Heparin Modulates the 99-Loop of Factor IXa
2006; Elsevier BV; Volume: 281; Issue: 32 Linguagem: Inglês
10.1074/jbc.m603743200
ISSN1083-351X
AutoresPierre F. Neuenschwander, Stephen R. Williamson, Armen Nalian, Kimberly J. Baker-Deadmond,
Tópico(s)Heparin-Induced Thrombocytopenia and Thrombosis
ResumoReactivity of factor IXa with basic pancreatic trypsin inhibitor is enhanced by low molecular weight heparin (enoxaparin). Previous studies by us have suggested that this effect involves allosteric modulation of factor IXa. We examined the reactivity of factor IXa with several isolated Kunitz-type inhibitor domains: basic pancreatic trypsin inhibitor, the Kunitz inhibitor domain of protease Nexin-2, and the first two inhibitor domains of tissue factor pathway inhibitor. We find that enhancement of factor IXa reactivity by enoxaparin is greatest for basic pancreatic trypsin inhibitor (>10-fold), followed by the second tissue factor pathway inhibitor domain (1.7-fold) and the Kunitz inhibitor domain of protease Nexin-2 (1.4-fold). Modeling studies of factor IXa with basic pancreatic trypsin inhibitor suggest that binding of this inhibitor is sterically hindered by the 99-loop of factor IXa, specifically residue Lys98. Slow-binding kinetic studies support the formation of a weak initial enzyme-inhibitor complex between factor IXa and basic pancreatic trypsin inhibitor that is facilitated by enoxaparin binding. Mutation of Lys98 to Ala in factor IXa results in enhanced reactivity with all inhibitors examined, whereas almost completely abrogating the enhancing effects of enoxaparin. The results implicate Lys98 and the 99-loop of factor IXa in defining enzyme inhibitor specificity. More importantly, these results demonstrate the ability of factor IXa to be allosterically modulated by occupation of the heparin-binding exosite. Reactivity of factor IXa with basic pancreatic trypsin inhibitor is enhanced by low molecular weight heparin (enoxaparin). Previous studies by us have suggested that this effect involves allosteric modulation of factor IXa. We examined the reactivity of factor IXa with several isolated Kunitz-type inhibitor domains: basic pancreatic trypsin inhibitor, the Kunitz inhibitor domain of protease Nexin-2, and the first two inhibitor domains of tissue factor pathway inhibitor. We find that enhancement of factor IXa reactivity by enoxaparin is greatest for basic pancreatic trypsin inhibitor (>10-fold), followed by the second tissue factor pathway inhibitor domain (1.7-fold) and the Kunitz inhibitor domain of protease Nexin-2 (1.4-fold). Modeling studies of factor IXa with basic pancreatic trypsin inhibitor suggest that binding of this inhibitor is sterically hindered by the 99-loop of factor IXa, specifically residue Lys98. Slow-binding kinetic studies support the formation of a weak initial enzyme-inhibitor complex between factor IXa and basic pancreatic trypsin inhibitor that is facilitated by enoxaparin binding. Mutation of Lys98 to Ala in factor IXa results in enhanced reactivity with all inhibitors examined, whereas almost completely abrogating the enhancing effects of enoxaparin. The results implicate Lys98 and the 99-loop of factor IXa in defining enzyme inhibitor specificity. More importantly, these results demonstrate the ability of factor IXa to be allosterically modulated by occupation of the heparin-binding exosite. Factor IXa (fIXa) 2The abbreviations used are: FIXa, factor IXa; BPTI, basic pancreatic trypsin inhibitor; PN2-KPI, the isolated Kunitz-type inhibitor domain from protease nexin-2; TFPI, tissue factor pathway inhibitor; TFPI-K1, the isolated first Kunitz-type inhibitor domain of TFPI; TFPI-K2, the isolated second Kunitz-type inhibitor domain of TFPI; WT, wild-type; MES, 4-morpholineethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.2The abbreviations used are: FIXa, factor IXa; BPTI, basic pancreatic trypsin inhibitor; PN2-KPI, the isolated Kunitz-type inhibitor domain from protease nexin-2; TFPI, tissue factor pathway inhibitor; TFPI-K1, the isolated first Kunitz-type inhibitor domain of TFPI; TFPI-K2, the isolated second Kunitz-type inhibitor domain of TFPI; WT, wild-type; MES, 4-morpholineethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. is a vitamin K-dependent blood coagulation factor that is essential for the amplification or "consolidation" phase of blood coagulatin (1Neuenschwander P. Laurant G. Shapiro S. Encylopedia of Respiratory Medicine. Elsevier Ltd., Oxford, UK2006: 509-514Google Scholar, 2Jesty J. Nemerson Y. Beutler E. Lichtman M.A. Collar B.S. Kipps T.J. Williams Hematology. 5th Ed. McGraw-Hill, Inc., New York1995: 1227-1238Google Scholar). As with other blood coagulation factors (namely factors VIIa, Xa, and thrombin) fIXa is a member of the serine protease family and shares a high degree of homology with trypsin. Despite this homology, the blood coagulation enzymes differ drastically from trypsin in that their activities are profoundly modulated by the binding of various protein and non-protein cofactors. In the case of fIXa, the ability of activated factor VIII (fVIIIa), anionic phospholipid, and ionic calcium to enhance the procoagulant activity of fIXa is well documented (3van Dieijen G. Tans G. Rosing J. Hemker H.C. J. Biol. Chem. 1981; 256: 3433-3442Abstract Full Text PDF PubMed Google Scholar, 4van Dieijen G. van Rijn J.L. Govers-Riemslag J.W. Hemker H.C. Rosing J. Thromb. Haemostasis. 1985; 53: 396-400Crossref PubMed Scopus (34) Google Scholar, 5Mertens K. Bertina R.M. Biochem. J. 1984; 223: 607-615Crossref PubMed Scopus (35) Google Scholar, 6Mertens K. van Wijngaarden A. Bertina R.M. Thromb. Haemostasis. 1985; 54: 654-660Crossref PubMed Scopus (57) Google Scholar); resulting in a 109-fold increase in activity of fIXa. The molecular details of this conversion have not been defined in total and are the subject of intense investigation by numerous groups.The major inhibitor of fIXa in plasma is antithrombin, whose reactivity with fIXa essentially requires heparin (7Rosenberg J.S. McKenna P.W. Rosenberg R.D. J. Biol. Chem. 1975; 250: 8883-8888Abstract Full Text PDF PubMed Google Scholar, 8Kurachi K. Fujikawa K. Schmer G. Davie E.W. Biochemistry. 1976; 15: 373-377Crossref PubMed Scopus (110) Google Scholar, 9DiScipio R.G. Kurachi K. Davie E.W. J. Clin. Investig. 1978; 60: 1528-1538Crossref Scopus (173) Google Scholar). Heparin is known to bind to antithrombin and sterically alter its conformation to allow this serpin to react with its target (10Villaneuva G.B. Danishefsky I. Biochem. Biophys. Res. Commun. 1977; 74: 803-809Crossref PubMed Scopus (92) Google Scholar, 11Villaneuva G.B. J. Biol. Chem. 1984; 259: 2531Abstract Full Text PDF PubMed Google Scholar, 12Olson S.T. Björk I. Adv. Exp. Med. Biol. 1992; 313: 155-165Crossref PubMed Scopus (34) Google Scholar, 13Olson 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). Heparin also binds to fIXa (14Yang L.K. Manithody C. Rezaie A.R. J. Biol. Chem. 2002; 277: 50756-50760Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) allowing long chains of heparin to additionally catalyze the interaction of fIXa with antithrombin via the formation of bridged complexes where heparin acts as a "template." Recently, we have shown that low molecular weight heparin binding to fIXa enhances reactivity of fIXa with the Kunitz-type inhibitor BPTI (15Neuenschwander P.F. Biochemistry. 2004; 43: 2978-2986Crossref PubMed Scopus (13) Google Scholar), suggesting that oligosaccharide binding can also allosterically modulate the fIXa active site region. In this study we examine in greater detail the ability of heparin to modulate fIXa reactivity toward several isolated Kunitz-type inhibitor domains. We show that the modulatory effect of heparin can be completely abrogated by mutating a single amino acid residue in the 99-loop region of the extended fIXa active site cleft outside of the heparin binding exosite.EXPERIMENTAL PROCEDURESMaterials—Factor IXaβ, factor VIIa, factor XIa, and the factor X activator from Russell's Viper venom were purchased from Hematologic Technologies Inc. (Essex Junction, VT). Recombinant soluble tissue factor (the extracellular domain of tissue factor) was expressed and purified from bacteria as previously described (16Rezaie A.R. Fiore M.M. Neuenschwander P.F. Esmon C.T. Morrissey J.H. Protein Expression Purif. 1992; 3: 453-460Crossref PubMed Scopus (77) Google Scholar). Factor Xa was prepared from plasma-derived factor X as previously described (17Neuenschwander P.F. Morrissey J.H. J. Biol. Chem. 1994; 269: 8007-8013Abstract Full Text PDF PubMed Google Scholar). Enoxaparin (Lovenox®) was purchased from Aventis Pharmaceuticals (Bridgewater, NJ). Purified heparin-derived oligosaccharides of 6, 10, 14, and 18 saccharide units (H6, H10, H14, and H18) were prepared and characterized essentially as described (18Olson S.T. Björk I. Shore J.D. Lorand L. Mann K.G. Methods in Enzymology. Academic Press, New York1993: 525-559Google Scholar, 19Lindahl U. Thunberg L. Bäckström G. Riesenfeld J. Nordling K. Björk I. J. Biol. Chem. 1984; 259: 12368-12376Abstract Full Text PDF PubMed Google Scholar, 20Olson S.T. J. Biol. Chem. 1988; 263: 1698-1708Abstract Full Text PDF PubMed Google Scholar) and were a generous gift of Dr. Steven T. Olson, University of Illinois, Chicago, IL. Bovine serum albumin (Fraction V, fatty acid free) was from Calbiochem (La Jolla, CA), and ethylene glycol was from Fisher Scientific. The chromogenic substrate CBS 31.39 (CH3SO2-d-LGR-pNA) was purchased from Diagnostica Stago (Parsippany, NJ). All other reagents were of the highest quality available.Construction and Expression of Recombinant Inhibitors—Appropriate expression clones encoded for: BPTI (59 amino acids) (21Anderer F.A. Z. Naturforsch. B. 1965; 20: 462-472Crossref PubMed Scopus (2) Google Scholar, 22Anderer F.A. Hoernle S. Z. Naturforsch. B. 1965; 20: 457-462Crossref PubMed Scopus (10) Google Scholar), PN2-KPI (61 amino acids corresponding to residues 285-344 of Protease Nexin-2) (23Wagner S.L. Siegel R.S. Vedvick T.S. Raschke W.C. Van Nostrand W.E. Biochem. Biophys. Res. Commun. 1992; 186: 1138-1145Crossref PubMed Scopus (59) Google Scholar), TFPI-K1 (58 amino acids corresponding to residues 50-107 of TFPI) (24Wun T.C. Kretzmer K.K. Girard T.J. Miletich J.P. Broze Jr., G.J. J. Biol. Chem. 1988; 263: 6001-6004Abstract Full Text PDF PubMed Google Scholar), and TFPI-K2 (59 amino acids corresponding to residues 121-178 of TFPI) (24Wun T.C. Kretzmer K.K. Girard T.J. Miletich J.P. Broze Jr., G.J. J. Biol. Chem. 1988; 263: 6001-6004Abstract Full Text PDF PubMed Google Scholar). Each construct was directionally cloned into pET11a (Novagen) and verified by sequencing. Inhibitors were expressed as inclusion bodies in Escherichia coli strain BL21(DE3). Transformed bacterial cells were first grown to log phase at 37 °C in TB media containing 50 μg/ml carbenicillin. Protein expression was induced by addition of isopropyl 1-thio-β-d-galactopyranoside to 0.5 mm (0.1 mm for TFPI-K1) and the cells were allowed to continue growing for 4 h at 37°C.Inclusion bodies were isolated essentially as described (25Steinle A. Li P. Morris D.L. Groh V. Lanier L.L. Strong R.K. Spies T. Immunogenetics. 2001; 53: 279-287Crossref PubMed Scopus (388) Google Scholar) and solubilized with 6 m guanidine HCl containing 20 mm dithiothreitol, 50 mm Tris-HCl, pH 8.0, and 1 mm EDTA to obtain a total protein concentration of roughly 20 mg/ml. The solution was then clarified by centrifugation (16,000 × g for 30 min) and oxidative refolding (26De Bernardez Clark E. Hevehan D. Szela S. Maachupalli-Reddy J. Biotechnol. Prog. 1998; 14: 47-54Crossref PubMed Scopus (115) Google Scholar) of each protein preparation was performed by rapid dilution into 20 volumes of buffer containing 50 mm Tris-HCl, pH 8.0, 1 m guanidine HCl, 1 mm EDTA, 2.5 mm oxidized glutathione (Sigma), and 1 mm dithiothreitol. The diluted protein solution was incubated at room temperature for 6 h with slow stirring for completion of protein refolding followed by exhaustive dialysis into an appropriate buffer for ionexchange chromatography.Construction and Expression of Wild-type and Mutant fIX—The coding sequence for wild-type fIX in pBR322 (27Kurachi K. Davie E.W. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 6461-6464Crossref PubMed Scopus (294) Google Scholar) was a generous gift of Dr. Earl Davie (University of Washington). The fIX coding sequence was removed into the mammalian expression vector pcDNA3 (Invitrogen) and sequenced to verify the correct orientation. This construct (pFN04) was used for expression of wild-type fIX as well as PCR-based mutagenesis (28Horton R.M. Cai Z. Ho S.N. Please L.R. BioTechniques. 1990; 8: 528-535PubMed Google Scholar) to generate fIXK98A essentially as previously described for constructing fVII mutants (29Neuenschwander P.F. Morrissey J.H. Biochemistry. 1995; 34: 8701-8707Crossref PubMed Scopus (33) Google Scholar). Expression constructs were transfected into human 293 cells using Lipofectin ® (Invitrogen) and high expressing clones isolated by limiting dilution.Protein Purifications—Purification of refolded BPTI was accomplished by ion-exchange chromatography using Mono-S HR 5/5 (Amersham Biosciences) in 20 mm Tris-HCl, pH 8.0. The column was developed with a 0-1 m NaCl gradient and BPTI eluted as a single peak at roughly 0.43 m NaCl. The specific activity of recombinant BPTI preparations was equivalent to or better than that of commercial preparations of aprotinin (not shown). Purification of refolded PN2-KPI and TFPI-K2 was accomplished by ion-exchange chromatography using Mono-Q HR 5/5 (Amersham Biosciences) in 20 mm MES, pH 6.0. In both cases the column was developed with a 0-0.5 m NaCl gradient. PN2-KPI eluted at roughly 90 mm NaCl and TFPI-K2 eluted at roughly 50 mm NaCl. Purification of refolded TFPI-K1 was accomplished by affinity chromatography over a trypsin-agarose column. Trypsin-agarose was prepared by coupling 20 mg of l-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (Worthington Biochemicals) to 2.5 ml of Affi-Gel 10 in 100 mm MOPS, pH 7.4, 10 mm CaCl2, and 100 μg/ml leupeptin overnight at 4 °C. This was followed by blocking non-reacted sites with 1 m ethanolamine-HCl, pH 8.0. Refolded TFPI-K1 was loaded onto the prepared trypsin-agarose column in 50 mm Tris-HCl, pH 7.5, 100 mm NaCl. The column was extensively washed with the same buffer before eluting the inhibitor with 10 mm HCl, pH 2.1, 100 mm NaCl. The pH of the eluted fractions was immediately neutralized with 0.02 volumes of 2 m Tris buffer. All inhibitors were judged >95% pure by SDS-PAGE.Recombinant human wild-type fIX and fIXK98A were isolated from 293 cell supernatants using a combination of ionexchange and heparin affinity chromatography. A 10-fold concentrate of cell supernatant was diluted 2-fold with deionized water to reduce the ionic strength before loading a 150-ml DEAE FF Sepharose (Amersham Biosciences) column equilibrated in 25 mm sodium citrate, pH 6.0, 33 mm NaCl, and 1 mm benzamidine. After loading, the column was extensively washed in the same buffer before elution of the fIX protein with a 0.033-0.4 m NaCl gradient over 10 column volumes. The fIX protein peak was identified by clotting activity, pooled, and dialyzed versus 50 mm Tris-HCl, pH 7.5, 100 mm NaCl before heparin affinity chromatography using either POROS ® HE2 (Applied Biosystems) or HiPrep™ Heparin FF 16/10 (Amersham Biosciences) and eluting with a NaCl gradient. Wild-type fIX and fIXK98A both eluted as single peaks at roughly 0.46 m NaCl.Wild-type and mutant fIX proteins were activated with the purified factor X activator from the venom of Russell's viper, which also cleaves fIX after Arg180 to generate active enzyme (fIXaα). The activated enzyme was purified away from the venom protease by subsequent heparin affinity chromatography essentially as described above using HiTrap™ Heparin HP (Amersham Biosciences). Although the activation peptide remains attached to the light chain of fIXaα, this enzyme retains 100% amidolytic activity compared with fIXaβ (activation peptide proteolytically removed) and is comparable with fIXaβ in kinetics of inhibition by Kunitz-type inhibitors (see "Results"). Unless indicated otherwise, the fIXaα form was used in experiments.Clotting Assays—Coagulant activities of wild-type and mutant fIXa proteins were assayed by a standard single-stage clotting assay using a Coag-a-mate XM (Organon Teknika) coagulometer, fIX-deficient plasma (George King Biomedical), and APTT Reagent (Sigma).Reactive Site Titration of Inhibitors—The active concentration of inhibitor preparations was determined by reactive site titration essentially as described (23Wagner S.L. Siegel R.S. Vedvick T.S. Raschke W.C. Van Nostrand W.E. Biochem. Biophys. Res. Commun. 1992; 186: 1138-1145Crossref PubMed Scopus (59) Google Scholar) using 10 nm active site-titrated trypsin (30Chase Jr., T. Shaw E. Biochem. Biophys. Res. Commun. 1967; 29: 508-514Crossref PubMed Scopus (779) Google Scholar) and S-2222 substrate (Chromogenix, Milano, Italy) to measure residual trypsin activity after a 15-min incubation period. This method assumes a 1:1 stoichiometry of inhibitor and trypsin. Amino acid analysis performed on an initial PN2-KPI preparation indicated an equivalent concentration as that determined by reactive site titration (not shown).Active Site Titration of fIXa Enzymes—Active concentrations of wild-type and mutant fIXa preparations were determined by active site titration using biotin-EGR-ck (Hematologic Technologies Inc.) essentially as described (31Mann K.G. Williams E.B. Krishnaswamy S. Church W. Giles A. Tracy R.P. Blood. 1990; 76: 755-766Crossref PubMed Google Scholar). Briefly, wild-type or mutant fIXa (roughly 5 μm) were incubated with 150 μm biotin-EGR-ck in 50 mm Tricine, pH 8.0, 200 mm NaCl, 10 mm CaCl2, and 30% ethylene glycol for 24 h at room temperature. Biotin-EGR-fIXa was then quantified by enzyme-linked immunosorbent assay using a goat anti-fIX polyclonal capture antibody and alkaline phosphatase-conjugated streptavidin. These were compared with a standard line made using native fIXaβ to determine active concentrations for all fIXa enzyme preparations.Equilibrium Enzyme Inhibition Assays—Reversible competitive inhibition of fIXa amidolytic activity at equilibrium was measured at 25 °C using final concentrations of 25 nm fIXa, 5 mm CaCl2 and the indicated concentrations of enoxaparin and inhibitor in the presence of 30% ethylene glycol as previously described (15Neuenschwander P.F. Biochemistry. 2004; 43: 2978-2986Crossref PubMed Scopus (13) Google Scholar). The addition of ethylene glycol enhances the enzymatic activity of fIXa toward CBS 31.39 roughly 20-fold (32Neuenschwander P.F. McCollough J. McCallum C.D. Johnson A.E. Thromb. Haemostasis. 1997; 78: 428Google Scholar). Previous studies by us as well as control experiments performed with each inhibitor examined showed that the inclusion of ethylene glycol in the assay has no effect on the reactivity of fIXa with these inhibitors (15Neuenschwander P.F. Biochemistry. 2004; 43: 2978-2986Crossref PubMed Scopus (13) Google Scholar). Values of the final equilibrium inhibition constant (Ki,eq) were estimated as described (15Neuenschwander P.F. Biochemistry. 2004; 43: 2978-2986Crossref PubMed Scopus (13) Google Scholar, 29Neuenschwander P.F. Morrissey J.H. Biochemistry. 1995; 34: 8701-8707Crossref PubMed Scopus (33) Google Scholar) using Equation 1. This equation describes simple reversible competitive inhibition where vo is the steady state rate obtained in the absence of inhibitor, vs is the steady state rate at each concentration of inhibitor, S is the experimental substrate concentration, and Km is the Michaelis constant for substrate hydrolysis.Vs=VoKi,eq(1+S/Km)I+Ki,eq(1+S/Km)(Eq. 1) Slow-binding Enzyme Inhibition Analysis—Kinetic parameters for slow-binding inhibition were obtained using Equations 2-5 as derived by others (33Morrison J.F. Walsh C.T. Adv. Enzymol. 1988; 61: 201-301PubMed Google Scholar, 34Copeland R.A. Enzymes: A Practical Introduction to Structure, Mechanism, and Data Analysis. VCH Publishers, Inc., New York1996: 225-261Google Scholar) and are described in Schemes Ia and Ib (Fig. 1). In both of these schemes the non-covalent enzyme-inhibitor complex, EI, isomerizes into EI*; either after significant formation of the EI complex (Scheme Ia) or without significant accumulation of the EI complex (Scheme Ib). In both cases, the overall inhibition constant describing generation of EI* is defined as Ki*, which is equivalent to Ki,eq obtained from equilibrium experiments (above). In cases following Scheme Ia, the parameter Ki* can be further broken down to obtain Ki, which describes the establishment of the initial "loose" EI complex, k5 and k6 (see below).Final reaction conditions were the same as described above for equilibrium studies. In these assays, however, the fIXa was preincubated with or without enoxaparin for 15 min at 25 °C in the reaction mixture before the combined addition of inhibitor and substrate at time 0. The absorbance at 405 nm was then monitored for up to 30 min in a Thermo-Max microplate reader (Molecular Devices) set at 25 °C to monitor substrate hydrolysis using KINEMAX software (written and kindly provided by Dr. Jolyon Jesty, SUSB, Stony Brook, NY). Data for each generated curve were fitted with the following integrated rate equation describing slow-binding inhibition,A=Vst+(Vo-Vs)(1-e-kobst)/kobs+Ao(Eq. 2) where A is the absorbance at 405 nm at any time, t. Fits of progress curves with Equation 2 yield values for Ao (the initial absorbance at t = 0), vo (the initial rate of substrate hydrolysis), vs (the steady-state rate of substrate hydrolysis), and kobs (the apparent first-order rate constant for inhibition).For analyses using Scheme Ia, values of k6 (the reverse rate constant for EI* isomerization) were determined from progress curves above using the following relationship.k6ork10=kobsVs/Vo(Eq. 3) Values of k5 and initial Ki (defined as k4/k3) were then obtained from secondary plots of kobs versus I using the following hyperbolic equation.kobs=k6+k5I/(I+Ki(1+S/Km))(Eq. 4) For analyses using Scheme Ib, values of k10 were obtained from progress curves also using Equation 3. However, in these cases vo does not vary with inhibitor concentration and a plot of kobs versus I yields a straight line, indicating conditions where Ki (1+ S/Km) >> I. Thus for Scheme Ib EI formation is insignificant and EI* can be considered formed directly from E + I. For these cases the following linear equation is applicable for obtaining an estimate of k9, the apparent second-order on-rate constant,kobs=k10+k9I/(1+S/Km)(Eq. 5) where the y intercept reflects k10 and the slope of the line is equal to k9/(1 + S/Km). Alternatively, k9 can be obtained from Ki*, which is equivalent to Ki,eq in Equation 1, using the relationship k9 = k10/Ki*.Although fits with Equations 4 or 5 yield estimates of k6 or k10, respectively, the values reported herein were obtained from Equation 3 using the more accurate fits of progress curves to Equation 2 and then verified in fits with Equations 4 or 5. Experimental values of S as well as experimentally determined values of Km (defined in the traditional manner as (k2 + k7)/k1 in Schemes Ia and Ib) were used as necessary in all fitting procedures. All regression procedures were performed using Slide-WritePlus 6.0 (Advanced Graphics Software), which uses the Levenberg-Marquardt algorithm.RESULTSPrevious studies (15Neuenschwander P.F. Biochemistry. 2004; 43: 2978-2986Crossref PubMed Scopus (13) Google Scholar) by us have shown that whereas fIXa is resistant to inhibition by the Kunitz-type inhibitor BPTI, this resistance is somewhat alleviated by enoxaparin, leading to a roughly 10-fold enhancement in the equilibrium inhibition constant. To gain further insight into mechanisms of fIXa selectivity and its modulation by heparin we undertook an examination of the reactivity of fIXa with several isolated Kunitz-type inhibitor domains: BPTI, PN2-KPI, TFPI-K1, and TFPI-K2. Each of these inhibitors was expressed in E. coli using standard recombinant techniques, purified to homogeneity and quantified by reactive site titration as described under "Experimental Procedures." The isolated PN2-KPI inhibitor domain was found to react with high affinity toward factor XIa, yielding a Ki,eq of roughly 400 pm (see supplemental data). Similarly, preliminary studies indicated that preparations of isolated TFPI-K1 domain inhibited the complex of factor VIIa and soluble tissue factor with high affinity (Ki,eq = 400 nm) and weakly inhibited factor Xa (Ki,eq > 1.5 μm). Conversely, the isolated TFPI-K2 domain inhibited factor Xa with high affinity (Ki,eq = 24 nm) and the factor VIIa-tissue factor complex with reduced affinity (Ki,eq = 7 μm). These results are consistent with the expected reactivity of the isolated inhibitor domains (35Van Nostrand W.E. Wagner S.L. Farrow J.S. Cunningham D.S. J. Biol. Chem. 1990; 265: 9591-9594Abstract Full Text PDF PubMed Google Scholar, 36Petersen L.C. Bjorn S.E. Olsen O.H. Nordfang O. Norris F. Norris K. Eur. J. Biochem. 1996; 235: 310-316Crossref PubMed Scopus (76) Google Scholar) and demonstrate the correct folding and inhibitor activity of the inhibitors examined.The abilities of these isolated Kunitz inhibitor domains to inhibit fIXaβ are compared in Fig. 2. As expected, fIXa exhibited remarkable specificity toward these inhibitors despite their high homology. Of the inhibitors examined, PN2-KPI showed the highest level of reactivity (Ki,eq = 10 μm), followed by TFPI-K2 (Ki,eq = 336 μm), BPTI (Ki,eq > 500 μm), and TFPI-K1 (Ki,eq > 1mm). Consistent with our previous observations, enoxaparin was able to enhance the reactivity of fIXa with BPTI more than 10-fold (Ki,eq = 46 μm). Surprisingly, however, this same level of enhancement by enoxaparin was not observed with any of the other inhibitors examined; TFPI-K2 and PN2-KPI each showed only a small, but consistent, enhancement in reactivity with enoxaparin (1.7- and 1.4-fold, respectively; Ki,eq values of 203 and 7 μm) and TFPI-K1 showed no measurable enhancement in reactivity with enoxaparin.FIGURE 2Inhibition of fIXaβ by isolated Kunitz-type inhibitor domains. FIXaβ (25 nm) was titrated with the indicated concentrations of BPTI (○), TFPI-K1 (•), TFPI-K2 (□), and PN2-KPI (▪). The remaining fIXa amidolytic activity at equilibrium was then measured and values were normalized to the activity of fIXa in the absence of inhibitor (100%). Data were fitted with Equation 1 (lines) to obtain values of Ki,eq (see text).View Large Image Figure ViewerDownload Hi-res image Download (PPT)The highly basic nature of BPTI compared with the other isolated Kunitz domains along with its ability to bind to heparin (albeit weakly; Kd = 172 μm (15Neuenschwander P.F. Biochemistry. 2004; 43: 2978-2986Crossref PubMed Scopus (13) Google Scholar)) raised the potential that enoxaparin, although short (15 saccharide units; H15), may retain some capacity to facilitate the interaction of BPTI with fIXa via a bridging-type mechanism. Although unlikely based on previous equilibrium kinetic studies and the level of enoxaparin used in these experiments (10 μm; or 0.06 × Kd for BPTI binding versus 78 × Kd for fIXa binding), this issue was examined by using increasing concentrations of enoxaparin as well as progressively smaller heparin oligosaccharides; H18, H14, H10, and H6 (18Olson S.T. Björk I. Shore J.D. Lorand L. Mann K.G. Methods in Enzymology. Academic Press, New York1993: 525-559Google Scholar, 19Lindahl U. Thunberg L. Bäckström G. Riesenfeld J. Nordling K. Björk I. J. Biol. Chem. 1984; 259: 12368-12376Abstract Full Text PDF PubMed Google Scholar, 20Olson S.T. J. Biol. Chem. 1988; 263: 1698-1708Abstract Full Text PDF PubMed Google Scholar). As shown in Fig. 3, the typical bell-shaped profile for bridging-type mechanisms was not observed at enoxaparin concentrations ranging from 1 nm to >100 μm. In addition, and of greater significance, is the observation that progressively smaller oligosaccharides do not lose the ability to enhance reactivity of fIXa. These results along with previous kinetic studies support the ability of heparin to modulate fIXa reactivity via a mechanism other than bridging, and are consistent with allosteric modulation of the fIXa protease domain.FIGURE 3Support of BPTI inhibition of fIXa by short chain oligosaccharides. The ability of heparin oligosaccharides with progressively shorter chain lengths to support enhancement of fIXa reactivity with BPTI was examined using 25 nm fIXaβ, 308 μm BPTI, and the indicated concentrations of heparin-derived oligosaccharides (18Olson S.T. Björk I. Shore J.D. Lorand L. Mann K.G. Methods in Enzymology. Academic Press, New York1993: 525-559Google Scholar, 19Lindahl U. Thunberg L. Bäckström G. Riesenfeld J. Nordling K. Björk I. J. Biol. Chem. 1984; 259: 12368-12376Abstract Full Text PDF PubMed Google Scholar, 20Olson S.T. J. Biol. Chem. 1988; 263: 1698-1708Abstract Full Text PDF PubMed Google Scholar) from 0.01 to 200 μm. Saccharide chain lengths were H18 (•), H14 (□), H10 (▪), and H6 (▿). Enoxaparin (H15) is shown for comparison (○). The fIXa activities were normalized to the activity of fIXa in the absence of added oligosaccharide.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To gain further insight into the potential mechanism of heparin modulation of fIXa, we prepared a rudimentary hypothetical mo
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