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• W4229675254 abstract "Incorporation of prothrombin into the prothrombinase complex is essential for rapid thrombin generation at sites of vascular injury. Prothrombin binds directly to anionic phospholipid membrane surfaces where it interacts with the enzyme, factor Xa, and its cofactor, factor Va. We demonstrate that HD1, a thrombin-directed aptamer, binds prothrombin and thrombin with similar affinities (Kd values of 86 and 34 nm, respectively) and attenuates prothrombin activation by prothrombinase by over 90% without altering the activation pathway. HD1-mediated inhibition of prothrombin activation by prothrombinase is factor Va-dependent because (a) the inhibitory activity of HD1 is lost if factor Va is omitted from the prothrombinase complex and (b) prothrombin binding to immobilized HD1 is reduced by factor Va. These data suggest that HD1 competes with factor Va for prothrombin binding. Kinetic analyses reveal that HD1 produces a 2-fold reduction in the kcat for prothrombin activation by prothrombinase and a 6-fold increase in the Km, highlighting the contribution of the factor Va-prothrombin interaction to prothrombin activation. As a high affinity, prothrombin exosite 1-directed ligand, HD1 inhibits prothrombin activation more efficiently than Hir54–65(SO-3). These findings suggest that exosite 1 on prothrombin exists as a proexosite only for ligands whose primary target is thrombin rather than prothrombin. Incorporation of prothrombin into the prothrombinase complex is essential for rapid thrombin generation at sites of vascular injury. Prothrombin binds directly to anionic phospholipid membrane surfaces where it interacts with the enzyme, factor Xa, and its cofactor, factor Va. We demonstrate that HD1, a thrombin-directed aptamer, binds prothrombin and thrombin with similar affinities (Kd values of 86 and 34 nm, respectively) and attenuates prothrombin activation by prothrombinase by over 90% without altering the activation pathway. HD1-mediated inhibition of prothrombin activation by prothrombinase is factor Va-dependent because (a) the inhibitory activity of HD1 is lost if factor Va is omitted from the prothrombinase complex and (b) prothrombin binding to immobilized HD1 is reduced by factor Va. These data suggest that HD1 competes with factor Va for prothrombin binding. Kinetic analyses reveal that HD1 produces a 2-fold reduction in the kcat for prothrombin activation by prothrombinase and a 6-fold increase in the Km, highlighting the contribution of the factor Va-prothrombin interaction to prothrombin activation. As a high affinity, prothrombin exosite 1-directed ligand, HD1 inhibits prothrombin activation more efficiently than Hir54–65(SO-3). These findings suggest that exosite 1 on prothrombin exists as a proexosite only for ligands whose primary target is thrombin rather than prothrombin. HD1, a thrombin-directed aptamer, binds exosite 1 on prothrombin with high affinity and inhibits its activation by prothrombinase.Journal of Biological ChemistryVol. 290Issue 8PreviewVOLUME 281 (2006) PAGES 37477–37485 Full-Text PDF Open Access Thrombin is the most versatile component of the hemostatic system, mediating procoagulant, anticoagulant, and anti-fibrinolytic pathways (1Di Cera E. Chest. 2003; 124: 11S-17SAbstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). The diverse activities of thrombin are regulated, at least in part, by electropositive exosites flanking its active site (2Lane D.A. Philippou H. Huntington J.A. Blood. 2005; 106: 2605-2612Crossref PubMed Scopus (271) Google Scholar). Exosite 1 binds ligands that interact with the active site of thrombin, including fibrinogen, heparin cofactor II, and protease-activated receptor, the major thrombin receptor on cells (2Lane D.A. Philippou H. Huntington J.A. Blood. 2005; 106: 2605-2612Crossref PubMed Scopus (271) Google Scholar). In contrast, exosite 2, which binds ligands such as heparin (3Fortenberry Y.M. Whinna H.C. Gentry H.R. Myles T. Leung L.L. Church F.C. J. Biol. Chem. 2004; 279: 43237-43244Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 4De Cristofaro R. De C ia E. Landolfi R. Circulation. 1998; 98: 1297-1301Crossref PubMed Scopus (29) Google Scholar) and platelet glycoprotein Ibα (5Adam F. Bouton M.C. Huisse M.G. Jandrot-Perrus M. Trends Mol. Med. 2003; 9: 461-464Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 7De Cristofaro R. De C ia E. Rutella S. Weitz J.I. J. Biol. Chem. 2000; 275: 3887-3895Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), serves to tether thrombin for subsequent interactions with substrates or inhibitors. Prothrombin, the precursor of thrombin, lacks an active site and has immature or inaccessible exosites (8Anderson P.J. Nesset A. Dharmawardana K.R. Bock P.E. J. Biol. Chem. 2000; 275: 16428-16434Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 10Kaczmarek E. Kaminski M. McDonagh J. Biochim. Biophys. Acta. 1987; 914: 275-282Crossref PubMed Scopus (9) Google Scholar). Because exosite 1 on prothrombin exhibits reduced affinity for certain ligands, it has been designated proexosite 1 (8Anderson P.J. Nesset A. Dharmawardana K.R. Bock P.E. J. Biol. Chem. 2000; 275: 16428-16434Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). This proexosite gains functional activity during prothrombin conversion to thrombin, as evidenced by fluorescent ligand binding studies (11Anderson P.J. Bock P.E. J. Biol. Chem. 2003; 278: 44489-44495Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Thus, Anderson and Bock (11Anderson P.J. Bock P.E. J. Biol. Chem. 2003; 278: 44489-44495Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar) reported that fluorescein-hirudin54–65(SO-3) (f-Hir54–65(SO-3)), 2The abbreviations used are: Hir54–65(SO-3), Tyr63-sulfated COOH-terminal hirudin peptide amino acids 54–65; FITC, fluorescein isothiocyanate; PCPS, l-α-phosphatidyl-choline and l-α-phosphatidyl-l-serine; pre1, prethrombin 1; pre2, prethrombin 2; mIIa, meizothrombin; mIIa(-F1), meizothrombin des F1; F1, prothrombin fragment 1; F2, prothrombin fragment 2; b-, biotin-; f-, fluorescein-; DAPA, dansylarginine N-(3-ethyl-1,5-pentanediyl)amide; Chz-Th, chromozym thrombin. the exosite 1-binding COOH terminus of hirudin, binds thrombin with an affinity 130-fold higher than that for prothrombin (Kd values of 25 nm and 3.2 μm, respectively). Prothrombin activation intermediates display intermediate affinities for f-Hir54–65(SO-3) that increase with the extent of activation (11Anderson P.J. Bock P.E. J. Biol. Chem. 2003; 278: 44489-44495Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 12Anderson P.J. Nesset A. Bock P.E. J. Biol. Chem. 2003; 278: 44482-44488Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Diminished affinity of other thrombin ligands for proexosite 1 on prothrombin also has been observed (13Fischer B.E. Schlokat U. Himmelspach M. Dorner F. Protein Eng. 1998; 11: 715-721Crossref PubMed Scopus (12) Google Scholar, 14Wu Q. Picard V. Aiach M. Sadler J.E. J. Biol. Chem. 1994; 269: 3725-3730Abstract Full Text PDF PubMed Google Scholar). In contrast to the progressive maturation of proexosite 1, exosite 2 displays more abrupt development. Exosite 2 is not accessible until fragment 2 (F2) is released from prothrombin. Thus, prethrombin 2 (pre2) and thrombin have similar affinities for heparin, whereas meizothrombin (mIIa) and meizothrombin des F1 [mIIa(-F1)], which retain the F2 domain, do not bind heparin (15Schoen P. Lindhout T. J. Biol. Chem. 1987; 262: 11268-11274Abstract Full Text PDF PubMed Google Scholar). Understanding the functional maturation of the exosites on thrombin has increased in importance with emerging evidence that the exosites serve not only as binding domains but also as allosteric regulators of thrombin activity (16Mengwasser K.E. Bush L.A. Shih P. Cantwell A.M. Di Cera E. J. Biol. Chem. 2005; 280: 26997-27003Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 18Fredenburgh J.C. Stafford A.R. Weitz J.I. J. Biol. Chem. 1997; 272: 25493-25499Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Numerous studies reveal that ligand binding to either exosite can modify the activity of thrombin. Thus, peptide (16Mengwasser K.E. Bush L.A. Shih P. Cantwell A.M. Di Cera E. J. Biol. Chem. 2005; 280: 26997-27003Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 19Hortin G.L. Trimpe B.L. J. Biol. Chem. 1991; 266: 6866-6871Abstract Full Text PDF PubMed Google Scholar), glycosaminoglycan (20Pike R.N. Buckle A.M. Le Bonniec B.F. Church F.C. FEBS J. 2005; 272: 4842-4851Crossref PubMed Scopus (109) Google Scholar, 21Cosmi B. Cini M. Legnani C. Pancani C. Calanni F. Coccheri S. Thromb. Res. 2003; 109: 333-339Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), and nucleotide (22Griffin L.C. Toole J.J. Leung L.L.K. Gene. 1993; 137: 25-31Crossref PubMed Scopus (49) Google Scholar) ligands have all been shown to modulate thrombin. Thrombin-binding DNA aptamers represent a unique class of ligand. These engineered oligonucleotides, which fold into characteristic secondary structures, form binding pockets for specific ligands (23Mao X.A. Gmeiner W.H. Biophys. Chem. 2005; 113: 155-160Crossref PubMed Scopus (39) Google Scholar, 24Schultze P. Macaya R.F. Feigon J. J. Mol. Biol. 1994; 235: 1532-1547Crossref PubMed Scopus (349) Google Scholar). In the case of thrombin, aptamer HD1 binds selectively to exosite 1 (25Bock L. Griffin L.C. Latham J.A. Vermaas E.H. Toole J.J. Nature. 1992; 355: 564-566Crossref PubMed Scopus (2117) Google Scholar), whereas aptamer HD22 binds to exosite 2 (26Dougan H. Weitz J.I. Stafford A.R. Gillespie K.D. Klement P. Hobbs J.B. Lyster D.M. Nucl. Med. Biol. 2003; 30: 61-72Crossref PubMed Scopus (31) Google Scholar). Because of their high affinity and selective binding, HD1 and HD22 serve as useful tools to probe the structure-function relationship of the exosites on thrombin. The crystal structure of the thrombin-HD1 complex has been defined (27Padmanabhan K. Padmanabhan K.P. Ferrara J.D. Sadler J.E. Tulinsky A. J. Biol. Chem. 1993; 268: 17651-17654Abstract Full Text PDF PubMed Google Scholar), as has the structure of the thrombin-Hir54–65(SO-3) complex (28Vijayalakshmi J. Padmanabhan K.P. Mann K.G. Tulinsky A. Protein Sci. 1994; 3: 2254-2271Crossref PubMed Scopus (152) Google Scholar). These structures suggest that the two exosite 1-directed ligands bind to overlapping, but discrete, subdomains. Thus, Hir54–65(SO-3) largely interacts with the hydrophobic cleft of exosite 1, whereas HD1 binds to charged residues surrounding this cleft. Given their distinct binding sites on thrombin, it is possible that HD1 and Hir54–65(SO-3) do not exhibit identical interactions with prothrombin and its intermediates. To explore this possibility, we used HD1 and Hir54–65(SO-3) to examine the functional maturation of exosite 1 and HD22, the exosite 2-binding DNA aptamer, to report exosite 2 maturation. Reagents—Human prothrombin, thrombin, and factor Xa were obtained from Enzyme Research Laboratories, Inc. (South Bend, IN). Factor Va and dansylarginine N-(3-ethyl-1,5-pentanediyl) amide (DAPA) were from Hematologic Technologies, Inc (Essex Junction, VT). d-Phe-Pro-Arg chloromethyl ketone and 1,5-dansyl-Glu-Gly-Arg chloromethyl ketone were obtained from Calbiochem. Fluorescein isothiocyanate (FITC) was from Sigma. HD1 (5′-GGTTGGTGTGGTTGG-3′), HD22 (5′-AGTCCGTGGTGGTAGGGCAGGTTGGGGTGACT-3′), the exosite 1 and 2-directed DNA aptamers, respectively, and HD23 (5′-AGTCCGTAAAGCAGGTTAAAATGACT-3′), a scrambled oligonucleotide sequence of HD22, and their 3′ FITC- or biotin-labeled counterparts, were synthesized by the Molecular Biology and Biotechnology Institute at McMaster University (Hamilton, Canada). Before use, all of the aptamers were subjected to renaturation by heating to 95 °C for 5 min followed by cooling on ice for 10 min (22Griffin L.C. Toole J.J. Leung L.L.K. Gene. 1993; 137: 25-31Crossref PubMed Scopus (49) Google Scholar). Ecarin, a snake venom protein derived from Echis carinatus, was from Pentapharm (Basel, Switzerland). DEAE-Sepharose, PD-10 Sephadex, G-10 Sephadex, Mono Q-Sepharose, and SP-C50 Sephadex were obtained from GE Healthcare (Dorval, Canada). Recombinant tick anticoagulant peptide, a factor Xa-directed inhibitor, was a generous gift from Dr. G. Vlasuk (Corvas International, Inc., San Diego, CA). Hirudin and its Tyr63-sulfated COOH-terminal peptide, Hir54–65(SO-3), were from Bachem (King of Prussia, PA). Chromozym thrombin (Chz-Th) was from Roche Applied Science, whereas S2765 and S2238 were from Chromogenix (Milano, Italy). l-α-Phosphatidyl-l-serine from bovine brain and l-α-phosphatidyl-choline type III-E from egg yolk were from Avanti Polar Lipids Inc. (Alabaster, AL) and Sigma, respectively. PCPS vesicles were synthesized using a modification of previously published methods (29Bloom J.W. Nesheim M.E. Mann K.G. Biochemistry. 1979; 18: 4419-4425Crossref PubMed Scopus (115) Google Scholar, 30Barenholz Y. Gibbes D. Litman B.J. Goll J. Thompson T.E. Carlson F.D. Biochemistry. 1977; 16: 2806-2810Crossref PubMed Scopus (729) Google Scholar). A phosphate assay was used to determine the concentration of PCPS vesicles (31Ames B.N. Methods Enzymol. 1966; VIII: 115-118Crossref Scopus (3023) Google Scholar). The vesicles were stored at –80 °C in 10% sucrose. Labeled Proteins—To label Hir54–65(SO-3) with FITC, 0.11 mg of peptide was dissolved in 250 μl of 0.2 m Na2HCO3 buffer, pH 9.0, and 20 μl of FITC (25.7 μm in Me2SO) was added to a final concentration of 2 μm. After wrapping the mixture in aluminum foil and mixing the sample end-over-end for 90 min at 23 °C, 20 μl of 1 m NH4Cl was added to stop the reaction. The sample was then applied to a 10-ml G10 Sephadex column that was pretreated with 5 mg/ml ovalbumin and washed with 20 mm Tris-HCl, 0.15 mm NaCl, pH 7.4 (Tris-buffered saline). 0.5-ml fractions were collected, and the fluorescent fraction, which was identified by monitoring the effluent with a UV light, was recovered in a single tube. Absorbance of the fractions was determined at 492 nm, and protein concentrations were calculated based on ϵ = 6.8 × 104 m–1 cm–1 (32Mercola D.A. Morris J.W. Arquilla E.R. Biochemistry. 1972; 11: 3860-3874Crossref PubMed Scopus (49) Google Scholar), assuming 1:1 incorporation of FITC into Hir54–65(SO-3). Preparation of Prothrombin Activation Intermediates—All of the prothrombin activation intermediates were prepared using modifications of published methods (33Mann K.G. Methods Enzymol. 1976; 45: 123-156Crossref PubMed Scopus (179) Google Scholar, 34Church W.R. Quellette L.A. Messier T.L. J. Biol. Chem. 1991; 266: 8384-8391Abstract Full Text PDF PubMed Google Scholar). The progress of each reaction was monitored using SDS-PAGE analysis (35Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207227) Google Scholar) on 4–15% acrylamide Ready Gels (Bio-Rad) under reducing and nonreducing conditions. Pre1 was prepared by incubating prothrombin (1 mg/ml) with 200 nm thrombin in 17 mm imidazole-HCl, 144 mm NaCl, pH 7.4, for 2 h at 37°C. The reaction was terminated by the addition of 500 nm FPR-ck, and the absence of residual thrombin activity was confirmed using Chz-Th. The sample was then subjected to chromatography on a 5-ml DEAE-Sepharose column. Pre2 was prepared by digesting pre1 with factor Xa. Briefly, 1 mg/ml pre1 in 25% trisodium citrate buffer, pH 8.8, was incubated with 50 nm factor Xa for 45 min at 37 °C. The reaction was terminated by the addition of 1 μm FPR-ck and 1 μm dansyl-Glu-Gly-Arg chloromethyl ketone, and inhibition of the residual thrombin or factor Xa activity was confirmed with Chz-Th or S2765, thrombin- and factor Xa-directed substrates, respectively. The mixture was then subjected to chromatography on a SP-C50 Sephadex column. mIIa and mIIa(-F1) were generated by treating prothrombin or pre1, respectively, with Ecarin. The reactions were conducted in the presence of FPR-ck to prevent autocatalytic cleavage. Prothrombin or pre1 (1 mg/ml in Tris-buffered saline) was incubated with 250 μg/ml Ecarin diluted in 10 mm HEPES, 150 mm NaCl, pH 7.0, containing 25 mm CaCl2 in the presence of 20 μm FPR-ck for 3 h at 37°C. The samples were then subjected to anion exchange chromatography on a Q-Sepharose column. Protein concentrations were determined at 280 nm using the following extinction coefficients: ϵ = 1.64, ϵ = 1.95, ϵ = 1.44, and ϵ = 1.64 ml·mg–1·cm–1 for pre1, pre2, mIIa, and mIIa(-F1), respectively (36Rabiet M.J. Blashill A. Furie B. Furie B.C. J. Biol. Chem. 1986; 261: 13210-13215Abstract Full Text PDF PubMed Google Scholar). All of the proteins were concentrated using an Amicon Centriprep YM-10 (Beverly, MA). The integrity of each of the prothrombin intermediates was assessed by SDS-PAGE, and aliquots were stored at –80 °C. Binding Studies—Functional assessment of exosites 1 and 2 was performed using fluorescein-labeled HD1 (f-HD1) and HD22 (f-HD22), respectively. f-HD1 or f-HD22 (30 nm), diluted in Tris-buffered saline containing 2 mm CaCl2, 5 mm KCl, 1 mm MgCl2, and 0.1% polyethylene glycol (aptamer buffer), was added to a 10 × 4-mm quartz cuvette maintained at 23 °C with a circulating water bath and stirred using a micro stir bar. Fluorescence was monitored at an emission wavelength of 535 nm (cut-off filter set at 520 nm) and slit width of 8 nm, with excitation wavelength at 492 nm and a slit width of 6 nm, using a PerkinElmer Life Sciences 50B luminescence spectrophotometer (Wellesley, MA). After allowing the base-line fluorescence (Io) to stabilize, aptamers were titrated with 2–20-μl aliquots of solutions containing 10 μm prothrombin, prothrombin intermediates, or thrombin allowing the fluorescence signal to stabilize between each titrant addition. To prevent fluorophore dilution, the titrant contained 30 nm fluorescent aptamer. Once the fluorescence signal reached a plateau, the intensity values (I) were obtained from time drive profiles. After plotting I/Io as a function of the protein titrant concentration, the data were fit by nonlinear regression using Table Curve (Jandel Scientific, San Rafael, CA) to the equation, II0=1+α2(1+KdAo-(1+Kd+PAo)2-4⋅PAo)(Eq. 1) where P is the concentration of the titrated protein, Ao is the concentration of aptamer, α is the maximal fluorescence change, and Kd is the dissociation constant. Competition experiments were performed to examine the capacity of Hir54–65(SO-3) to displace f-HD1 from thrombin or prothrombin. f-HD1 (60 nm), in 900 μl of aptamer buffer, was added to the cuvette, and fluorescence was monitored. Prothrombin or thrombin was then added to final concentrations of 220 or 110 nm, respectively. After the fluorescence signal stabilized, the samples were titrated with 10–25-μl aliquots of 30 μm Hir54–65(SO-3). For all titrations, titrant solutions contained 60 nm f-HD1 to prevent fluorophore dilution. Reciprocal titrations were performed in a similar fashion where 1.1 μm prothrombin or 110 nm thrombin was added to 30 nm f-Hir54–65(SO-3), and the samples were then titrated with aliquots of 10 μm HD1 containing 30 nm f-Hir54–65(SO-3). Surface Plasmon Resonance—Surface plasmon resonance was used as an independent method to examine the interaction of HD1 with prothrombin. Biotinylated HD1 (b-HD1) was immobilized on a SA sensor chip, which is coated with streptavidin. To increase streptavidin reactivity, the flow cells were first washed three times with 1 m NaCl, 50 mm NaOH at a flow rate of 10 μl/min using a BIAcore 1000 (Piscataway, NJ). b-HD1 (250 nm) was then passed through the flow cells at 5 μl/min for 10 min, after which the cells were washed with BIAcore aptamer buffer at 10 μl/min for 2 min. To examine whether factor Va competes with b-HD1 for prothrombin binding, a sample containing 300 nm prothrombin was injected into a b-HD1-coated flow cell in the absence or presence of 1000 nm factor Va, and Req (response units at equilibrium) values were recorded under “kinetic” or “quick inject” modes. To correct for nonspecific DNA-protein binding, the Req values obtained when samples were passed over a b-HD23 flow cell were subtracted. Binding of prothrombin to HD1 in the presence of factor Va was then expressed as a percentage of that measured in its absence. Prothrombin Activation Experiments—A discontinuous assay system was used to examine the effect of HD1 or Hir54–65(SO-3) on prothrombin activation by prothrombinase using a modification of a previously published method (37Anderson J.A.M. Fredenburgh J.C. Stafford A.R. Guo Y.S. Hirsh J. Ghazarossian V. Weitz J.I. J. Biol. Chem. 2001; 276: 9755-9761Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). A 10× stock solution of prothrombinase, consisting of 60 μm PCPS, 2.5 nm factor Xa, and 6 nm factor Va, diluted in aptamer buffer was preincubated for 10 min at 23 °C. 1 μm prothrombin was incubated with HD1 or Hir54–65(SO-3), in concentrations ranging from 0 to 2.5 μm, for 5 min in a series of wells in a 96-well plate (90-μl volume). To start the reactions, 10 μl of the prothrombinase stock solution was added to each prothrombin-containing well. At intervals up to 10 min, individual reactions were terminated by addition of 4.5 μl of a solution containing 5.5 μm tick anticoagulant peptide and 200 mm EDTA. Generated thrombin was quantified by measuring hydrolysis of 600 μm Chz-Th at 405 nm for 10 min using a SpectraMax 340 plate reader (Molecular Devices, Sunnyvale, CA). Rates of substrate cleavage (mOD/min), as determined by instrument software, were used to calculate thrombin concentration based on the specific activity of thrombin cleavage of Chz-Th, as determined in a separate experiment. By plotting thrombin concentration versus time, rates of prothrombin activation were determined. In some experiments, the effect of HD1 on prothrombin activation by factor Xa was measured in the absence of factor Va or PCPS. For these studies, 1 μm prothrombin was activated either with 50 nm factor Xa and 6 μm PCPS, 0.25 nm factor Xa and 0.3 nm factor Va, or with 2 nm factor Xa and 20 nm factor Va. As a control, the effect of HD1 on Ecarin-mediated prothrombin activation also was examined. For these studies, 1 μm prothrombin was activated with 100 μg/ml Ecarin in the absence or presence of 10 μm HD1, and thrombin generation was measured as described above. To examine the effect of HD1 on the kinetics of prothrombin activation by prothrombinase, prothrombin (in concentrations ranging from 0 to 8 μm) was incubated with prothrombinase (0.25 nm factor Xa, 0.6 nm factor Va, and 5 μm PCPS) in the absence or presence of 25 μm HD1 for varying intervals up to 60 s. Initial rates of thrombin production (nm/s) were plotted versus prothrombin concentration (nm) and fit by nonlinear regression to the Michaelis-Menten equation, V=(Vmax)×(S)/(Km+S)(Eq. 2) where Km is the Michaelis-Menten constant, and Vmax is the maximum rate of prothrombin activation (nm IIa/s). kcat was calculated by dividing Vmax by the factor Xa concentration. SDS-PAGE Analysis of Prothrombin Activation—To examine the effect of HD1 on the prothrombin activation pathway, activation intermediates generated in the absence or presence of HD1 were assessed by SDS-PAGE. Prothrombin (14 μm) was incubated with 70 μm DAPA in the absence or presence of 50 μm HD1. The reactions were initiated by the addition of the preassembled prothrombinase complex, consisting of 2 nm factor Xa, 20 nm factor Va, and 6 μm PCPS vesicles (final concentrations) diluted in aptamer buffer. At intervals, 5-μl aliquots were removed into sample buffer, boiled for 2 min, and subjected to SDS-PAGE analysis under reducing and nonreducing conditions. Statistical Methods—Unless otherwise indicated, the experiments were performed at least three times. The results are presented as the means ± S.E. Competitive Binding of Exosite 1 Ligands to Thrombin and Prothrombin—Inspection of the crystal structures of thrombin in complex with Hir54–65(SO-3) or with HD1 suggests that these exosite 1-directed ligands interact with distinct but partially overlapping domains on thrombin (27Padmanabhan K. Padmanabhan K.P. Ferrara J.D. Sadler J.E. Tulinsky A. J. Biol. Chem. 1993; 268: 17651-17654Abstract Full Text PDF PubMed Google Scholar, 28Vijayalakshmi J. Padmanabhan K.P. Mann K.G. Tulinsky A. Protein Sci. 1994; 3: 2254-2271Crossref PubMed Scopus (152) Google Scholar). Whether these ligands interact with the same domains on prothrombin as they do on thrombin is currently unknown. To begin to address this, we first examined the capacity of Hir54–65(SO-3) to displace f-HD1 from prothrombin or thrombin. As illustrated in Fig. 1A, the fluorescence intensity increases by 17 ± 1.1% when 110 nm thrombin is added to a cuvette containing 30 nm f-HD1. This increase in fluorescence intensity is negated when unlabeled HD1 is added (data not shown), consistent with reversible binding. The fluorescence intensity value also returns to base line when the f-HD1-thrombin complex is titrated with Hir54–65(SO-3), suggesting that the Hir54–65(SO-3)-binding site on thrombin overlaps with that of HD1 (Fig. 1A). In the reciprocal experiments, the addition of thrombin to f-Hir54–65(SO-3) results in a 10 ± 2.8% reduction in fluorescence intensity (Fig. 1B). When the f-Hir54–65(SO-3)-thrombin complex is titrated with HD1, the fluorescence intensity increases but does not return to base line. These findings suggest that the HD1-binding site on thrombin only partially overlaps with the Hir54–65(SO-3)-binding site. Studies were then repeated using prothrombin in place of thrombin. As illustrated in Fig. 1C, the addition of prothrombin to f-HD1 results in a 16 ± 0.5% increase in fluorescence intensity. Upon titration with Hir54–65(SO-3), the fluorescence intensity decreases but does not reach base-line levels. In the reverse experiment, fluorescence intensity decreases by 5 ± 0.4% when prothrombin is added to f-Hir54–65(SO-3). There is no change in fluorescence intensity when the f-Hir54–65(SO-3)-prothrombin complex is titrated with HD1, suggesting that the HD1-binding site on prothrombin does not overlap with the f-Hir54–65(SO-3)-binding site. The addition of unlabeled Hir54–65(SO-3) to the cuvette resulted in fluorescence returning to base line, confirming reversibility (data not shown). These data suggest that prothrombin binds both exosite 1 ligands and that the Hir54–65(SO-3)-binding site on prothrombin only partially overlaps with the HD1-binding site. Binding of Aptamers to Prothrombin Derivatives—Because the change in fluorescence intensity that occurs when f-HD1 complexes with prothrombin is similar in magnitude to that which occurs when it binds thrombin, we measured the affinity of f-HD1 for prothrombin, prothrombin intermediates, and thrombin. The fluorescence intensity of 30 nm f-HD1 was monitored before and after titration with prothrombin, prothrombin intermediates, or thrombin, and the relative changes in intensity signal were plotted versus protein concentration. The addition of prothrombin to f-HD1 results in a maximal 33 ± 1.7% increase in fluorescence intensity and yields a saturable curve with a Kd value of 86 ± 8.4 nm (Fig. 2). Thus, the affinity of f-HD1 for prothrombin is much higher than that of Hir54–65(SO-3) (below) and is comparable with the Kd value of 40 nm reported for domain 2 of staphylocoagulase (38Panizzi P. Friedrich R. Fuentes-Prior P. Kroh H.K. Briggs J. Tans G. Bode W. Bock P.E. J. Biol. Chem. 2006; 281: 1169-1178Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Binding experiments were subsequently performed to measure the affinity of f-HD1 for prothrombin derivatives. As outlined in Table 1, f-HD1 binds mIIa, mIIa(-F1), pre1, and pre2 with Kd values of 35 ± 5.5, 45 ± 0.3, 86 ± 1.0, and 66 ± 16 nm, respectively. f-HD1 binds thrombin with a Kd value of 34 ± 4.8 nm. Thus, f-HD1 binds prothrombin and all its activation intermediates with an affinity similar to that for thrombin. These findings suggest that the HD1-binding site on prothrombin undergoes little structural change during prothrombin conversion to thrombin.TABLE 1Kd values of f-HD1, f-Hir54–65(SO-3), and f-HD22 for prothrombin, prothrombin activation intermediates and thrombinProthrombin derivativeKdf-HD1f-Hir54-65(SO-3)f-HD22nmProthrombin86 ± 8.43000 ± 1400No bindingMeizothrombin35 ± 5.5N.D.aN.D. indicates that values were not determined.No bindingMeizothrombin des F145 ± 0.3N.D.No bindingPrethrombin 186 ± 1.0N.D.No bindingPrethrombin 266.1 ± 15.8N.D.42 ± 6.8Thrombin34 ± 4.868 ± 5.229 ± 3.1a N.D. indicates that values were not determined. Open table in a new tab Studies were then done using f-Hir54–65(SO-3) in place of HD1. Consistent with previous reports (11Anderson P.J. Bock P.E. J. Biol. Chem. 2003; 278: 44489-44495Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 12Anderson P.J. Nesset A. Bock P.E. J. Biol. Chem. 2003; 278: 44482-44488Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar), Hir54–65(SO-3) binds prothrombin with an affinity 44-fold lower than that for thrombin (Kd values of 3000 ± 1400 and 68 ± 5.2 nm, respectively; Table 1). These data suggest that, unlike the HD1-binding site on prothrombin, the f-Hir54–65(SO-3)-binding site on prothrombin undergoes changes during prothrombin conversion to thrombin that heighten its affinity for the ligand. Previous structural (39Arni R.K. Padmanabhan K. Padmanabhan K.P. Wu T.P. Tulinsky A. Chem. Phys. Lipids. 1994; 67–68: 59-66Crossref PubMed Scopus (29) Google Scholar) and functional (40Cote H.C. Bajzar L. Stevens W.K. Samis J.A. Morser J. MacGillivray R.T. Nesheim M" @default.
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• W4229675254 date "2006-12-01" @default.
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• W4229675254 title "HD1, a Thrombin-directed Aptamer, Binds Exosite 1 on Prothrombin with High Affinity and Inhibits Its Activation by Prothrombinase" @default.
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