William H. Kane

Crystal structures of the membrane-binding C2 domain of human coagulation factor V

Rapid and controlled clot formation is achieved through sequential activation of circulating serine proteinase precursors on phosphatidylserine-rich procoagulant membranes of activated platelets and endothelial cells. The homologous complexes Xase and prothrombinase, each consisting of an active proteinase and a non-enzymatic cofactor, perform critical steps within this coagulation cascade. The activated cofactors VIIIa and Va, highly specific for their cognate proteinases, are each derived from precursors with the same A1-A2-B-A3-C1-C2 architecture. Membrane binding is mediated by the C2 domains of both cofactors. Here we report two crystal structures of the C2 domain of human factor Va. The conserved β-barrel framework provides a scaffold for three protruding loops, one of which adopts markedly different conformations in the two crystal forms. We propose a mechanism of calcium-independent, stereospecific binding of factors Va and VIIIa to phospholipid membranes, on the basis of (1) immersion of hydrophobic residues at the apices of these loops in the apolar membrane core; (2) specific interactions with phosphatidylserine head groups in the groove enclosed by these loops; and (3) favourable electrostatic contacts of basic side chains with negatively charged membrane phosphate groups.

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.

Characterization of an acquired inhibitor to coagulation factor V. Antibody binding to the second C-type domain of factor V inhibits the binding of factor V to phosphatidylserine and neutralizes procoagulant activity.

Coagulation Factor V is an essential component of the prothrombinase complex, which activates the zymogen prothrombin to thrombin. A patient was described who developed a Factor V inhibitor that neutralized the procoagulant activity of Factor V and resulted in a fatal hemorrhagic diathesis (Coots, M. C., A. F. Muhleman, and H. I. Glueck. 1978. Am. J. Hematol. 4:193-206). This inhibitor was shown to be an IgG antibody that bound to the light chain of Factor V. Using a series of light chain deletion mutants, we have found that this antibody binds to the second C-type domain of the light chain. Both inhibitor IgG and Fab fragments rapidly neutralized the procoagulant activity of Factor Va, implying that the neutralization resulted from specific binding to the C2 domain. We have previously demonstrated that deletion of the C2 domain results in loss of procoagulant activity, as well as loss of phosphatidylserine-specific binding. Confirming these results, both inhibitor IgG and Fab fragments interfered with phosphatidylserine-specific binding of Factor V. Conversely, preincubation of Factor Va with procoagulant phospholipids protected the cofactor from inactivation by the inhibitor. Our results suggest that this inhibitor neutralizes the procoagulant activity of Factor Va by interfering with the C2-mediated interaction with phospholipid surfaces, thereby disrupting formation of the prothrombinase complex.

Mutation of hydrophobic residues in the factor Va C1 and C2 domains blocks membrane-dependent prothrombin activation

Summary.  The binding of factor (FVa) to phosphatidylserine (PS) membranes regulates assembly of the prothrombinase complex. Two pairs of solvent‐exposed amino acids, Tyr1956/Leu1957 in the C1 domain and Trp2063/Trp2064 in the C2 domain, each make significant contributions to the affinity of FVa for PS membranes, but individually neither pair of amino acids is required for prothrombinase assembly on 25% PS membranes. In this study we characterize a FVa mutant with alanine substitutions in both the C1 and C2 domains: (Y1956,L1957,W2063,W2064)A. We conclude that: (i) prothrombinase assembly on PS membranes requires Trp2063/Trp2064 and/or Tyr1956/Leu1957; (ii) combined mutation of Trp2063/Trp2064 and Tyr1956/Leu1957 results in only a modest 4‐fold decrease in the rate of thrombin generation in the absence of membranes; (iii) the present data provide experimental support for the joint participation of the C1 and C2 domains in the binding of FVa to phospholipid membranes as suggested by the recently solved structure for FVai (A1/A3‐C1‐C2).

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 α-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.

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.