Thrombomodulin-independent Activation of Protein C and Specificity of Hemostatically Active Snake Venom Serine Proteinases

Protein C activation initiated by the thrombin-thrombomodulin complex forms the major physiological anticoagulant pathway. Agkistrodon contortrix contortrix protein C activator, a glycosylated single-chain serine proteinase, activates protein C without relying on thrombomodulin. The crystal structures of native and inhibited Agkistrodon contortrix contortrix protein C activator determined at 1.65 and 1.54 Å resolutions, respectively, indicate the pivotal roles played by the positively charged belt and the strategic positioning of the three carbohydrate moieties surrounding the catalytic site in protein C recognition, binding, and activation. Structural changes in the benzamidine-inhibited enzyme suggest a probable function in allosteric regulation for the anion-binding site located in the C-terminal extension, which is fully conserved in snake venom serine proteinases, that preferentially binds Cl1- instead of SO42−. Protein C activation initiated by the thrombin-thrombomodulin complex forms the major physiological anticoagulant pathway. Agkistrodon contortrix contortrix protein C activator, a glycosylated single-chain serine proteinase, activates protein C without relying on thrombomodulin. The crystal structures of native and inhibited Agkistrodon contortrix contortrix protein C activator determined at 1.65 and 1.54 Å resolutions, respectively, indicate the pivotal roles played by the positively charged belt and the strategic positioning of the three carbohydrate moieties surrounding the catalytic site in protein C recognition, binding, and activation. Structural changes in the benzamidine-inhibited enzyme suggest a probable function in allosteric regulation for the anion-binding site located in the C-terminal extension, which is fully conserved in snake venom serine proteinases, that preferentially binds Cl1- instead of SO42−. Hemostasis, a complex system responsible for maintaining the fluidity of blood under physiological conditions, is primed to react rapidly to vascular injury, stemming blood loss by locally sealing the injured vessel wall (1Colman R.W. Marder V.J. Salzman E.W. Hirsh J. Colman R.W. Hemostasis and Thrombosis: Basic Principles and Clinical Practice. 3rd Ed. J. B. Lippincott Company, Philadelphia1994: 3-18Google Scholar). The highly regulated hemostatic system functions in equilibrium between two extremes: coagulation and fibrinolysis (2Davie E.W. Fujikawa K. Kisiel W. Biochemistry. 1991; 30: 10363-10370Crossref PubMed Scopus (1609) Google Scholar). On one hand by initiating the coagulation cascade, which comprises a network of highly, correlated and controlled reactions and is triggered either by the extrinsic (tissue factor) or the intrinsic pathways (Factor XIa), activating thrombin to generate cross-linked fibrin polymers (1Colman R.W. Marder V.J. Salzman E.W. Hirsh J. Colman R.W. Hemostasis and Thrombosis: Basic Principles and Clinical Practice. 3rd Ed. J. B. Lippincott Company, Philadelphia1994: 3-18Google Scholar). On the other hand, in the fibrinolytic system, the protein C pathway comprises the major physiological anticoagulant mechanism and is activated on the surface of endothelial cells by the thrombin-thrombomodulin complex (3Esmon C.T. Fukudome K. Semin. Cell Biol. 1995; 6: 259-268Crossref PubMed Scopus (60) Google Scholar). Activated protein C subsequently binds protein S and inhibits coagulation by degrading FVIIIa and FVa on the surface of negatively charged membranes by stimulating fibrinolytic activity (4Walker F.J. Sexton P.W. Esmon C.T. Biochim. Biophys. Acta. 1979; 571: 333-342Crossref PubMed Scopus (304) Google Scholar, 5Fulcher C.A. Gardiner J.E. Griffin J.H. Zimmerman T.S. Blood. 1984; 63: 486-489Crossref PubMed Google Scholar). Deficiencies or defects in the protein C (PC) 3The abbreviations used are:PCprotein CTSV-PATrimeresurus stejnegeri venom plasminogen activatorACC-Cprotein C activator from Agkistrodon contortrix contortrix venomcmkchloromethyl ketoneTME45thrombomodulin epidermal growth factor-like domains 4 and 5NAP5nematode anticoagulant protein 5 from Ancylostoma caniniumdansyl5-dimethylaminonaphthalene-1-sulfonylr.m.s.root mean square 3The abbreviations used are:PCprotein CTSV-PATrimeresurus stejnegeri venom plasminogen activatorACC-Cprotein C activator from Agkistrodon contortrix contortrix venomcmkchloromethyl ketoneTME45thrombomodulin epidermal growth factor-like domains 4 and 5NAP5nematode anticoagulant protein 5 from Ancylostoma caniniumdansyl5-dimethylaminonaphthalene-1-sulfonylr.m.s.root mean square anticoagulant pathway are associated with the increased risk of venous thromboembolism (6Schwarz H.P. Fischer M. Hopmeier P. Batard M.A. Griffin J.H. Blood. 1984; 64: 1297-1300Crossref PubMed Google Scholar). The PC pathway also plays an important role in inflammatory processes and activated PC has been implicated in anti-apoptotic and neuroprotective activities (7Griffin J.H. Zlokovic B. Fernandez J.A. Semin. Hematol. 2002; 39: 197-205Crossref PubMed Scopus (82) Google Scholar). protein C Trimeresurus stejnegeri venom plasminogen activator protein C activator from Agkistrodon contortrix contortrix venom chloromethyl ketone thrombomodulin epidermal growth factor-like domains 4 and 5 nematode anticoagulant protein 5 from Ancylostoma caninium 5-dimethylaminonaphthalene-1-sulfonyl root mean square protein C Trimeresurus stejnegeri venom plasminogen activator protein C activator from Agkistrodon contortrix contortrix venom chloromethyl ketone thrombomodulin epidermal growth factor-like domains 4 and 5 nematode anticoagulant protein 5 from Ancylostoma caninium 5-dimethylaminonaphthalene-1-sulfonyl root mean square Snake venom serine proteinases belong to the trypsin subfamily of enzymes, share significant sequence identity (50-70%), and display high specificity toward macromolecular substrates. These enzymes interfere in the control and regulation of the hemostatic system at different key points ranging from the coagulation cascade to the fibrinolytic feedback system (8Tu A.T. Tu A.T. Handbook of Natural Toxins. 1st Ed. Marcel Dekker, New York1991: 827-830Google Scholar) and are characterized as activators of the fibrinolytic system (plasminogen activators), procoagulant (thrombin-like enzymes), anti-coagulant (PC activators), and platelet-aggregating enzymes (9Marsh N. Williams V. Toxicon. 2005; 45: 1171-1181Crossref PubMed Scopus (148) Google Scholar). Trimeresurus stejnegeri venom plasminogen activator converts plasminogen to plasmin by cleavage of the peptide bond Arg561-Val562 with high substrate specificity and is resistant to inhibition (10Zhang Y. Wisner A. Maroun R.C. Choumet V. Xiong Y. Bon C. J. Biol. Chem. 1997; 272: 20531-20537Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 11Parry M.A. Jacob U. Huber R. Wisner A. Bon C. Bode W. Structure (Camb.). 1998; 6: 1195-1206Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Batroxobin, a thrombin-like enzyme from Bothrops atrox venom used for the treatment of thrombotic diseases (12Stocker K. Markwardt F. Handbook of Experimental Pharmacology. 1st Ed. Springer-Verlag, Berlin1978: 451-484Google Scholar), converts fibrinogen to fibrin by cleaving fibrinopeptide A (Aα 1-16) at the N-terminal portion of the Aα-chain forming a non-cross-linked “soft clot” that is rapidly removed from the circulatory system by the fibrinolytic mechanism resulting in a defibrinogenerating effect. The PC activator from Agkistrodon contortrix contortrix venom, commercially referred to as Protac®, specifically converts PC to activated PC by hydrolyzing the Arg169-Leu170 bond, functioning independently of plasmatic factors in comparison to the physiological activation of PC by thrombin which is dependent on the participation of thrombomodulin (13Kisiel. W. Kondo S. Smith K.J. McMullen B.A. Smith L.F. J. Biol. Chem. 1987; 262: 12607-12613Abstract Full Text PDF PubMed Google Scholar). The PC activator, from A. contortrix contortrix venom, induced zymogen activation is clinically used in assays of functional PC determination, total protein S content, and other protein S functional assays (14Gempeler-Messina P.M. Volz K. Bühler B. Muller C. Haemostasis. 2001; 31: 266-272PubMed Google Scholar) in plasma, since catalytically active PC can easily be detected by coagulation tests or by utilizing chromogenic substrates. We present the first crystal structures of the PC activator from the venom of the copperhead snake A. contortrix contortrix (ACC-C), a single-chain glycosylated serine proteinase that is a fast-acting PC activator, both in the native and inhibited states at 1.65 and 1.54 Å resolutions, respectively. These results provide information at the molecular level concerning the alternative PC activation pathway that is independent of thrombomodulin, the central physiological barrier against thrombosis and could serve as a basis for the structure-based design of clinically useful molecules and in the treatment of thromboembolism. ACC-C, provided by Pentapharm (Basel, Switzerland), was dissolved to a concentration of 10 mg/ml in a 20 mm Hepes (pH 7.5) buffer that also contained 10 mm sodium chloride and 1 mm dithiothreitol. Crystals were obtained at 18 °C by equilibration of the protein solution (1 μl) against a reservoir solution containing 2.0 m ammonium sulfate and 100 mm sodium acetate (pH 4.6) utilizing the hanging-drop vapor diffusion method. The benzamidine-inhibited ACC-C complex was obtained by soaking crystals for 45 s in a cryo-protectant solution (20% (v/v) glycerol) that additionally contained 50 mm benzamidine. Dose-dependent x-ray diffraction data were collected from cryo-protected crystals at 100 K at a synchrotron radiation source (CPr Beamline-Laboratório Nacional de Luz Síncrotron, Campinas, Brazil) where the wavelength was fixed at 1.438 Å, diffraction intensities were measured utilizing a Mar CCD165 detector (Mar Inc.), and the diffraction intensities were reduced and scaled using the DENZO/SCALEPACK suite of programs (15Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38253) Google Scholar). Both the native and benzamidine-inhibited crystals are isomorphous, belong to the monoclinic space group C2, and contain one protein molecule in the asymmetric unit. The crystal structure of ACC-C was solved by molecular replacement (AMoRe) (16Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5026) Google Scholar) initially using the atomic coordinates of T. stejnegeri venom plasminogen activator (Protein Data Bank entry: 1BQY) (11Parry M.A. Jacob U. Huber R. Wisner A. Bon C. Bode W. Structure (Camb.). 1998; 6: 1195-1206Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) as a search model. Positional and restrained isotropic B-factor refinements were performed using REF-MAC5 (17Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13712) Google Scholar), all model building was carried out utilizing TURBO-FRODO (18Jones T.A. Methods Enzymol. 1985; 115: 157-171Crossref PubMed Scopus (934) Google Scholar), and PROCHECK (19Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar) was utilized to evaluate the stereochemistry of the final models. The data collection, processing, and refinement statistics are presented in TABLE ONE.TABLE ONEData collection and refinement statisticsNative stateInhibited stateCrystal preparationCryoprotectant solutionMother liquor + 20% glycerolMother liquor + 20% glycerol + 50 mm benzamidineSoaking time30 s45 sData collectionWavelength (Å)1.4381.438Temperature (K)100100DetectorMARCCDMARCCDSynchrotron radiation sourceCPr beamline/LNLS-BrazilCPr beamline/LNLS-BrazilSpace groupC 2C 2Unit cell parameters (Å, °)a = 79.87, b = 63.30, and c = 48.24; β = 99.80a = 80.52, b = 63.46, and c = 48.22; β = 99.85Number of molecules in the asymmetric unit11Solvent content (%)42.441.7VM (Å/Da)2.152.12Number of reflections487,208409,068Number of unique reflections28,664 (2835)35,447 (5817)〈I/σ(I) 〉20.0 (2.9)18.3 (3.5)Multiplicity7.8 (7.5)6.4 (5.8)Completeness (%)99.8 (99.6)99.9 (99.8)RmergeaRmerge=∑|I(h)I−{I(h)}|/∑{I(h)}, where Ih is the observed intensity of the ih measurement of reflection h, and {I(h)} is the mean intensity of reflection h calculated after scaling. (%)9.0 (64.5)8.4 (49.2)Refinement statisticsRfactor (%)16.716.7Rfree (%)19.619.1r.m.s. deviation bond distances (Å)0.0110.010r.m.s. deviation bond angles (°)1.4921.453Average B-factors (Å2)21.217.3Ramachandran plot analysisMost favored regions (%)83.982.8Allowed regions (%)15.116.1Generously allowed regions (%)1.01.0a Rmerge=∑|I(h)I−{I(h)}|/∑{I(h)}, where Ih is the observed intensity of the ih measurement of reflection h, and {I(h)} is the mean intensity of reflection h calculated after scaling. Open table in a new tab The crystal structures of both native and benzamidine-inhibited ACC-C have been refined at 1.65 and 1.54 Å resolutions to crystallographic residuals of 16.7% (Rfree = 19.6%) and 16.7% (Rfree = 19.1%), respectively. An analysis of the stereochemistry of the final models indicates that the main-chain dihedral angles for all residues are located in the permitted regions of the Ramachandran diagram and that the root mean square (r.m.s.) deviations from ideal values are distributed within the expected ranges for well refined structures (TABLE ONE). In analogy to the structures of trypsin-like serine proteinases, the structure of ACC-C consists of two domains (S and S′) each containing a six-stranded β-barrel and two short α-helices (residues: 165-173 and 235-244, sequence numbering is based on chymotrypsinogen) (20Harley B.S. Shotton D.M. Boyer P.D. The Enzymes. 1st Ed. Academic Press, New York1971: 323-373Google Scholar) (Fig. 1, A and B). The catalytic triad (His57, Asp102, and Ser195) is located at the junction of both barrels and is surrounded by the conserved 70-, 148-, and 218-loops, and the non-conserved 37-, 60-, 99-, and 174-loops (Fig. 1, A and B). ACC-C contains 16% carbohydrate, including glucosamine, neuraminic acid, and neutral hexose (13Kisiel. W. Kondo S. Smith K.J. McMullen B.A. Smith L.F. J. Biol. Chem. 1987; 262: 12607-12613Abstract Full Text PDF PubMed Google Scholar, 21McMullen B.A. Fujikawa. J. Kisiel W. Biochemistry. 1988; 28: 674-679Crossref Scopus (50) Google Scholar), the consensus signal sequence for the attachment of carbohydrate moieties to asparagines (Asn-X-Thr/Ser, where X represents any amino acid) was identified at positions 38, 96A, and 148. In both the structures, clear electron density was observed for the N-acetyl-d-glucosamine monosaccharide, which is N-linked to Asn96a and Asn148; however, the moiety linked to Asn38 was inserted based on the presence of diffuse electron density (Fig. 1B). The three carbohydrate moieties are strategically positioned at the tips of the 37-, 99-, and 148-loops, which form the entrance to the active-site pocket (Fig. 1B) and modulate the selectivity toward macromolecular substrates (discussed below). Two snake venom serine proteinase isoforms, AaV-SP-I and AaV-SP-II, from Agkistrodon acutus, also posses an N-linked carbohydrate group (Asn35) that is considered to interfere with the binding of macromolecular inhibitors (22Zhu Z. Liang Z. Zhang T. Zhu Z. Xu W. Teng M. Niu L. J. Biol. Chem. 2005; 280: 10524-10529Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). In contrast, T. stejnegeri venom plasminogen activator (TSV-PA) has a unique glycosylation site at Asn178 located on the opposite face (23Zhang Y. Wisner A. Xiong Y. Bon C. J. Biol. Chem. 1995; 270: 10246-10255Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar) and apparently does a role in the binding macromolecular substrates at the The electron density for the molecule to the of is (Fig. and indicates a of to that observed in the structures of benzamidine-inhibited trypsin-like enzymes W. P. J. Biol. PubMed Scopus Google Scholar). A sulfate is by and two molecules both in the native and benzamidine-inhibited states (Fig. which that the catalytic triad is by binding and could for the activity observed in benzamidine-inhibited ACC-C toward substrates J. U. J. Toxicon. 1991; PubMed Scopus Google Scholar). The binding of at the results in in the C-terminal and and in the deviations and The anion-binding pocket between the highly conserved C-terminal and the by a and a and binds either or Cl1- on the presence or of a molecule at the active site and In native ACC-C, indicates amino from a also in the of the sulfate by forming a pocket and and binds a In benzamidine-inhibited ACC-C, also coordinates the Cl1- a different the The of functioning as a a that the binding of the These changes suggest the of an allosteric mechanism functioning between the active-site pocket and the C-terminal that preferentially binds Cl1- instead of in to substrate or binding at the of the structure of ACC-C with results in positions with deviations of Å, a high of in with the observed sequence identity of (Fig. The S is highly conserved and the are in the surface surrounding the active-site the 37-, 60-, and which form the (Fig. The located at of the is by a a at and a N-linked at 38, which results in the of a with deviation The is positively charged in ACC-C to and in is negatively charged (Fig. The positioning of and a between the S and domains (Fig. as acid, that contain a group and a could to and the and to the catalytic of the active-site pocket for the structures of ACC-C and ketone and rapidly both the and anticoagulant activities of ACC-C (13Kisiel. W. Kondo S. Smith K.J. McMullen B.A. Smith L.F. J. Biol. Chem. 1987; 262: 12607-12613Abstract Full Text PDF PubMed Google Scholar). The structure of ACC-C was on the structure of the single-chain plasminogen with (Protein Data Bank entry: M. R.A. Huber R. Fischer S. U. Bode W. J. 1997; PubMed Scopus Google Scholar), and the of was to indicates that the between from and at the is by between the of and the group of and The forms with the of and These could for the of ACC-C by ketone of macromolecular inhibitors trypsin and trypsin be by the by the of in ACC-C and by the of in (Fig. of inhibitors also be by the carbohydrate moieties present on the surface of ACC-C (Fig. 1B) and from Agkistrodon venom (22Zhu Z. Liang Z. Zhang T. Zhu Z. Xu W. Teng M. Niu L. J. Biol. Chem. 2005; 280: 10524-10529Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). significant of ACC-C, from the three carbohydrate moieties distributed strategically the active-site is the significant present on the surface (Fig. to the presence of residues to the that could The activity of ACC-C is inhibited by (13Kisiel. W. Kondo S. Smith K.J. McMullen B.A. Smith L.F. J. Biol. Chem. 1987; 262: 12607-12613Abstract Full Text PDF PubMed Google Scholar) as a of binding at site is negatively charged in (Fig. and for the role of in the selectivity of snake venom serine physiological conditions, the binding site of the and of PC zymogen in the thrombin-thrombomodulin complex is by a of and charged which form an on thrombomodulin epidermal growth factor-like domains 4 and 5 P. Y. Huber R. R. M. J. Bode W. PubMed Scopus Google Scholar). The negatively charged activation peptide of PC was based on the crystal structure of activated PC (Protein Data Bank entry: T. V. P. Huber R. S. Esmon C. Bode W. J. PubMed Scopus Google Scholar) and the by nematode anticoagulant protein 5 from Ancylostoma caninium with In between the positively charged surface of ACC-C by the residues positions and and the residues of the activation peptide and the of (discussed that forms the with are (Fig. C and the three carbohydrate moieties that form on the surface of ACC-C are important in the and of PC zymogen activation. In physiological PC the carbohydrate moieties are considered to be important and are either for thrombin binding or for activity as by the activity of thrombomodulin in M. J. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar, M. J.H. J. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). The enzyme PC J. J. Biol. Chem. Full Text PDF PubMed Google Scholar) is activated by cleavage of the Arg169-Leu170 bond by the complex and functions as an anticoagulant enzyme by factors and C.T. J. Biol. Chem. Full Text PDF PubMed Google Scholar). assays of PC activation are either by activation by thrombin or by the of thrombin in chromogenic The ACC-C by Pentapharm as does on other plasmatic factors and is clinically used for PC assays by the chromogenic by the chromogenic based on activated or by a functional (13Kisiel. W. Kondo S. Smith K.J. McMullen B.A. Smith L.F. J. Biol. Chem. 1987; 262: 12607-12613Abstract Full Text PDF PubMed Google Scholar, P.M. Volz K. Bühler B. Muller C. Haemostasis. 2001; 31: 266-272PubMed Google Scholar). the activation of ACC-C is by the of PC to PC are (14Gempeler-Messina P.M. Volz K. Bühler B. Muller C. Haemostasis. 2001; 31: 266-272PubMed Google Scholar). of the crystal structures of native and inhibited ACC-C indicates that binding of does the residues of the catalytic which could for the activity observed and are used as inhibitors J. U. J. Toxicon. 1991; PubMed Scopus Google Scholar). binding significant the a different the anion-binding site located at the C by and a preferentially binds Cl1- instead of and the and of the the of the between and the and of the Cl1- the of out and the resulting at site a which the binding of a These changes indicate the of an allosteric The of the of the active site observed in the crystal structure of ACC-C indicates that inhibitors as with the and with and respectively, to substrate The on both and anticoagulant activities induced by and as and are to the to and and with and which the and modulate to the catalytic The two significant between ACC-C and the positively charged surface surrounding the catalytic site and the three carbohydrate moieties that on the surface of ACC-C, which a pivotal role in macromolecular substrate or Both ACC-C and share a and high to macromolecular for substrates as trypsin and trypsin inhibitors are to to the positioning of the of and in to the presence of N-linked carbohydrate in the In ACC-C, both are utilized is by a the of and the N-linked are positioned to with macromolecular physiological conditions, thrombin on thrombomodulin to between functioning as a or anticoagulant enzyme and the domains form the binding site of the PC the of the bond between and In the activation of PC by ACC-C the present on the surface and carbohydrate moieties are important that could roles in PC We are to T. for the of ACC-C