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. We incorporated a FRET pair with donor (fluorescein) at the active site and acceptor (Alexa Fluor® 555) at the FXa N-terminus near the membrane. The results demonstrated that FXa structure changes upon binding of C6PS to two sites: a regulatory site at the N-terminus [identified previously as involving the Gla (γ-carboxyglutamic acid) and EGFN (N-terminus of epidermal growth factor) domains] and a presumptive protein-recognition site in the catalytic domain. Binding of C6PS to the regulatory site increased the interprobe distance by ~3 Å (1 Å=0.1 nm), whereas saturation of both sites increased the distance by a further ~6.4 Å. FXa binding to a membrane produced a smaller increase in length (~1.4 Å), indicating that FXa has a somewhat different structure on a membrane from when bound to C6PS in solution. However, when both FVa2 (a FVa glycoform) and either C6PS- or PS-containing membranes were bound to FXa, the overall change in length was comparable (~5.6-5.8 Å), indicating that C6PS- and PS-containing membranes in conjunction with FVa2 have comparable regulatory effects on FXa. We conclude that the similar functional regulation of FXa by C6PS or membranes in conjunction with FVa2 correlates with similar structural regulation. The results demonstrate the usefulness of FRET in analysing structure-function relationships in FXa and in the FXa·FVa2 complex.

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. Although reduced interfacial packing does contribute to factor Va binding in the absence of PS, it does not correlate with the enhanced activity of the Xa-Va complex assembled on PE-containing membranes. Instead, specific protein-PE interactions appear to contribute to the effects of PE. In support of this, soluble C6PE binds to recombinant factor Va(2) (K(d) = ∼6.5 µm) and to factor Xa (K(d) = ∼91 µm). C6PE and C6PS binding sites of factor Xa are specific, distinct, and linked, because binding of one lipid enhances the binding and activity effects of the other. C6PE triggers assembly (K(d)(app) = ∼40 nm) of a partially active prothrombinase complex between factor Xa and factor Va(2), compared with K(d)(app) for C6PS ∼2 nm. These findings provide new insights into the possible synergistic roles of platelet PE and PS in regulating thrombin formation, particularly when exposed membrane PS may be limiting.

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. Mutations in the C1 and C1-C2 domains of rFVa reduced the rate of activation of prothrombin to thrombin by FXa in the presence of 400 muM C6PS by 14 000- to 15 000-fold relative to either wild-type or C2 mutant factor rFVa. The K(d')s of FXa binding with rFVa (wild-type, C2 mutant, C1 mutant, and C1-C2 mutant) were 3, 4, 564, and 624 nM, respectively. Equilibrium dialysis experiments detected binding of 4, 3, and 2 molecules of C6PS to wild-type rFVa, C1-mutated, and C1,C2-mutated rFVa, respectively. Because FVa heavy chain binds 2 molecules of C6PS, we conclude that both C2 and C1 domains bind one C6PS, with binding to the C1 domain regulating prothrombinase complex assembly.

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 homologous structures and roles of the FVa light chain C1 and C2 domains led us to investigate if the C1 domain also contains a MMRN1 binding site. The MMRN1 binding properties of FV constructs were tested by modified enzyme-linked immunoassays, before and after thrombin activation. The constructs tested included the combined C1 and C2 domain deleted FV, and B-domain deleted forms of FV containing C1 domain point mutations or combined C1 and C2 domain phospholipid binding site mutations. The MMRN1 binding site in FV/FVa was mapped to a large region that included the C1 domain phospholipid binding residues Y1956 and L1957. The FV construct with combined C1 and C2 domain phospholipid binding site mutations had no MMRN1 binding, highlighting the critical role of the FV C1 and C2 domain phospholipid binding residues in MMRN1 binding. Our data update the information on the structural features of FV and FVa important for MMRN1 binding, and suggest that the extended MMRN1 binding site in the C1 and C2 domains is important for the storage of FV-MMRN1 complexes in platelets.

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. To address both the roles of these tryptophans in C6PS or membrane binding and the role of the C2 domain lipid binding site in regulation of FV(a) cofactor activity, we expressed W(2063,2064)A mutants of the recombinant C2 domain (rFV(a2)-C2) and of a B domain-deleted factor V light isoform (rFV(a2)) in Hi-5 and COS cells, respectively. Intrinsic fluorescence showed that wild-type rFV(a2)-C2 binds to C6PS and to 20% PS/PC membranes with apparent K(d) values of 2.8 microM and 9 nM, respectively, while mutant rFV(a2)-C2 does not. Equilibrium dialysis confirmed that mutant rFV(a2)-C2 does not bind to C6PS. Mutant rFV(a2) binds to C6PS (K(d) approximately 37 microM) with an affinity comparable to that of wild-type rFV(a2) (K(d) approximately 20 microM), although it does not bind to PS/PC membranes to which wild-type rFV(a2) binds with native affinity (K(d) approximately 3 nM). Both wild-type and mutant rFV(a2) bind to active site-labeled FX(a) (DEGR-X(a)) in the presence of 400 microM C6PS with native affinity (K(d) approximately 3-4 nM) to produce a solution rFV(a2)-FX(a) complex of native activity. We conclude that (1) the C2 domain PS site provides all but approximately 1 kT of the free energy of FV(a) membrane binding, (2) tryptophans lining the C2 lipid binding pocket are critical to C6PS and membrane binding and insert into the bilayer interface during membrane binding, (3) occupancy of the C2 lipid binding pocket is not necessary for C6PS-induced formation of the FX(a)-FV(a) complex or its activity, but (4) another PS site on FV(a) does have a regulatory role.

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. Upon reduction, the unusually large, variably-sized forms of platelet factor V liberated components that comigrated with other forms of platelet factor V, indicating that they contained factor V in interchain disulfide-linkages. With thrombin cleavage, factor Va heavy and light chain domains, but not B-domains,were liberated from the components linked by interchain disulfide bonds, indicating that the single cysteine in the B-domain at position 1085 was the site of disulfide linkage. Since unusually large factor V had a variable size and included forms larger than factor V dimers, the data suggested disulfide-linkage with another platelet protein, possibly multimerin. Immunoprecipitation experiments confirmed that unusually large factor V was associated with multimerin and it remained associated in 0.5 M salt. Moreover, platelets contained a subpopulation of multimerin polymers that resisted dissociation from factor V by denaturing detergent and comigrated with unusually large platelet factor V, before and after thrombin cleavage. The disulfide-linked complexes of multimerin and factor V in platelets, which are cleaved by thrombin to liberate factor Va, could be important for modulating the function of platelet factor V and its delivery onto activated platelets. Factor Va generation and function from unusually large platelet factor V is only speculative at this time.

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. Factor V constructs lacking the C2 region or entire light chain had impaired and absent multimerin 1 binding, respectively, whereas the B domain deleted construct had modestly reduced binding. Analyses of point mutated constructs indicated that the multimerin 1 binding site in the C2 domain of factor V partially overlaps the phosphatidylserine binding site and that the factor V B domain enhances multimerin 1 binding. Multimerin 1 did not inhibit factor V phosphatidylserine binding, and it bound to phosphatidylserine independently of factor V. There was a reduction in factor V in complex with multimerin 1 after activation, and thrombin cleavage significantly reduced factor V binding to multimerin 1. In molar excess, multimerin 1 minimally reduced factor V procoagulant activity in prothrombinase assays and only if it was added before factor V activation. The dissociation of factor V-multimerin 1 complexes following factor V activation suggests a role for multimerin 1 in delivering and localizing factor V onto platelets prior to prothrombinase assembly.

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 %). 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.

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. The aromatic and hydrophobic side chains of Tyr (1956) /Leu (1957) in fVa-C1 are located at the predicted apex of spike-3. Exposed charged and hydrophobic residues in fVa-C1 were changed to alanine in clusters of 1-3 mutations per construct. The resultant 20 mutants were expressed in COS cells and screened for binding to immobilized PS and prothrombinase activity on phospholipid vesicles containing either 25% or 5% PS. Two mutants, (Y1956,L1957)A, and (R2023,R2027)A showed both decreased binding to immobilized PS and a selective decrease in prothrombinase activity on membranes containing 5% PS. The interaction of purified (Y1956,L1957)A with phospholipid vesicles was studied using fluorescence resonance energy transfer and prothrombinase assays. The affinity of (Y1956,L1957)A binding to 25% PS membranes was reduced 12-fold compared to rHFVa. Prothrombin activation in the presence of (Y1956,L1957)A was markedly impaired on phos-pholipid vesicles containing 10% or less PS. We conclude that solvent exposed hydrophobic and aromatic amino acids in both fVa-C1 and fVa-C2 contribute to the interaction of factor V with PS membranes.