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The Contribution of Factor Xa to Exosite-dependent Substrate Recognition by Prothrombinase*

  • Matthias Wilkens
    Affiliations
    From the Joseph Stokes Research Institute, Children's Hospital of Philadelphia, and Department of Pediatrics, University of Pennsylvania, Philadelphia, Pennsylvania 19104
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  • Sriram Krishnaswamy
    Correspondence
    To whom correspondence should be addressed: Joseph Stokes Research Institute, Children's Hospital of Philadelphia, 310 Abramson, 3516 Civic Center Blvd., Philadelphia, PA 19104. Tel.: 215-590-3346; Fax: 215-590-2320
    Affiliations
    From the Joseph Stokes Research Institute, Children's Hospital of Philadelphia, and Department of Pediatrics, University of Pennsylvania, Philadelphia, Pennsylvania 19104
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  • Author Footnotes
    * This work was supported by National Institutes of Health Grants HL-47465 and HL-62523 (to S. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:January 08, 2002DOI:https://doi.org/10.1074/jbc.M110848200
      Kinetic studies support the concept that protein substrate recognition by the prothrombinase complex of coagulation is achieved by interactions at extended macromolecular recognition sites (exosites), distinct from the active site of factor Xa within the complex. We have used this formal kinetic model and a monoclonal antibody directed against Xa (αBFX-2b) to investigate the contributions of surfaces on the proteinase to exosite-mediated protein substrate recognition by prothrombinase. αBFX-2b bound reversibly to a fluorescent derivative of factor Xa (K d = 17.1 ± 5.6 nm) but had no effect on active site function of factor Xa or factor Xa saturably assembled into prothrombinase. In contrast, αBFX-2b was a slow, tight binding inhibitor of the cleavage of either prethrombin 2 or meizothrombin des-fragment 1 by prothrombinase (K i* = 0.55 ± 0.05 nm). Thus, αBFX-2b binding to factor Xa within prothrombinase selectively leads to the inhibition of protein substrate cleavage without interfering with active site function. Inhibition kinetics could adequately be accounted for by a kinetic model in which prethrombin 2 and αBFX-2b bind in a mutually exclusive way to prothrombinase. These are properties expected of an exosite-directed inhibitor. The site(s) on factor Xa responsible for antibody binding were evaluated by identification of immunoreactive fragments following chemical digestion of human and bovine Xa and were further confirmed with a series of recombinantly expressed fragments. These approaches suggest that residues 82–91 and 102–116 in the proteinase domain contribute to αBFX-2b binding. The data establish this antibody as a prototypic exosite-directed inhibitor of prothrombinase and suggest that the occlusion of a surface on factor Xa, spatially removed from the active site, is sufficient to block exosite-dependent recognition of the protein substrate by prothrombinase.
      SpXa
      methoxycarbonyl-d-cyclohexylglycyl-glycyl-l-arginine-p-nitroanilide
      mIIaΔF1
      meizothrombin des-fragment 1
      mIIaΔF1F
      mIIaΔF1 inactivated with acetothioacetyld-phenylalanyl-l-prolyl-l-arginine chloromethyl ketone and modified with 6-(iodoacetamido)fluorescein following thioester hydrolysis
      OG488-Xa
      Xa inactivated with acetothioacetyll-glutamyl-glycyl-l-arginine chloromethyl ketone and modified with Oregon Green488 following thioester hydrolysis
      PAB
      4-aminobenzamidine
      PC
      l-α-phosphatidylcholine
      PS
      l-α-phosphatidylserine
      PEG
      polyethylene glycol with average molecular weight of 8000
      rTAP
      recombinant tick anticoagulant peptide
      Caps
      3-(cyclohexylamino)propanesulfonic acid
      Maintenance of hemostasis depends on the function of a series of membrane-bound enzyme complexes of which prothrombinase may be considered an archetype (
      • Mann K.G.
      • Jenny R.J.
      • Krishnaswamy S.
      ,
      • Kalafatis M.
      • Swords N.A.
      • Rand M.D.
      • Mann K.G.
      ,
      • Davie E.W.
      • Fujikawa K.
      • Kisiel W.
      ). Prothrombinase, which assembles through reversible interactions between the serine proteinase, factor Xa, the cofactor, factor Va, and membranes, catalyzes the conversion of prothrombin to thrombin by the cleavage of two peptide bonds (
      • Mann K.G.
      • Jenny R.J.
      • Krishnaswamy S.
      ,
      • Kalafatis M.
      • Swords N.A.
      • Rand M.D.
      • Mann K.G.
      ,
      • Jackson C.M.
      • Nemerson Y.
      ).
      Although factor Xa can activate prothrombin by itself, the catalytic efficiency for prothrombin activation is increased by a factor of ∼300,000 upon the incorporation of the proteinase into the prothrombinase complex (
      • Mann K.G.
      • Jenny R.J.
      • Krishnaswamy S.
      ,
      • Kalafatis M.
      • Swords N.A.
      • Rand M.D.
      • Mann K.G.
      ,
      • Jackson C.M.
      • Nemerson Y.
      ). Factor Va is considered to contribute in a major way to the enhanced catalytic efficiency for prothrombin activation by prothrombinase (
      • Mann K.G.
      • Jenny R.J.
      • Krishnaswamy S.
      ,
      • Mann K.G.
      • Nesheim M.E.
      • Church W.R.
      • Haley P.
      • Krishnaswamy S.
      ). However, it appears increasingly improbable that the role of the cofactor is primarily realized by perturbing the catalytic site of factor Xa, as has been tacitly assumed (
      • Mann K.G.
      • Jenny R.J.
      • Krishnaswamy S.
      ,
      • Mann K.G.
      • Nesheim M.E.
      • Church W.R.
      • Haley P.
      • Krishnaswamy S.
      ,
      • Esmon C.T.
      • Mather T.
      ). Instead, the data are more consistent with the idea that factor Va either contributes additional binding sites for the protein substrate and/or perturbs sites on factor Xa removed from the catalytic site, leading to enhanced extended interactions between the protein substrate and prothrombinase (
      • Krishnaswamy S.
      • Walker R.K.
      ,
      • Walker R.K.
      • Krishnaswamy S.
      ). It therefore follows that ground state rather than transition state effects likely play a significant role in the enhanced catalytic efficiency of prothrombinase toward its protein substrate.
      Support for the latter concept has accumulated from mechanistic studies of the individual half-reactions of prothrombin activation (
      • Krishnaswamy S.
      • Betz A.
      ,
      • Boskovic D.S.
      • Krishnaswamy S.
      ). In these approaches, the kinetics of substrate recognition and cleavage at the two sites has been discretely assessed using proteolytic derivatives of prothrombin containing one of the two cleavage sites as substrate analogs. In either case, the bimolecular reaction between the protein substrate and prothrombinase results from interactions between extended macromolecular interaction sites (exosites) on the enzyme removed from the catalytic site and substrate sites distinct from structures surrounding the scissile bond (
      • Krishnaswamy S.
      • Betz A.
      ,
      • Boskovic D.S.
      • Krishnaswamy S.
      ). Docking of substrate structures at the active site of the enzyme occurs in a second unimolecular binding step preceding scissile bond cleavage. The exosite-binding step dominates the perceived affinity of the enzyme for the protein substrate (
      • Krishnaswamy S.
      • Betz A.
      ,
      • Boskovic D.S.
      • Krishnaswamy S.
      ,
      • Betz A.
      • Krishnaswamy S.
      ).
      The contributions of extended surfaces in factors Xa and Va within prothrombinase toward exosite interactions with the protein substrate remain uncertain. An important direct role for factor Va in binding the substrate is implied by the established ability of the cofactor to bind the fragment 2 domain of prothrombin (
      • Esmon C.T.
      • Owen W.G.
      • Duiguid D.L.
      • Jackson C.M.
      ,
      • Esmon C.T.
      • Jackson C.M.
      ,
      • Luckow E.A.
      • Lyons D.A.
      • Ridgeway T.M.
      • Esmon C.T.
      • Laue T.M.
      ), and by evidence documenting an interaction between the fibrinogen-binding site in the proteinase domain of thrombin and factor Va (
      • Dharmawardana K.R.
      • Olson S.T.
      • Bock P.E.
      ,
      • Dharmawardana K.R.
      • Bock P.E.
      ,
      • Anderson P.J.
      • Nesset A.
      • Dharmawardana K.R.
      • Bock P.E.
      ). However, a dominant role for such interactions in exosite-dependent protein substrate recognition is neither supported by kinetic studies using substrate derivatives lacking the fragment 2 domain nor by inhibition studies with proteolytic derivatives of thrombin lacking part or all of the fibrinogen-binding site (
      • Betz A.
      • Krishnaswamy S.
      ).
      Selective inhibition of protein substrate cleavage without restricting access of small ligands to the active site of factor Xa within prothrombinase or interfering with the assembly of the enzyme complex is the hallmark of an exosite-directed inhibitor (
      • Krishnaswamy S.
      • Betz A.
      ,
      • Boskovic D.S.
      • Krishnaswamy S.
      ,
      • Baugh R.J.
      • Dickinson C.D.
      • Ruf W.
      • Krishnaswamy S.
      ). A monoclonal antibody directed against the proteinase domain of factor Xa that exhibits some of these properties has been described in previous work (
      • Church W.R.
      • Messier T.L.
      • Tucker M.M.
      • Mann K.G.
      ). We have further characterized the properties of this antibody to probe the contributions of extended surfaces in factor Xa toward exosite-dependent macromolecular substrate recognition by prothrombinase.

      DISCUSSION

      A series of studies have established a primary role for exosite interactions in determining the affinity and binding specificity of prothrombinase for its protein substrate (
      • Krishnaswamy S.
      • Betz A.
      ,
      • Boskovic D.S.
      • Krishnaswamy S.
      ,
      • Betz A.
      • Krishnaswamy S.
      ). Ligands targeting such exosites within prothrombinase are expected to compete with protein substrate binding to the enzyme complex without interfering with complex assembly or active site function (
      • Krishnaswamy S.
      • Betz A.
      ,
      • Boskovic D.S.
      • Krishnaswamy S.
      ,
      • Betz A.
      • Krishnaswamy S.
      ). Evidence developed in the present work establishes αBFX-2b as a prototypic exosite-directed inhibitor of prothrombinase. Our findings also suggest that the occlusion of sites on the surface of factor Xa within prothrombinase is sufficient to interfere with exosite-mediated recognition of the protein substrate.
      Recent high resolution x-ray structures of analogous or related enzyme complexes in coagulation and fibrinolysis have provided indications that the cofactor or accessory protein likely provides an extended surface for binding the protein substrate (
      • Parry M.A.
      • Fernandez-Catalan C.
      • Bergner A.
      • Huber R.
      • Hopfner K.P.
      • Schlott B.
      • Guhrs K.H.
      • Bode W.
      ,
      • Fuentes-Prior P.
      • Iwanaga Y.
      • Huber R.
      • Pagila R.
      • Rumennik G.
      • Seto M.
      • Morser J.
      • Light D.R.
      • Bode W.
      ,
      • Banner D.W.
      • D'Arcy A.
      • Chene C.
      • Winkler F.K.
      • Guha A.
      • Konigsberg W.H.
      • Nemerson Y.
      • Kirchhofer D.
      ). In most of these cases, such inferences remain to be correlated with functional studies assessing the precise energetic contributions of extended surfaces in the cofactor to the productive interaction between the protein substrate and the enzyme complex. In the case of prothrombinase, early studies (
      • Esmon C.T.
      • Jackson C.M.
      ) suggested a requisite interaction between the fragment 2 domain of the substrate and factor Va for efficient cleavage of prothrombin by the enzyme complex. Subsequent work with prothrombin derivatives lacking the fragment 2 domain has indicated that a direct binding interaction, of the type initially proposed, is unlikely to play a major contributing role to the function of factor Va within the prothrombinase complex (
      • Krishnaswamy S.
      • Walker R.K.
      ). More recently, a series of studies (
      • Anderson P.J.
      • Nesset A.
      • Dharmawardana K.R.
      • Bock P.E.
      ) have provided evidence for an interaction between the substrate/product and factor Va, mediated by the fibrinogen-binding site present in the proteinase domain. However, proteolytic derivatives of prothrombin, lacking the fibrinogen-binding site, retain the ability to bind to prothrombinase with comparable affinity as the substrate and block exosite-mediated substrate binding (
      • Betz A.
      • Krishnaswamy S.
      ). Consequently, the precise contribution of Va-substrate interactions to the productive pathway of substrate recognition by prothrombinase is uncertain. Our results now indicate that extended surfaces on the proteinase domain of factor Xa within the enzyme complex, and removed from the active site, play a significant role in the exosite-dependent binding of the protein substrate to prothrombinase. Although these findings do not rule out a contribution from direct interactions between the protein substrate and factor Va, it would appear that extended surfaces on factor Xa, within the enzyme complex, participate in determining the binding specificity for the protein substrate.
      The findings are consistent with the interpretation that the binding of αBFX-2b to factor Xa within prothrombinase is sufficient to exclude protein substrate binding. As competitive inhibition of protein substrate cleavage can only be achieved by interfering with the bimolecular interaction between the substrate and the enzymic exosite (
      • Krishnaswamy S.
      • Betz A.
      ,
      • Betz A.
      • Krishnaswamy S.
      ), one possible interpretation is that residues included in the binding site identified for αBFX-2b directly contribute to interactions with the protein substrate. However, alternative interpretations include the possibility that the binding of αBFX-2b perturbs residues distant from the antibody-binding site that are directly involved in interactions with the substrate or that selective inhibition of protein substrate cleavage arises from steric effects associated with the binding of this large probe to the enzyme. The finding that the Fab fragment of the antibody acts in an equivalent manner to the intact IgG (
      • Church W.R.
      • Messier T.L.
      • Tucker M.M.
      • Mann K.G.
      ) and the fact that αBFX-2b does not alter the binding of large ligands such as rTAP that involve extended interactions with the proteinase domain (
      • Wei A.
      • Alexander R.S.
      • Duke J.
      • Ross H.
      • Rosenfeld S.A.
      • Chang C.H.
      ) can be offered as arguments against generalized steric phenomena. However, these arguments are not compelling, and we are presently unable to convincingly rule out such alternative interpretations. The present findings, however, provide a reasonable starting point for the further systematic investigation of structures present in the proteinase domain of factor Xa that may play a role in binding the protein substrate.
      Despite the fact that the heavy chain of factors X and Xa and their proteolytic digestion products are all readily detected by αBFX-2b by Western blotting following disulfide bond reduction, this antibody surprisingly appears to bind to a discontinuous epitope on the protein. This point has adversely affected our ability to establish the structural basis for the interaction between αBFX-2b and factor Xa in a quantitative way. Thus, it is possible that additional structures neighboring residues 82–91 and 102–116 also contribute significantly to the interaction between αBFX-2b and factor Xa or that the antibody epitope is considerably smaller than proposed by our studies. The identified sequences are immediately adjacent to the Ca2+-binding site in the proteinase domain (
      • Brandstetter H.
      • Kuhne A.
      • Bode W.
      • Huber R.
      • von der Saal W.
      • Wirthensohn K.
      • Engh R.A.
      ,
      • Sabharwal A.K.
      • Padmanabhan K.
      • Tulinsky A.
      • Mathur A.
      • Gorka J.
      • Bajaj S.P.
      ). Such proximity provides a plausible explanation for the Ca2+dependence of αBFX-2b binding observed in this and prior work (
      • Church W.R.
      • Messier T.L.
      • Tucker M.M.
      • Mann K.G.
      ). In the event that this region includes residues that directly contribute to the interaction with the protein substrate, it is possible that Ca2+ binding to the proteinase domain of factor Xa may also modulate exosite-dependent tethering of the protein substrate to the enzyme complex.
      Evidence has also been developed for a major contribution of exosite interactions in determining the productive recognition of factor X by the VIIa-tissue factor complex (
      • Baugh R.J.
      • Dickinson C.D.
      • Ruf W.
      • Krishnaswamy S.
      ). An extensive body of information indicates that extended surfaces in both VIIa and tissue factor contribute to factor X binding. Structural studies of VIIa in complex with an inhibitory polypeptide have implicated extended surfaces that include the autolysis loop of VIIa in factor X activation (
      • Dennis M.S.
      • Eigenbrot C.
      • Skelton N.J.
      • Ultsch M.H.
      • Santell L.
      • Dwyer M.A.
      • O'Connell M.P.
      • Lazarus R.A.
      ). However, this inhibitor appears to modulate catalytic function rather than compete with the binding of the protein substrate (
      • Dennis M.S.
      • Eigenbrot C.
      • Skelton N.J.
      • Ultsch M.H.
      • Santell L.
      • Dwyer M.A.
      • O'Connell M.P.
      • Lazarus R.A.
      ). Additional evidence for a role of extended surfaces in VIIa in binding factor X has resulted from mutagenesis studies used to map the binding determinants of antibody probes that function as exosite-directed inhibitors of factor X activation (
      • Dickinson C.D.
      • Shobe J.
      • Ruf W.
      ). The residues identified are generally localized to the same face of the proteinase domain and include or are in the vicinity of the residues implicated in the present work for the binding of αBFX-2b to factor Xa. Thus, it is possible that both VIIa and Xa within their respective enzyme complexes employ equivalent structures in the proteinase domain to mediate exosite interactions with their protein substrates.
      The incorporation of factor Xa into prothrombinase was found to enhance detectably the binding of αBFX-2b. This observation is in line with the suggestions of previous studies (
      • Krishnaswamy S.
      • Vlasuk G.P.
      • Bergum P.W.
      ,
      • Betz A.
      • Vlasuk G.P.
      • Bergum P.W.
      • Krishnaswamy S.
      ) that surfaces in factor Xa, distinct from the active site, are modulated following the interaction between proteinase and cofactor. Such perturbations have been shown previously to lead to large changes in the affinity and kinetic mechanism of the interaction of macromolecular inhibitor probes directed toward Xa (
      • Krishnaswamy S.
      • Vlasuk G.P.
      • Bergum P.W.
      ,
      • Betz A.
      • Vlasuk G.P.
      • Bergum P.W.
      • Krishnaswamy S.
      ). Although the significance of such changes to protein substrate recognition remains to be established, the data are consistent with the hypothesis that one consequence of the interaction of factor Xa with factor Va on the membrane surface is the modulation of extended macromolecular recognition sites on the proteinase that at least partly accounts for the exosite-mediated binding of the protein substrate to prothrombinase. These ideas and the present findings indicate a significant role for exosites present in the proteinase domain of factor Xa in determining binding affinity and specificity for the recognition of the protein substrate by prothrombinase.

      Acknowledgments

      We are grateful to Dr. William Church, University of Vermont, for the generous gift of monoclonal antibody αBFX-2b. We are also grateful to Dr. George Vlasuk for critical reading of the manuscript and to Dr. Jan Pohl, Emory University Microchemical Facility, for N-terminal sequence analysis.

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