Phosphatidylserine and FVa regulate FXa structure.

Human coagulation FXa (Factor Xa) plays a key role in blood coagulation by activating prothrombin to thrombin on 'stimulated' platelet membranes in the presence of its cofactor FVa (Factor Va). PS (phosphatidylserine) exposure on activated platelet membranes promotes prothrombin activation by FXa by allosterically regulating FXa. To identify the structural basis of this allosteric regulation, we used FRET to monitor changes in FXa length in response to (i) soluble short-chain PS [C6PS (dicaproylphosphatidylserine)], (ii) PS membranes, and (iii) FVa in the presence of C6PS and membranes.

Modulation of prothrombinase assembly and activity by phosphatidylethanolamine.

Constituents of platelet membranes regulate the activity of the prothrombinase complex. We demonstrate that membranes containing phosphatidylcholine and phosphatidylethanolamine (PE) bind factor Va with high affinity (K(d) = ∼10 nm) in the absence of phosphatidylserine (PS). These membranes support formation of a 60-70% functional prothrombinase complex at saturating factor Va concentrations.

A phosphatidylserine binding site in factor Va C1 domain regulates both assembly and activity of the prothrombinase complex.

Tightly associated factor V(a) (FVa) and factor X(a) (FXa) serve as the essential prothrombin-activating complex that assembles on phosphatidylserine (PS)-containing platelet membranes during blood coagulation. We have previously shown that (1) a soluble form of PS (C6PS) triggers assembly of a fully active FVa-FXa complex in solution and (2) that 2 molecules of C6PS bind to FVa light chain with one occupying a site in the C2 domain. We expressed human factor V(a) (rFVa) with mutations in either the C1 domain (Y1956,L1957)A, the C2 domain (W2063,W2064)A, or both C domains (Y1956,L1957,W2063,W2064)A.

Location of the multimerin 1 binding site in coagulation factor V: an update.

Activated coagulation factor V (FVa) is an important cofactor that accelerates thrombin production. In human blood, 25% of the factor V (FV) is stored in platelets, complexed to the polymeric, FV binding protein multimerin 1 (MMRN1). The light chain of FV is required for MMRN1 binding, and its C2 domain contains a MMRN1 binding site that overlaps phospholipid binding residues essential for FVa procoagulant function.

The phosphatidylserine binding site of the factor Va C2 domain accounts for membrane binding but does not contribute to the assembly or activity of a human factor Xa-factor Va complex.

Factors V(a) and X(a) (FV(a) and FX(a), respectively) assemble on phosphatidylserine (PS)-containing platelet membranes to form the essential "prothrombinase" complex of blood coagulation. The C-terminal domain (C2) of FV(a) (residues 2037-2196 in human FV(a)) contains a soluble phosphatidylserine (C6PS) binding pocket flanked by a pair of tryptophan residues, Trp(2063) and Trp(2064). Mutating these tryptophans abolishes FV(a) membrane binding.

Human platelets contain forms of factor V in disulfide-linkage with multimerin.

Factor V is an essential cofactor for blood coagulation that circulates in platelets and plasma. Unlike plasma factor V, platelet factor V is stored complexed with the polymeric alpha-granule protein multimerin. In analyses of human platelet factor V on nonreduced denaturing multimer gels, we identified that approximately 25% was variable in size and migrated larger than single chain factor V, the largest form in plasma.

Identification of the MMRN1 binding region within the C2 domain of human factor V.

In platelets, coagulation cofactor V is stored in complex with multimerin 1 in alpha-granules for activation-induced release during clot formation. The molecular nature of multimerin 1 factor V binding has not been determined, although multimerin 1 is known to interact with the factor V light chain. We investigated the region in factor V important for multimerin 1 binding using modified enzyme-linked immunoassays and recombinant factor V constructs.

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.

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 %).

The factor V C1 domain is involved in membrane binding: identification of functionally important amino acid residues within the C1 domain of factor V using alanine scanning mutagenesis.

The contribution of the factor Va C1 domain (fVa-C1) to assembly of the prothrombinase complex has not been previously investigated. The homologous fVa-C2 domain contains a binding site for phosphatidylserine (PS) that includes the indole moieties of Trp(2063)/Trp(2064) at the apex of spike-1. In order to investigate the structure and function of fVa-C1 a molecular model was constructed based on the structure of fVa-C2.