Structural and Energetic Characteristics of the Heparin-binding Site in Antithrombotic Protein C

Abstract

Human activated protein C (APC) is a key component of a natural anticoagulant system that regulates blood coagulation. In vivo, the catalytic activity of APC is regulated by two serpins, α1-antitrypsin and the protein C inhibitor (PCI), the inhibition by the latter being stimulated by heparin. We have identified a heparin-binding site in the serine protease domain of APC and characterized the energetic basis of the interaction with heparin. According to the counter-ion condensation theory, the binding of heparin to APC is 66% ionic in nature and comprises four to six net ionic interactions. To localize the heparin-binding site, five recombinant APC variants containing amino acid exchanges in loops 37, 60, and 70 (chymotrypsinogen numbering) were created. As demonstrated by surface plasmon resonance, reduction of the electropositive character of loops 37 and 60 resulted in complete loss of heparin binding. The functional consequence was loss in heparin-induced stimulation of APC inhibition by PCI, whereas the PCI-induced APC inhibition in the absence of heparin was enhanced. Presumably, the former observations were due to the inability of heparin to bridge some APC mutants to PCI, whereas the increased inhibition of certain APC variants by PCI in the absence of heparin was due to reduced repulsion between the enzymes and the serpin. The heparin-binding site of APC was also shown to interact with heparan sulfate, albeit with lower affinity. In conclusion, we have characterized and spatially localized the functionally important heparin/heparan sulfate-binding site of APC. Human activated protein C (APC) is a key component of a natural anticoagulant system that regulates blood coagulation. In vivo, the catalytic activity of APC is regulated by two serpins, α1-antitrypsin and the protein C inhibitor (PCI), the inhibition by the latter being stimulated by heparin. We have identified a heparin-binding site in the serine protease domain of APC and characterized the energetic basis of the interaction with heparin. According to the counter-ion condensation theory, the binding of heparin to APC is 66% ionic in nature and comprises four to six net ionic interactions. To localize the heparin-binding site, five recombinant APC variants containing amino acid exchanges in loops 37, 60, and 70 (chymotrypsinogen numbering) were created. As demonstrated by surface plasmon resonance, reduction of the electropositive character of loops 37 and 60 resulted in complete loss of heparin binding. The functional consequence was loss in heparin-induced stimulation of APC inhibition by PCI, whereas the PCI-induced APC inhibition in the absence of heparin was enhanced. Presumably, the former observations were due to the inability of heparin to bridge some APC mutants to PCI, whereas the increased inhibition of certain APC variants by PCI in the absence of heparin was due to reduced repulsion between the enzymes and the serpin. The heparin-binding site of APC was also shown to interact with heparan sulfate, albeit with lower affinity. In conclusion, we have characterized and spatially localized the functionally important heparin/heparan sulfate-binding site of APC. activated protein C protein C mutant with E60aS plus S61R replacements polyacrylamide gel electrophoresis wild type protein C inhibitor basic fibroblast growth factor dissociation constant morpholinoethanesulfonic acid streptavidin sensor chips The protein C pathway is a functionally important anticoagulant system that regulates blood coagulation in vivo. The key component of this pathway is the vitamin K-dependent protein C (1Davie E.W. Fujikawa K. Kisiel W. Biochemistry. 1991; 30: 10363-10370Crossref PubMed Scopus (1675) Google Scholar, 2Esmon C.T. Ding W. Yasuhiro K. Gu J.M. Ferrell G. Regan L.M. Stearns-Kurosawa D.J. Kurosawa S. Mather T. Laszik Z. Esmon N.L. Thromb. Haemostasis. 1997; 78: 70-74Crossref PubMed Scopus (108) Google Scholar, 3Dahlbäck B. Lancet. 2000; 355: 1627-1632Abstract Full Text Full Text PDF PubMed Scopus (607) Google Scholar). Protein C circulates in plasma as a zymogen to a serine protease that has anticoagulant properties. Protein C is a multidomain molecule that is composed of two disulfide-linked chains. The light chain comprises a γ-carboxyglutamatic acid domain and two epidermal growth factor (EGF)-like domains, whereas the heavy chain is composed of the activation peptide and a serine protease domain (4Furie B. Furie B.C. Cell. 1988; 53: 505-518Abstract Full Text PDF PubMed Scopus (1073) Google Scholar). Protein C is activated on endothelial cells by thrombin bound to thrombomodulin. Activated protein C (APC)1 regulates blood coagulation by cleaving and inhibiting two cofactors, activated factor V (FVa) and activated factor VIII (FVIIIa) (5Kalafatis M. Rand M.D. Mann K.G. J. Biol. Chem. 1994; 269: 31869-31880Abstract Full Text PDF PubMed Google Scholar), which serve as phospholipid-membrane-bound cofactors to factor Xa (FXa) and factor IXa (FIXa), respectively. FXa is the enzyme that activates prothrombin to thrombin, whereas FIXa converts FX to its active form (1Davie E.W. Fujikawa K. Kisiel W. Biochemistry. 1991; 30: 10363-10370Crossref PubMed Scopus (1675) Google Scholar, 6Rosing J. Tans G. Thromb. Haemostasis. 1997; 78: 427-433Crossref PubMed Scopus (76) Google Scholar). In vivo, the proteolytic activity of APC is regulated by two serpins, namely α1-antitrypsin and protein C inhibitor (PCI) (7Heeb M.J. Griffin J.H. J. Biol. Chem. 1988; 263: 11613-11616Abstract Full Text PDF PubMed Google Scholar, 8Hermans J.M. Stone S.R. Biochem. J. 1993; 295: 239-245Crossref PubMed Scopus (54) Google Scholar). Inhibition of APC by PCI is potentiated by the glycosaminoglycan heparin, whereas the inhibition by α1-antitrypsin is insensitive to the presence of heparin. Structurally, heparin is heterogeneous in nature and is composed of long, highly negatively charged, unbranched polysaccharide chains. It is hypothesized that heparin binds to both PCI and APC during PCI-mediated inhibition of APC, thus guiding the encounter of these proteins via a template mechanism (9Aznar J. Espana F. Estelles A. Royo M. Thromb. Haemostasis. 1996; 76: 983-988Crossref PubMed Scopus (19) Google Scholar, 10Neese L.L. Wolfe C.A. Church F.C. Arch. Biochem. Biophys. 1998; 355: 101-108Crossref PubMed Scopus (33) Google Scholar). We therefore expect the formation of a ternary complex similar to the one suggested between antithrombin, heparin, and thrombin (11Olson S.T. Bjork I. J. Biol. Chem. 1991; 266: 6353-6364Abstract Full Text PDF PubMed Google Scholar, 12Olson S.T. Bjork I. Semin. Thromb. Hemostasis. 1994; 20: 373-409Crossref PubMed Scopus (132) Google Scholar). Recently some residues in APC were implied to interact directly with heparin during the PCI-induced inhibition of APC (13Shen L. Villoutreix B.O. Dahlbäck B. Thromb. Haemostasis. 1999; 82: 72-79Crossref PubMed Scopus (32) Google Scholar, 14Shen L. Dahlbäck B. Villoutreix B.O. Biochemistry. 2000; 39: 2853-2860Crossref PubMed Scopus (17) Google Scholar), but a more complete definition of a heparin-binding site(s) in APC and energetic characteristics of the heparin interaction with APC were lacking. Binding of heparin to proteins is usually ionic in nature, involving the side chains of clustered basic amino acids on the protein and negatively charged groups on the heparin molecule. Amino acid sequence patterns such as XBBXBX and XBBBXXBX (B denotes basic, and X denotes nonbasic residues) are potential heparin recognition sites (15Cardin A.D. Weintraub H.J. Arteriosclerosis. 1989; 9: 21-32Crossref PubMed Google Scholar, 16Montserret R. Aubert-Foucher E. McLeish M.J. Hill J.M. Ficheux D. Jaquinod M. van der Rest M. Deleage G. Penin F. Biochemistry. 1999; 38: 6479-6488Crossref PubMed Scopus (29) Google Scholar). Alternatively, the basic residues may be located far apart in the linear sequence but are topological neighbors in the three-dimensional structure (17Sali A. Matsumoto R. McNeil H.P. Karplus M. Stevens R.L. J. Biol. Chem. 1993; 268: 9023-9034Abstract Full Text PDF PubMed Google Scholar, 18Matsumoto R. Sali A. Ghildyal N. Karplus M. Stevens R.L. J. Biol. Chem. 1995; 270: 19524-19531Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 19Murakami M. Matsumoto R. Urade Y. Austen K.F. Arm J.P. J. Biol. Chem. 1995; 270: 3239-3246Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 20Ghildyal N. Friend D.S. Stevens R.L. Austen K.F. Huang C. Penrose J.F. Sali A. Gurish M.F. J. Exp. Med. 1996; 184: 1061-1073Crossref PubMed Scopus (75) Google Scholar). In the three-dimensional structure of APC, a basic cluster of amino acids is located on loops 37, 60, and 70 (chymotrypsinogen nomenclature) (see Fig. 1) (21Wacey A.I. Pemberton S. Cooper D.N. Kakkar V.V. Tuddenham E.G. Br. J. Haematol. 1993; 84: 290-300Crossref PubMed Scopus (27) Google Scholar, 22Fisher C.L. Greengard J.S. Griffin J.H. Protein Sci. 1994; 3: 588-599Crossref PubMed Scopus (40) Google Scholar, 23Mather T. Oganessyan V. Hof P. Huber R. Foundling S. Esmon C. Bode W. EMBO J. 1996; 15: 6822-6831Crossref PubMed Scopus (194) Google Scholar). Heparin-binding sites sharing similar distribution of charged amino acids are present on the surface of many proteins that bind heparin, e.g.hepatocyte growth factor (24Zhou H. Casas-Finet J.R. Heath Coats R. Kaufman J.D. Stahl S.J. Wingfield P.T. Rubin J.S. Bottaro D.P. Byrd R.A. Biochemistry. 1999; 38: 14793-14802Crossref PubMed Scopus (51) Google Scholar). Multiple-sequence and structural alignments of APC with other serine proteases formed the basis for our mutagenesis strategy, aimed at identification of the APC heparin-binding site. In the present study, site-directed mutagenesis and recombinant human protein C expression were used to localize the heparin-binding site in APC. A cluster of lysines located on loops 37 and 60 was found to be crucial for heparin binding. Characterization of the binding of heparin to APC under different salt concentrations provided evidence for the electrostatic signature of the interaction. Reduction of the electropositive character of these loops resulted in lost heparin binding and reduced heparin stimulation of APC inhibition by PCI. Restriction endonucleases were obtained from New England Biolabs or Fermentas. Dulbecco's modified Eagle's medium, Waymouth's medium, and fetal calf serum were obtained from Life Technologies, Inc. Cell culture ware was purchased from Falcon, vitamin K1 was from Hoffmann La Roche, hygromycin B was from Calbiochem. Q-Sepharose Fast flow, sulfopropyl-Sepharose, heparin-Sepharose, and PD-10 columns were from Amersham Pharmacia Biotech. The chromogenic substrate S-2366 (l-pyroglutamyl-l-prolyl-l-arginine-p-nitroanilin), specific for protein C, was obtained form Chromogenix (Sweden), and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium chloride (NBT) were from ICN. Human α-thrombin was prepared from plasma-purified prothrombin, as described (25Dahlbäck B. Hildebrand B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1396-1400Crossref PubMed Scopus (344) Google Scholar). Basic fibroblast growth factor (bFGF) was from Sigma. Heparan sulfate (bovine kidney) was from Seikagaku (Japan). The EZ-link biotin hydrazide was from Pierce. Streptavidin sensor chips (SA-chip) were from Biacore (Sweden). Three new recombinant human protein C variants, K37S/K38Q/K39Q, K37S/K38Q/K39Q/K62N/K63D, and R74Q, were created by site-directed in vitro mutagenesis using the polymerase chain reaction technique. In brief, full-length human protein C WT cDNA (1425 base pairs) was inserted into theBcII site of the eukaryotic expression vector pGT-hyg (Eli Lilly). The following primers were used to introduce the mutations into the human protein C WT cDNA: sense primer, 5′-CTG CTG GAC TCAAGC CAG CAG CTG GCC TGC GGG for variant K37S/K38Q/K39Q; sense primer, 5′-GAG TAT GAC CTGCAG CGC TGG GAG AAG for variant R74Q. The construction of variants E60aS/S61R and K62N/K63D have been previously described (13Shen L. Villoutreix B.O. Dahlbäck B. Thromb. Haemostasis. 1999; 82: 72-79Crossref PubMed Scopus (32) Google Scholar, 14Shen L. Dahlbäck B. Villoutreix B.O. Biochemistry. 2000; 39: 2853-2860Crossref PubMed Scopus (17) Google Scholar). The human protein C variant K37S/K38Q/K39Q-cDNA and sense primer 5′-TGC ATG GAT GAG TCCAAC GAC CTC CTT GTC AGG CTT was used to construct mutant K37S/K38Q/K39Q/K62N/K63D. Exchanged nucleotides as listed above are marked in bold, and codons corresponding to the exchanged amino acids are The sequence of for WT protein C and variants were using Life human was in Dulbecco's modified Eagle's with fetal calf and at 37 in The complete human protein cDNA corresponding to WT and variants K37S/K38Q/K39Q, K37S/K38Q/K39Q/K62N/K63D, and R74Q, inserted into the eukaryotic expression vector were into cells using B was used for of characterized by the protein as by were and K62N/K63D were as previously described (13Shen L. Villoutreix B.O. Dahlbäck B. Thromb. Haemostasis. 1999; 82: 72-79Crossref PubMed Scopus (32) Google Scholar, 14Shen L. Dahlbäck B. Villoutreix B.O. Biochemistry. 2000; 39: 2853-2860Crossref PubMed Scopus (17) Google Scholar). were in Technologies, with vitamin the was and the recombinant human protein C was from the by and as previously described U. B. G. H. Thromb. Haemostasis. 1994; PubMed Scopus Google Scholar). The and of the protein C was by gel electrophoresis and Protein C concentrations were by of at using of In activated protein C concentrations were by a chromogenic the of S-2366 to were by or to for of the proteins to the were with containing and and with The proteins were using a human protein C by and blue tetrazolium chloride (NBT) substrate was used for WT protein C or protein C variants were with human α-thrombin in at 37 for in the presence of A was used to thrombin from the reaction APC concentrations were by of at were using chromogenic substrate S-2366 at 37 in a from and in at The concentrations of chromogenic substrate S-2366 between and and the concentrations of APC between and and were obtained from and from the G. Scopus Google Scholar, W. PubMed Scopus Google Scholar). or heparan sulfate of were in of EZ-link biotin hydrazide were to a of and respectively. a at with constant the were on a PD-10 Pharmacia in and in Binding of WT and variant to heparin and heparan sulfate was by surface plasmon using heparin and heparan sulfate were in cells and of a streptavidin sensor of the heparan sulfate was shown by the that bound with affinity. APC was at a of at concentrations into cells containing heparin. In APC was at into a containing heparan glycosaminoglycan was used as To heparan sulfate with heparin for the binding to APC, heparan sulfate was in the The were with to and dissociation WT and variant in were on heparin-Sepharose, and bound APC was with a linear from to APC and concentrations were by of chromogenic substrate S-2366 and (13Shen L. Villoutreix B.O. Dahlbäck B. Thromb. Haemostasis. 1999; 82: 72-79Crossref PubMed Scopus (32) Google Scholar). The side chains of variant APC were using and and were using the The electrostatic potential were with as of the ionic for APC and for the highly negatively charged heparin molecule. The of inhibition of WT and mutant by PCI in the absence of heparin or heparan sulfate was under In brief, WT and variant were to with a of human plasma PCI at in containing serum substrate S-2366 was to a of and the of substrate was with a were as a is the protease activity to the is the of and is the PCI In the presence of heparin to the of APC was to be under the that are described PCI at was with APC and to heparin in a of the reaction was by the of The APC were by of S-2366 was and the at was The following was used to the and were the concentrations of APC and PCI, and was the of formed was from were in To the of heparan sulfate on the of inhibition of APC by PCI, PCI was with APC and concentrations of heparan sulfate in a of these stimulation of inhibition was at concentrations the were by the of activity of APC was by the of of and the at was were as described for heparin from were in To the of charged amino acid residues in loops 37, 60, and 70 in the binding of heparin to APC, five protein C variants were by site-directed mutagenesis According to our structural four of the variants were to have reduced heparin K37S/K38Q/K39Q/K62N/K63D, whereas one mutant was created with the of the heparin the amino acid residues are on and in serine be that the above amino acid the of APC or its catalytic the residues are similar in to the these mutations be as and be in the structure for of to five residues a to form with or due to the of this amino The protein C cDNA variants were used to the eukaryotic and were The different protein C variants demonstrated similar expression to The proteins were with of and the proteins were more as by the protein C variants at similar as WT protein C the different protein C variants as reduction of the heavy chains and the light chains were in to the chain of protein C. for recombinant proteins a of chain protein C the different recombinant protein variants demonstrated similar and The expression for the different protein C variants and activation with the above structural that the recombinant proteins were and be used for functional of chromogenic substrate S-2366 by WT APC and the APC in a new Binding of WT APC and APC variants to heparin was with surface plasmon using a the dissociation constant of WT APC for heparin was The variant lower whereas E60aS/S61R bound more to heparin WT APC Fig. was binding of K37S/K38Q/K39Q, and to heparin To the obtained by WT APC and the five APC variants were on heparin-Sepharose, and bound protein was with a linear The with obtained by the of the charged residues at or or or binding to the In the E60aS/S61R variant bound to the heparin WT APC and was at that the heparin-binding site of APC is on the presence of a charged residues on loops 37 and 60 and for interaction of APC with binding of APC to heparin was with surface plasmon resonance, and was used to and dissociation from the APC variants K37S/K38Q/K39Q, and demonstrated binding to the and be for these APC in a new surface potential of APC. A for heparin containing was APC loops 37 and potential are shown at and for WT and variant APC and at for heparin. The electropositive of loops 37 and 60 in and are with or binding of heparin. is or in variants K37S/K38Q/K39Q, and K37S/K38Q/K39Q/K62N/K63D, which bind heparin or at The binding of APC to heparin was with surface plasmon resonance, and was used to and dissociation from the APC variants K37S/K38Q/K39Q, and demonstrated binding to the and be for these APC is from and is that APC with heparin under heparin is as a Heparan sulfate present on the surface of endothelial cells is the glycosaminoglycan that is more to interact with APC. this we the binding of APC to heparan sulfate in the under similar as for heparin. In to the binding with heparin, binding of APC to heparan sulfate be In this be that is to characterized by many be by this To heparan sulfate was to interact with APC, heparan sulfate was with APC in the containing heparin. these complete inhibition of binding of APC to heparin was that APC may interact with heparan sulfate, albeit with heparin. was used to the characteristics of the binding of WT APC to heparin at salt the being to into the of ionic to the interaction The were using the counter-ion condensation theory, that heparin as a by a counter-ion condensation Biophys. PubMed Scopus Google Scholar, Biophys. PubMed Scopus Google Scholar). interact with heparin by electrostatic and by binding to ionic of heparin binding to protein has been demonstrated H. M.D. Biochemistry. 2000; 39: PubMed Scopus Google Scholar). In a of is the of the to the of heparin its binding to a has been used to many such as the between heparin and thrombin or (11Olson S.T. Bjork I. J. Biol. Chem. 1991; 266: 6353-6364Abstract Full Text PDF PubMed Google Scholar, Biochemistry. 1994; PubMed Scopus Google Scholar). The were was that of the binding between and heparin resulted from ionic interactions. In this the of was linear and was that net ionic were between the two was at a by of the complex S. J.R. 1996; PubMed Scopus Google of on the of complex binding of WT APC to heparin was with surface plasmon and were by of the in in a new binding of WT APC to heparin was with surface plasmon and were by of the in To the the following was The is to the dissociation constant the The of is and the of be from the The formation of electrostatic between WT APC and heparin is by the of from the heparin. is the of counter-ion bound heparin which has been to be (11Olson S.T. Bjork I. J. Biol. Chem. 1991; 266: 6353-6364Abstract Full Text PDF PubMed Google Scholar). The the of ionic formed between WT APC and heparin. ionic to be in this ionic were and which we to be the for the interaction at ionic was 66% of the binding was to be with the The of WT APC binding to heparin at salt was to be with the and ionic to this interaction and respectively. the WT APC binding to heparin is electrostatic in nature the binding of heparin to thrombin (11Olson S.T. Bjork I. J. Biol. Chem. 1991; 266: 6353-6364Abstract Full Text PDF PubMed Google or to inhibitor B. Y. D. Biochemistry. PubMed Scopus Google Scholar). is a of salt on the interaction and the corresponding from at to at whereas the is that electrostatic are important for the formation of encounter salt are the ionic interactions. We also that the interaction is the of is the for of in which is Z. Chem. Google Scholar). inhibition of APC by PCI, heparin or heparan sulfate may as to PCI into the active site of APC during the formation of the the identified heparin/heparan sulfate-binding site on APC to be functionally the recombinant APC are to the heparin/heparan sulfate stimulation of inhibition by PCI. was found to be the to the of PCI-mediated inhibition of WT APC whereas heparin in the of inhibition of the variant The E60aS/S61R which bound heparin with WT APC, demonstrated stimulation of inhibition by heparin Heparan sulfate was in the and was found to a increased of inhibition of WT APC and a increased inhibition of the E60aS/S61R variant but a increased of inhibition of K62N/K63D and a increased of inhibition of the variant that heparan sulfate is to interact with the site as heparin on APC and that heparin and heparan sulfate similar functional on the of inhibition by PCI. on both and the PCI inhibition heparin to bind APC with heparan for PCI-mediated inhibition of APC variants in the presence and absence of of heparin, of heparin, of APC in a new In the absence of heparin/heparan sulfate, E60aS/S61R and variants were by PCI as WT APC, whereas the of PCI inhibition was increased for the K37S/K38Q/K39Q, and variants The was shown for the variant K37S/K38Q/K39Q/K62N/K63D. more of the variant was whereas WT APC activity was reduced by that the cluster in loops 37 and 60 PCI in the absence of heparin. The stimulation of PCI inhibition of the K37S/K38Q/K39Q, K62N/K63D and is in with the involving heparin or heparan sulfate binding to the cluster of APC as as to PCI. In with this theory, heparin and heparan sulfate were found to increased stimulation to the PCI inhibition of the E60aS/S61R which demonstrated increased for heparin It is that the demonstrated heparin/heparan sulfate-binding site in APC is located on of the protease domain as with the heparin binding of thrombin PCI binds heparin on the other side as with the heparin binding of L.L. Wolfe C.A. Church F.C. Arch. Biochem. Biophys. 1998; 355: 101-108Crossref PubMed Scopus (33) Google J.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google Scholar, E. A. Y. V. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). the of PCI be the topological of APC loops 37 and 60 in the In conclusion, we have characterized the structural and energetic basis of a functionally important heparin-binding site in the serine protease domain of APC. binding site is functionally important in the heparin/heparan inhibition of APC by its inhibitor PCI. We for for the of PCI, for the heparin, and for