Human Plasmin Enzymatic Activity Is Inhibited by Chemically Modified Dextrans
2000; Elsevier BV; Volume: 275; Issue: 38 Linguagem: Inglês
10.1074/jbc.m000837200
ISSN1083-351X
AutoresDominique Ledoux, Dulce Papy‐Garcia, Quentin Escartin, Marie‐Astride Sagot, Yihai Cao, Denis Barritault, Josiane Courtois, William Hornebeck, Jean‐Pierre Caruelle,
Tópico(s)Wound Healing and Treatments
ResumoSome synthetic dextran derivatives that mimic the action of heparin/heparan sulfate were shown to promote in vivo tissue repair when added alone to wounds. These biofunctional mimetics were therefore designated as "regenerating agents" in regard to their in vivo properties.In vitro, these biopolymers were able to protect various heparin-binding growth factors against proteolytic degradation as well as to inhibit the enzymatic activity of neutrophil elastase. In the present work, different dextran derivatives were tested for their capacity to inhibit the enzymatic activity of human plasmin. We show that dextran containing carboxymethyl, sulfate as well as benzylamide groups (RG1192 compound), was the most efficient inhibitor of plasmin amidolytic activity. The inhibition of plasmin by RG1192 can be classified as tight binding hyperbolic noncompetitive. One molecule of RG1192 bound 20 molecules of plasmin with a Ki of 2.8 × 10−8m. Analysis with an optical biosensor confirmed the high affinity of RG1192 for plasmin and revealed that this polymer equally binds plasminogen with a similar affinity (Kd = 3 × 10−8m). Competitive experiments carried out with 6-aminohexanoic acid and kringle proteolytic fragments identified the lysine-binding site domains of plasmin as the RG1192 binding sites. In addition, RG1192 blocked the generation of plasmin from Glu-plasminogen and inhibited the plasmin-mediated proteolysis of fibronectin and laminin. Data from the present in vitroinvestigation thus indicated that specific dextran derivatives can contribute to the regulation of plasmin activity by impeding the plasmin generation, as a result of their binding to plasminogen and also by directly affecting the catalytic activity of the enzyme. Some synthetic dextran derivatives that mimic the action of heparin/heparan sulfate were shown to promote in vivo tissue repair when added alone to wounds. These biofunctional mimetics were therefore designated as "regenerating agents" in regard to their in vivo properties.In vitro, these biopolymers were able to protect various heparin-binding growth factors against proteolytic degradation as well as to inhibit the enzymatic activity of neutrophil elastase. In the present work, different dextran derivatives were tested for their capacity to inhibit the enzymatic activity of human plasmin. We show that dextran containing carboxymethyl, sulfate as well as benzylamide groups (RG1192 compound), was the most efficient inhibitor of plasmin amidolytic activity. The inhibition of plasmin by RG1192 can be classified as tight binding hyperbolic noncompetitive. One molecule of RG1192 bound 20 molecules of plasmin with a Ki of 2.8 × 10−8m. Analysis with an optical biosensor confirmed the high affinity of RG1192 for plasmin and revealed that this polymer equally binds plasminogen with a similar affinity (Kd = 3 × 10−8m). Competitive experiments carried out with 6-aminohexanoic acid and kringle proteolytic fragments identified the lysine-binding site domains of plasmin as the RG1192 binding sites. In addition, RG1192 blocked the generation of plasmin from Glu-plasminogen and inhibited the plasmin-mediated proteolysis of fibronectin and laminin. Data from the present in vitroinvestigation thus indicated that specific dextran derivatives can contribute to the regulation of plasmin activity by impeding the plasmin generation, as a result of their binding to plasminogen and also by directly affecting the catalytic activity of the enzyme. regenerating agent(s) p-nitroanilide polyacrylamide gel electrophoresis urokinase plasminogen activator tissue-type plasminogen activator 6-aminohexanoic acid lysine-binding sites degree of substitution We have previously reported that some dextran derivatives could stimulate tissue repair when applied at the site of the injury in various in vivo models such as skin (1Meddahi A. Blanquaert F. Saffar J.L. Colombier M.L. Caruelle J.P. Jozefonvicz J. Barritault D. Pathol. Res. Pract. 1994; 190: 923-928Crossref PubMed Scopus (40) Google Scholar), bone (2Blanquaert F. Saffar J.L. Colombier M.L. Carpentier G. Barritault D. Caruelle J.P. Bone. 1995; 17: 499-506Crossref PubMed Scopus (83) Google Scholar), colon (3Meddahi A. Benoit J. Ayoub N. Sezeur A. Barritault D. J. Biomed. Mater. Res. 1996; 31: 293-297Crossref PubMed Scopus (50) Google Scholar), cornea (4Fredj-Reygrobellet D. Hriskova D.L. Ettaı̈che M. Meddahi A. JozefonVicz J. Barritault D. Ophtalmic Res. 1994; 26: 325-331Crossref PubMed Scopus (25) Google Scholar), and muscle (5Desgranges P. Barbaud C. Caruelle J.P. Barritault D. Gautron J. FASEB J. 1999; 13: 761-766Crossref PubMed Scopus (63) Google Scholar). These biopolymers were obtained by controlled chemical substitution of dextran polymers by defined amounts of carboxymethyl, sulfate as well as hydrophobic groups such as benzylamide. As regards their in vivo properties, these biopolymers were denominated regenerating agents (RGTA).1 Our initial interpretation of the ability of these biopolymers to stimulate tissue repair was to postulate that these molecules acted as functional mimetics of heparin/heparan sulfate in terms of stabilizers, protectors, and potentiators of endogenously released heparin-binding growth factors. This hypothesis was supported by in vitroexperiments, which showed that these biopolymers protected some heparin-binding growth factors such as fibroblast growth factors and transforming growth factor β against proteolytic degradation and enhanced their bioavailability (3Meddahi A. Benoit J. Ayoub N. Sezeur A. Barritault D. J. Biomed. Mater. Res. 1996; 31: 293-297Crossref PubMed Scopus (50) Google Scholar, 6Tardieu M. Gamby C. Avramoglou T. Jozefonvicz J. Barritault D. J. Cell. Physiol. 1992; 150: 194-203Crossref PubMed Scopus (116) Google Scholar). A second interpretation of thein vivo wound healing properties of these polymers, which does not exclude the first, is that they could also act on some of the proteinases involved in tissue remodeling. This led us to report in a previous study that human neutrophil elastase was inhibited by specific dextran derivatives (7Meddahi A. Lemdjabar H. Caruelle J.P. Barritault D. Hornebeck W. Int. J. Biol. Macromol. 1996; 18: 141-145Crossref PubMed Scopus (51) Google Scholar). Among other known proteinases involved in tissue remodeling, plasmin plays a key role, since it acts directly by hydrolyzing components of the basement membrane such as fibrin, fibronectin, and laminin and also acts indirectly by activating other enzymes such as matrix metalloproteases (8Alexander C.M. Werb Z. Curr. Opin. Cell Biol. 1989; 1: 974-982Crossref PubMed Scopus (192) Google Scholar, 9Werb Z. Cell. 1997; 91: 439-442Abstract Full Text Full Text PDF PubMed Scopus (1131) Google Scholar). As regards the pivotal role of this enzyme in tissue remodeling, we have further investigated the effect of these dextran derivatives on the enzymatic activity of plasmin. We report that as for neutrophil elastase, human plasmin activity is inhibited by specific dextran derivatives that contained the aromatic residue benzylamide. Complementary studies revealed that this type of biopolymer also bind plasminogen and modulate plasmin activity in a noncompetitive manner via regulatory sites involving the lysine-binding site (LBS) domains of plasmin. The chromogenic substratesd-Val-Leu-Lys-pNA (S-2251), pyro-Glu-Gly-Arg-pNA (S-2444), andd-Ile-Pro-Arg-pNA (S-2258) as well as human tissue-type plasminogen activator (tPA) were purchased from Chromogenix (Mölndal, Sweden). Ac-Arg-pNA and Suc-Gly-Gly-Phe-pNA were obtained from Bachem (Budendorf, Switzerland). Human plasmin, human urokinase plasminogen activator (uPA), bovine pancreatic trypsin, α-chymotrypsin, bovine kidney heparan sulfate, aprotinin, human Glu-plasminogen, human fibronectin, Engelbreth-Holm-Swarm mouse sarcoma laminin, polyclonal rabbit anti-fibronectin or laminin Ig, human kringle 1–3, human kringle 4, 6-aminohexanoic acid, streptavidin, and biocytin hydrazide were obtained from Sigma. Horseradish peroxidase-conjugated goat anti-rabbit Ig were from Diagnostic-Pasteur (Marne la Coquette, France). Immobilon P and ECL chemiluminescence were purchased from Millipore Corp. (Saint Quentin en Yvelines, France), and Superblock® blocking buffer was from Pierce. Recombinant mouse kringle 5 was prepared as described previously (10Cao Y. Chen A. An S. Ji R. Davidson D. Cao Y. Llinas M. J. Biol. Chem. 1997; 272: 22924-22928Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). Porcine intestine heparin (H108; averageMr 12,000) with specific anticoagulant activity of 173 IU/mg was kindly provided by M. Petitou from Sanofi (France). The BIAcoreTM instrument, CM5 sensor chip, surfactant P-20, ethanolamine hydrochloride, N-hydroxysuccinimide, andN-ethyl-N′-(3-diethylaminopropyl)carbodiimide hydrochloride were acquired from Amersham Pharmacia Biotech (Uppsala, Sweden). Plasmin active site titration was determined withp-nitrophenyl p′-guanidinobenzoate (Sigma) as described by Chase and Shaw (11Chase T. Shaw E. Biochemistry. 1969; 8: 2212-2224Crossref PubMed Scopus (380) Google Scholar). Water-soluble modified dextrans were prepared from T40 dextran (average Mr 37,000; Amersham Pharmacia Biotech) according to the method described by Mauzac et al. (12Mauzac M. Jozefonvicz J. Biomaterials. 1984; 5: 301-304Crossref PubMed Scopus (111) Google Scholar). Compound RG1100 (Fig. 1) was synthesized from dextran T40 by carboxymethylation of OH residues with monochloroacetic acid treatment in aqueous NaOH at 50 °C, pH 10, for 20 min with constant stirring. The product was then precipitated with methanol and dried under vacuum. The presence of carboxymethyl groups was confirmed by infrared spectroscopy with the appearance of an absorption band at 1650 cm−1. Compound RG1503 (Fig. 1) was synthesized from a carboxymethylated dextran by O-sulfonation with 2 eq of chlorosulfonic acid in dry dichloromethane at room temperature for 2 h with constant stirring. The dichloromethane was eliminated by filtration, and the powdered O-sulfonated product was dissolved in water, adjusted to pH 7.3 with 3 m NaOH, ultrafiltered with a PLCGC 10 K membrane (Millipore, France), and freeze-dried. The presence of sulfate groups was indicated by infrared spectroscopy with the appearance of two absorption bands at 1250 and 1025 cm−1. Compound RG1192 (Fig. 1) was synthesized from a carboxymethylated dextran by amidation of the carboxylate residues with benzylamine followed byO-sulfonation. Briefly, carboxymethylated dextran was dissolved in H2O/EtOH, and carboxylate functions were activated withN-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline for 30 min at pH 3.5 at room temperature. 2 eq of free benzylamine were then added, and the reaction mixture was kept at room temperature overnight. The product was precipitated and washed with methanol and then dried under vacuum. The infrared spectrum confirmed the presence of benzylamide functions with the appearance of a new absorption band at 1750 cm−1, corresponding to the carbonyl bond of the amide. This derivatized carboxymethyl-benzylamide compound was thenO-sulfonated according to the above protocol. In addition, we elaborated a dextran sulfate (RG1003 compound, Fig.1) by directly substituting T40 dextran with sulfate groups according to the above O-sulfonation protocol. It is noteworthy that the conditions of O-sulfonation we used were originally described for sulfonation of aromatic hydrocarbon. It has been also reported that under the same conditions, hydroxyl groups can compete with aromatic groups to generate a mixture ofO- and C-sulfonates (13De Wit P. Woldhuis A.F. Cerfontain H. Recl. Trav. Chim. Pays-Bas. 1988; 107: 668-675Crossref Scopus (10) Google Scholar). This is illustrated during the course of the RG1192 synthesis, where total reactive groups present in its precursor form were distributed among 87 and 13% of hydroxyl and aromatic groups, respectively. To thus establish whether RG1192 contained a mixture of O- andC-sulfonates, a 1H NMR (200 MHz, D2O) spectroscopy study was undertaken. In the aromatic region spectrum, only a broad 1H peak at 7.15 ppm, assigned to the aromatic protons of unsubstituted benzylamide, was detected. As control, a C-sulfonated product was synthesized, and its analysis by 1H NMR showed two broad multiplets at 7.3 and 7.6 ppm. The absence of C-sulfonate groups was confirmed by13C NMR (75 MHz, D2O) spectrum, in which the13C signal at 142 ppm, present in theC-sulfonated product was not detected in the RG1192 product. We then concluded that as described previously for other dextran derivatives (14Maiga-Revel O. Chaubet F. Jozefonvicz J. Carbohydr. Polym. 1997; 32: 89-93Crossref Scopus (27) Google Scholar), C-sulfonate groups were undetectable in the RG1192 compound and that O-sulfonation mainly occurred during treatment of derivatized carboxymethyl-benzylamide dextrans with chlorosulfonic acid. This dominant O-sulfonate formation may thus be explained by the higher content of OH groups (87%) as compared with aromatic groups (13%) present in the RG1192 precursor. The chemical characterization of all dextran derivatives was based on the degree of substitution (d.s.) of each individual group per glucosidic unit (Table I). Each d.s. value was determined by acidimetric titration for CH2COONa and SO3Na content and elementary analysis for CH2CONHCH2C6H5 and SO3Na content. All of these values were confirmed by1H NMR. Distribution of each group among the three reactive OH groups was also reported in Table I. Results showed that reactions of carboxymethylation and sulfonation on hydroxyl functions preferentially occurred on the C-2 position. These results are in agreement with those showing that the OH on C-2 position displayed the higher rate coefficient of dextran carboxymethylation (15Krenstsel L. Chaubet F. Rebrov A. Champion J. Ermakov I. Bittoun P. Fermandjian S. Litmanovich A. Plate N. Jozefonvicz J. Carbohydr. Polym. 1997; 33: 63-71Crossref Scopus (25) Google Scholar). The average molecular weight of each polymer (Table I) was determined by high performance size exclusion chromatography in 0.1 mNaNO3, using KB-804 and KB-805 aqueous gel filtration columns (Shodex, Japan) applied in series. The effluent was monitored with a mini Dawn light scattering detector and a RID 10 A refractometer (Touzard & Matignon, France). The flow rate was 0.7 ml/min. All of these polymers did not present any significant anticoagulant activity (less than 5 IU/mg as compared with 173 IU/mg for heparin) (6Tardieu M. Gamby C. Avramoglou T. Jozefonvicz J. Barritault D. J. Cell. Physiol. 1992; 150: 194-203Crossref PubMed Scopus (116) Google Scholar).Table IChemical characterization of modified dextransPolymerd.s.ad.s., degree of substitution of individual group in one glucosidic unit.Position of groups expressed in d.s.Average MrCMbCM, CH2COONa.SucSu, SO3Na.CMBndCMBn, CH2CONHCH2C6H5.HeH, Nonreacted hydroxyl groups, calculated as the residual d.s. value as compared with the total d.s. value (= 3).CMSuCMBnC-2C-3 + C-4fC-3 + C-4, global substitution on C-3 + C-4 positions, calculated for each group as the difference between its total d.s. value and the one determined on the C-2 position.C-2C-3 + C-4C-2C-3 + C-4RG11000.492.510.290.2074,000RG15030.261.920.820.160.100.621.3047,000RG11920.311.380.390.920.190.120.450.930.230.16140,000RG10032.260.741.001.2640,000Chemical characterization of each polymer is represented through the d.s. of individual group per glucosidic unit. Each d.s. value was determined and confirmed by acidimetric titration, elementary analysis, and 1H NMR. A d.s. value of 3 represents the maximum of substitution, since one glucosidic unit contains three reactive OH groups on C-2, C-3, and C-4 positions. The position of each group on the C-2 versus C-3 + C-4 positions was also determined by analyzing the anomeric proton signal in 1H NMR. In this representation, a d.s. value of 1 and 2 represents the maximum of substitution for C-2 and C-3 + C-4 positions, respectively. S.D. of d.s. values were less than 5% (n = 3).a d.s., degree of substitution of individual group in one glucosidic unit.b CM, CH2COONa.c Su, SO3Na.d CMBn, CH2CONHCH2C6H5.e H, Nonreacted hydroxyl groups, calculated as the residual d.s. value as compared with the total d.s. value (= 3).f C-3 + C-4, global substitution on C-3 + C-4 positions, calculated for each group as the difference between its total d.s. value and the one determined on the C-2 position. Open table in a new tab Chemical characterization of each polymer is represented through the d.s. of individual group per glucosidic unit. Each d.s. value was determined and confirmed by acidimetric titration, elementary analysis, and 1H NMR. A d.s. value of 3 represents the maximum of substitution, since one glucosidic unit contains three reactive OH groups on C-2, C-3, and C-4 positions. The position of each group on the C-2 versus C-3 + C-4 positions was also determined by analyzing the anomeric proton signal in 1H NMR. In this representation, a d.s. value of 1 and 2 represents the maximum of substitution for C-2 and C-3 + C-4 positions, respectively. S.D. of d.s. values were less than 5% (n = 3). Enzymatic kinetics were monitored with a Philips PU8740 spectrophotometer equipped with a thermostated cell holder. The progress curves were recorded for 0.3–5 min, depending upon the reaction velocity, and less than 5% of the substrate was hydrolyzed during the rate measurement. Plasmin, trypsin, and α-chymotrypsin enzymatic activities were determined in 50 mm Tris/HCl buffer, pH 7.4, containing 50 mm NaCl at 37 °C and steady-state velocities were measured by following the release ofp-nitroaniline at 410 nm (ε = 8800m−1cm−1). Human tPA and uPA enzymatic activities were assayed in similar conditions at pH 8.8. To determine the kinetic constants kcat and Km, initial rates were measured as a function of substrate concentrations, and the data were fitted to the Michaelis-Menten rate equation using the GraphPad Prism software (San Diego, CA). Thekcat and Km values for the plasmin/S-2251 system are 14 s−1 and 0.4 mm, and those for the plasmin/S-2444 system are 9 s−1 and 5.3 mm, respectively. The effect of various polymers on the different enzymatic activities was determined by reacting constant concentration of enzyme with increasing concentrations of polymers for 15 min at 37 °C and measuring the residual enzymatic activity with a synthetic substrate. For plasmin, the equilibrium dissociation constants (Ki) were measured with 8 nm of enzyme and 0.4 mm of substrate (S-2251). The Kivalues and their S.E. values were calculated by nonlinear regression using the integral equation editor of the GraphPad Prism software. For the determination of inhibitor-plasmin binding stoichiometries, 750 nm plasmin was used, and the substrate was S-2444 (0.4 mm). RG1192 polymer was labeled by reaction of its aldehydic reducing group with the hydrazide functional group of biocytin hydrazide using a modified method described by Nadkarni et al. (16Nadkarni V. Pervin A. Linhardt R.J. Anal. Biochem. 1994; 222: 59-67Crossref PubMed Scopus (40) Google Scholar). Briefly, 15 mg of polymer were dissolved in 50 μl of formamide containing 50 mmbiocytin hydrazide and heat at 37 °C for 24 h. Free biocytin hydrazide and formamide were removed by gel filtration chromatography (Econo-Pac 10 DG) (Bio-Rad), and homogeneity of the labeled polymer was checked by gel permeation chromatography (TSK gel G4000 PWXL) (Tosohaas, Montgomeryville, PA). The immobilization procedure was carried out at 25 °C in 10 mm HEPES, pH 7.4, 150 mm NaCl, 1 mm EDTA, and 0.005% P-20 (Amersham Pharmacia Biotech) at a constant flow rate of 5 μl/min according to the BIApplications handbook (Amersham Pharmacia Biotech). 25 μl of a mixture of 0.1 m N-hydroxysuccinimide/N-ethyl-N′-(3-diethylaminopropyl)carbodiimide hydrochloride was injected over the surface of a carboxymethyl dextran surface sensor chip (CM5) to activate carboxyl groups. Then 50 μl of streptavidin (200 μg/ml in 10 mm sodium acetate, pH 4.5) was injected, followed by 25 μl of ethanolamine to block remaining active succinimide groups and a pulse of 10 mm HCl. Onto the streptavidin-immobilized surface, 10 μl of biotinylated RG1192 (1 mg/ml) was injected, followed by two pulses of 10 mm HCl to remove noncovalently attached ligand. The amount of RG1192 immobilized on the surface was 100 resonance units. Binding reactions were carried out at a flow rate of 10 μl/min at 25 °C. Various concentrations of plasmin or Glu-plasminogen in 50 mm Tris-HCl, pH 7.4, 50 mm NaCl, 0.005% P-20 were injected over the RG1192-immobilized surface, and change in resonance signal was monitored according to time. Equal volumes of each protein were also injected over a streptavidin-immobilized surface to serve as a blank for subtraction of nonspecific binding of analyte. The sensor surfaces were regenerated with a pulse of 2 m NaCl between each injection of analyte. Equilibrium dissociation constants were determined as described in the BIAtechnology handbook (Amersham Pharmacia Biotech). At steady state, the following is true,dRdt=ka×C(Rmax−Req)−kd×Req=0(Eq. 1) which may be rearranged as follows,Req=C×RmaxKd+C(Eq. 2) where Kd =kd/ka is the equilibrium dissociation constant, Req is the response value at steady state, Rmax is the maximal capacity of the sensor chip for binding analyte, and C is the molar concentration of analyte. Kd was calculated by nonlinear regression analysis by fitting the (Req, C) pairs to Equation 2 using the GraphPad Prism software. Plasmin (1.6 μm) and RG1192 (0.05–1 μm) were incubated at 37 °C for 30 min in 50 mm Tris/HCl, pH 7.4, 50 mm NaCl, 5 mm CaCl2, 0.01% Triton X-100 prior to the addition of 10 ng of fibronectin (0.5 nm). Following 4 h of incubation, the reaction was stopped by the addition of 1.6 μm of aprotinin, and samples were separated by SDS-PAGE (7% (w/v) acrylamide) under reducing conditions. Proteins were electrophoretically transferred overnight at 4 °C to Immobilon-P membrane in 25 mm Tris, pH 8.3, 192 mm glycine. Membranes saturated with Superblock® blocking buffer were incubated with polyclonal rabbit anti-human fibronectin Ig at a 1:1000 dilution in phosphate-buffered saline containing 0.02% (v/v) Tween 20 and 0.3% Superblock® blocking buffer. Antibodies were detected by using horseradish peroxidase-conjugated goat anti-rabbit IgG and ECL chemiluminescence according to the manufacturer's recommendations. Plasmin (0.8 μm) and RG1192 (0.05–2.5 μm) were incubated at 37 °C for 30 min in 50 mm Tris/HCl, pH 7.4, 50 mm NaCl, 0.01% Triton X-100. 50 ng of laminin (2 nm) was then added and the reaction mixture was kept for additional 60 min. The reaction was stopped with 0.8 μm aprotinin, and samples were separated by SDS-PAGE (4–15% (w/v) acrylamide gradient) under reducing conditions. Transfer and hybridization were performed in the same way as for fibronectin, except that the first incubation was performed with polyclonal rabbit anti-mouse laminin Ig at a 1:1000 dilution. The generation of plasmin from Glu-plasminogen in the presence of RG1192 was analyzed in 50 mm Tris/HCl, pH 7.4, 50 mm NaCl, 0.01% Triton X-100 as follows. 3 μg of Glu-plasminogen (0.8 μm) was preincubated for 30 min at 37 °C with increasing concentrations of RG1192 (0.05–1 μm) before the addition of uPA (20 nm). After 2 h of incubation, samples were separated by SDS-PAGE (10% (w/v) acrylamide) under reducing conditions. Gels were then fixed and stained with Coomassie Brillant Blue R-250. Fig.2 shows the influence of increasing concentrations of various dextran derivatives obtained by sequential chemical substitutions of dextran polymers on the amidolytic activity of plasmin. Dextran substituted with carboxymethyl functions (RG1100) did not affect the activity of plasmin even for concentrations greater than 1 μm. Following O-sulfonation, the resulting derivatized polymer (RG1503) showed an inhibitory potential toward human plasmin (IC50 = 20 nm) with 70% of residual enzyme activity. This potential was, however, better than the one obtained with a dextran sulfate (RG1003), indicating a beneficial contribution of the carboxymethyl groups with respect to the antiproteinase activity of this polymer. Interestingly, when both carboxymethyl, sulfate and benzylamide groups were coupled to dextran glucosidic units (RG1192), a potent inhibitory activity of plasmin was observed. The IC50 value was 2 nm with 20% of residual enzyme activity. In contrast, heparin and heparan sulfate did not affect the activity of plasmin. Among the various dextran derivatives tested, RG1192 was the most efficient inhibitor of plasmin activity. The antiproteinase potency of this polymer was therefore investigated toward other serine proteinases such as trypsin, α-chymotrypsin, and the two human plasminogen activators, uPA and tPA (Fig.3). In the same concentration range used for plasmin, trypsin and uPA were insensitive to RG1192. α-Chymotrypsin activity was slightly affected by the presence of RG1192, whereas 70% of the tPA activity was inhibited at a saturating concentration of inhibitor. However, the inhibitory potential of RG1192 on tPA was lower than that of obtained with plasmin: IC50equal to 34 nm as compared with 2 nm for plasmin. The mechanism of the inhibition pattern of a single-substrate reaction can be schematically represented as follows, nE+nS⇄Ksn(ES)→kcatnE+nP++IIKi⇅⇅Ki′=αKiEnI+nS⇄αKs(ES)nI→βkcatEnI+nPSCHEME I where n is the number of free or substrate-bound plasmin molecules per molecule of inhibitor, Ki is the equilibrium dissociation constant of the enzyme-inhibitor complex, α is a dimensionless number that affects the binding of substrate to the plasmin-inhibitor complex or that of inhibitor to enzyme-substrate complex, and β is a dimensionless factor that affects the catalytic constant kcat. Hyperbolic noncompetitive inhibition is characterized by α = 1 and 0 < β < 1. While classical inhibition does not depend upon the total enzyme concentration, tight binding inhibition does (17Bieth J. Methods Enzymol. 1995; 248: 59-84Crossref PubMed Scopus (186) Google Scholar), since the concentration of the bound inhibitor [EI] is no longer negligible with respect to that of total inhibitor concentration [I]0. We therefore analyzed whether RG1192 and RG1503 behaved as tight binding inhibitors by measuring the inhibition of plasmin activity using different enzyme concentrations. The inhibition profiles obtained with RG1192 (Fig. 4) indicated tight binding inhibition, since plasmin inhibition decreased with the total enzyme concentration [E]0; IC50 = 2 and 8 nm for [E]0 = 8 and 150 nm, respectively. At high enzyme concentration that ensured pseudoirreversible binding of plasmin to inhibitor, the tight binding inhibitor titrated the enzyme (17Bieth J. Methods Enzymol. 1995; 248: 59-84Crossref PubMed Scopus (186) Google Scholar). Indeed, at [E]0 = 750 nm(Fig. 4, see inset), RG1192 titrated the enzyme with an equivalence point corresponding to the binding of about 20 molecules of plasmin per molecule of RG1192. Similar experiments demonstrated tight binding inhibition of plasmin by RG1503 and a 1:6 inhibitor/plasmin binding stoichiometry (data not shown). Since these two polymers behave as tight binding inhibitors, the analysis of steady-state velocities data and Kideterminations by means of the Dixon (18Dixon M. Biochem. J. 1953; 55: 170-171Crossref PubMed Scopus (3280) Google Scholar) and/or Cornish-Bowden (19Cornish-Bowden A. Biochem. J. 1974; 137: 143-144Crossref PubMed Scopus (777) Google Scholar) representation is unsuitable. We therefore used the complex steady-state rate equation (Equation 3) derived by Szedlacsek et al. (20Szedlacsek S.E. Ostafe V. Serban M. Vlad M.O. Biochem. J. 1988; 254: 311-312Crossref PubMed Scopus (23) Google Scholar), which takes into account both the tightly binding behavior of the inhibitors and the residual enzymatic activity of the plasmin/inhibitor complexes,νiν0=ν0−ν∞2ν0(((A+n[I]0[E]0−1)2+4A)1/2+ν0+ν∞ν0−ν∞−A−n[I]0[E]0)(Eq. 3) withA=1+[S]0/Kmα+[S]0/Km×αKi[E]0(Eq. 4) andν∞=βkcat[E]0[S]0[S]0+αKm(Eq. 5) where νi/ν0 = enzyme velocity in the presence of inhibitor/enzyme velocity in the absence of inhibitor, ν∞ = limit of νi for saturating concentrations of inhibitor, and [I]0, [S]0, and [E]0 are the total concentration of inhibitor, substrate and plasmin, respectively. To calculate α and β, we measured and compared the kinetic parameters of the enzymatic reaction in the absence (kcat,Km) and in the presence (βkcat, αKm) of a saturating concentration of inhibitors. The Kivalues were calculated by nonlinear regression analysis by fitting the (νi/ν0, [I]0) pairs to Equation3, in which kcat, [S]0/Km, [E]0, α, β, and n were set as fixed parameters. The inhibition curves obtained with RG1503 (Fig.2) and RG1192 (Fig.4) have been calculated using 8 nm of plasmin, 0.4 mm of S-2251, and the best estimates ofKi. A good fit was obtained between the oretical curves and experimental data (R>0.99). Table II summarizes the parameters describing the inhibition of plasmin by RG1503 and RG1192. These two compounds behaved as tight binding hyperbolic noncompetitive inhibitors (α=1, β≠0) with Ki values in the 30 nm range for RG1192, whereas that for RG1503 is 3.7-fold higher. All measurements reported here were performed in 50mm Tris-HCl buffer containing 50mm NaCl. Increasing the ionic strength by the addition of NaCl resulted in a progressive reduction of the inhibitor potency of all dextran derivatives tested. At physiological ionic strength, only RG1192 retained an inhibitory capacity toward plasmin with 30% of residual enzymatic activity (data notshown). In these conditions, the new binding characteristics of RG1192-plasmin complex were α = 1, β=0.3, and Ki =
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