrated role for residues of spike 2.…”

Modulation of Prothrombinase Assembly and Activity by Phosphatidylethanolamine

“…Recombinant Proteins-Native recombinant factor Va 2 (rHFVa) was expressed in COS-7 and purified using established methods (11).…”

“…Phospholipid Preparations-Phospholipid (both synthetic and natural) vesicles were prepared by sonication as previously described (11). Measured suspensions of C6PS and C6PE were prepared as previously described (12).…”

“…Experiments were performed using either all bovine coagulation proteins or all human coagulation proteins. Rates of thrombin generation were measured using the substrate S2238 as described previously (11). Thrombin generation was also assayed in some cases using DAPA exactly as described (12) in the presence of 3 mM CaCl 2 .…”

“…Membrane Binding of Factors Xa and Va-Fluorescence energy transfer from Trp to a membrane-located dansyl probe was measured using either an SLM-8100 or a SPEX FluoroLog-3 fluorescence spectrophotometer, as described by Gilbert et al (13) and modified by Peng et al (11).…”