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• W2310565721 abstract "Thrombin is produced from the C-terminal half of prothrombin following its proteolytic activation. The N-terminal half, released as the propiece Fragment 12 (F12), is composed of an N-terminal γ-carboxyglutamate domain (Gla) followed by two kringles (K1 and K2). The propiece plays essential roles in regulating prothrombin activation and proteinase function. The latter results from the ability of F12 to reversibly bind to the (pro)catalytic domain through K2 with high affinity and highly favorable thermodynamic constants when it is a zymogen in comparison to proteinase. Such discrimination is lost for K2 binding after proteolytic removal of the N-terminal Gla-K1 region of F12. The Ca2+-stabilized structure of the Gla domain is not required for F12 to bind the zymogen form more favorably. Enhanced binding to zymogen versus proteinase correlates with the ability of the propiece to enforce zymogen-like character in the proteinase. This is evident in variants of meizothrombin, an intermediate of prothrombin activation that contains the propiece covalently attached. This phenomenon is also independent of the Gla domain. Thus, the presence of K1 in covalent linkage with K2 in the propiece governs the ability of K2 to bind the (pro)catalytic domain in favor of zymogen, thereby enforcing zymogen-like character in the proteinase. Thrombin is produced from the C-terminal half of prothrombin following its proteolytic activation. The N-terminal half, released as the propiece Fragment 12 (F12), is composed of an N-terminal γ-carboxyglutamate domain (Gla) followed by two kringles (K1 and K2). The propiece plays essential roles in regulating prothrombin activation and proteinase function. The latter results from the ability of F12 to reversibly bind to the (pro)catalytic domain through K2 with high affinity and highly favorable thermodynamic constants when it is a zymogen in comparison to proteinase. Such discrimination is lost for K2 binding after proteolytic removal of the N-terminal Gla-K1 region of F12. The Ca2+-stabilized structure of the Gla domain is not required for F12 to bind the zymogen form more favorably. Enhanced binding to zymogen versus proteinase correlates with the ability of the propiece to enforce zymogen-like character in the proteinase. This is evident in variants of meizothrombin, an intermediate of prothrombin activation that contains the propiece covalently attached. This phenomenon is also independent of the Gla domain. Thus, the presence of K1 in covalent linkage with K2 in the propiece governs the ability of K2 to bind the (pro)catalytic domain in favor of zymogen, thereby enforcing zymogen-like character in the proteinase. Thrombin, a trypsin-like serine proteinase, plays a preeminent role in hemostasis and the prevention of blood loss (1Mann K.G. Jenny R.J. Krishnaswamy S. Cofactor proteins in the assembly and expression of blood clotting enzyme complexes.Annu. Rev. Biochem. 1988; 57: 915-956Crossref PubMed Scopus (449) Google Scholar, 2Mann K.G. Nesheim M.E. Church W.R. Haley P. Krishnaswamy S. Surface-dependent reactions of the vitamin K-dependent enzyme complexes.Blood. 1990; 76: 1-16Crossref PubMed Google Scholar). It activates platelets and cleaves soluble fibrinogen to form insoluble fibrin, both essential components of the blood clot (3Mann K.G. Thrombin formation.Chest. 2003; 124: 4S-10SAbstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 4Jenny N.S. Lundblad R.L. Mann K.G. Thrombin.in: Colman R.W. Marder V.J. Clowes A.J. George J.N. Goldhaber S.Z. In Hemostasis and Thrombosis. Basic Principles and Clinical Practice. Lippincott Williams & Wilkins, Philadelphia2006: 193-213Google Scholar). It acts as a feedback activator of steps in the coagulation cascade to enhance flux toward its own formation (3Mann K.G. Thrombin formation.Chest. 2003; 124: 4S-10SAbstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 4Jenny N.S. Lundblad R.L. Mann K.G. Thrombin.in: Colman R.W. Marder V.J. Clowes A.J. George J.N. Goldhaber S.Z. In Hemostasis and Thrombosis. Basic Principles and Clinical Practice. Lippincott Williams & Wilkins, Philadelphia2006: 193-213Google Scholar). When bound to the cofactor, thrombomodulin on the endothelium, it catalyzes the activation of protein C and initiates the anticoagulant reactions responsible for decreasing flux through the coagulation cascade (5Esmon C.T. The protein C pathway.Chest. 2003; 124: 26S-32SAbstract Full Text Full Text PDF PubMed Scopus (671) Google Scholar). Thus, thrombin functions as a central regulator of coagulation that derives from its ability to act on multiple biological substrates (6Lane D.A. Philippou H. Huntington J.A. Directing thrombin.Blood. 2005; 106: 2605-2612Crossref PubMed Scopus (270) Google Scholar). Two anion binding exosites (ABE1 2The abbreviations used are: ABE1anion binding exosite 1ABE2anion binding exosite 2DAPAdansylarginine-N-(3-ethyl-1,5-pentanediyl)-amidedG-F12F12 lacking γ-carboxyglutamate modificationsdG-IIprothrombin lacking γ-carboxyglutamate modificationsmIIameizothrombinIIQQQprothrombin variant containing Gln in place of Arg155, Arg271, and Arg284mIIaQQQmeizothrombin prepared from IIQQQdG-mIIaQQQmeizothrombin lacking γ-carboxyglutamate modifications prepared from IIQQQdG-mIIaA195meizothrombin lacking γ-carboxyglutamate modifications prepared from IIA195FPRckd-phenylalanyl-l-proline-l-arginine chloromethyl ketoneF12fragment 1.2IIA195prothrombin variant containing Ala in place of catalytic Ser195IIaA195thrombin prepared from IIA195I-2581Nα-dansyl-(p-guanidino)-phenylalanine-piperidideITCisothermal titration calorimetrymIIa-ΔF1meizothrombin variant lacking the fragment 1 regionmIIa-Δ43meizothrombin variant lacking residues 1–43P2prethrombin 2P2A195prethrombin 2 prepared from IIA195GlaN-terminal 4-carboxyglutamate domainBistris propane1,3-bis[tris(hydroxymethyl)methylamino]propane. and ABE2) play important roles in the binding of substrates and ligands to thrombin (7Bock P.E. Panizzi P. Verhamme I.M. Exosites in the substrate specificity of blood coagulation reactions.J. Thromb. Haemost. 2007; 5: 81-94Crossref PubMed Scopus (120) Google Scholar, 8Huntington J.A. Molecular recognition mechanisms of thrombin.J. Thromb. Haemost. 2005; 3: 1861-1872Crossref PubMed Scopus (208) Google Scholar). The role played by ABE1 in ligating a range of thrombin substrates and ligands has been established by numerous biochemical and structural studies (6Lane D.A. Philippou H. Huntington J.A. Directing thrombin.Blood. 2005; 106: 2605-2612Crossref PubMed Scopus (270) Google Scholar, 7Bock P.E. Panizzi P. Verhamme I.M. Exosites in the substrate specificity of blood coagulation reactions.J. Thromb. Haemost. 2007; 5: 81-94Crossref PubMed Scopus (120) Google Scholar). In contrast, far fewer ligands have been identified for ABE2, found approximately on the opposite face of the proteinase domain from ABE1 (6Lane D.A. Philippou H. Huntington J.A. Directing thrombin.Blood. 2005; 106: 2605-2612Crossref PubMed Scopus (270) Google Scholar). Although ABE2 was originally defined on the basis of heparin binding, the propiece of the zymogen precursor that is cleaved away when thrombin is produced represents an important protein ligand for this site (9Arni R.K. Padmanabhan K. Padmanabhan K.P. Wu T.P. Tulinsky A. Structures of the noncovalent complexes of human and bovine prothrombin fragment 2 with human PPACK-thrombin.Biochemistry. 1993; 32: 4727-4737Crossref PubMed Scopus (100) Google Scholar, 10Adams T.E. Huntington J.A. Structural transitions during prothrombin activation: on the importance of fragment 2.Biochimie. 2016; 122: 235-242Crossref PubMed Scopus (11) Google Scholar). anion binding exosite 1 anion binding exosite 2 dansylarginine-N-(3-ethyl-1,5-pentanediyl)-amide F12 lacking γ-carboxyglutamate modifications prothrombin lacking γ-carboxyglutamate modifications meizothrombin prothrombin variant containing Gln in place of Arg155, Arg271, and Arg284 meizothrombin prepared from IIQQQ meizothrombin lacking γ-carboxyglutamate modifications prepared from IIQQQ meizothrombin lacking γ-carboxyglutamate modifications prepared from IIA195 d-phenylalanyl-l-proline-l-arginine chloromethyl ketone fragment 1.2 prothrombin variant containing Ala in place of catalytic Ser195 thrombin prepared from IIA195 Nα-dansyl-(p-guanidino)-phenylalanine-piperidide isothermal titration calorimetry meizothrombin variant lacking the fragment 1 region meizothrombin variant lacking residues 1–43 prethrombin 2 prethrombin 2 prepared from IIA195 N-terminal 4-carboxyglutamate domain 1,3-bis[tris(hydroxymethyl)methylamino]propane. Prothrombin, the zymogen precursor, requires cleavage at two sites to be converted to thrombin (Scheme 1). Cleavage at Arg271 releases the N-terminal propiece (F12), whereas cleavage at Arg320 is analogous to the cleavage at Arg15 in chymotrypsinogen and yields the proteinase. Thrombin is composed of the catalytic domain in disulfide linkage with a short light chain (Scheme 1). When Arg320 is not cleaved, the C-terminal procatalytic domain corresponds to the zymogen, prethrombin 2 (P2). The F12 propiece is composed of an N-terminal domain (Gla), bearing γ-carboxyglutamic acid residues, which is stabilized by Ca2+ and allows for membrane binding. Two kringle domains (K1 and K2) follow separated by a linker (Scheme 1). The presence of a thrombin-sensitive cleavage site at position 155, within the linker, facilitates the separation of F12 into the N-terminal fragment 1 (F1, Gla-K1) and fragment 2 (F2) bearing K2. A series of x-ray structures establish that it is F2 that contacts the ABE2 site within the (pro)catalytic domain, and the F1 region extends away as schematically illustrated (Scheme 1)(10Adams T.E. Huntington J.A. Structural transitions during prothrombin activation: on the importance of fragment 2.Biochimie. 2016; 122: 235-242Crossref PubMed Scopus (11) Google Scholar, 11Pozzi N. Chen Z. Pelc L.A. Shropshire D.B. Di Cera E. The linker connecting the two kringles plays a key role in prothrombin activation.Proc. Natl. Acad. Sci. U.S.A. 2014; 111: 7630-7635Crossref PubMed Scopus (35) Google Scholar, 12Pozzi N. Chen Z. Di Cera E. How the Linker Connecting the two kringles influences activation and conformational plasticity of prothrombin.J. Biol. Chem. 2016; 291: 6071-6082Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Thrombin and P2 can reversibly bind F12 despite prior cleavage at Arg271 (Scheme 1) (9Arni R.K. Padmanabhan K. Padmanabhan K.P. Wu T.P. Tulinsky A. Structures of the noncovalent complexes of human and bovine prothrombin fragment 2 with human PPACK-thrombin.Biochemistry. 1993; 32: 4727-4737Crossref PubMed Scopus (100) Google Scholar, 13Kamath P. Krishnaswamy S. Fate of membrane-bound reactants and products during the activation of human prothrombin by prothrombinase.J. Biol. Chem. 2008; 283: 30164-30173Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). F12 binds the zymogen P2 with ∼30-fold higher affinity than it does to thrombin (13Kamath P. Krishnaswamy S. Fate of membrane-bound reactants and products during the activation of human prothrombin by prothrombinase.J. Biol. Chem. 2008; 283: 30164-30173Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Binding of F12 imparts membrane binding function and accounts for the membrane-dependent cleavage of the F12-P2 complex to thrombin by prothrombinase, the membrane-bound enzyme complex responsible for prothrombin activation (13Kamath P. Krishnaswamy S. Fate of membrane-bound reactants and products during the activation of human prothrombin by prothrombinase.J. Biol. Chem. 2008; 283: 30164-30173Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). The ∼30-fold weaker interaction of F12 with thrombin releases the bulk of the proteinase product from the membrane surface freeing it to act on its multiple substrates in a membrane-independent manner (13Kamath P. Krishnaswamy S. Fate of membrane-bound reactants and products during the activation of human prothrombin by prothrombinase.J. Biol. Chem. 2008; 283: 30164-30173Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). The differential affinity of F12 for zymogen and proteinase is dwarfed by profound and compensating differences in enthalpy and entropy for the binding of the propiece to P2 and thrombin (14Kamath P. Huntington J.A. Krishnaswamy S. Ligand binding shuttles thrombin along a continuum of zymogen- and proteinase-like states.J. Biol. Chem. 2010; 285: 28651-28658Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Thus, F12 binding to the zymogen is highly thermodynamically favored in comparison to the proteinase. This is evident from the finding that F12 binding to ABE2 enforces zymogen-like character in thrombin (14Kamath P. Huntington J.A. Krishnaswamy S. Ligand binding shuttles thrombin along a continuum of zymogen- and proteinase-like states.J. Biol. Chem. 2010; 285: 28651-28658Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Despite irreversible cleavage at Arg320, thrombin can reversibly interconvert between zymogen-like and proteinase-like states depending on the complement of ligands bound to ABE2, the active site, and the Na+ binding site in the proteinase domain (14Kamath P. Huntington J.A. Krishnaswamy S. Ligand binding shuttles thrombin along a continuum of zymogen- and proteinase-like states.J. Biol. Chem. 2010; 285: 28651-28658Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Although these ideas provide a unifying framework for the consideration of thrombin allostery, their physiologic relevance is questionable because of the modest affinity of F12 for the proteinase (13Kamath P. Krishnaswamy S. Fate of membrane-bound reactants and products during the activation of human prothrombin by prothrombinase.J. Biol. Chem. 2008; 283: 30164-30173Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 14Kamath P. Huntington J.A. Krishnaswamy S. Ligand binding shuttles thrombin along a continuum of zymogen- and proteinase-like states.J. Biol. Chem. 2010; 285: 28651-28658Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Binding affinity is not at issue in the case of meizothrombin (mIIa) that is cleaved only at Arg320 and retains covalent linkage with F12 (Scheme 1). Accordingly, despite cleavage at the equivalent of Arg15 in chymotrypsinogen, mIIa displays prominent zymogen-like character (15Bradford H.N. Krishnaswamy S. Meizothrombin is an unexpectedly zymogen-like variant of thrombin.J. Biol. Chem. 2012; 287: 30414-30425Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). It exists in approximately equally populated zymogen-like and proteinase-like forms that interconvert slowly (15Bradford H.N. Krishnaswamy S. Meizothrombin is an unexpectedly zymogen-like variant of thrombin.J. Biol. Chem. 2012; 287: 30414-30425Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). This has major implications for the sequential cleavage of prothrombin activation by prothrombinase in which mIIa is produced as a major intermediate and for the understanding of the basis of its skewed spectrum of biological functions in comparison to thrombin (15Bradford H.N. Krishnaswamy S. Meizothrombin is an unexpectedly zymogen-like variant of thrombin.J. Biol. Chem. 2012; 287: 30414-30425Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 16Krishnaswamy S. The transition of prothrombin to thrombin.J. Thromb. Haemost. 2013; 11: 265-276Crossref PubMed Scopus (95) Google Scholar). Surprisingly, despite the fact that it is K2 in the propiece that contacts ABE2, F2 binding to the zymogen and proteinase are energetically equivalent and weak (13Kamath P. Krishnaswamy S. Fate of membrane-bound reactants and products during the activation of human prothrombin by prothrombinase.J. Biol. Chem. 2008; 283: 30164-30173Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Thus, provided the thermodynamic arguments have merit, F2 will not be expected to elicit zymogen-like features in the proteinase. This discrepancy in the binding characteristics of F12 and F2 suggests that the N-terminal F1 domain somehow enforces the zymogen favoring ability of K2 and the following residues in their interaction with ABE2. The possibility that the F2 domain is fundamentally altered when F12 is cleaved at Arg155 is not obvious from available x-ray structures (10Adams T.E. Huntington J.A. Structural transitions during prothrombin activation: on the importance of fragment 2.Biochimie. 2016; 122: 235-242Crossref PubMed Scopus (11) Google Scholar). It is also inconsistent with evidence suggesting that F1 and F2 are independent folding domains within F12 or prothrombin (17Bloom J.W. Mann K.G. Prothrombin domains: circular dichroic evidence for a lack of cooperativity.Biochemistry. 1979; 18: 1957-1961Crossref PubMed Scopus (15) Google Scholar). On the other hand, several lines of evidence suggest that the Ca2+-dependent folding of the Gla domain has distant effects on the proteinase domain (18Armstrong S.A. Husten E.J. Esmon C.T. Johnson A.E. The active site of membrane-bound meizothrombin. A fluorescence determination of its distance from the phospholipid surface and its conformational sensitivity to calcium and factor Va.J. Biol. Chem. 1990; 265: 6210-6218Abstract Full Text PDF PubMed Google Scholar, 19Doyle M.F. Mann K.G. Multiple active forms of thrombin. IV. Relative activities of meizothrombins.J. Biol. Chem. 1990; 265: 10693-10701Abstract Full Text PDF PubMed Google Scholar, 20Côté H.C. Stevens W.K. Bajzar L. Banfield D.K. Nesheim M.E. MacGillivray R.T. Characterization of a stable form of human meizothrombin derived from recombinant prothrombin (R155A, R271A, and R284A).J. Biol. Chem. 1994; 269: 11374-11380Abstract Full Text PDF PubMed Google Scholar). It, therefore, seems plausible that the folded structure of the Gla domain regulates the binding of F12 through the F2 region to the proteinase domain. The thermodynamic and kinetic approaches presented here shed unexpected light on the function of the F1 domain as zymogenizer within the propiece. Human plasma used for protein isolation was a gift of the Plasmapheresis Unit of the Hospital of the University of Pennsylvania. d-Phenylalanyl-l-proline-l-arginine chloromethyl ketone (FPRck, Calbiochem), dansylarginine-N-(3-ethyl-1,5-pentanediyl)-amide (DAPA, Hematologic Technologies), and Nα-dansyl-(p-guanidino)-phenylalanine-piperidide (I-2581,DiaPharma), tosyl-l-lysine chloromethyl ketone, and tosyl-l-phenylalanine chloromethyl ketone (Sigma) were from the indicated suppliers. Concentrations of stock solutions prepared in water were determined using E330m = 4010 m−1·cm−1 (DAPA) and E342m = 4105 m−1·cm−1 (I-2581). Prothrombin was isolated from human plasma by established procedures (21Baugh R.J. Krishnaswamy S. Role of the activation peptide domain in human factor X activation by the extrinsic Xase complex.J. Biol. Chem. 1996; 271: 16126-16134Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 22Orcutt S.J. Pietropaolo C. Krishnaswamy S. Extended interactions with prothrombinase enforce affinity and specificity for its macromolecular substrate.J. Biol. Chem. 2002; 277: 46191-46196Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Bovine chymotrypsin (Worthington) was treated with tosyl-lysine chloromethyl ketone dialyzed into 20 mm Hepes, 0.15 m NaCl, pH 7.4, and stored frozen in aliquots. Recombinant ecarin was expressed and purified as described (23Bradford H.N. Orcutt S.J. Krishnaswamy S. Membrane binding by prothrombin mediates its constrained presentation to prothrombinase for cleavage.J. Biol. Chem. 2013; 288: 27789-27800Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Recombinant prothrombin with alanine substituting the catalytic serine (IIA195, chymotrypsin numbering) or bearing glutamine substitutions for arginine as positions 155, 271, and 284 (IIQQQ, sequential numbering of the mature protein) were expressed as either fully carboxylated or uncarboxylated (dG) forms and purified as before (13Kamath P. Krishnaswamy S. Fate of membrane-bound reactants and products during the activation of human prothrombin by prothrombinase.J. Biol. Chem. 2008; 283: 30164-30173Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 23Bradford H.N. Orcutt S.J. Krishnaswamy S. Membrane binding by prothrombin mediates its constrained presentation to prothrombinase for cleavage.J. Biol. Chem. 2013; 288: 27789-27800Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). The stated degree of carboxylation was confirmed by amino acid analysis of γ-carboxyglutamate content after base hydrolysis and ion exchange HPLC with post-column derivatization (24Price P.A. Analysis for γ-carboxyglutamic acid.Methods Enzymol. 1983; 91: 13-17Crossref PubMed Scopus (37) Google Scholar). Preparative proteolysis of plasma prothrombin was used to generate F12, F2, and prethrombin 1 followed by their purification (25Mann K.G. Elion J. Butkowski R.J. Downing M. Nesheim M.E. Prothrombin.Methods Enzymol. 1981; 80: 286-302Crossref PubMed Scopus (97) Google Scholar). Uncarboxylated F12 (dG-F12), P2A195, and IIaA195 were generated from dG-IIA195 and purified as described (14Kamath P. Huntington J.A. Krishnaswamy S. Ligand binding shuttles thrombin along a continuum of zymogen- and proteinase-like states.J. Biol. Chem. 2010; 285: 28651-28658Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 23Bradford H.N. Orcutt S.J. Krishnaswamy S. Membrane binding by prothrombin mediates its constrained presentation to prothrombinase for cleavage.J. Biol. Chem. 2013; 288: 27789-27800Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). IIQQQ lacking the first 43 residues (IIQQQ-Δ43) was generated by preparative cleavage of fully carboxylated IIQQQ with chymotrypsin (26Pollock J.S. Shepard A.J. Weber D.J. Olson D.L. Klapper D.G. Pedersen L.G. Hiskey R.G. Phospholipid binding properties of bovine prothrombin peptide residues 1–45.J. Biol. Chem. 1988; 263: 14216-14223Abstract Full Text PDF PubMed Google Scholar). The cleavage mixture (11 ml) contained 13.9 μm IIQQQ and 140 nm chymotrypsin in 20 mm Tris, 0.15 m NaCl, pH 7.4. After digestion for 1 h at 25 °C, chymotrypsin was inactivated by the addition of 20 μm tosyl-l-phenylalanine chloromethyl ketone, diluted with 50 ml of 20 mm Tris, pH 7.4, and applied at 5 ml/min to a 10 × 100-mm column of Poros HQM (Thermo Fisher). After washing with the same buffer, bound protein was eluted with a gradient of increasing NaCl (0–600 mm NaCl in 20 mm Tris, pH 7.4, 5 ml/min, 12 min). Fractions containing IIQQQ-Δ43 free of precursor and the cleaved peptide were pooled and precipitated with solid (NH4)2SO4 to 80% saturation. Precipitated protein was collected by centrifugation, dissolved in 50% (v/v) glycerol, and stored at −20 °C. The chemical identity of IIQQQ-Δ43 was established by N-terminal sequence analysis performed by Dr. Jan Pohl (Centers for Disease Control). While the bulk of the product was cleaved at Tyr43, a minor second sequence (∼10% yield) was observed consistent with some cleavage at Trp41. Protein concentrations were determined using the following molecular weights and extinction coefficients (E2800.1%): all full-length prothrombin variants, 72,000, 1.47 (25Mann K.G. Elion J. Butkowski R.J. Downing M. Nesheim M.E. Prothrombin.Methods Enzymol. 1981; 80: 286-302Crossref PubMed Scopus (97) Google Scholar); IIaA195 or P2A195, 37,500, 1.89 (27Lundblad R.L. Kingdon H.S. Mann K.G. Thrombin.Methods Enzymol. 1976; 45: 156-176Crossref PubMed Scopus (233) Google Scholar); F12 or dG-F12, 34,800, 1.2 (25Mann K.G. Elion J. Butkowski R.J. Downing M. Nesheim M.E. Prothrombin.Methods Enzymol. 1981; 80: 286-302Crossref PubMed Scopus (97) Google Scholar); F2, 1.28, 12,800 (25Mann K.G. Elion J. Butkowski R.J. Downing M. Nesheim M.E. Prothrombin.Methods Enzymol. 1981; 80: 286-302Crossref PubMed Scopus (97) Google Scholar); prethrombin 1, 1.78, 50,000 (25Mann K.G. Elion J. Butkowski R.J. Downing M. Nesheim M.E. Prothrombin.Methods Enzymol. 1981; 80: 286-302Crossref PubMed Scopus (97) Google Scholar); IIQQQ-Δ43, 1.69, 66,600; ecarin, 88,000, 1.0. Isothermal titration calorimetry (ITC) was performed using an iTC200 (Microcal) at 25 °C. All protein species were treated with 10 μm p-amidinophenylmethylsulfonyl fluoride (Sigma) before dialysis for 18 h versus 2 changes of 4 liters of buffer composed of 20 mm Hepes, 0.15 m NaCl containing either 5 mm Ca2+ or 5 mm EDTA, pH 7.4, at 4 °C. For two experiments, buffering in the second dialysis step was done with 20 mm Bistris propane instead of Hepes. After dialysis the samples were concentrated by centrifugal ultrafiltration if needed (Ultracel-10K, Amicon) and centrifuged to remove particulates before use. The cell (204 μl) contained either P2A195 or IIaA195 (14.6–30 μm), and the injection syringe (45 μl) contained F12 or F2 (224–431 μm). Heat flow was measured with continuous stirring, with injections of titrant at 180-s intervals. Typically, the first injection was 0.5 μl followed by 20 injections of 2 μl each. Heat flow due to ligand dilution and buffer mismatch was determined from an identical series of injections of titrant into dialysate. Samples were taken both before and after the experiment for analysis by SDS-PAGE to confirm that the heat flow measurements were not compromised by proteolytic degradation of the proteins. Stopped flow fluorescence measurements were performed at 25 °C using a SX20 stopped flow instrument with a 20-μl cell (Applied Photophysics). The binding of DAPA or I-2581 to mIIa variants was measured after rapid mixing of probe with proteinase using λEX = 280 nm and monitoring broadband fluorescence (λEX > 500 nm) using a long pass filter (LWP-500, Miles Girot) in the emission beam. As previously described, mIIa variants were prepared by cleaving the appropriate prothrombin variants with recombinant ecarin in situ and maintaining the cleaved product on ice for the ∼4-h experiment duration (15Bradford H.N. Krishnaswamy S. Meizothrombin is an unexpectedly zymogen-like variant of thrombin.J. Biol. Chem. 2012; 287: 30414-30425Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). This approach minimizes possible perturbations in the zymogen/proteinase equilibrium that could result from the use of ion exchange to repurify the cleaved product (15Bradford H.N. Krishnaswamy S. Meizothrombin is an unexpectedly zymogen-like variant of thrombin.J. Biol. Chem. 2012; 287: 30414-30425Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). IIA195 or IIQQQ were used to generate mIIaA195 or mIIaQQQ and dG-IIA195 or dG-IIQQQ to yield the corresponding uncarboxylated mIIa forms. Prethrombin 1 was cleaved by ecarin to yield mIIa-ΔF1, and IIQQQ-Δ43 was used to produce mIIaQQQ-Δ43. For cleavage, 5 μm concentrations of the zymogen variant in 20 mm Hepes, 0.15 m NaCl, 5 mm Ca2+, 0.1% (w/v) PEG-8000, 5 μm CoCl2, pH 7.4, was treated with 100 nm ecarin for 10 min at 25 °C and transferred to ice. For experiments done in EDTA, the cleavage mixture lacked Ca2+, and the reaction mixture was supplemented with 5 mm EDTA after cleavage was complete. SDS-PAGE analysis confirmed quantitative conversion to the appropriate mIIa species, and stability over the experimental period was established by gel analysis after the ∼4-h experiment duration. Stopped flow measurements were conducted by rapid mixing of equal volumes of enzyme solution (0.6 μm mIIa variant) with 10 different concentrations of either DAPA or I-2581 (1–16 μm) in the same buffer. Traces were acquired over 2 time scales, typically 2000 points in 0.2 s followed by 1000 points in 6.0 s, to permit description of the rapid and slow phases of the transient. Probe dissociation was measured by trapping free proteinase using FPRck (15Bradford H.N. Krishnaswamy S. Meizothrombin is an unexpectedly zymogen-like variant of thrombin.J. Biol. Chem. 2012; 287: 30414-30425Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). For these studies, 0.6 μm mIIa variant was premixed with 4 μm probe and reacted with equal volumes of 400 or 800 μm FPRck in the same buffer. Traces describing the decrease in fluorescence associated with probe dissociation were collected using 10,000 data points over ∼10 t½ of the transient. Despite the fact that mIIaA195 cannot covalently react with FPRck, the inhibitor evidently binds with sufficient affinity to trap free enzyme. Successful isolation of the intrinsic dissociation rate constant for the probe was inferred from the independence of the time constant for fluorescence decay on the concentration of FPRck. Heat traces from ITC measurements were initially analyzed by singular value decomposition using the program NITPICK to normalize for baseline variation and noise and yield integrated areas for each injection (28Keller S. Vargas C. Zhao H. Piszczek G. Brautigam C.A. Schuck P. High-precision isothermal titration calorimetry with automated peak shape analysis.Anal. Chem. 2012; 84: 5066-5073Crossref PubMed Scopus (354) Google Scholar). After subtraction of the small integrated heats obtained from the control experiment, the isotherms were analyzed using SEDPHAT (29Houtman J.C. Brown P.H. Bowden B. Yamaguchi H. Appella E. Samelson L.E. Schuck P. Studying multisite binary and ternary protein interactions by global analysis of isothermal titration calorimetry data in SEDPHAT: application to adaptor protein complexes in cell signaling.Protein Sci. 2007; 16: 30-42Crossref PubMed Scopus (244) Google Scholar). Fitted values of Kd, ΔH, and baseline offset as well as calculated values of ΔG and ΔS were obtained using the A + B hetero-associatio" @default.
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