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Zinc Modulates the Interaction of Protein C and Activated Protein C with Endothelial Cell Protein C Receptor*

  • Prosenjit Sen
    Footnotes
    Affiliations
    From the Center for Biomedical Research, The University of Texas Health Science Center at Tyler, Tyler, Texas 75708
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  • Sanghamitra Sahoo
    Footnotes
    Affiliations
    From the Center for Biomedical Research, The University of Texas Health Science Center at Tyler, Tyler, Texas 75708
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  • Usha R. Pendurthi
    Affiliations
    From the Center for Biomedical Research, The University of Texas Health Science Center at Tyler, Tyler, Texas 75708
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  • L. Vijaya Mohan Rao
    Correspondence
    To whom correspondence should be addressed: Center for Biomedical Research, The University of Texas Health Science Center at Tyler, 11937 U.S. Highway 271, Tyler, TX 75708. Tel.: 903-877-7332; Fax: 903-877-7426;
    Affiliations
    From the Center for Biomedical Research, The University of Texas Health Science Center at Tyler, Tyler, Texas 75708
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grants HL58869 (to L. V. M. R.) and HL65500 (to U. R. P.).
    1 Both authors contributed equally to this work.
Open AccessPublished:April 22, 2010DOI:https://doi.org/10.1074/jbc.M110.111575
      Zinc is an essential trace element for human nutrition and is critical to the structure, stability, and function of many proteins. Zinc ions were shown to enhance activation of the intrinsic pathway of coagulation but down-regulate the extrinsic pathway of coagulation. The protein C pathway plays a key role in blood coagulation and inflammation. At present there is no information on whether zinc modulates the protein C pathway. In the present study we found that Zn2+ enhanced the binding of protein C/activated protein C (APC) to endothelial cell protein C receptor (EPCR) on endothelial cells. Binding kinetics revealed that Zn2+ increased the binding affinities of protein C/APC to EPCR. Equilibrium dialysis with 65Zn2+ revealed that Zn2+ bound to the Gla domain as well as sites outside of the Gla domain of protein C/APC. Intrinsic fluorescence measurements suggested that Zn2+ binding induces conformational changes in protein C/APC. Zn2+ binding to APC inhibited the amidolytic activity of APC, but the inhibition was reversed by Ca2+. Zn2+ increased the rate of APC generation on endothelial cells in the presence of physiological concentrations of Ca2+ but did not further enhance increased APC generation obtained in the presence of physiological concentrations of Mg2+ with Ca2+. Zn2+ had no effect on the anticoagulant activity of APC. Zn2+ enhanced APC-mediated activation of protease activated receptor 1 and p44/42 MAPK. Overall, our data show that Zn2+ binds to protein C/APC, which results in conformational changes in protein C/APC that favor their binding to EPCR.

      Introduction

      Calcium is an obligatory cofactor in the initiation and regulation of blood coagulation. All vitamin K-dependent coagulation proteins have calcium-binding sites and thus presumably could interact to some degree with other divalent cations. A number of studies have reported that divalent metal ions other than Ca2+, such Mn2+, Mg2+, and Zn2+, bind to various coagulation factors and thereby modulate blood coagulation (
      • Byrne R.
      • Amphlett G.W.
      • Castellino F.J.
      ,
      • Shore J.D.
      • Day D.E.
      • Bock P.E.
      • Olson S.T.
      ,
      • Greengard J.S.
      • Heeb M.J.
      • Ersdal E.
      • Walsh P.N.
      • Griffin J.H.
      ,
      • Church W.R.
      • Boulanger L.L.
      • Messier T.L.
      • Mann K.G.
      ,
      • Butenas S.
      • Lawson J.H.
      • Kalafatis M.
      • Mann K.G.
      ,
      • Sekiya F.
      • Yoshida M.
      • Yamashita T.
      • Morita T.
      ,
      • Liaw P.C.
      • Neuenschwander P.F.
      • Smirnov M.D.
      • Esmon C.T.
      ,
      • Petersen L.C.
      • Olsen O.H.
      • Nielsen L.S.
      • Freskgård P.O.
      • Persson E.
      ,
      • Pedersen A.H.
      • Lund-Hansen T.
      • Komiyama Y.
      • Petersen L.C.
      • Oestergard P.B.
      • Kisiel W.
      ,
      • Bajaj S.P.
      • Schmidt A.E.
      • Agah S.
      • Bajaj M.S.
      • Padmanabhan K.
      ,
      • Tubek S.
      • Grzanka P.
      • Tubek I.
      ). Zinc deficiency has been associated with bleeding tendencies and defective platelet aggregation, suggesting an important role for zinc in hemostasis (
      • Tubek S.
      • Grzanka P.
      • Tubek I.
      ,
      • Gordon P.R.
      • Woodruff C.W.
      • Anderson H.L.
      • O'Dell B.L.
      ). Zinc is an essential trace element, and the concentration of Zn2+ in plasma ranges between 10 and 25 μm in healthy individuals (
      • Vallee B.L.
      • Falchuk K.H.
      ). In addition, platelets store, mainly in their cytoplasm and α-granules, up to 30 to 60 times (
      • Gorodetsky R.
      • Mou X.
      • Blankenfeld A.
      • Marx G.
      ) more Zn2+ than is present in plasma. These reserves could be released upon platelet activation (
      • Gorodetsky R.
      • Mou X.
      • Blankenfeld A.
      • Marx G.
      ).
      Zinc ions were shown to enhance activation of the intrinsic pathway of coagulation by increasing the binding of high molecular weight kininogen and factor XII to negatively charged surfaces (
      • Bernardo M.M.
      • Day D.E.
      • Olson S.T.
      • Shore J.D.
      ) or platelets (
      • Greengard J.S.
      • Heeb M.J.
      • Ersdal E.
      • Walsh P.N.
      • Griffin J.H.
      ,
      • Greengard J.S.
      • Griffin J.H.
      ) and by accelerating the activation of factor XII and prekallikrein (
      • Shore J.D.
      • Day D.E.
      • Bock P.E.
      • Olson S.T.
      ,
      • Shimada T.
      • Kato H.
      • Iwanaga S.
      ,
      • Schousboe I.
      ). In contrast, zinc ions have been found to inhibit the extrinsic pathway of coagulation by attenuating FVIIa
      The abbreviations used are: FVIIa
      activated factor VII
      APC
      activated protein C
      GD
      Gla domain-deleted
      EPCR
      endothelial cell protein C receptor
      PAR
      protease-activated receptor
      HUVEC
      human umbilical vein endothelial cell(s)
      AP
      alkaline phosphatase
      mAb
      monoclonal antibody
      CHO
      Chinese hamster ovary.
      and FVIIa/tissue factor activity (
      • Petersen L.C.
      • Olsen O.H.
      • Nielsen L.S.
      • Freskgård P.O.
      • Persson E.
      ,
      • Pedersen A.H.
      • Lund-Hansen T.
      • Komiyama Y.
      • Petersen L.C.
      • Oestergard P.B.
      • Kisiel W.
      ). Recent crystallography studies identified a pair of Zn2+-binding sites in the FVIIa protease domain (
      • Bajaj S.P.
      • Schmidt A.E.
      • Agah S.
      • Bajaj M.S.
      • Padmanabhan K.
      ). Except for Glu220, all of the side chains involved in both Zn1 and Zn2 coordination in FVIIa are unique to it and not present in other vitamin K-dependent clotting factors (
      • Bajaj S.P.
      • Schmidt A.E.
      • Agah S.
      • Bajaj M.S.
      • Padmanabhan K.
      ). Nonetheless, Zn2+ may bind to other vitamin K-dependent clotting factors as found recently with protein S (
      • Heeb M.J.
      • Prashun D.
      • Griffin J.H.
      • Bouma B.N.
      ).
      Zinc is a multi-functional element and plays an important role in many biological functions primarily by binding to proteins at specific sites, thereby stabilizing the conformation and proper function of a particular protein (
      • Coleman J.E.
      ,
      • Berg J.M.
      • Shi Y.
      ,
      • Brewer G.J.
      • Hill G.M.
      • Prasad A.S.
      • Cossack Z.T.
      ). Zinc can also relocate calcium from its natural binding site and thus modify calcium-dependent processes (
      • Csermely P.
      • Sándor P.
      • Radics L.
      • Somogyi J.
      ). All of the vitamin K-dependent coagulation proteins have calcium-binding sites and could interact to some extent with both calcium and zinc (
      • Petersen L.C.
      • Olsen O.H.
      • Nielsen L.S.
      • Freskgård P.O.
      • Persson E.
      ,
      • Pedersen A.H.
      • Lund-Hansen T.
      • Komiyama Y.
      • Petersen L.C.
      • Oestergard P.B.
      • Kisiel W.
      ,
      • Bajaj S.P.
      • Schmidt A.E.
      • Agah S.
      • Bajaj M.S.
      • Padmanabhan K.
      ,
      • Heeb M.J.
      • Prashun D.
      • Griffin J.H.
      • Bouma B.N.
      ). The protein C pathway plays key roles in the regulation of blood coagulation and inflammation (
      • Esmon C.T.
      ,
      • Esmon C.T.
      ). At present, there is no information on whether Zn2+ binds to protein C/APC and how this affects protein C/APC binding to EPCR and their subsequent functions. The data presented in this manuscript show that Zn2+ binds to protein C/APC, induces conformational changes in the protein, promotes protein C/APC binding to EPCR and thereby enhances the activation of protein C and APC-mediated cell signaling.

      DISCUSSION

      Zinc is essential for growth, development, and the transmission of the genetic message (
      • Vallee B.L.
      • Falchuk K.H.
      ,
      • Falchuk K.H.
      ). Earlier studies have suggested that zinc ions may also play a role in hemostasis by modulating the activity of plasma clotting factors, platelet aggregation, and platelet interaction with endothelial cells (
      • Tubek S.
      • Grzanka P.
      • Tubek I.
      ). The current study describes the influence of zinc ions on the protein C pathway. Our data show that zinc ions promote the binding of protein C and activated protein C to their receptor, EPCR. Zn2+-mediated enhancement of protein C/APC binding to EPCR results in a net increase in internalization of the ligands. The enhanced binding of APC to EPCR on the endothelial cell surface in the presence of zinc ions may also enhance APC-mediated PAR1 activation.
      The Gla domain of protein C is responsible for much of the binding energy and specificity of the protein C-EPCR interaction. This binding is dependent on the divalent metal ion Ca2+ (
      • Regan L.M.
      • Mollica J.S.
      • Rezaie A.R.
      • Esmon C.T.
      ). The data presented in this manuscript show that the presence of physiological concentrations of Zn2+ (25 μm) markedly increases the Ca2+-dependent protein C and APC binding to EPCR. Zn2+, however, does not replace Ca2+ as a mandatory cofactor for protein C/APC binding to EPCR. The presence of physiological amounts of Mg2+ had no effect on Zn2+-mediated enhancement of protein C and APC binding to EPCR. The kinetic analysis of protein C and APC binding to EPCR suggests that Zn2+ promotes protein C/APC binding to EPCR by increasing the binding affinity (i.e. lowering Kd) of protein C/APC for its receptor. The Zn2+-mediated increase in protein C and APC binding to EPCR led to increased endocytosis of protein C/APC, suggesting that Zn2+ may facilitate the clearance of protein C/APC.
      Zn2+ modulation of Gla domain-mediated protein C/APC binding to EPCR and the Gla domain-independent APC amidolytic activity suggest that Zn2+, like Ca2+, may bind to the Gla domain as well as the protease domain of protein C. Data from the equilibrium binding studies performed with 65Zn2+ provide strong support for this hypothesis. These data show that Zn2+ binds to both GD-APC and APC but that the amount of Zn2+ bound to full-length APC was 3–4-fold higher than the amount bound to GD-APC. Kinetic analysis of equilibrium binding studies suggests that two Zn2+ atoms bind to APC outside the Gla domain with relatively high affinity (∼70 μm). At least one of the Zn2+-binding sites may overlap with the Ca2+-binding site because the Zn2+ binding to GD-APC was inhibited by ∼50% in the presence of saturating concentrations of Ca2+. These data were consistent with earlier findings suggesting that Zn2+ probably binds to a high affinity Ca2+-binding site in GD-protein C (
      • Johnson A.E.
      • Esmon N.L.
      • Laue T.M.
      • Esmon C.T.
      ). However, at present there is no direct evidence that Zn2+ and Ca2+ may have overlapping sites on the protease domain. It is possible that the two metal sites are energetically linked, and thus Ca2+ binding to the protease domain may allosterically regulate Zn2+ binding to the protease domain as observed with sodium binding (
      • He X.
      • Rezaie A.R.
      ). Irrespective of the mechanism by which Ca2+ reduces Zn2+ binding to the protease domain, these data suggest that at physiological concentrations of Ca2+, only one Zn2+ atom may bind to the protease domain of APC. The substantially increased Zn2+ binding to the full-length APC compared with GD-APC suggests that the N terminus of the Gla domain of protein C contains multiple Zn2+-binding sites. Interestingly, Zn2+ bound to full-length APC and GD-APC with a similar affinity, suggesting that the Gla domain, as well the protease domain, may contain relatively high affinity binding sites for Zn2+. A majority of the Zn2+-binding sites in the Gla domain appear to be distinct from the Ca2+-binding sites, because less than 40% of Zn2+ was replaced by Ca2+. The intrinsic fluorescence quenching of both GD-APC and APC upon the addition of Zn2+ suggests that Zn2+ binding induces conformational changes in protein C/APC. Overall our observations suggest that Zn2+ binds to protein C/APC in both the Gla and the protease domains. Zn2+ binding to the Gla domain of protein C/APC stabilizes the conformation of the Gla domain, thereby favoring its binding to EPCR, whereas Zn2+ binding to the protease domain destabilizes the catalytic triad and thereby inhibits the amidolytic activity of APC. However, the occupancy of the protease domain by Ca2+ protects against the destabilizing effect of Zn2+.
      The locations of zinc coordination sites in protein C/APC are uncertain at this time. An examination of the protein C/APC primary structure revealed no consensus canonical zinc binding sequences homologous to other known zinc-binding proteins. The most common amino acids found in coordination sites for Zn2+ are His, Glu, Asp, and Cys (
      • Auld D.S.
      ). Although the protease domain of APC contains multiple His, Glu, Asp, and Cys residues, they are not present in any significant abundance compared with other amino acids. Recent crystallography studies of FVIIa revealed that the Zn1 site involves the side chains of His216, Glu220, and Ser222, and the Zn2 site involves the side chains of His257, Asp219, and Lys161 (
      • Bajaj S.P.
      • Schmidt A.E.
      • Agah S.
      • Bajaj M.S.
      • Padmanabhan K.
      ). Interestingly, none of these residues, except for Glu220, are conserved in APC. Therefore, it seems unlikely that Zn2+ binds to APC at the Ca2+ loop as it does in FVIIa. However, as observed with FVIIa, Ca2+ partly reverses the inhibitory effect of Zn2+ on APC amidolytic activity, raising the possibility that the Zn2+- and Ca2+-binding sites in the protease domain are either overlapping or energetically linked. Analysis of the protein C sequence with a recently developed program (PREDZINC) that predicts zinc-binding sites from amino acid sequences by combining a support vector machine (SVM) and homology-based predictions (
      • Shu N.
      • Zhou T.
      • Hovmöller S.
      ) identified one potential Zn2+-binding site in the protease domain involving Cys141, His246, Cys331, and Asp359 (analysis was performed by Nanjiang Shu, Structural Chemistry, Stockholm University, Stockholm, Sweden). Scanning the crystal structure of GD-APC from the Protein Data Bank for zinc-binding sites using the recently developed FEATURE algorithm yielded a cluster of hits in the protease domain. Analysis of the cluster for potential coordinating residues revealed that Zn2+-binding sites may involve the side chains of Asp254, His212, Cys211, and Cys96.
      Although we expected Zn2+ to bind to the Gla domain as it modulated the Gla domain-dependent protein C/APC binding to EPCR, it was surprising for us to find that the Gla domain of protein C contains as many as six to eight Zn2+-binding sites. Although this observation is unexpected, it is unlikely that this is an experimental artifact because the data were reproduced with two different batches of APC. Moreover, consistent with earlier studies (
      • Petersen L.C.
      • Olsen O.H.
      • Nielsen L.S.
      • Freskgård P.O.
      • Persson E.
      ,
      • Bajaj S.P.
      • Schmidt A.E.
      • Agah S.
      • Bajaj M.S.
      • Padmanabhan K.
      ), we noted only two Zn2+ atoms binding to full-length factor VIIa. Furthermore, 100-fold molar excess of unlabeled zinc completely abrogated 65Zn2+ binding to APC, which confirms the specificity of the observed Zn2+ binding. Residues 1–41 of the Gla domain of protein C, a fragment that was removed from APC by selective proteolysis with chymotrypsin, contain one His residue (His10) and two Cys residues (Cys17 and Cys22), the two predominant residues that participate in zinc coordination. Both Cys17 and Cys22 are predicted as zinc-binding sites in protein C by the PREDZINC program (
      • Shu N.
      • Zhou T.
      • Hovmöller S.
      ). One could speculate that other residues outside of the N-terminal Gla domain region (residues 1–41) may contribute to Zn2+ binding. It is possible that the removal of amino acids 1–41 may change the conformation of the protein and abolish the Zn2+ binding to the region outside of the N terminus of the Gla domain. It may be pertinent to note here that PREDZINC identified several residues outside of the N-terminal Gla region as putative zinc-binding residues with high binding score (>0.45). They include His44, Cys50, Cys63, Cys89, Cys98, and Cys105. FEATURE also recognized one or more of these residues as potential coordinating residues for zinc binding. Although most of the zinc-binding sites are coordinated by three or four amino acid residues, in rare cases zinc atoms could be coordinated by only one or two amino acid residues (
      • Vallee B.L.
      • Auld D.S.
      ). In light of multiple Zn2+ binding to the Gla domain of protein C, it is likely that zinc binding to the Gla domain may be coordinated by a single zinc-binding residue. Further studies are needed to identify zinc-binding sites in protein C.
      The full physiological significance of Zn2+ binding to protein C/APC remains to be elucidated. Our present data using the endothelial cell model system show that Zn2+-mediated increase in protein C binding to EPCR results in increased APC generation. However, it is difficult to judge the physiological significance of Zn2+ effect on APC generation because physiological concentrations of Mg2+ also enhanced APC generation, and Zn2+ did not further enhance APC generation in the presence of Mg2+. Although Zn2+ inhibits the amidolytic activity of APC, the physiological concentrations of Zn2+ will have no significant effect on the anticoagulant function of APC, because high concentrations of zinc ions were required to inhibit the amidolytic activity of APC, and this inhibition was attenuated by the presence of physiological levels of Ca2+. Consistent with this, we found that Zn2+ had no effect on the anticoagulant activity of APC. Recent studies suggest that APC bound to EPCR activates PAR1-mediated cell signaling, and this may be responsible for some of the nonhemostatic functions of EPCR (
      • Ludeman M.J.
      • Kataoka H.
      • Srinivasan Y.
      • Esmon N.L.
      • Esmon C.T.
      • Coughlin S.R.
      ,
      • Riewald M.
      • Petrovan R.J.
      • Donner A.
      • Mueller B.M.
      • Ruf W.
      ,
      • Feistritzer C.
      • Riewald M.
      ,
      • Cheng T.
      • Liu D.
      • Griffin J.H.
      • Fernández J.A.
      • Castellino F.
      • Rosen E.D.
      • Fukudome K.
      • Zlokovic B.V.
      ). Zn2+ binding to APC could promote APC-induced cell signaling because it increases the formation of EPCR-APC complexes on cell surfaces, which results in increased activation of PAR1.
      Overall our present data show that Zn2+ binds to protein C/APC and induces conformational changes in the protein. Zn2+ binding to protein C/APC increases their affinity for EPCR, which in turn may result in increased APC generation and APC-mediated cell signaling. These observations suggest that zinc ions may play an important and physiologically relevant role in modulating the protein C/APC pathway. Further studies are needed to map the specific Zn2+-binding sites in protein C/APC and to elucidate the specific physiological functions mediated by the interaction of Zn2+ with protein C and APC.

      Acknowledgments

      We thank C. T. Esmon (Oklahoma Medical Research Foundation) for providing EPCR mAb, A. Rezaie (St. Louis University) for providing soluble thrombomodulin, P. Neuenschwander (The University of Texas Health Science Center at Tyler) for helping in preparation of Gla domain-deleted APC, N. Shu (Stockholm University) for predicting potential zinc-binding sites in protein C, and Russ Altman and Jessica Ebert (Stanford University) for helpful discussion on the use of FEATURES web interface.

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