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• W1996624351 abstract "A conformationally altered prelatent form of antithrombin that possesses both anticoagulant and antiangiogenic activities is produced during the conversion of native to latent antithrombin (Larsson, H., Akerud, P., Nordling, K., Raub-Segall, E., Claesson-Welsh, L., and Björk, I. (2001) J. Biol. Chem. 276, 11996–12002). Here, we show that the previously characterized prelatent antithrombin is a mixture of native antithrombin and a modified, true prelatent antithrombin that are resolvable by heparin-agarose chromatography. Kinetic analyses revealed that prelatent antithrombin is an intermediate in the conversion of native to latent antithrombin whose formation is favored by stabilizing anions of the Hofmeister series. Purified prelatent antithrombin had reduced anticoagulant function compared with native antithrombin, due to a reduced heparin affinity and consequent impaired ability of heparin to either bridge prelatent antithrombin and coagulation proteases in a ternary complex or to induce full conformational activation of the serpin. Significantly, prelatent antithrombin possessed an antiangiogenic activity more potent than that of latent antithrombin, based on the relative abilities of the two forms to inhibit endothelial cell growth. The prelatent form was conformationally altered from native antithrombin as judged from an attenuation of tryptophan fluorescence changes following heparin activation and a reduced thermal stability. The alterations are consistent with the limited structural changes involving strand 1C observed in a prelatent form of plasminogen activator inhibitor-1 (Dupont, D. M., Blouse, G. E., Hansen, M., Mathiasen, L., Kjelgaard, S., Jensen, J. K., Christensen, A., Gils, A., Declerck, P. J., Andreasen, P. A., and Wind, T. (2006) J. Biol. Chem. 281, 36071–36081), since the 1H NMR spectrum, electrophoretic mobility, and proteolytic susceptibility of prelatent antithrombin most resemble those of native rather than those of latent antithrombin. Together, these results demonstrate that limited conformational alterations of antithrombin that modestly reduce anticoagulant activity are sufficient to generate antiangiogenic activity. A conformationally altered prelatent form of antithrombin that possesses both anticoagulant and antiangiogenic activities is produced during the conversion of native to latent antithrombin (Larsson, H., Akerud, P., Nordling, K., Raub-Segall, E., Claesson-Welsh, L., and Björk, I. (2001) J. Biol. Chem. 276, 11996–12002). Here, we show that the previously characterized prelatent antithrombin is a mixture of native antithrombin and a modified, true prelatent antithrombin that are resolvable by heparin-agarose chromatography. Kinetic analyses revealed that prelatent antithrombin is an intermediate in the conversion of native to latent antithrombin whose formation is favored by stabilizing anions of the Hofmeister series. Purified prelatent antithrombin had reduced anticoagulant function compared with native antithrombin, due to a reduced heparin affinity and consequent impaired ability of heparin to either bridge prelatent antithrombin and coagulation proteases in a ternary complex or to induce full conformational activation of the serpin. Significantly, prelatent antithrombin possessed an antiangiogenic activity more potent than that of latent antithrombin, based on the relative abilities of the two forms to inhibit endothelial cell growth. The prelatent form was conformationally altered from native antithrombin as judged from an attenuation of tryptophan fluorescence changes following heparin activation and a reduced thermal stability. The alterations are consistent with the limited structural changes involving strand 1C observed in a prelatent form of plasminogen activator inhibitor-1 (Dupont, D. M., Blouse, G. E., Hansen, M., Mathiasen, L., Kjelgaard, S., Jensen, J. K., Christensen, A., Gils, A., Declerck, P. J., Andreasen, P. A., and Wind, T. (2006) J. Biol. Chem. 281, 36071–36081), since the 1H NMR spectrum, electrophoretic mobility, and proteolytic susceptibility of prelatent antithrombin most resemble those of native rather than those of latent antithrombin. Together, these results demonstrate that limited conformational alterations of antithrombin that modestly reduce anticoagulant activity are sufficient to generate antiangiogenic activity. Antithrombin and its glycosaminoglycan activators, heparin and heparan sulfate, are well established anticoagulant regulators of blood clotting proteases (1Olson S.T. Chuang Y.-J. Trends Cardiovasc. Med. 2002; 12: 331-338Crossref PubMed Scopus (91) Google Scholar, 2Olson S.T. Björk I. Bock S.C. Trends Cardiovasc. Med. 2002; 12: 198-205Crossref PubMed Scopus (62) Google Scholar, 3Church F.C. Pike R.N. Tollefsen D.M. Buckle A.M. Ciaccia A.V. Olson S.T. Silverman G.A. Lomas D.A. Molecular and Cellular Aspects of the Serpinopathies and Disorders of Serpin Activity. World Scientific Publishing, Singapore2007: 509-554Google Scholar). Antithrombin acts as an anticoagulant by irreversibly inhibiting clotting proteases through a conformational trapping mechanism that is unique to the serpin superfamily of proteins of which antithrombin is a member (4Huntington J.A. Read R.J. Carrell R.W. Nature. 2000; 407: 923-926Crossref PubMed Scopus (948) Google Scholar, 5Dementiev A. Dobó J. Gettins P.G.W. J. Biol. Chem. 2006; 281: 3452-3457Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Heparin and heparan sulfate are required to activate antithrombin to ensure that clotting proteases are inhibited at a physiologically significant rate. This activating effect is the basis for the widespread clinical use of heparin for anticoagulant therapy. The activation results from heparin binding to antithrombin through a specific pentasaccharide sequence and inducing a conformational change in the serpin (6Choay J. Petitou M. Lormeau J.C. Sinay P. Casu B. Gatti G. Biochem. Biophys. Res. Commun. 1983; 116: 492-499Crossref PubMed Scopus (596) Google Scholar, 7Olson S.T. Björk I. Sheffer R. Craig P.A. Shore J.D. Choay J. J. Biol. Chem. 1992; 267: 12528-12538Abstract Full Text PDF PubMed Google Scholar). Conformational activation greatly enhances the affinity of antithrombin for heparin and exposes exosites on the inhibitor that promote its interaction with the target proteases, factor Xa and factor IXa (8Johnson D.J. Li W. Adams T.E. Huntington J.A. EMBO J. 2006; 25: 2029-2037Crossref PubMed Scopus (151) Google Scholar, 9Izaguirre G. Zhang W. Swanson R. Bedsted T. Olson S.T. J. Biol. Chem. 2003; 278: 51433-51440Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 10Izaguirre G. Swanson R. Raja S.M. Rezaie A.R. Olson S.T. J. Biol. Chem. 2007; 282: 33609-33622Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 11Izaguirre G. Olson S.T. J. Biol. Chem. 2006; 281: 13424-13432Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Heparin additionally accelerates antithrombin-protease reactions by providing a bridging exosite for the protease to bind next to antithrombin and thereby promote its interaction with the serpin in a ternary complex with heparin (12Olson S.T. Björk I. J. Biol. Chem. 1991; 266: 6353-6364Abstract Full Text PDF PubMed Google Scholar, 13Dementiev A. Petitou M. Herbert J. Gettins P.G.W. Nat. Struct. Mol. Biol. 2004; 11: 863-867Crossref PubMed Scopus (126) Google Scholar, 14Li W. Johnson D.J.D. Esmon C.T. Huntington J.A. Nat. Struct. Mol. Biol. 2004; 11: 857-862Crossref PubMed Scopus (310) Google Scholar). The latter is the predominant mechanism involved in accelerating antithrombin inhibition of thrombin. Antithrombin has more recently been shown to express a potent antiangiogenic activity after having undergone conformational alterations induced either by limited proteolysis in a reactive protease binding loop or by mild heating (15O'Reilly M.S. Pirie-Shepherd S. Lane W.S. Folkman J. Science. 1999; 285: 1926-1928Crossref PubMed Scopus (424) Google Scholar, 16Larsson H. Sjoblom T. Dixelius J. Ostman A. Ylinenjärvi K. Björk I. Claesson-Welsh L. Cancer Res. 2000; 60: 6723-6729PubMed Google Scholar). Such conformational alterations transform the native metastable protein to a much more stable but inactive form in which the reactive loop has inserted into the major β-sheet, the A-sheet, of the serpin (17Wardell M.R. Chang W.S.W. Bruce D. Skinner R. Lesk A.M. Carrell R.W. Biochemistry. 1997; 36: 13133-13142Crossref PubMed Scopus (77) Google Scholar, 18Skinner R. Abrahams J.-P. Whisstock J.C. Lesk A.M. Carrell R.W. Wardell M.R. J. Mol. Biol. 1997; 266: 601-609Crossref PubMed Scopus (189) Google Scholar). These conformationally altered forms of antithrombin are produced under physiologic conditions (19Zhou A. Huntington J.A. Carrell R.W. Blood. 1999; 94: 3388-3396Crossref PubMed Google Scholar) and have antiangiogenic activity comparable with that of other naturally produced angiogenesis inhibitors (20Prox D. Becker C. Pirie-Shepherd S.R. Celik I. Folkman J. Kisker O. World J. Surg. 2003; 68: 47-54Google Scholar). The requirement for conformational change to generate antiangiogenic activity sets antithrombin apart from other serpins, such as pigment epithelium-derived factor, maspin, and kallistatin, which have been shown to also possess antiangiogenic activity but without the need for conformational change (21Dawson D.W. Volpert O.V. Gillis P. Crawford S.E. Xu H. Benedict W. Bouck N.P. Science. 1999; 285: 245-248Crossref PubMed Scopus (1401) Google Scholar, 22Zhang M. Volpert O. Shi Y.H. Bouck N. Nat. Med. 2000; 6: 196-199Crossref PubMed Scopus (410) Google Scholar, 23Miao R.Q. Agata J. Chao L. Chao J. Blood. 2002; 100: 3245-3252Crossref PubMed Scopus (163) Google Scholar). Interestingly, mild heat treatment was also found to produce a distinct form of antithrombin, termed the prelatent form, which possessed antiangiogenic activity (24Larsson H. Akerud P. Nordling K. Raub-Segall E. Claesson-Welsh L. Björk I. J. Biol. Chem. 2001; 276: 11996-12002Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). However, unlike the cleaved and latent forms of antithrombin that have lost their ability to inhibit clotting proteases, prelatent antithrombin was found to retain clotting protease inhibitory activity and to have its reaction with these proteases accelerated by heparin. The only reported difference between the native and prelatent forms of antithrombin was a greater susceptibility of the latter to proteolysis by nontarget proteases. Since these findings suggested that an antiangiogenic epitope may be generated by more limited conformational changes than those having occurred in the cleaved and latent forms of antithrombin, it has been of interest to characterize the nature of these conformational differences between then native and prelatent forms of the serpin. In the present report, we show that prelatent antithrombin generated as in past studies is actually a mixture of a novel antiangiogenically active species of antithrombin, the true prelatent form, and the antiangiogenically inactive native serpin. The purified prelatent antithrombin has a more potent antiangiogenic activity than latent or cleaved antithrombins but retains the anticoagulant functions of the native serpin. It is shown to be generated as an intermediate on the pathway to latent antithrombin in the presence of stabilizing anions of the Hofmeister series (25von Hippel P.H. Schleich T. Acc. Chem. Res. 1969; 2: 257-265Crossref Scopus (488) Google Scholar, 26Busby T.F. Atha D.H. Ingham K.C. J. Biol. Chem. 1981; 256: 12140-12147Abstract Full Text PDF PubMed Google Scholar). Significantly, only limited conformational alterations are involved in transforming native to prelatent antithrombin, as judged from the modest changes in heparin affinity, heparin-induced conformational activation, thermal stability, electrophoretic mobility, proteolytic susceptibility, and 1H NMR spectrum. Overall, our findings suggest that limited conformational changes, comparable with those recently demonstrated in a prelatent form of the serpin, plasminogen activator inhibitor-1 (27Dupont D.M. Blouse G.E. Hansen M. Mathiasen L. Kjelgaard S. Jensen J.K. Christensen A. Gils A. Declerck P.J. Andreasen P.A. Wind T. J. Biol. Chem. 2006; 281: 36071-36081Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), are required to express antiangiogenic activity in antithrombin. Proteins—Human α-antithrombin was purified from blood plasma, as previously described (28Olson S.T. Björk I. Shore J.D. Methods Enzymol. 1993; 222: 525-560Crossref PubMed Scopus (267) Google Scholar, 29Turk B. Brieditis I. Bock S.C. Olson S.T. Björk I. Biochemistry. 1997; 36: 6682-6691Crossref PubMed Scopus (104) Google Scholar). The concentration of the protein was determined from the 280 nm absorbance based on a molar absorption of 37,700 m−1 cm−1 (30Nordenman B. Nyström C. Björk I. Eur. J. Biochem. 1977; 78: 195-203Crossref PubMed Scopus (193) Google Scholar). Reactive loop-cleaved antithrombin was prepared by incubating native antithrombin with human neutrophil elastase (Athens Research Technology) as in previous studies (31Zhang W. Swanson R. Izaguirre G. Xiong Y. Lau L.F. Olson S.T. Blood. 2005; 106: 1621-1628Crossref PubMed Scopus (36) Google Scholar, 32Jordan R.E. Kilpatrick J. Nelson R.M. Science. 1987; 237: 777-780Crossref PubMed Scopus (88) Google Scholar). Human thrombin was prepared from plasma-purified prothrombin (33Miletich J.P. Broze Jr., G.J. Majerus P.W. Methods Enzymol. 1981; 80: 221-228Crossref Scopus (61) Google Scholar) by activating the zymogen and purifying the active protease as described (34Owen W.G. Jackson C.M. Thromb. Res. 1973; 3: 705-714Abstract Full Text PDF Scopus (79) Google Scholar). Human factor Xa was purchased from Enzyme Research (South Bend, IN). Concentrations of proteases were assessed from their activities in standard assays with peptidyl-p-nitroanilide chromogenic substrates and were based on calibration of these assays with active site-titrated enzymes (10Izaguirre G. Swanson R. Raja S.M. Rezaie A.R. Olson S.T. J. Biol. Chem. 2007; 282: 33609-33622Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Heparin—Full-length heparin chains of ∼26 saccharides or ∼50 saccharides with reduced polydispersity and with high affinity or low affinity for antithrombin were isolated from commercial heparin, as described (35Streusand V.J. Björk I. Gettins P.G.W. Petitou M. Olson S.T. J. Biol. Chem. 1995; 270: 9043-9051Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). The synthetic pentasaccharide corresponding to the antithrombin binding sequence in high affinity heparin chains was generously provided by Maurice Petitou (Sanofi-Aventis, Toulouse, France). Concentrations of high affinity heparins were determined from stoichiometric titrations of antithrombin with the heparins as described previously (28Olson S.T. Björk I. Shore J.D. Methods Enzymol. 1993; 222: 525-560Crossref PubMed Scopus (267) Google Scholar). Low affinity heparin concentrations were measured by Azure A dye binding assays (35Streusand V.J. Björk I. Gettins P.G.W. Petitou M. Olson S.T. J. Biol. Chem. 1995; 270: 9043-9051Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Preparation of Prelatent and Latent Forms of Antithrombin— Two slightly different procedures were used for isolating prelatent antithrombin. In the first, antithrombin (∼10 mg at 3 mg/ml) in 10 mm Tris-HCl, 0.5 m sodium citrate, pH 7.4, was incubated at 60 °C for 24 h as in previous studies (24Larsson H. Akerud P. Nordling K. Raub-Segall E. Claesson-Welsh L. Björk I. J. Biol. Chem. 2001; 276: 11996-12002Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The protein was then dialyzed against 20 mm sodium phosphate, 0.02 m NaCl, 0.1 mm EDTA, pH 7.4, buffer, and the dialyzed protein was applied to a 5-ml Hi-Trap heparin column at 0.5 ml/min. The column was subsequently washed with buffer with no NaCl at 0.5–2 ml/min until the protein fluorescence, detected continuously with a Shimadzu fluorescence monitor (280-nm excitation and 340-nm emission wavelengths), reached base-line level. A linear salt gradient from 0 to 2 m NaCl in sodium phosphate buffer was then applied over 45 min at 1 ml/min to elute the antithrombin. The low salt-eluting peak (∼0.2 m) comprising latent antithrombin was pooled, concentrated, and dialyzed. The high salt-eluting peak, previously designated as prelatent antithrombin, was subdivided into leading and trailing edge pools (labeled A + B and C, respectively) at the center of the peak (see Fig. 1). The leading edge pool was concentrated by ultrafiltration, dialyzed into sodium phosphate buffer, pH 7.4, containing 20 mm NaCl, and rechromatographed as above except that the protein was eluted with a 0–2 m convex NaCl gradient over 50 min at 1 ml/min. The two partly resolved peaks that eluted were separately pooled by subdividing at the trough between the two peaks and designated pools A and B in order of their elution. In the second procedure, later adopted for optimizing the chromatographic separation and yield of prelatent antithrombin, the preparation was done as above except that antithrombin was incubated at 60 °C for 30 h instead of 24 h prior to dialysis and Hi-Trap heparin chromatography. Moreover, after washing of the column, the NaCl concentration was increased to 0.1 m, and the flow rate was increased to 2.5 ml/min to elute latent antithrombin and allow the fluorescence to return to base line. The prelatent antithrombin was then eluted using the following linear gradient program at 2.5 ml/min: 0.1 m NaCl wash for 0–5 min, 0.1–0.88 m NaCl gradient from 5 to 30 min, hold at 0.88 m NaCl for 10 min, and 0.88–3 m NaCl gradient from 40 to 65 min. The shoulder on the high salt-eluting peak was pooled, concentrated, and dialyzed into low salt buffer and then rechromatographed using the same program. In some cases, two separate samples were successively processed as above, and the shoulders from both runs were combined prior to the second chromatography step. The peak eluting prior to the major protein peak was pooled to obtain prelatent antithrombin. Kinetics of Prelatent Antithrombin Formation—The kinetics of formation of prelatent and latent antithrombins from native antithrombin were assessed by heating the native serpin in the Tris/citrate buffer at 60 °C as above for varying times and then chromatographing samples on the Hi-Trap heparin column using the convex salt gradient. Corrections for background fluorescence were made by subtracting a buffer blank run from all chromatograms. The fluorescence peaks corresponding to native, prelatent, and latent antithrombins were then integrated using Millenium software (Waters Corp.) to quantitate their relative amounts. Minor fluorescence peaks corresponding to protein that was not bound or weakly bound to the column were integrated and summed to account for a nonbinding fraction. To determine whether prelatent antithrombin was an intermediate in the formation of latent antithrombin, purified prelatent antithrombin (0.16 mg/ml) was dialyzed into the Tris/citrate buffer and incubated under the same conditions used to form the prelatent species. Samples were taken at varying times, dialyzed, and chromatographed on the Hi-Trap heparin column as in the studies with native antithrombin. The kinetic stability of purified latent antithrombin was analyzed similarly. Reaction progress curves were fit by the minimal reaction model of Fig. 4 by numerical integration of the differential equations for the model using Scientist software (Micromath, Inc.). Alternative conditions that were tested for producing prelatent antithrombin involved (i) incubating 3 mg/ml antithrombin samples in 10 mm MES, 2The abbreviations used are: MES, 4-morpholineethanesulfonic acid; TAPS, 3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}-1-propanesulfonic acid; HUVECs, human umbilical vein endothelial cells; FGF-2, human fibroblast growth factor-2. pH 6, 10 mm Tris, pH 7.4, or 10 mm TAPS, pH 9, buffers, each containing 0.5 or 1 m sodium citrate, at60 °C for 30 h at pH 6 and 7.4 or 7 h at pH 9; (ii) incubating 0.5 mg/ml antithrombin samples in 50 mm Tris, 50 mm NaCl, pH 7.5, buffer containing 20% glycerol at 60 °C for 24–48 h (19Zhou A. Huntington J.A. Carrell R.W. Blood. 1999; 94: 3388-3396Crossref PubMed Google Scholar); (iii) incubating 0.2 mg/ml antithrombin samples at 25 °C for 24 h in Tris/sodium citrate buffer containing 0, 0.5, 1, 3, or 6 m guanidine HCl followed by dialysis into ionic strength (I) 0.15 sodium phosphate buffer, pH 7.4 (36Fish W.W. Danielsson Å. Nordling K. Miller S.H. Lam C.F. Björk I. Biochemistry. 1985; 24: 1510-1517Crossref PubMed Scopus (29) Google Scholar); (iv) incubating 1–3 mg/ml antithrombin samples in 10 mm Tris, pH 7.4, containing 0.1, 0.25, 0.5, or 1 m citrate, 0.5 m sodium phosphate, 0.5 m EDTA, 3 m NaCl, or 100 μm heparin pentasaccharide at 60 °C for 30 h (26Busby T.F. Atha D.H. Ingham K.C. J. Biol. Chem. 1981; 256: 12140-12147Abstract Full Text PDF PubMed Google Scholar); and (v) incubating 2 μm antithrombin samples in a physiologic buffer consisting of 9.47 mm sodium phosphate, 137 mm NaCl, 2.5 mm KCl, 1 mm CaCl2, pH 7.4 (37Lewis S.D. Shields P.P. Shafer J.A. J. Biol. Chem. 1985; 260: 10192-10199Abstract Full Text PDF PubMed Google Scholar), in the absence or presence of 1 μm 50-saccharide high affinity heparin or 15 μg/ml heparan sulfate (bovine kidney; Sigma) at 60 °C for a time yielding ∼50% inactivation of antithrombin. Samples were diluted 50–100-fold into sodium phosphate buffer with no salt before chromatography of 20–50 μg as above except for samples containing heparin. The latter were first chromatographed on the monoQ column to remove heparin (29Turk B. Brieditis I. Bock S.C. Olson S.T. Björk I. Biochemistry. 1997; 36: 6682-6691Crossref PubMed Scopus (104) Google Scholar) and then, after concentration and dilution, chromatographed on the Hi-Trap heparin column. Experimental Conditions—Experiments were conducted at 25 °C in I 0.15, pH 7.4 buffers consisting of either (i) 20 mm sodium phosphate, 0.1 m NaCl, 0.1 mm EDTA, 0.1% polyethylene glycol 8000 or (ii) 0.1 m Hepes, 0.1 m NaCl, 5 mm CaCl2, 0.1% polyethylene glycol 8000, except where otherwise noted. Some experiments were performed in the sodium phosphate buffer but with no added NaCl or with the addition of 0.25 m NaCl to yield ionic strengths of 0.05 or 0.3, respectively. PAGE—Native PAGE and SDS-PAGE analysis of proteins was done as in past studies using the Laemmli discontinuous buffer system (38Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207218) Google Scholar). The susceptibility of different antithrombin forms to proteolysis by nontarget proteases was analyzed essentially according to previous work (24Larsson H. Akerud P. Nordling K. Raub-Segall E. Claesson-Welsh L. Björk I. J. Biol. Chem. 2001; 276: 11996-12002Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) by incubation of 200 μg/ml serpin with 20 μg/ml chymotrypsin or thermolysin in 10 mm Tris, 5 mm CaCl2, pH 7.4, buffer for varying times, followed by quenching with 1.5 mm phenylmethylsulfonyl fluoride for chymotrypsin reactions or 30 mm EDTA for thermolysin reactions. Samples were denatured by the addition of SDS treatment buffer and boiling and were then analyzed for proteolysis by SDS-PAGE. Affinity and Kinetics of Antithrombin-Heparin Interactions— Binding of pentasaccharide or ∼26-saccharide high affinity heparins to antithrombin was analyzed by fluorescence titrations in which the enhancement in protein tryptophan fluorescence accompanying polysaccharide binding was used to monitor the interaction (28Olson S.T. Björk I. Shore J.D. Methods Enzymol. 1993; 222: 525-560Crossref PubMed Scopus (267) Google Scholar). Titrations were done with 50–200 nm antithrombin in I 0.15 or I 0.3 sodium phosphate buffers for measurements of KD or in I 0.05 sodium phosphate buffer for measurements of binding stoichiometry. Measurements were made on a SLM 8000 spectrofluorometer, at an excitation wavelength of 280 nm and an emission wavelength of 340 nm. Titrations were analyzed by computer fitting to the quadratic binding equation with KD, the maximal fluorescence change, and the stoichiometry as the fitted parameters (28Olson S.T. Björk I. Shore J.D. Methods Enzymol. 1993; 222: 525-560Crossref PubMed Scopus (267) Google Scholar). For measurements of KD, the stoichiometry was fixed at the average fitted value obtained in titrations at low ionic strength. The kinetics of heparin pentasaccharide binding to antithrombin were analyzed under pseudo-first order conditions with an Applied Photophysics SX-17MV stopped-flow fluorometer as in past studies (7Olson S.T. Björk I. Sheffer R. Craig P.A. Shore J.D. Choay J. J. Biol. Chem. 1992; 267: 12528-12538Abstract Full Text PDF PubMed Google Scholar, 29Turk B. Brieditis I. Bock S.C. Olson S.T. Björk I. Biochemistry. 1997; 36: 6682-6691Crossref PubMed Scopus (104) Google Scholar). Variable concentrations of pentasaccharide were mixed with antithrombin at a concentration at least 5-fold lower than that of the saccharide in I 0.3 buffer. Pentasaccharide binding to the protein was monitored from increases in protein fluorescence, and the observed pseudo-first order rate constant (kobs) was determined by computer fitting of the progress curves to a single exponential function. Stoichiometries of Protease Inhibition—Fixed concentrations of protease (50 nm) were incubated with variable concentrations of antithrombin ranging from substoichiometric to approximately equimolar in I 0.15, pH 7.4, sodium phosphate buffer. After incubation for a time sufficient to yield >90% inactivation based on measured inhibition rate constants, residual enzyme activities were determined as in the kinetic assays described below. Plots of residual enzyme activity versus the molar ratio of inhibitor to enzyme were fit by linear regression to obtain the inhibition stoichiometry from the abscissa intercept. Kinetics of Protease Inhibition—The kinetics of high affinity heparin and low affinity heparin acceleration of antithrombin-protease reactions were measured under pseudo-first order conditions as in past studies (35Streusand V.J. Björk I. Gettins P.G.W. Petitou M. Olson S.T. J. Biol. Chem. 1995; 270: 9043-9051Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Antithrombin (20 or 50 nm) was reacted at 25 °C with protease at one-tenth (factor Xa reactions) or one-twentieth (thrombin reactions) the concentration of inhibitor in the presence of variable concentrations of heparin. The buffers employed were either I 0.15 sodium phosphate (thrombin reactions) or I 0.15 Hepes/CaCl2 (factor Xa reactions), pH 7.4. Reactions with thrombin and high affinity heparin additionally contained 2 mm p-aminobenzamidine to decrease the rate of the reaction so as to allow accurate kinetic measurements by the discontinuous sampling method. Reaction mixtures (50–100 μl) were incubated for fixed times (2-min reactions with thrombin and high affinity heparin, 5-min reactions with thrombin and low affinity heparin, 30-s reactions with factor Xa and high affinity heparin and 5-min reactions with factor Xa and low affinity heparin) or for variable times to obtain full progress curves (factor Xa reactions with low affinity heparin) in polyethylene glycol-coated polystyrene cuvettes. The reactions were then quenched with substrate (900–950 μl), either 50 μm tosyl-Gly-Pro-Arg-7-amido-4-methylcoumarin (Sigma) for thrombin reactions or 100 μm Spectrozyme FXa (American Diagnostica) for factor Xa reactions. The substrate was in a high salt buffer consisting of 20 mm sodium phosphate, 0.1 mm EDTA or 100 mm Hepes, both at pH 7.4, and with 1 m NaCl, 0.1% polyethylene glycol 8000, 0.1 mg/ml Polybrene, to ensure quenching of the heparin-catalyzed reaction. The residual enzyme activity was determined by monitoring for several minutes the initial linear rate of substrate hydrolysis either from the increase in 7-amido-4-methylcoumarin fluorescence at λex 380 nm, λem 440 nm (for thrombin reactions) or the increase in p-nitroaniline absorbance at 405 nm (for factor Xa reactions). Observed pseudo-first order rate constants (kobs) were obtained from the fractional decrease of the enzyme activity from the starting activity measured in the absence of inhibitor by assuming an exponential loss of activity with zero end point (28Olson S.T. Björk I. Shore J.D. Methods Enzymol. 1993; 222: 525-560Crossref PubMed Scopus (267) Google Scholar). Apparent second order rate constants (kapp) were then calculated from kobs by dividing by the functional inhibitor concentration. The latter was determined by multiplying the inhibitor concentration measured from the 280 nm absorbance by the reciprocal of the inhibition stoichiometry (25von Hippel P.H. Schleich T. Acc. Chem. Res. 1969; 2: 257-265Crossref Scopus (488) Google Scholar). The functional inhibitor concentration thus obtained agreed with the 0.8–0.9-mol fraction of antithrombin found to be active in binding heparin from stoichiometric heparin binding titrations (Table 2). The dependence of kapp on heparin concentration was fit by the ternary complex bridging model for all reactions (35Streusand V.J. Björk I. Gettins P.G.W. Petitou M. Olson S.T. J. Biol. Chem. 1995; 270: 9043-9051Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) except for the low affinity heparin-catalyzed reaction of antithrombin with factor Xa. The latter was fit by a rectangular hyperbolic function, based on a model in which heparin catalysis results solely from conformational activation of antithrombin with no contribution of bridging (35Streusand V.J. Björk I. Gettins P.G.W. Petitou M. Olson S.T. J. Bi" @default.
• W1996624351 created "2016-06-24" @default.
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• W1996624351 date "2008-05-01" @default.
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• W1996624351 title "Characterization of the Conformational Alterations, Reduced Anticoagulant Activity, and Enhanced Antiangiogenic Activity of Prelatent Antithrombin" @default.
• W1996624351 cites W1486977846 @default.
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