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• W2068600199 abstract "Although fibrin-bound thrombin is resistant to inactivation by heparin·antithrombin and heparin·heparin cofactor II complexes, indirect studies in plasma systems suggest that the dermatan sulfate·heparin cofactor II complex can inhibit fibrin-bound thrombin. Herein we demonstrate that fibrin monomer produces a 240-fold decrease in the heparin-catalyzed rate of thrombin inhibition by heparin cofactor II but reduces the dermatan sulfate-catalyzed rate only 3-fold. The protection of fibrin-bound thrombin from inhibition by heparin·heparin cofactor II reflects heparin-mediated bridging of thrombin to fibrin that results in the formation of a ternary heparin·thrombin·fibrin complex. This complex, formed as a result of three binary interactions (thrombin·fibrin, thrombin·heparin, and heparin·fibrin), limits accessibility of heparin-catalyzed inhibitors to thrombin and induces conformational changes at the active site of the enzyme. In contrast, dermatan sulfate binds to thrombin but does not bind to fibrin. Although a ternary dermatan sulfate· thrombin·fibrin complex forms, without dermatan sulfate-mediated bridging of thrombin to fibrin, only two binary interactions exist (thrombin·fibrin and thrombin· dermatan sulfate). Consequently, thrombin remains susceptible to inactivation by heparin cofactor II. This study explains why fibrin-bound thrombin is susceptible to inactivation by heparin cofactor II in the presence of dermatan sulfate but not heparin. Although fibrin-bound thrombin is resistant to inactivation by heparin·antithrombin and heparin·heparin cofactor II complexes, indirect studies in plasma systems suggest that the dermatan sulfate·heparin cofactor II complex can inhibit fibrin-bound thrombin. Herein we demonstrate that fibrin monomer produces a 240-fold decrease in the heparin-catalyzed rate of thrombin inhibition by heparin cofactor II but reduces the dermatan sulfate-catalyzed rate only 3-fold. The protection of fibrin-bound thrombin from inhibition by heparin·heparin cofactor II reflects heparin-mediated bridging of thrombin to fibrin that results in the formation of a ternary heparin·thrombin·fibrin complex. This complex, formed as a result of three binary interactions (thrombin·fibrin, thrombin·heparin, and heparin·fibrin), limits accessibility of heparin-catalyzed inhibitors to thrombin and induces conformational changes at the active site of the enzyme. In contrast, dermatan sulfate binds to thrombin but does not bind to fibrin. Although a ternary dermatan sulfate· thrombin·fibrin complex forms, without dermatan sulfate-mediated bridging of thrombin to fibrin, only two binary interactions exist (thrombin·fibrin and thrombin· dermatan sulfate). Consequently, thrombin remains susceptible to inactivation by heparin cofactor II. This study explains why fibrin-bound thrombin is susceptible to inactivation by heparin cofactor II in the presence of dermatan sulfate but not heparin. heparin cofactor II anilinonaphthalene-6-sulfonic acid dermatan sulfate fluorescein-labeled soluble fibrin fibrin monomer fluorescein-5′-isothiocyanate d-Phe-Pro-Arg d-Phe-Pro-Arg-chloromethyl ketone Gly-Pro-Arg-Pro-amide N-p-tosyl-Gly-Pro-Arg-p-nitroanilide acetate N-(acetylthio)acetyl Tris-buffered saline polyethylene glycol Heparin, a sulfated polysaccharide, acts as an anticoagulant by accelerating the inhibition of thrombin and factor Xa by antithrombin (1Rosenberg R.D. Colman R.W. Hirsh J. Marder V.J. Salzman E.W. Haemostasis and Thrombosis: Basic Principles and Clinical Practice. J. B. Lippincott, Philadelphia1987: 1377-1382Google Scholar). Although heparin is widely used for the treatment of acute coronary ischemic syndromes, it has limitations in patients undergoing percutaneous coronary interventions (2Popma J.J. Ohman E.M. Weitz J. Lincoff A.M. Harrington R.A. Berger P. Chest. 2001; 119: 321S-336SAbstract Full Text Full Text PDF PubMed Google Scholar) or when used as an adjunct to thrombolytic therapy (3The International Study Group, Lancet, 336, 1990, 71, 75.Google Scholar, 4ISIS-3 Collaborative Group, Lancet, 339, 1992, 753, 770.Google Scholar, 5Collins R. MacMahon S. Flather M. Baigent C. Remvig L. Mortensen S. Appleby P. Godwin J. Yusuf S. Peto R. Br. Med. J. 1996; 313: 652-659Crossref PubMed Scopus (182) Google Scholar). These limitations have been attributed to the inability of the antithrombin·heparin complex to inactivate clotting enzymes bound to components of the thrombus, particularly thrombin bound to fibrin (6Hogg P.J. Jackson C.M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3619-3623Crossref PubMed Scopus (318) Google Scholar, 7Weitz J.I. Hudoba M. Massel D. Maraganore J. Hirsh J. J. Clin. Invest. 1990; 86: 385-391Crossref PubMed Scopus (1080) Google Scholar). Resistance of fibrin-bound thrombin to inactivation by the antithrombin·heparin complex reflects the incorporation of thrombin into a ternary heparin·thrombin·fibrin complex (8Hogg P.J. Jackson C.M. J. Biol. Chem. 1990; 265: 241-247Abstract Full Text PDF PubMed Google Scholar, 9Hogg P.J. Jackson C.M. J. Biol. Chem. 1990; 265: 248-255Abstract Full Text PDF PubMed Google Scholar, 10Hogg 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 (50) Google Scholar). To form this complex, heparin interacts with both exosite II on thrombin (11Church F.C. Pratt C.W. Noyes C.M. Kalayanamit T. Sherrill G.B. Tobin R.B. Meade J.B. J. Biol. Chem. 1989; 264: 18419-18425Abstract Full Text PDF PubMed Google Scholar, 12Gan Z.R. Li Y. Chen Z. Lewis S.D. Shafer J.A. J. Biol. Chem. 1994; 269: 1301-1305Abstract Full Text PDF PubMed Google Scholar, 13Sheehan J.P. Sadler J.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5518-5522Crossref PubMed Scopus (181) Google Scholar) and the D domain of fibrin (14Odrljin T. Shainoff J.R. Lawrence S.O. Simpson-Haidaris P.J. Blood. 1996; 88: 2050-2061Crossref PubMed Google Scholar), thereby bridging thrombin to fibrin via exosite II (8Hogg P.J. Jackson C.M. J. Biol. Chem. 1990; 265: 241-247Abstract Full Text PDF PubMed Google Scholar). This heightens exosite I-mediated binding of thrombin to fibrin and likely increases the overall affinity of thrombin for fibrin. The formation of the ternary complex, therefore, is a consequence of the presence of two exosites on thrombin, which independently bind fibrin and heparin. Recently, we demonstrated that protection requires ligation of both of thrombin's exosites within the ternary heparin·thrombin·fibrin complex, a process that impairs access of inhibitor-bound heparin to exosite II on thrombin (15Becker 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). Thrombin within the ternary complex is protected from inactivation by the heparin·heparin cofactor II (HCII)1 complex to a greater extent than the heparin·antithrombin complex (15Becker 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), because access of the amino terminus of HCII to exosite I on thrombin, an obligatory part of the HCII inhibitory mechanism (16Tollefsen D.M. Thromb. Haemost. 1995; 74: 1209-1214Crossref PubMed Scopus (99) Google Scholar, 17Liaw P.C. 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), is reduced. Allosteric changes in the active site of thrombin induced upon formation of the ternary heparin·thrombin·fibrin complex may also contribute to the protection of fibrin-bound thrombin from inactivation by heparin·antithrombin and heparin·HCII complexes by limiting inhibitor reactivity with fibrin-bound thrombin (18Hogg P.J. Bock P.E. Thromb. Haemost. 1997; 77: 424-433Crossref PubMed Scopus (42) Google Scholar). Dermatan sulfate (DS), a sulfated glycosaminoglycan that has antithrombotic activity in laboratory animals (19Merton R.E. Thomas D.P. Thromb. Haemost. 1987; 58: 839-842Crossref PubMed Scopus (50) Google Scholar, 20Van Ryn-McKenna J. Ofosu F.A. Gray E. Hirsh J. Buchanan M.R. Ann. N. Y. Acad. Sci. 1989; 556: 304-312Crossref PubMed Scopus (39) Google Scholar) and in humans (21Lane D.A. Ryan K. Ireland H. Curtis J.R. Nurmohamed M.T. Krediet R.T. Roggekamp M.C. Stevens P. ten Cate J.W. Lancet. 1992; 339: 334-335Abstract PubMed Scopus (25) Google Scholar, 22Prandoni P. Meduri F. Cuppini S. Toniato A. Zangrandi F. Polistena P. Gianese F. Maffei Faccioli A. Br. J. Surg. 1992; 79: 505-509Crossref PubMed Scopus (36) Google Scholar, 23Agnelli G. Cosmi B. Di Filippo P. Ranucci V. Veschi F. Longetti M. Renga C. Barzi F. Gianese F. Lupattelli L. Thromb. Haemost. 1992; 67: 203-208Crossref PubMed Scopus (89) Google Scholar), acts as an anticoagulant by catalyzing only HCII. Because thrombin is the exclusive plasma target of HCII, DS is considered a selective inhibitor of thrombin (16Tollefsen D.M. Thromb. Haemost. 1995; 74: 1209-1214Crossref PubMed Scopus (99) Google Scholar). Although fibrin-bound thrombin is protected from inactivation by the heparin·HCII complex (15Becker 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), indirect studies done in plasma systems suggest that fibrin-bound thrombin is susceptible to inactivation by the DS·HCII complex (24Bendayan P. Boccalon H. Dupouy D. Boneu B. Thromb. Haemost. 1994; 71: 576-580Crossref PubMed Scopus (60) Google Scholar,25Okwusidi J.I. Anvari N. Kulczycky M. Blajchman M.A. Buchanan M.R. Ofosu F.A. J. Lab. Clin. Med. 1991; 117: 359-364PubMed Google Scholar). The purpose of this study was to confirm these findings using purified reagents and to elucidate the mechanism by which the DS·HCII complex, but not the heparin·HCII complex, inactivates fibrin-bound thrombin. Human HCII, isolated from plasma by affinity chromatography, was from Affinity Biologicals Inc. (Hamilton, Ontario, Canada). Human α- and γ-thrombin and fibrinogen were from Enzyme Research Laboratories (South Bend, IN). Dermatan sulfate (DS) was from Mediolanum Farmaceutici (Milan, Italy). Heparin,N-p-tosyl-Gly-Pro-Arg-p-nitroanilide acetate (tGPR-pNA), gelatin-agarose, and Gly-Pro-Arg-Pro-amide (GPRP-NH2) were from Sigma Chemical Co. (St. Louis, MO). Enoxaparin, a commercial low-molecular-weight heparin, was from Rhône-Poulenc Rorer Canada (Montreal). Based on high performance liquid chromatography gel filtration analysis, the mean molecular masses of DS, heparin, and enoxaparin are 20 kDa (range 7–34 kDa), 15 kDa (range 5–30 kDa), and 4.5 kDa (range 3.5–5.5 kDa), respectively. Fluorescein-5′-isothiocyanate (FITC) was from Molecular Probes Inc. (Eugene, OR). d-Phe-Pro-Arg-chloromethyl ketone (FPRCK) was from Calbiochem Novabiochem Corp. (San Diego, CA). FITC-FPRCK was from Hematologic Technologies, Inc. (Essex Junction, VT). Hexadimethrine bromide (Polybrene) was obtained from Aldrich Chemical Co. (Milwaukee, WI). Cyanogen bromide-activated Sepharose 4B was fromAmersham Pharmacia Biotech (Dorval, Quebec). Human fibrinogen, treated with gelatin-agarose to remove fibronectin, was used to prepare soluble fibrin (SF), as described previously (15Becker 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). Briefly, fibrin clots were centrifuged and dialyzed versus water. The fibrin was then dissolved by dialysis versus 20 mm acetic acid, aliquoted, and stored at a concentration of about 100 μmat −70 °C. Polymerization of SF was blocked by addition of 5 mm GPRP-NH2 (26Kawasaki K. Hirase K. Miyano M. Tsuji T. Iwamoto M. Chem. Pharm. Bull. (Tokyo). 1992; 40: 3253-3260Crossref PubMed Scopus (17) Google Scholar), and the material was neutralized with 1 m Tris-HCl, pH 7.5, in a volume corresponding to 40% of the volume of SF, just prior to use. A molecular weight of 340,000 and ε 2801% value of 14.0 were used to calculate the soluble fibrin concentration. Fibrinogen was coupled to cyanogen bromide-activated Sepharose 4B, treated with thrombin to convert it to fibrin monomer (FM), and washed with 20 mm Tris-HCl, pH 7.4, 150 nm NaCl (TBS) as described previously (15Becker 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). The influence of varying concentrations of heparin or DS on the second-order rate constants (k 2) for inhibition of thrombin by HCII were determined under pseudo-first-order conditions in the absence or presence of SF. Thrombin (10 nm) was incubated for 5 min at room temperature in TBS containing 0.6% PEG 8000 in the presence of various concentrations of heparin or DS (0–11 μm), SF (0–4 μm), and 10 mm GPRP-NH2. Reaction mixtures (10 μl) were aliquoted to 96-well round bottom microtiter plates and an equal volume of HCII (in a concentration at least 10-fold higher than that of thrombin) was added to each well at time intervals ranging from 2 s to 5 min. All reactions were terminated concomitantly by the addition of 200 μmchromogenic substrate (tGPR-pNA) in 200 μl of TBS containing 10 mg/ml Polybrene. Residual thrombin activity was calculated by measuring absorbance at 405 nm for 5 min using a Spectra Max 340 Microplate Reader (Molecular Devices, Menlo Park, CA). The pseudo-first-order rate constants (k 1) for thrombin inhibition were determined by fitting the data to the equationk 1·t = ln([P]o/[P] t ), where [P]o is initial thrombin activity and [P] t is thrombin activity at time t. The second-order rate constant,k 2, was then determined by dividingk 1 by the HCII concentration (27Olson S.T. Björk I. Shore J.D. Methods Enzymol. 1993; 222: 525-560Crossref PubMed Scopus (264) Google Scholar). Thrombin blocked at its active site with FPRCK (FPR-thrombin) was radiolabeled with Na125I as described previously (28Fredenburgh J.C. Stafford A.R. Weitz J.I. J. Biol. Chem. 1997; 272: 25493-25499Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). The binding of125I-FPR-thrombin to FM-Sepharose in the absence or presence of either heparin or DS (from 0 to 10 μm) was studied in TBS containing 0.6% PEG 8000 and 0.01% Tween 20. FM-Sepharose (9 μm) in 0.5 ml of buffer was incubated with 50 nm125I-FPR-thrombin by inverted mixing for 2 min. The tube was centrifuged in a microcentrifuge for 2 min at 15,000 × g, and a 100-μl aliquot of supernatant was removed for gamma counting. The aliquot was returned to the tube. This procedure was repeated following each addition of heparin or DS to the tube. The fraction of 125I-FPR-thrombin bound to FM-Sepharose as a function of the total amount of125I-FPR-thrombin present was calculated. DS (3 mg) was labeled with 1.0 mCi of Na125I (PerkinElmer Life Sciences, Markham, Ontario, Canada) in a volume of 300 μl using the IODO-BEAD iodination reagent as described by the manufacturer (Pierce, Rockford, IL). Labeled DS catalyzed the inhibition of thrombin by HCII to the same extent as its unlabeled counterpart, indicating that the labeling procedure did not influence DS activity. A mixture of 500 nm125I-labeled DS and 5 μmFM-Sepharose was titrated with increasing concentrations of thrombin or γ-thrombin (from 0 to 3 μm). After centrifugation, residual 125I-labeled DS in the supernatant was quantified by gamma counting and used to determine the fraction of125I-labeled DS bound to FM-Sepharose. Thrombin labeled at the active site with anilinonaphthalene-6-sulfonic acid (ANS-FPR-thrombin) was prepared using ATA-FPR (N-(acetylthio)acetyl-d-Phe-Pro-Arg-CH2Cl) as described by the supplier (Molecular Innovations Inc., Royal Oak, MI). Briefly, 10.6 μm thrombin was incubated for 30 min at 23 °C with a 2.5-fold molar excess of ATA-FPR in 20 mm HEPES-NaOH, pH 7.0, 100 mm NaCl, 1 mm EDTA (HBSE buffer). The reaction mixture was dialyzed against HBSE buffer, incubated with a 10-fold molar excess of 2-(4-(iodoacetamide)anilino)naphthalene-6-sulfonic acid (Molecular Probes Inc.) in the presence of hydroxylamine for 60 min at 23 °C in the dark and then redialyzed. 800 μl of 140 nmANS-FPR-thrombin in 50 mm HEPES, pH 7.5, 0.125m NaCl, 1 mg/ml PEG 8000, 5 mmGPRP-NH2 was added to a semi-micro quartz cuvette. Using a PerkinElmer Life Sciences LS50B luminescence spectrometer with excitation wavelength set to 334 nm and excitation and emission slit widths set to 5 nm, the fluorescence emission spectrum from 380–580 nm of ANS-FPR-thrombin was monitored before and after the addition of 310 nm SF and/or 100 nm heparin or 3 μm DS. Soluble fibrin was neutralized by the addition of 40% v/v 1 m Tris-HCl, pH 7.5, just prior to use. Addition of 1 m Tris-HCl, pH 7.5, had no effect on the fluorescence spectrum of ANS-FPR-thrombin (data not shown). Fluorescent derivatives of heparin and DS were prepared as follows (29Nagasawa K. Uchiyama H. Biochim. Biophys. Acta. 1978; 544: 430-440Crossref PubMed Scopus (29) Google Scholar). 10 mg of heparin or DS were incubated with 15 mg of FITC in 2.5 ml of 1.0 mNa2CO3, pH 9.0 for 5 h at 23 °C. After centrifugation at 13,000 × g for 5 min, 1 ml of the supernatant was applied to duplicate PD-10 gel filtration columns (Millipore Corp., Bedford, MA), equilibrated with H2O, and eluted with H2O under gravity. 0.5-ml fractions were collected, frozen, and lyophilized. Recovered material was pooled, weighed, and dissolved in TBS to a stock concentration of 10 mg/ml. FITC-labeled active site-blocked thrombin (f-FPR-thrombin) was prepared as previously described (28Fredenburgh J.C. Stafford A.R. Weitz J.I. J. Biol. Chem. 1997; 272: 25493-25499Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Briefly, thrombin was incubated with fluorescein-d-Phe-Pro-Arg chloromethyl ketone (f-FPRCK) until no residual thrombin chromogenic activity was detected. After dialysis to remove unincorporated f-FPRCK, the concentration of fluorescently labeled thrombin was determined by measuring absorbance using ε 2801% of 1.8 after correction for light scatter at 320 nm using the relationship A 280corr=A 280 − 1.7 ×A 320. The affinity of FITC-labeled ligands for fibrin was determined by measuring unbound ligand in supernatants of clots prepared by clotting varying concentrations of fibrinogen with 1 nm thrombin. Briefly, 200 μl of 2 mm CaCl2, 100 nm FITC-labeled ligand, various concentrations of fibrinogen (30–3000 nm), and 1 nm thrombin were mixed in a series of microcentrifuge tubes. After 1-h incubation at 23 °C, fibrin was pelleted by centrifugation for 5 min at 15,000 × g and 100 μl of supernatant was removed and added to 300 μl of TBS. The fluorescence intensity of the samples was monitored with excitation and emission wavelengths set to 492 and 522 nm, respectively, and excitation and emission slit widths both set to 15 nm. K d values were calculated by plottingI/I o versus fibrinogen concentration, where I o and Irepresent the fluorescence intensities in the absence and presence of the varying concentrations of fibrinogen, respectively. The parametersK d and ΔI were calculated by nonlinear regression (“Tablecurve,” Jandel Scientific, San Rafael, CA) using the equation, II0=1+1+Kd+LP−1+Kd+LP2−4×LP×ΔI2Equation 1 where ΔI is the maximum fluorescence change,P is the initial concentration of f-heparin, f-DS, or f-FPR-thrombin, L is the total fibrinogen concentration, and a stoichiometry of 1 is assumed (28Fredenburgh J.C. Stafford A.R. Weitz J.I. J. Biol. Chem. 1997; 272: 25493-25499Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). The affinities of heparin or DS for thrombin were determined by monitoring changes in intrinsic thrombin fluorescence when the enzyme was titrated with heparin or DS. The initial intensity reading of 100 nm thrombin in a 2-ml quartz cuvette (I o) was determined with excitation and emission wavelengths set to 280 and 340 nm, respectively, and excitation and emission slit widths set to 6 nm. Aliquots of either heparin or DS were then added to the cuvette, and, after mixing, changes in fluorescence were monitored (I). K d values were calculated by plotting I/I o versus glycosaminoglycan concentration and the data were fit by nonlinear regression to the equation given above. The association between HCII and either heparin, DS, or fibrin was monitored by the ligand-dependent fluorescence intensity change of anilinonaphthalene-6-sulfonic acid labeled HCII (ANS-HCII). ANS-HCII, prepared as previously described (30Weitz J.I. Young E. Johnston M. Stafford A.R. Fredenburgh J.C. Hirsh J. Circulation. 1999; 99: 682-689Crossref PubMed Scopus (32) Google Scholar), was added to a concentration of 100 nm to a quartz cuvette. The initial fluorescence intensity (I o) of ANS-HCII was determined at excitation and emission wavelengths set to 280 and 437 nm, respectively, and excitation and emission slit widths set to 10 nm, and an emission filter of 290 nm. Known quantities of either heparin, DS, or SF were then added to the cuvette and, after mixing, changes in fluorescence was monitored (I).K d values were calculated by plottingI/I o versus ligand concentration, and the data were fit by nonlinear regression to the equation given above. 500-μl suspensions of FM-Sepharose (9 μm FM) containing 50 nm125I-FPR-thrombin without glycosaminoglycan or with 2.5 μm DS in TBS/0.6% PEG/0.01% Tween 20 were mixed for 2 min. The amount of unbound 125I-FPR-thrombin in the suspension after each addition of an aliquot of 80 μmHCII was determined as described above. To verify the protective effect of SF on thrombin inhibition by HCII, we determined the rates of thrombin inhibition in the absence or presence of SF and heparin. As shown in Fig. 1 A, SF caused a dose-dependent decrease in the heparin-catalyzed rates of thrombin inhibition by HCII, which by two-way analysis of variance performed using Minitab (software version 11, State College, PA), was highly significant (p < 0.001). At 1 μmheparin and 4 μm SF, a maximal 240-fold decrease in the rate was observed, a value consistent with that reported previously (15Becker 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). In contrast, at a concentration of 4 μm SF, a significant (p < 0.001) but modest 3-fold decrease in the DS-catalyzed rates of thrombin inhibition by HCII was observed (Fig. 1 B). The assembly of the ternary heparin·thrombin·fibrin complex is postulated to occur through a series of binary interactions between thrombin·fibrin, thrombin·heparin, and fibrin·heparin (8Hogg P.J. Jackson C.M. J. Biol. Chem. 1990; 265: 241-247Abstract Full Text PDF PubMed Google Scholar, 10Hogg 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 (50) Google Scholar, 18Hogg P.J. Bock P.E. Thromb. Haemost. 1997; 77: 424-433Crossref PubMed Scopus (42) Google Scholar). In this study, we determined the dissociation constants for these interactions, as well as those involving DS. The affinities of DS and heparin for thrombin were determined by monitoring changes in intrinsic protein fluorescence of thrombin when titrated with DS or heparin (not shown). DS and heparin bind saturably to thrombin with K d values of 2600 and 116 nm, respectively (TableI). The K d value for the thrombin·heparin interaction is in agreement with that determined by titration of ANS-labeled thrombin with increasing concentrations of heparin (K d = 59 nm) (10Hogg 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 (50) Google Scholar).Table IDissociation constants for the binary interactions constituting ternary thrombin · fibrin · heparin and thrombin · fibrin · DS complexesInteractionK dnmThrombin and heparin117Thrombin and DS2,600Fibrin and f-thrombin1,500Fibrin and f-heparin187Fibrin and f-DSNo bindingANS-HCII and heparin13,000ANS-HCII and DS71,000ANS-HCII and fibrinNo bindingK d values were determined as described under “Methods.” Interactions where no binding could be detected under the given conditions are indicated. Open table in a new tab K d values were determined as described under “Methods.” Interactions where no binding could be detected under the given conditions are indicated. Current evidence suggests that both heparin and DS bind to exosite II on thrombin (11Church F.C. Pratt C.W. Noyes C.M. Kalayanamit T. Sherrill G.B. Tobin R.B. Meade J.B. J. Biol. Chem. 1989; 264: 18419-18425Abstract Full Text PDF PubMed Google Scholar, 12Gan Z.R. Li Y. Chen Z. Lewis S.D. Shafer J.A. J. Biol. Chem. 1994; 269: 1301-1305Abstract Full Text PDF PubMed Google Scholar, 13Sheehan J.P. Sadler J.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5518-5522Crossref PubMed Scopus (181) Google Scholar, 31Sheehan J.P. Tollefsen D.M. Sadler J.E. J. Biol. Chem. 1994; 269: 32747-32751Abstract Full Text PDF PubMed Google Scholar). This was confirmed in two ways. First, competitive binding studies were performed where thrombin-bound f-heparin was displaced by DS (Fig. 2). In this experiment, addition of thrombin to f-heparin resulted in an approximate 3.5% decrease in fluorescence intensity. Subsequent titration of the sample with DS resulted in a dose-dependent increase of fluorescence intensity to the value observed for f-heparin prior to binding to thrombin (I/I o of 1). Likewise, in the reciprocal experiment, the fluorescence decrease that occurred upon addition of thrombin to f-DS was negated in a dose-dependent fashion by titration with heparin (not shown). In a second approach, we used thrombin variants with impaired exosites to identify the DS binding site (not shown). DS binds to γ-thrombin, a proteolytic derivative of α-thrombin lacking exosite I (11Church F.C. Pratt C.W. Noyes C.M. Kalayanamit T. Sherrill G.B. Tobin R.B. Meade J.B. J. Biol. Chem. 1989; 264: 18419-18425Abstract Full Text PDF PubMed Google Scholar), with a 2.6-fold lower affinity than α-thrombin (K d values of 6.8 μm and 2.6 μm, respectively). In contrast, DS binds RA-thrombin, a thrombin variant with three point mutations in exosite II that lower its affinity for heparin 20-fold (32Ye J. Rezaie A.R. Esmon C.T. J. Biol. Chem. 1994; 269: 17965-17970Abstract Full Text PDF PubMed Google Scholar), with a 7-fold lower affinity (K d = 17 μm). These studies confirm that, like heparin, DS also binds to exosite II on thrombin, albeit with lower affinity (Table I). The affinities of f-FPR-thrombin, f-heparin, and f-DS for fibrin were monitored by clotting varying concentrations of fibrinogen with a catalytic amount of thrombin in the presence of a fluorescently labeled ligand, and quantifying unbound ligand in the clot supernatant (not shown). As listed in Table I, f-FPR-thrombin and f-heparin bind to fibrin with K d values of 1500 and 187 nm, respectively, values consistent with previous reports (8Hogg P.J. Jackson C.M. J. Biol. Chem. 1990; 265: 241-247Abstract Full Text PDF PubMed Google Scholar, 10Hogg 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 (50) Google Scholar). In contrast, f-DS does not bind to fibrin. Both heparin and DS bind to ANS-HCII, but the affinity of heparin for HCII is 5-fold higher (13 and 71 μm, respectively). Thus, DS binds to both HCII and thrombin with lower affinity than heparin. No binding of ANS-HCII to fibrin was detected (Table I). It has been shown previously that heparin enhances the binding of thrombin to fibrin, an effect that occurs regardless of whether heparin has high or low affinity for antithrombin, but occurring only with heparin chains of 11,200 Da or more (8Hogg P.J. Jackson C.M. J. Biol. Chem. 1990; 265: 241-247Abstract Full Text PDF PubMed Google Scholar). In this study, we compared the ability of DS to promote thrombin binding to FM-Sepharose with that of heparin and low-molecular-weight heparin. As shown in Fig.3, DS has little effect on125I-FPR-thrombin binding to FM-Sepharose, even at concentrations up to 10 μm. In contrast, at concentrations up to 250 nm, heparin enhances125I-FPR-thrombin binding to FM-Sepharose in a dose-dependent manner. At heparin concentrations above 250 nm, 125I-FPR-thrombin binding to fibrin monomer decreases, likely reflecting the accumulation of distinct heparin·fibrin and heparin·thrombin populations (8Hogg P.J. Jackson C.M. J. Biol. Chem. 1990; 265: 241-247Abstract Full Text PDF PubMed Google Scholar). When thrombin and FM-Sepharose were titrated with low-molecular-weight heparin (enoxaparin), there was only a small increase in the amount of thrombin bound, comparable to that observed with DS. Similar results were obtained with fibrin clots in place of FM-Sepharose (not shown). Our binding studies indicate that thrombin can interact with both fibrin and DS, via exosites I and II, respectively. These findings suggest that, like heparin, DS can form a three-component complex with thrombin and fibrin, even though DS does not bind fibrin. To explore this possibility, a mixture of 500 nm125I-labeled DS and 5 μm FM-Sepharose was titrated with increasing concentrations of thrombin or γ-thrombin (Fig.4). After centrifugation, residual125I-labeled DS in the supernatant was quantified and used to determine the fraction of 125I-labeled DS bound to FM-Sepharose. In the absence of thrombin, minimal amounts of125I-labeled DS bind to FM-Sepharose. With thrombin addition, 125I-labeled DS binds to FM-Sepharose in a concentration-dependent fashion. In contrast, when γ-thrombin, a proteolytic derivative of thrombin that lacks exosite 1, is substituted for thrombin, there is no increase in the amount of125I-labeled DS that bind" @default.
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• W2068600199 title "Molecular Basis for the Susceptibility of Fibrin-bound Thrombin to Inactivation by Heparin Cofactor II in the Presence of Dermatan Sulfate but Not Heparin" @default.
• W2068600199 cites W1487224455 @default.
• W2068600199 cites W1517896461 @default.
• W2068600199 cites W1518473002 @default.
• W2068600199 cites W1520224518 @default.
• W2068600199 cites W1542079164 @default.
• W2068600199 cites W1553129544 @default.
• W2068600199 cites W1585928558 @default.
• W2068600199 cites W1587033309 @default.
• W2068600199 cites W1833880006 @default.
• W2068600199 cites W1887504786 @default.
• W2068600199 cites W1972083038 @default.
• W2068600199 cites W1990715644 @default.
• W2068600199 cites W1994999842 @default.
• W2068600199 cites W1999450294 @default.
• W2068600199 cites W2002600260 @default.
• W2068600199 cites W2004821315 @default.
• W2068600199 cites W2006029112 @default.
• W2068600199 cites W2011085863 @default.
• W2068600199 cites W2015367504 @default.
• W2068600199 cites W2017757304 @default.
• W2068600199 cites W2028146514 @default.
• W2068600199 cites W2032834267 @default.
• W2068600199 cites W2033525600 @default.
• W2068600199 cites W2067327367 @default.
• W2068600199 cites W2085873519 @default.
• W2068600199 cites W2099813241 @default.
• W2068600199 cites W2102430103 @default.
• W2068600199 cites W2138849164 @default.
• W2068600199 cites W2343792664 @default.
• W2068600199 cites W2411245891 @default.
• W2068600199 cites W2411742966 @default.
• W2068600199 cites W2413015655 @default.
• W2068600199 cites W2417999300 @default.
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• W2068600199 cites W3170680033 @default.
• W2068600199 cites W4251417417 @default.
• W2068600199 cites W92034159 @default.
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