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The Preferred Pathway of Glycosaminoglycan-accelerated Inactivation of Thrombin by Heparin Cofactor II

2004; Elsevier BV; Volume: 279; Issue: 11 Linguagem: Inglês

10.1074/jbc.m313962200

ISSN

1083-351X

Autores

Ingrid M. Verhamme, Paul Bock, Craig M. Jackson,

Tópico(s)

Proteoglycans and glycosaminoglycans research

Resumo

Thrombin (T) inactivation by the serpin, heparin cofactor II (HCII), is accelerated by the glycosaminoglycans (GAGs) dermatan sulfate (DS) and heparin (H). Equilibrium binding and thrombin inactivation kinetics at pH 7.8 and ionic strength (I) 0.125 m demonstrated that DS and heparin bound much tighter to thrombin (KT(DS) 1–5.8 μm; KT(H) 0.02–0.2 μm) than to HCII (KHCII(DS) 236–291 μm; KHCII(H) 25–35 μm), favoring formation of T·GAG over HCII·GAG complexes as intermediates for T·GAG·HCII complex assembly. At [GAG] ≪ KHCII(GAG) the GAG and HCII concentration dependences of the first-order inactivation rate constants (kapp) were hyperbolic, reflecting saturation of T·GAG complex and formation of the T·GAG·HCII complex from T·GAG and free HCII, respectively. At [GAG] ≫ KHCII(GAG), HCII·GAG complex formation caused a decrease in kapp. The bell-shaped logarithmic GAG dependences fit an obligatory template mechanism in which free HCII binds GAG in the T·GAG complex. DS and heparin bound fluorescently labeled meizothrombin(des-fragment 1) (MzT(-F1)) with KMzT(-F1)(GAG) 10 and 20 μm, respectively, demonstrating a binding site outside of exosite II. Exosite II ligands did not attenuate the DS-accelerated thrombin inactivation markedly, but DS displaced thrombin from heparin-Sepharose, suggesting that DS and heparin share a restricted binding site in or nearby exosite II, in addition to binding outside exosite II. Both T·DS and MzT(-F1)·DS interactions were saturable at DS concentrations substantially below KHCII(DS), consistent with DS bridging T·DS and free HCII. The results suggest that GAG template action facilitates ternary complex formation and accommodates HCII binding to GAG and thrombin exosite I in the ternary complex. Thrombin (T) inactivation by the serpin, heparin cofactor II (HCII), is accelerated by the glycosaminoglycans (GAGs) dermatan sulfate (DS) and heparin (H). Equilibrium binding and thrombin inactivation kinetics at pH 7.8 and ionic strength (I) 0.125 m demonstrated that DS and heparin bound much tighter to thrombin (KT(DS) 1–5.8 μm; KT(H) 0.02–0.2 μm) than to HCII (KHCII(DS) 236–291 μm; KHCII(H) 25–35 μm), favoring formation of T·GAG over HCII·GAG complexes as intermediates for T·GAG·HCII complex assembly. At [GAG] ≪ KHCII(GAG) the GAG and HCII concentration dependences of the first-order inactivation rate constants (kapp) were hyperbolic, reflecting saturation of T·GAG complex and formation of the T·GAG·HCII complex from T·GAG and free HCII, respectively. At [GAG] ≫ KHCII(GAG), HCII·GAG complex formation caused a decrease in kapp. The bell-shaped logarithmic GAG dependences fit an obligatory template mechanism in which free HCII binds GAG in the T·GAG complex. DS and heparin bound fluorescently labeled meizothrombin(des-fragment 1) (MzT(-F1)) with KMzT(-F1)(GAG) 10 and 20 μm, respectively, demonstrating a binding site outside of exosite II. Exosite II ligands did not attenuate the DS-accelerated thrombin inactivation markedly, but DS displaced thrombin from heparin-Sepharose, suggesting that DS and heparin share a restricted binding site in or nearby exosite II, in addition to binding outside exosite II. Both T·DS and MzT(-F1)·DS interactions were saturable at DS concentrations substantially below KHCII(DS), consistent with DS bridging T·DS and free HCII. The results suggest that GAG template action facilitates ternary complex formation and accommodates HCII binding to GAG and thrombin exosite I in the ternary complex. The blood clotting proteinase, α-thrombin (T), 1The abbreviations used are: T, human α-thrombin; HCII, heparin cofactor II; AT, antithrombin; MzT, meizothrombin; MzT(-F1), meizothrombin(des-fragment 1); GAG, glycosaminoglycan; DS, dermatan sulfate; H, heparin; FPR-CH2Cl, d-Phe-Pro-Arg-CH2Cl; FFR-CH2Cl, d-Phe-Phe-Arg-CH2Cl; [6F]FPR-T, [6-(acetamido)fluorescein]-d-Phe-Pro-Arg-thrombin; [6F]FFR-T, [6-(acetamido)fluorescein]-d-Phe-Phe-Arg-thrombin; [6F]FPR-MzT(-F1), [6-(acetamido)fluorescein]-d-Phe-Pro-Arg-meizothrombin(desF1); HD22, 5′-AGTCCGTGGTAGGGCAGGTTGGGGTGACT-3′; F2, prothrombin fragment 2; mAb, antiexosite II monoclonal antibody; PEG, polyethylene glycol 8000; Hir54-65(SO3-) Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr(SO3-)-Leu-Gln; [5F]Hir54-65(SO3-), Hir54-65(SO3-) labeled at the N terminus with 5-carboxy(fluorescein); QAC, quantitative affinity chromatography; Taps, 3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}-1-propanesulfonic acid. possesses two electropositive sites, exosites I and II (1Stubbs M.T. Bode W. Thromb. Res. 1993; 69: 1-58Abstract Full Text PDF PubMed Scopus (446) Google Scholar, 2Bode W. Turk D. Karshikov A. Protein Sci. 1992; 1: 426-471Crossref PubMed Scopus (648) Google Scholar), that play distinct roles in its covalent inactivation by the serine proteinase inhibitors (serpins), antithrombin (AT), and heparin cofactor II (HCII), in the absence and presence of rate-accelerating glycosaminoglycans (GAGs) (3Bock P.E. Olson S.T. Björk I. J. Biol. Chem. 1997; 272: 19837-19845Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 4Olson S.T. J. Biol. Chem. 1988; 263: 1698-1708Abstract Full Text PDF PubMed Google Scholar, 5Tollefsen D.M. Pestka C.A. Monafo W.J. J. Biol. Chem. 1983; 258: 6713-6716Abstract Full Text PDF PubMed Google Scholar, 6Van Deerlin V.M.D. Tollefsen D.M. J. Biol. Chem. 1991; 266: 20223-20231Abstract Full Text PDF PubMed Google Scholar, 7Rogers S.J. Pratt C.W. Whinna H.C. Church F.C. J. Biol. Chem. 1992; 267: 3613-3617Abstract Full Text PDF PubMed Google Scholar, 8Becker D.L. Fredenburgh J.C. Stafford A.R. Weitz J.I. J. Biol. Chem. 1999; 274: 6226-6233Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Exosite I, containing the basic residues Lys36, Lys70, Lys81, Lys109, Lys110, Lys149E and Arg35, Arg67, Arg73, Arg75, Arg77A (chymotrypsin numbering), is inactivated during formation of the stable T·AT complex (3Bock P.E. Olson S.T. Björk I. J. Biol. Chem. 1997; 272: 19837-19845Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), while exosite II, containing Lys169, Lys235, Lys236, Lys240, and Arg93, Arg126, Arg165, Arg173, Arg175, Arg233, may still be available for ligand binding (9Shi F. Hogg P.J. Winzor D.J. Jackson C.M. Biophys. Chem. 1998; 75: 187-199Crossref PubMed Scopus (9) Google Scholar, 10Fredenburgh J.C. Stafford A.R. Weitz J.I. J. Biol. Chem. 2001; 276: 44828-44834Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Inactivation by AT is accelerated up to 50,000-fold by high molecular weight heparin, which acts as a template, by binding to thrombin exosite II and AT helices A and D (4Olson S.T. J. Biol. Chem. 1988; 263: 1698-1708Abstract Full Text PDF PubMed Google Scholar, 11van Boeckel C.A.A. Grootenhuis P.D.J. Visser A.A. Nature Struct. Biol. 1994; 1: 423-425Crossref PubMed Google Scholar). Whereas heparin accelerates thrombin inactivation by both AT and HCII, dermatan sulfate (DS), and other polyanions (5Tollefsen D.M. Pestka C.A. Monafo W.J. J. Biol. Chem. 1983; 258: 6713-6716Abstract Full Text PDF PubMed Google Scholar, 12Church F.C. Meade J.B. Treanor R.E. Whinna H.C. J. Biol. Chem. 1989; 264: 3618-3623Abstract Full Text PDF PubMed Google Scholar, 13Verhamme I.M.A. Hogg P.J. Jackson C.M. Arch. Intl. Physiol. Biochim. 1989; 97: B117PubMed Google Scholar, 14Church F.C. Treanor R.E. Sherrill G.B. Whinna H.C. Biochem. Biophys. Res. Commun. 1987; 148: 362-368Crossref PubMed Scopus (20) Google Scholar, 15Church F.C. Pratt C.W. Treanor R.E. Whinna H.C. FEBS Lett. 1988; 237: 26-30Crossref PubMed Scopus (23) Google Scholar, 16Hayakawa Y. Hayashi T. Hayashi T. Niiya K. Sakuragawa N. Blood Coag. Fibrinol. 1995; 6: 643-679Crossref PubMed Scopus (10) Google Scholar, 17Hayakawa Y. Hayashi T. Lee J.-B. Ozawa T. Sakuragawa N. J. Biol. Chem. 2000; 275: 11379-11382Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) selectively enhance HCII inhibitory action. The HCII mechanism was initially thought to utilize GAGs as templates between exosite II and HCII helix D (5Tollefsen D.M. Pestka C.A. Monafo W.J. J. Biol. Chem. 1983; 258: 6713-6716Abstract Full Text PDF PubMed Google Scholar, 12Church F.C. Meade J.B. Treanor R.E. Whinna H.C. J. Biol. Chem. 1989; 264: 3618-3623Abstract Full Text PDF PubMed Google Scholar, 13Verhamme I.M.A. Hogg P.J. Jackson C.M. Arch. Intl. Physiol. Biochim. 1989; 97: B117PubMed Google Scholar), but later hypothesized to involve allosteric, GAG-induced interaction of an acidic N-terminal domain of HCII with exosite I (6Van Deerlin V.M.D. Tollefsen D.M. J. Biol. Chem. 1991; 266: 20223-20231Abstract Full Text PDF PubMed Google Scholar, 7Rogers S.J. Pratt C.W. Whinna H.C. Church F.C. J. Biol. Chem. 1992; 267: 3613-3617Abstract Full Text PDF PubMed Google Scholar, 18Hortin G.L Tollefsen D.M. Benutto B.M. J. Biol. Chem. 1989; 264: 13979-13982Abstract Full Text PDF PubMed Google Scholar, 19Ragg H. Ulshöfer T. Gerewitz J. J. Biol. Chem. 1990; 265: 22386-22391Abstract Full Text PDF PubMed Google Scholar, 20Myles T. Church F.C. Whinna H.C. Monard D. Stone S.R. J. Biol. Chem. 1998; 273: 31203-31208Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 21Mitchell J.W. Church F.C. J. Biol. Chem. 2002; 277: 19823-19830Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). A synthetic HCII-(54–75) peptide was shown to compete with hirudin-(54–65) for thrombin binding (18Hortin G.L Tollefsen D.M. Benutto B.M. J. Biol. Chem. 1989; 264: 13979-13982Abstract Full Text PDF PubMed Google Scholar). Residues 52–75 in HCII contain two hirudin-like repeats, the first of which interacts directly with exosite I (21Mitchell J.W. Church F.C. J. Biol. Chem. 2002; 277: 19823-19830Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). The crystal structure of the HCII·S195A-thrombin Michaelis complex confirmed this exosite I interaction (22Baglin T.P. Carrell R.W. Church F.C. Esmon C.T. Huntington J.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11079-11084Crossref PubMed Scopus (186) Google Scholar). Deletion of the first HCII acidic repeat (6Van Deerlin V.M.D. Tollefsen D.M. J. Biol. Chem. 1991; 266: 20223-20231Abstract Full Text PDF PubMed Google Scholar, 19Ragg H. Ulshöfer T. Gerewitz J. J. Biol. Chem. 1990; 265: 22386-22391Abstract Full Text PDF PubMed Google Scholar), and mutations in exosite I residues Arg67 and Arg73 (20Myles T. Church F.C. Whinna H.C. Monard D. Stone S.R. J. Biol. Chem. 1998; 273: 31203-31208Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) caused large decreases in the rate constants for the heparin- and DS-catalyzed inactivation, further suggesting the importance of the direct HCII-exosite I interaction. Selective thrombin exosite II mutations did not affect the DS-catalyzed inactivation rate significantly (23Sheehan J.P. Tollefsen D.M. Sadler J.E. J. Biol. Chem. 1994; 269: 32747-32751Abstract Full Text PDF PubMed Google Scholar, 24Cooper S.T. Rezaie A.R. Esmon C.T. Church F.C. Thromb. Res. 2002; 107: 67-73Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). Blocking exosite II with HD22, a single-stranded DNA aptamer only had a modest effect on this inactivation rate, and DS with a chain length of 12–20 saccharide units was found to be as effective as higher Mr DS in catalyzing thrombin inactivation (25Liaw P.C.Y. Austin R.C. Fredenburgh J.C. Stafford A.R. Weitz J.I. J. Biol. Chem. 1999; 274: 27597-27604Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Whereas some studies report DS binding to thrombin (13Verhamme I.M.A. Hogg P.J. Jackson C.M. Arch. Intl. Physiol. Biochim. 1989; 97: B117PubMed Google Scholar, 23Sheehan J.P. Tollefsen D.M. Sadler J.E. J. Biol. Chem. 1994; 269: 32747-32751Abstract Full Text PDF PubMed Google Scholar, 26Liaw P.C.Y. Becker D.L. Stafford A.R. Fredenburgh J.C. Weitz J.I. J. Biol. Chem. 2001; 276: 20959-20965Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), others postulate that DS does not interact with thrombin (27Maaroufi R.M. Jozefowicz M. Tapon-Bretaudière J. Jozefonvicz J. Fischer A.-M. Biomaterials. 1997; 18: 359-366Crossref PubMed Scopus (30) Google Scholar, 28Huntington J.A. J. Thromb. Haemostas. 2003; 1: 1535-1549Crossref PubMed Scopus (129) Google Scholar). These findings, and the assumption that DS template action, like heparin, engages exosite II, led to the proposal of a mechanism in which direct interaction of the HCII N-terminal domain with exosite I is predominant, and GAG template action may be less important, or not even obligatory. In the mechanism of high affinity heparin-catalyzed thrombin inactivation by AT, tight binding was shown to occur between AT and the heparin molecule, containing high affinity pentasaccharide repeats, with an apparent dissociation constant KAT(H) of 0.01–0.02 μm (29Nordenman B. Danielsson A.Å. Björk I. Eur. J. Biochem. 1978; 90: 1-6Crossref PubMed Scopus (92) Google Scholar), and this binary complex bound free thrombin to form the productive ternary complex. By contrast, low affinity heparin with Mr 7,900 bound AT with KAT(H) 19 μm, and thrombin with KT(H) 0.69 μm, resulting in preferred formation of the T·H binary complex, which bound free AT (4Olson S.T. J. Biol. Chem. 1988; 263: 1698-1708Abstract Full Text PDF PubMed Google Scholar, 30Streusand 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). Preferred formation of AT·H and T·H complexes as intermediates on the pathway to ternary complex formation is governed by the differences in the dissociation constants of the binary complexes. Heparin acts as a template in the ternary T·H·AT complexes with high and low affinity heparin, bridging AT helices A and D, and thrombin exosite II. No acidic N-terminal sequence is present in AT, and the heparin-catalyzed thrombin inactivation mechanism is driven by template formation, as evidenced by the bell-shaped, logarithmic dependence of the inactivation rate constant on the heparin concentration (30Streusand 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). Bell-shaped inactivation profiles have also been reported for GAG-catalyzed thrombin inactivation (4Olson S.T. J. Biol. Chem. 1988; 263: 1698-1708Abstract Full Text PDF PubMed Google Scholar, 12Church F.C. Meade J.B. Treanor R.E. Whinna H.C. J. Biol. Chem. 1989; 264: 3618-3623Abstract Full Text PDF PubMed Google Scholar, 31Bauman S.J. Church F.C. J. Biol. Chem. 1999; 274: 34556-34565Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 32Colwell N.S. Grupe M.J. Tollefsen D.M. Biochim. Biophys. Acta. 1999; 1431: 148-156Crossref PubMed Scopus (27) Google Scholar), meizothrombin (MzT), and meizothrombin (des-fragment 1) (MzT(-F1)) inactivation by HCII (33Han J.-H. Côté H.C.F. Tollefsen D.M. J. Biol. Chem. 1997; 272: 28660-28665Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). However, unlike for the heparin-catalyzed thrombin-AT reaction, no mechanism is available for the GAG-catalyzed thrombin-HCII reaction that simultaneously describes the template and allosteric behavior, and fits the GAG and HCII dependences of the inactivation rate constant, consistent with independently obtained protein-GAG binding constants. The aim of the present work was to characterize the roles of heparin and DS in the inactivation of thrombin by HCII, and, for the first time, to define a mechanism that fits both the HCII and GAG concentration dependences of thrombin inactivation kinetics, and is consistent with binary T·GAG and HCII·GAG complex dissociation constants obtained independently by equilibrium binding. The results support the conclusions that the preferred pathway for ternary complex formation with thrombin, HCII and GAGs proceeds via the T·GAG binary intermediate, and that the inactivation mechanism combines GAG template action and allosteric regulation via direct HCII binding to thrombin exosite I in the T·GAG complex. Although the proposed mechanism involves obligatory ternary complex formation by GAG template action, it does not contradict the allosteric interaction between the HCII N-terminal sequence and thrombin exosite I, but it places this interaction in a preferred pathway of T·GAG interacting with HCII, rather than HCII·GAG interacting with thrombin. The present study also demonstrated a novel binding site for DS on thrombin, outside exosite II, which may participate in GAG template formation, and could explain the limited attenuating effect of exosite II ligands and thrombin exosite II mutants in the DS-catalyzed inactivation. Materials, Proteins, and Glycosaminoglycans—Human HCII was purified from plasma by a modification of published procedures (34Tollefsen D.M. Majerus D.W. Blank M.K. J. Biol. Chem. 1982; 257: 2162-2169Abstract Full Text PDF PubMed Google Scholar, 35Griffith M.J. Noyes C.M. Tyndall J.A. Church F.C. Biochemistry. 1985; 24: 6777-6782Crossref PubMed Scopus (39) Google Scholar, 36Tollefsen D.M. Peacock M.E. Monafo W.J. J. Biol. Chem. 1986; 261: 8854-8858Abstract Full Text PDF PubMed Google Scholar). Plasma was treated with soybean trypsin inhibitor, phenylmethanesulfonyl fluoride, and peptide chloromethylketones (FPR-CH2Cl, FFR-CH2Cl), and subjected to barium citrate precipitation. The 8–22% PEG 8000 cut of the supernatant was resuspended and chromatographed on Q-Sepharose, in 50 mm Hepes, 0.1 mm EDTA, 0.02% azide, 0.1 μm FPR-CH2Cl, and FFR-CH2Cl buffer pH 7.5 using a 0.1–0.4 m NaCl gradient. Active fractions were chromatographed on heparin-Sepharose in the same buffer, using a 0.1–0.5 m NaCl gradient. Traces of remaining heparin and impurities were removed by consecutive Hi Trap Q-Sepharose and concanavalin A-Sepharose FPLC. Human α-thrombin was a gift from Dr. J. Fenton II, New York State Department of Health, Albany, NY, or was prepared as described previously (37Bock P.E. J. Biol. Chem. 1992; 267: 14963-14973Abstract Full Text PDF PubMed Google Scholar). MzT(-F1) (38Mann K.G. Elion J. Butkowski R.J. Downing M. Nesheim M.E. Methods Enzymol. 1981; 80: 286-302Crossref PubMed Scopus (97) Google Scholar), and prothrombin fragment 2 (F2) (39Bock P.E. J. Biol. Chem. 1992; 267: 14974-14981Abstract Full Text PDF PubMed Google Scholar, 40Carlisle T.L. Bock P.E. Jackson C.M. J. Biol. Chem. 1990; 265: 22044-22055Abstract Full Text PDF PubMed Google Scholar) were prepared as described previously. Enzyme preparations were ≥90% active as determined by active site titration (41Chase Jr., T. Shaw E. Biochemistry. 1969; 8: 2212-2224Crossref PubMed Scopus (381) Google Scholar). Protein concentrations were determined by absorbance at 280 nm with the absorption coefficients and molecular weights (Mr) of 1.83 (mg/ml)–1 cm–1 in 0.1 m NaOH or 1.74 in buffer, and 36,700, thrombin (42Fenton II, J.W. Fasco M.J. Stackrow A.B. Aronson D.L. Young A.M. Finlayson J.S. J. Biol. Chem. 1977; 252: 3587-3598Abstract Full Text PDF PubMed Google Scholar); 1.78 and 49,900, MzT(-F1) (38Mann K.G. Elion J. Butkowski R.J. Downing M. Nesheim M.E. Methods Enzymol. 1981; 80: 286-302Crossref PubMed Scopus (97) Google Scholar); 1.25 and 12,900, F2 (42Fenton II, J.W. Fasco M.J. Stackrow A.B. Aronson D.L. Young A.M. Finlayson J.S. J. Biol. Chem. 1977; 252: 3587-3598Abstract Full Text PDF PubMed Google Scholar); 0.59 and 65,600, HCII (43Church F.C. Meade J.B. Pratt C.W. Arch. Biochem. Biophys. 1987; 259: 331-340Crossref PubMed Scopus (19) Google Scholar). The active inhibitor concentration of HCII was determined by stoichiometric titration with active site-titrated thrombin in the presence of molar excess DS. A monoclonal antibody against thrombin exosite II, with Mr 150,000 and absorption coefficient of 1.35 (mg/ml)–1 cm–1 (44Colwell N.S. Tollefsen D.M. Blinder M.A. Br. J. Haematol. 1997; 97: 219-226Crossref PubMed Scopus (42) Google Scholar) was a gift from Dr. Douglas M. Tollefsen, Washington University, St. Louis, MO. The thrombin exosite II DNA aptamer, identical to HD22 (25Liaw P.C.Y. Austin R.C. Fredenburgh J.C. Stafford A.R. Weitz J.I. J. Biol. Chem. 1999; 274: 27597-27604Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), with the sequence 5′-AGTCCGTGGTAGGGCAGGTTGGGGTGACT-3′, was synthesized by Midland. Heparin-Sepharose was prepared following published procedures (45March S.C. Parikh I. Cuatrecasas P. Anal. Biochem. 1974; 60: 149-152Crossref PubMed Scopus (1430) Google Scholar), or purchased from Amersham Biosciences. Porcine intestinal mucosa heparins of Mr 12,000 and 16,000 were from Sigma, and a gift of Dr. G. van Dedem, Diosynth BV, Oss, The Netherlands, respectively. Heparin fraction D with high affinity for AT, and Mr 20,300 (46Sache E. Maillard M. Bertrand H. Maman M. Kunz M. Choay J. Fareed J. Messmore H. Thromb. Res. 1982; 25: 443-458Abstract Full Text PDF PubMed Scopus (26) Google Scholar) was a gift of Dr. E. Sache, Institut Choay, Paris, France. Hirudin peptide (Hir54-65(SO3-)) and porcine skin DS (Mr 18,000) were purchased from Sigma. Porcine intestinal mucosa low Mr heparin (Mr 5,000) and porcine skin DS of Mr 30,000 were from Calbiochem. DS of Mr 45,000 was from Celsus. DS was treated with NaNO2 in H2SO4 to degrade contaminating heparin (47Shively J.E. Conrad H.E. Biochemistry. 1976; 15: 3932-3942Crossref PubMed Scopus (667) Google Scholar), inspected for molecular integrity on Stains-All©-stained gradient SDS gels (48Bahr V. Stierhof Y.D. Ilg T. Demar M. Quinten M. Overath P. Mol. Biochem. Parasitol. 1993; 58: 107-122Crossref PubMed Scopus (115) Google Scholar), and tested for absence of catalytic effect in the Xa-AT assay. Chromogenic substrates H-d-Ile-Pro-Arg-p-nitroanilide (-pNA) (S2288), Tos-Gly-Pro-Arg-pNA (Chromozym TH), and methoxycarbonyl-d-cyclohexyl-glycyl-Gly-Arg-pNA (Spectrozyme FXa) were purchased from Chromogenix, Pentapharm and American Diagnostica. Equilibrium binding studies were performed at 25 °C, in 20 mm Hepes, 20 mm Taps, 0.1 m NaCl, 1 mg/ml PEG 8000, pH 7.8 buffer, or in 50 mm Hepes, 0.11 m NaCl, 1 mg/ml PEG 8000, pH 7.4 buffer. Buffers contained 0.1–5 μm FPR-CH2Cl in titrations. Equilibrium Binding of Thrombin and HCII to DS and Heparin, Measured by Quantitative Affinity Chromatography—Binding of thrombin and HCII to Mr 18,000 DS and Mr 16,000 and 20,300 heparin was analyzed by quantitative affinity chromatography (QAC) under nonsaturating matrix conditions (49Nichol L.W. Ward L.D. Winzor D.J. Biochemistry. 1981; 20: 4856-4860Crossref PubMed Scopus (58) Google Scholar), using low substitution grade heparin-Sepharose (Amersham Biosciences) for thrombin binding, or heparin-Sepharose prepared by coupling unfractionated porcine heparin to CNBr-activated Sepharose 4B, at 2 mg of heparin/ml drained gel (45March S.C. Parikh I. Cuatrecasas P. Anal. Biochem. 1974; 60: 149-152Crossref PubMed Scopus (1430) Google Scholar), for HCII binding. QAC allows the quantitation of competitive displacement of thrombin or HCII by heparin or DS from heparin-Sepharose. To determine kax[X]0, the product of the concentration of matrix binding sites [X]0 and the association constant kax for protein and matrix-bound heparin, binding isotherms of bound versus free protein were established in the absence of GAG, under nonsaturating thrombin and HCII conditions. Thrombin and HCII binding experiments were performed in separate PEG-coated microcentrifuge tubes with continuous mixing, or, for HCII, in a 10-ml Amicon concentrator cell, using the recycling partition method (50Hogg P.J. Winzor D.J. Arch. Biochem. Biophys. 1985; 240: 70-76Crossref PubMed Scopus (19) Google Scholar) and sequential additions of small aliquots of concentrated ligand solution. Increasing amounts of thrombin or HCII were incubated with a constant volume of matrix, <5% of the total, constant incubation volume (1 ml, microcentrifuge tubes; 6.5 ml, recycling partition method). Residual thrombin in the supernatant was assayed by hydrolysis of S2288 at 405 nm. Residual HCII was determined by protein absorbance of the supernatant at 280 nm, and by GAG-catalyzed thrombin inactivation in the presence of competing chromogenic substrate as described below. Appropriate thrombin and HCII concentration ranges for GAG binding were limited to nonsaturation matrix conditions defined by the initial, quasi-linear portion of the hyperbolic bound versus free protein binding isotherms to heparin-Sepharose matrix in the absence of competing GAG. In separate experiments, thrombin and HCII at constant concentrations (12 nm thrombin, and 3.4, 7.6, and 8.6 μm HCII, in the heparin binding experiments; 0.46 μm thrombin and 3 μm HCII, in the DS binding experiments), were incubated with a constant amount of heparin-Sepharose matrix, and increasing concentrations of GAG. In the heparin binding experiment, initially 51% thrombin and 54–58% HCII were bound to the matrix; in the DS binding experiment, initially 97% thrombin and 56% HCII were bound, at total protein concentrations well below matrix saturation. Bound thrombin ([T]bound) and HCII ([HCII]bound) as a function of the increasing total GAG concentration ([GAG]o) was analyzed using hyperbolic Equations 1 and 2, and the mass balance for total T or HCII ([T or HCII]o = [T or HCII]bound + [T or HCII]free), to obtain KT(H), KHCII(H), KT(DS), and KHCII(DS) (55Tian W.-X. Tsou C.-L. Biochemistry. 1982; 21: 1028-1032Crossref PubMed Scopus (305) Google Scholar, 56Hogg P.J. Jackson C.M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3619-3623Crossref PubMed Scopus (319) Google Scholar), R=[TorHCII]bound[TorHCII]free=kax[X]o1+[GAG]oKTorHCII(GAG)(Eq. 1) [TorHCII]free=[TorHCII]o1+R(Eq. 2) where kax is the association constant for T or HCII and matrix-bound heparin, [X]o is the concentration of matrix binding sites, and GAG denotes either heparin or DS. Fluorescence Equilibrium Binding of DS and Heparin to Thrombin and MzT(-F1), and Involvement of Exosite I—Active site-labeled fluorescent derivatives of thrombin and MzT(-F1) (39Bock P.E. J. Biol. Chem. 1992; 267: 14974-14981Abstract Full Text PDF PubMed Google Scholar) were prepared following published methods (37Bock P.E. J. Biol. Chem. 1992; 267: 14963-14973Abstract Full Text PDF PubMed Google Scholar, 39Bock P.E. J. Biol. Chem. 1992; 267: 14974-14981Abstract Full Text PDF PubMed Google Scholar). Hir54-65(SO3-) was labeled at the N terminus with 5-carboxy-fluorescein ([5F]Hir54-65(SO3-)) (3Bock P.E. Olson S.T. Björk I. J. Biol. Chem. 1997; 272: 19837-19845Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Measurements were made with an SLM 8100 spectrofluorometer, using acrylic cuvettes coated with polyethylene glycol 20,000 to minimize protein adsorption. Titrations were performed as described previously (53Verhamme I.M. Olson S.T. Tollefsen D.M. Bock P.E. J. Biol. Chem. 2002; 277: 6788-6798Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), at the following excitation and emission wavelengths: [6F]FPR-T with heparin or DS, 495 and 520 nm; [6F]FFR-T with DS, 498 and 518 nm; [6F]FPR-MzT(-F1) with heparin or DS, 499 and 514 nm; [5F]Hir54-65(SO3-)) with thrombin, in the absence or presence of GAGs, 491 and 520 nm; with 4–8-nm band passes. Results were expressed as the fractional change in the initial fluorescence ((Fobs – Fo)/Fo = ΔF/Fo) as a function of total titrant concentration, and were fit by the appropriate binding equation for each experimental setup, as described below. [6F]FFR-T was titrated with Mr 30,000 DS in pH 7.8 buffer, in the absence and the presence of 5 μm Hir54-65(SO3-). [6F]FPR-T was titrated with DS in pH 7.4 and 7.8 buffer. In separate experiments, [6F]FPR-T was titrated with high (12,000) and low Mr (5,000) heparin, in the absence and presence of 5 μm Hir54-65(SO3-). Labeled thrombin concentrations were below or at KT(H), to avoid heparin-induced thrombin aggregation (54Hogg P.J. Jackson C.M. Labanowski J.K. Bock P.E. J. Biol. Chem. 1996; 271: 26088-26095Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). [6F]FPR-MzT(-F1) was titrated with DS and high Mr heparin. In separate experiments, [5F]Hir54-65(SO3-) was titrated with thrombin, in the absence and presence of 176 μm Mr 30,000 DS; 27.6 and 206 μm Mr 12,000 heparin; and 117 μm Mr 5,000 heparin. Direct binding of GAGs to labeled thrombin and MzT(-F1), expressed as the fractional change in initial fluorescence (ΔF/Fo), was analyzed by fitting of the quadratic equation for binding of a single ligand (39Bock P.E. J. Biol. Chem. 1992; 267: 14974-14981Abstract Full Text PDF PubMed Google Scholar), to obtain values of the maximum fluorescence intensity change (ΔFmax/Fo) and the dissociation constant (KD), with one binding site assumed on thrombin, MzT(-F1) or MzT (n = 1). The observed fluorescence change is given by the contribution of the T*·GAG complex, weighted by the maximum fluorescence change associated with its formation (Equation 3), ΔFFo=([T*·GAG]n[T*]o)ΔFmax,T(GAG)Fo(Eq. 3) in which [T*]0 is the total concentration of the labeled thrombin or MzT(-F1), and n is the number of equivalent and independent binding sites for GAG. Binding of thrombin to [5F]Hir54-65(SO3-) was analyzed similarly, where the fractional change in fluorescence was given by Equation 4, ΔFFo=([T·Hir*]n[Hir*]o)ΔFmax,T(Hir)Fo(Eq. 4) in which [Hir*]0 is the total concentration of the labeled peptide (Hir*), T is thrombin, and n is the number of binding sites for GAG. [6F]FPR-T reported GAG and Hir54-65(SO3-) binding by unequal fluorescence changes, while GAG binding to thrombin had no effect on ΔFmax/Fo in the [5F]Hir54-65(SO3-) titrations. Experiments with simultaneous GAG and peptide binding to thrombin were analyzed by Equations 5 and 6 for the ternary complex model (see Scheme 1), as described previously (53Verhamme I.M. Olson S.T. Tollefsen D.M. Bock P.E. J.

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