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• W2087317222 abstract "The effect of 95- (HRgpA) and 50-kDa gingipain R (RgpB), arginine-specific cysteine proteinases from periodontopathogenic bacterium Porphyromonas gingivalis on human prothrombin activation was investigated. Each enzyme released thrombin from prothrombin in a dose- and time-dependent manner with the former enzyme, containing adhesion domains, being 17-fold more efficient than the single chain RgpB. A close correlation between the generation of fibrinogen clotting activity and amidolytic activity indicated that α-thrombin was produced by gingipains R, and this was confirmed by SDS-polyacrylamide gel electrophoresis, thrombin active site labeling, and amino-terminal sequence analysis of prothrombin digestion fragments. Significantly, the catalytic efficiency of HRgpA to generate thrombin (k cat /K m = 1.2 × 106m−1s−1) was 100-fold higher than that of RgpB (k cat /K m = 1.2 × 104m−1s−1). The superior prothrombinase activity of HRgpA over RgpB correlates with the fact that only the former enzyme was able to clot plasma, and kinetic data indicate that prothrombin activation can occur in vivo. At P. gingivalis-infected periodontitis sites HRgpA may be involved in the direct production of thrombin and, therefore, in the generation of prostaglandins and interleukin-1, both have been found to be associated with the development and progression of the disease. Furthermore, by taking into account that the P. gingivalis bacterium has been immunolocalized in carotid atherosclerotic plaques at thrombus formation sites (Chiu, B. (1999) Am. Heart J. 138, S534–S536), our results indicate that bacterial proteinases may potentially participate in the pathogenesis of cardiovascular disease associated with periodontitis. The effect of 95- (HRgpA) and 50-kDa gingipain R (RgpB), arginine-specific cysteine proteinases from periodontopathogenic bacterium Porphyromonas gingivalis on human prothrombin activation was investigated. Each enzyme released thrombin from prothrombin in a dose- and time-dependent manner with the former enzyme, containing adhesion domains, being 17-fold more efficient than the single chain RgpB. A close correlation between the generation of fibrinogen clotting activity and amidolytic activity indicated that α-thrombin was produced by gingipains R, and this was confirmed by SDS-polyacrylamide gel electrophoresis, thrombin active site labeling, and amino-terminal sequence analysis of prothrombin digestion fragments. Significantly, the catalytic efficiency of HRgpA to generate thrombin (k cat /K m = 1.2 × 106m−1s−1) was 100-fold higher than that of RgpB (k cat /K m = 1.2 × 104m−1s−1). The superior prothrombinase activity of HRgpA over RgpB correlates with the fact that only the former enzyme was able to clot plasma, and kinetic data indicate that prothrombin activation can occur in vivo. At P. gingivalis-infected periodontitis sites HRgpA may be involved in the direct production of thrombin and, therefore, in the generation of prostaglandins and interleukin-1, both have been found to be associated with the development and progression of the disease. Furthermore, by taking into account that the P. gingivalis bacterium has been immunolocalized in carotid atherosclerotic plaques at thrombus formation sites (Chiu, B. (1999) Am. Heart J. 138, S534–S536), our results indicate that bacterial proteinases may potentially participate in the pathogenesis of cardiovascular disease associated with periodontitis. arginine-specific gingipains, products of rgpA andrgpB genes, respectively prothrombin time tosyl-l-lysine chloromethyl ketone polyacrylamide gel electrophoresis activated partial thromboplastin time t-butyloxycarbonyl-l-Val-l-Pro-l-Arg-4-methylcoumaryl-7-amide Blood coagulation is an important defense system, protecting the body against blood loss from injured vessels. The process is initiated by the binding of factor VII to tissue factor (1Nemerson Y. Blood. 1988; 71: 1-8Crossref PubMed Google Scholar), present in tissues surrounding vessels (2Drake T.E. Morrissey J.H. Edgington T.S. Am. J. Pathol. 1989; 134: 1087-1097PubMed Google Scholar), followed by proteolytic activation of plasma coagulation factors in a cascade pathway (3Colman R.W. Marder V.J. Salzman E.W. Hirsh J. Colman R.W. Hirsh J. Marder V.J. Salzman E.W. Hemostasis and Thrombosis: Basic Principles and Clinical Practice. 2nd Ed. J. B. Lippincott, Philadelphia, PA1987: 3-17Google Scholar, 4Mann K.G.R. Lundblad L. Colman R.W. Hirsh J. Marder V.J. Salzman E.W. Hemostasis and Thrombosis : Basic Principles and Clinical Practice. 2nd Ed. J. B. Lippincott, Philadelphia, PA1987: 148-161Google Scholar, 5Furie B. Furie B.C. Cell. 1988; 53: 505-518Abstract Full Text PDF PubMed Scopus (991) Google Scholar). Thrombin, the ultimate product of these reactions, is an extremely potent platelet activator (6Coughlin S.R. Thromb. Haemostasis. 1999; 82: 356-363Google Scholar, 7Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11023-11027Crossref PubMed Scopus (524) Google Scholar) and converts fibrinogen to a fibrin clot (8Fenton J.W., II Ann. N. Y. Acad. Sci. 1986; 485: 5-15Crossref PubMed Scopus (193) Google Scholar), thus plugging damaged vessels. Besides its central role in hemostasis, thrombin also enhances vascular permeability (9DeMichele M.A.A. Moon D.G. Fenton J.W., II Minnear F.L. J. Appl. Physiol. 1990; 69: 1599-1606Crossref PubMed Scopus (46) Google Scholar), induces leukocyte chemotaxis (10Bizios R. Lai L. Fenton J.W., II Malik A.B. J. Cell. Physiol. 1986; 128: 485-490Crossref PubMed Scopus (174) Google Scholar,11Bar-Shavit R. Kahn A. Fenton L.W., II Wilner G.D. Science. 1983; 220: 728-730Crossref PubMed Scopus (260) Google Scholar), and potentiates lipopolysaccharide-stimulated interleukin-1 production by macrophages (12Jones A. Geczy C.L. Immunology. 1990; 71: 236-241PubMed Google Scholar). These data, and the fact that prothrombin activation in vivo is known to be associated with inflammatory conditions, implicate thrombin as a major player in inflammation. The deposition of fibrin is a common feature at the site of bacterial infection (13Menkin V. Physiol. Rev. 1938; 18: 366-418Crossref Google Scholar). Endotoxin can induce fibrin accumulation in vivo through the Shwartzman reaction (14Lipinski B. Nowak A. Gurewich V. Br. J. Haematol. 1974; 28: 221-231Crossref PubMed Scopus (18) Google Scholar), presumably by activating monocytes to express tissue factor (15Rivers R.P. Hathaway W.E. Weston W.L. Br. J. Haematol. 1975; 30: 311-316Crossref PubMed Scopus (302) Google Scholar). For this reason it is recognized as the component primarily responsible for blood coagulation associated with bacterial infections. Proteinases from such foreign sources are also thought to be virulence factors involved in various inflammatory events occurring at infected sites (16Imamura T. Pike R.N. Potempa J. Travis J. J. Clin. Invest. 1994; 94: 361-367Crossref PubMed Scopus (156) Google Scholar). For example, many of these enzymes present in snake venoms are known to activate prothrombin (17Speijer H. Govers-Riemslag J.W.P. Zwaal R.F.A. Rosing J. J. Biol. Chem. 1986; 261: 13258-13267Abstract Full Text PDF PubMed Google Scholar, 18Tans G. Govers-Riemslag J.W.P. van Rijin J.M.L. Rosing J. J. Biol. Chem. 1985; 260: 9366-9372Abstract Full Text PDF PubMed Google Scholar); however, whereas bacterial proteinases may be able to convert prothrombin to thrombin, such a process has not been studied in detail. A close relationship between Porphyromonas gingivalis(formerly Bacteroides gingivalis) and adult periodontitis has been reported (19Slots J. Genco R.J. Mergenhagen S.E. Host-Parasite Interactions in Periodontal Diseases. American Society of Microbiology, Washington. D. C.1982: 27-45Google Scholar, 20Holt S.C. Ebersole J. Fenton J. Brunsvold M. Kornmann K.S. Science. 1987; 239: 55-57Crossref Scopus (376) Google Scholar, 21Zambon J.J. Genco R.J. Goldman H.M. Cohen D.W. Contemporary Periodontics. Mosby, St. Louis, MO1990: 147-160Google Scholar), with proteolytic enzymes that are known to be produced in large quantity by this microorganism and have been shown to act as important pathogenic agents (22Grenier D. Mayrand D. J. Clin. Microbiol. 1987; 25: 738-740Crossref PubMed Google Scholar, 23Smalley J.W. Birss A.J. Kay H.M. McKee A.S. Marsh P.D. Oral Microbiol. Immunol. 1989; 4: 178-181Crossref PubMed Scopus (62) Google Scholar, 24Marsh P.D. McKee A.S. McDermid A.S. Dowsett A.B. FEMS Microbiol. Lett. 1989; 59: 181-185Crossref Scopus (45) Google Scholar). From the culture medium of P. gingivalis HG66 we have purified previously two major forms of arginine-specific cysteine proteinases, HRgpA1 and RgpB, formerly referred to as high molecular mass gingipain R (95-kDa gingipain R1 or HRGP) and 50-kDa gingipain R2 (RGP-2), respectively (24Marsh P.D. McKee A.S. McDermid A.S. Dowsett A.B. FEMS Microbiol. Lett. 1989; 59: 181-185Crossref Scopus (45) Google Scholar, 26Potempa J. Mikolajczyk-Pawlinska J. Brassell D. Nelson D. Thogersen I.B. Enghild J.J. Travis J. J. Biol. Chem. 1998; 273: 21648-21657Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Both of these enzymes are products of two distinct but related genes (27Nakayama K. Kadowaki T. Okamoto K. Yamamoto K. J. Biol. Chem. 1995; 270: 23619-23626Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar).rgpA encodes a polyprotein which, after post-translational processing/modifications, yields three different forms of the enzyme (28Pavloff N. Potempa J. Pike R.N. Prochazka V. Kiefer M.C. Travis J. Barr P.J. J. Biol. Chem. 1995; 270: 1007-1010Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 29Rangarajan M. Aduse-Opoku J. Slaney J.M. Young K.A. Curtis M.A. Mol. Microbiol. 1987; 23: 955-965Crossref Scopus (75) Google Scholar); the major one is a non-covalent complex containing separate catalytic and adhesion/hemagglutinin domains (HRgpA). In contrast, the fragment encoding the latter domain(s) is missing in thergpB gene structure, and its translation product is a single protein with a primary structure essentially identical to the catalytic domain of HRgpA (30Mikolajczyk-Pawlinska J. Kordula T. Pavloff N. Pemberton P.A. Chen W.C.A. Travis J. Potempa J. Biol. Chem. 1998; 379: 205-211Crossref PubMed Scopus (64) Google Scholar, 31Nakayama K. Microbiol. Immunol. 1997; 41: 185-196Crossref PubMed Scopus (68) Google Scholar). Despite both a structural similarity and a specificity restricted to Arg-Xaa peptide bonds, HRgpA and RgpB show considerable differences in catalytic potency (26Potempa J. Mikolajczyk-Pawlinska J. Brassell D. Nelson D. Thogersen I.B. Enghild J.J. Travis J. J. Biol. Chem. 1998; 273: 21648-21657Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar) which is most profoundly manifested in their ability to activate factor X (32Imamura T. Potempa J. Tanase S. Travis J. J. Biol. Chem. 1997; 272: 16062-16067Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar) and protein C (33Hosotaki K. Imamura T. Potempa J. Travis J. Hiraoka T. Kitamura N. Biol. Chem. 1999; 380: 75-80Crossref PubMed Scopus (21) Google Scholar). In addition to activation of these members of the coagulation cascade pathway, it was also shown that RgpB was capable of generating kallikrein from plasma prekallikrein (16Imamura T. Pike R.N. Potempa J. Travis J. J. Clin. Invest. 1994; 94: 361-367Crossref PubMed Scopus (156) Google Scholar). Thus, it may be anticipated that gingipains R could activate other coagulation cascade proenzymes in this pathway, since each of these processes requires cleavage of peptide bonds at the carboxyl-terminal side of specific arginine residues (34Halkier T. Mechanisms in Blood Coagulation, Fibrinolysis and the Complement System. Cambridge University Press, UK1991Google Scholar). In the present study, we describe the results of experiments designed to investigate the ability of two forms of gingipains R to convert prothrombin to thrombin, an enzyme known to have multiple functions in both coagulation and pro-inflammatory processes. Benzoyl-l-arginine-p-nitroanilide, tosyl-l-lysine chloromethyl-ketone (TLCK), leupeptin, and fibrinogen were purchased from Sigma. Factor X-deficient plasma was obtained from George King Bio-Medical, Inc. (Overland Park, KS). Purified human prothrombin, α-, μ-, and γ-thrombins, and biotinylated Phe-Pro-Arg-chloromethyl ketone were purchased from Hematologie Technologies, Inc. (Essex Junction, VT).t-Butyloxycarbonyl-l-Val-l-Pro-l-Arg-4-methylcoumaryl-7-amide (Boc-Val-Pro-Arg-MCA) was obtained from the Peptide Institute (Minoh, Japan); p-nitrophenyl-p′-guanidinobenzoate was a product from Nacalai Tesque (Kyoto, Japan), and DX-9065a, a specific factor Xa inhibitor, was obtained from Daiichi Pharmaceutical Co., Ltd. (Tokyo, Japan). The purified human factor X was purchased from Enzyme Research Laboratories, Inc. (South Bend, IN). Factor X-, IX-, and XI-deficient plasmas and Platelin® (rabbit brain phospholipids) were obtained from George King Biomedical (Overland Park, KS), and from Organon Teknika (Durham, NC), respectively. Normal human plasma was prepared by centrifugation of a mixture of 9 volumes of freshly drawn blood from healthy volunteers and 1 volume of 3.8% (w/v) sodium citrate. RgpB and HRgpA were isolated according to the method described by Potempa et al. (26Potempa J. Mikolajczyk-Pawlinska J. Brassell D. Nelson D. Thogersen I.B. Enghild J.J. Travis J. J. Biol. Chem. 1998; 273: 21648-21657Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). The amount of active enzyme in each purified proteinase was determined by active site titration using Phe-Pro-Arg-chloromethyl ketone (35Potempa J. Pike R. Travis J. Biol. Chem. 1997; 378: 223-230Crossref PubMed Scopus (147) Google Scholar), with the concentration of active gingipain R being calculated from the amount of inhibitor needed for complete inactivation of the proteinase. Each P. gingivalisproteinase was activated with 10 mm cysteine in 0.2m HEPES buffer, pH 8.0, containing 5 mmCaCl2 at 37 °C for 10 min. The activated proteinase (2 μm) was diluted with 10 mm Tris-HCl, pH 7.3, containing 150 mm NaCl (TBS) and 5 mmCaCl2 prior to use. The molar concentration of purified prothrombin was calculated using A280nm1%= 13.8 and a molecular mass of 72 kDa (36Kisiel W. Hanahan D.J. Biochim. Biophys. Acta. 1973; 304: 103-113Crossref PubMed Scopus (59) Google Scholar). The fibrinogen clotting activity of released thrombin was measured by incubating 90 μl of prothrombin (90 μg/ml) with 10 μl of a given proteinase at 37 °C for 3 min. One hundred μl of fibrinogen (3 mg/ml), prewarmed to 37 °C, was then added to the mixture, and the clotting time was measured with a Coagulometer KC 1A (Amelung, Lemgo, Germany). For plasma clotting time assays, 90 μl of factor X-deficient plasma supplemented with 4 μmfactor X-specific inhibitor (DX-9065a) or the same plasma reconstituted with 10 μl of factor X (100 μg/ml) in the absence of DX-9065a were prewarmed to 37 °C, and then 10 μl of a proteinase was added and the clotting time measured. For activated partial thromboplastin time (APTT) assay, 90 μl of citrated plasma was mixed with 90 μl of PTT-LT® (cephalin 1.2 mg/ml, silica 1 mg/ml) (Roche Molecular Biochemicals) and preheated to 37 °C in a plastic cell for 1 min. Then, 20 μl of HRgpA was added, and the mixture was incubated at 37 °C for 2 min. After adding 100 μl of 25 mmCaCl2, the clotting time was measured with a Coagulometer KC 1A (Amelung, Lemgo, Germany). Prothrombin, dissolved in 50 μl of 0.1 m Tris-HCl, pH 7.6, containing 0.15 m NaCl and 5 mm CaCl2, was incubated with the same volume of either gingipain R (0.1 nm HRgpA or 0.4 nm RgpB final concentration) dissolved in the same buffer supplemented with 80 μg/ml phospholipids at 37 °C for 30, 60, 90, or 120 s. Then, 50 μl of 6 μm leupeptin in the same buffer was added, to inhibit completely the cysteine proteinase activity. At this concentration leupeptin does not affect the amidolytic activity of thrombin. To this mixture 50 μl of a thrombin-specific substrate, Boc-Val-Pro-Arg-MCA (0.4 mm), in the same buffer was added. Substrate cleavage and the release of AMC by thrombin was monitored by the relative fluorescence increase at 440 ± 20 nm after excitation at 380 ± 20 nm, using a microplate fluorescence spectrophotometer (CytoFluor Series 4000, Perspective Biosystems). To calculate concentrations of thrombin produced by either gingipain R, the amidolytic activity of purified α-thrombin, which had been titrated withp-nitrophenyl-p′-guanidinobenzoate (37Chase Jr., T. Shaw E. Biochem. Biophys. Res. Commun. 1967; 29: 508-514Crossref PubMed Scopus (781) Google Scholar), was used as a standard. The initial velocity of thrombin production at various prothrombin concentrations (final concentrations: 50, 100, 150, 200, 300, 400, 600, and 1000 nm for HRgpA and 1, 2, 3, 4, 5, 7, and 10 μm for RgpB) was determined by the best fit line after incubation for various periods. The values forK m and V max were extracted by direct fit of the Michaelis-Menten equation to experimental data using non-linear curve fitting employing the method of least squares with Taylor expansion (38Sakoda M. Hiromi K. J. Biochem. (Tokyo). 1976; 80: 547-555Crossref PubMed Scopus (192) Google Scholar). Moreover, because the values generated in this way were very similar to the ones obtained by three transformations of the Michaelis-Menten equation ([S]0/v versus [S]0, 1/v versus1/[S]0 and v versus v/[S]0, where v and [S]0 denote the catalytic rate and the initial substrate concentration, respectively), the means ± S.D. derived from four independent experiments and four different transformations of the Michaelis-Menten equation were calculated and presented in Table IV.Table IVKinetic constants for the activation of prothrombinEnzymesK mk catk cat/K mms−1m−1s−1HRgpA2.6 ± 0.1 × 10−73.2 ± 0.2 × 10−11.2 × 106(9.8 × 10−8) aData in parentheses for factor X activation were taken from Ref. 34. All values were measured in the presence of calcium ions and phospholipids. Factor Xa + factor Va indicates activated factor X in the presence of activated factor V. OSV-PTA indicates prothrombin activator from O. scutellatus venom. NSSV-PTA + factor Va indicates prothrombin activator from N. scutatus scutatus in the presence of activated factor V.(4 × 10−1)(4.1 × 106)RgpB6.6 ± 0.4 × 10−67.6 ± 0.5 × 10−21.2 × 104(1.4 × 10−7)(1.2 × 10−2)(8.9 × 104)Factor Xa + factor Va bData were obtained from Ref. 44.1.2 × 10−7119.2 × 107OSV-PTAcData were obtained from Ref. 19.3.4 × 10−7361.0 × 108NSSV-PTA + factor VadData were obtained from Ref. 20.1.6 × 10−72.5 × 10−21.6 × 105The mean ± S.D. of the K m andk cat values for prothrombin activation by gingipains were calculated from data obtained in four independent experiments. For details see under “Experimental Procedures.”4-a Data in parentheses for factor X activation were taken from Ref. 34Halkier T. Mechanisms in Blood Coagulation, Fibrinolysis and the Complement System. Cambridge University Press, UK1991Google Scholar. All values were measured in the presence of calcium ions and phospholipids. Factor Xa + factor Va indicates activated factor X in the presence of activated factor V. OSV-PTA indicates prothrombin activator from O. scutellatus venom. NSSV-PTA + factor Va indicates prothrombin activator from N. scutatus scutatus in the presence of activated factor V.4-b Data were obtained from Ref. 44Pike R.N. Potempa J. McGraw W. Coetzer T.H.T. Travis J. J. Bacteriol. 1996; 178: 2876-2882Crossref PubMed Google Scholar.4-c Data were obtained from Ref. 19Slots J. Genco R.J. Mergenhagen S.E. Host-Parasite Interactions in Periodontal Diseases. American Society of Microbiology, Washington. D. C.1982: 27-45Google Scholar.4-d Data were obtained from Ref. 20Holt S.C. Ebersole J. Fenton J. Brunsvold M. Kornmann K.S. Science. 1987; 239: 55-57Crossref Scopus (376) Google Scholar. Open table in a new tab The mean ± S.D. of the K m andk cat values for prothrombin activation by gingipains were calculated from data obtained in four independent experiments. For details see under “Experimental Procedures.” Eighteen microliters of activated HRgpA or RgpB (3.6 pmol) were added to 162 μl of prothrombin (3.68 nmol in 0.1m Tris-HCl, pH 7.6, 150 mm NaCl, 5 mm CaCl2, and 0.5 mm benzamidine), and the mixture (20 nm and 20 μm final concentration of HRgpA or RgpB and prothrombin, respectively) was incubated at 37 °C. At 0.5 mm concentration, benzamidine inhibits thrombin activity but not that of gingipains, and it was included into the assay buffer to avoid autocatalytic cleavage. At specific time intervals, aliquots were withdrawn, and 1.5 μl of d-Phe-Phe-Arg-chloromethyl ketone (10 mm) was added to terminate the reaction. Samples were boiled in reducing treatment buffer and applied for SDS-PAGE with 10% slab gels, according to the method of Laemmli (39Laemmli U.K. Nature. 1970; 227: 680-684Crossref PubMed Scopus (207231) Google Scholar). For Western blot analysis 2-μl aliquots of the prothrombin/gingipain incubation mixture were transferred to 8 μl of HEPES, pH 7.6, containing 10 μm biotinylated Phe-Pro-Arg-chloromethyl ketone, incubated for 10 min at room temperature, and boiled in reducing treatment buffer. After SDS-PAGE the separated protein fragments were electroblotted onto a polyvinylidene difluoride membrane (Hybond-P membrane from Amersham Pharmacia Biotech). The membrane was incubated with streptavidin-horseradish peroxidase conjugate, and bands were developed by enhanced chemiluminescence (Amersham Pharmacia Biotech). Automatic sequence analysis was performed with a pulse liquid-phase sequencer (model 477A Protein Sequencer, PerkinElmer Life Sciences/Applied Biosystems Inc.). To determine the amino-terminal sequence of prothrombin-derived fragments, the mixture was separated by SDS-PAGE and transferred to ImmobilonTM polyvinylidene difluoride transfer membrane (Millipore Co., Ltd., Bedford, MA). The transferred proteins were visualized by staining with Coomassie Brilliant Blue R-250. Excised bands were placed on a Polybrene-treated glass filter prior to sequence analysis. In order to determine whether gingipains R activate prothrombin, each bacterial proteinase was incubated with the human zymogen, and the release of thrombin activity was measured. Both proteinases caused prothrombin activation in a dose- and incubation time-dependent manner (Fig. 1, A and B), with HRgpA being nearly 17 times more potent. At conditions used for SDS-PAGE analysis of the prothrombin degradation pattern (Fig. 3), the thrombin activity released from 20 μm prothrombin by 20 nm of either gingipain transiently reached a peak after 10 min of incubation and then slowly disappeared during prolonged enzyme exposure (Fig. 2). Significantly, no generation of thrombin activity was observed if prothrombin was incubated with TLCK-treated gingipains R (Fig. 1).Figure 3Cleavage of prothrombin by gingipains R. A, prothrombin (20 μm) and gingipains R (20 nm) were incubated together as described in Fig. 2 legend in the buffer supplemented with 0.5 mm benzamidine. At specific time intervals, aliquots were withdrawn, and 1.5 μl ofd-Phe-Phe-Arg-chloromethyl ketone (10 mm) was added to terminate the reaction. Samples were boiled in sample treatment buffer and analyzed by SDS-PAGE. Lane a, prothrombin alone (3.5 μl loaded); lanes b–f, prothrombin incubated with HRgpA for 1, 2, 5, 15, and 60 min, respectively;lanes g and n, α-thrombin (1.3 μg loaded);lanes h–m, prothrombin incubated with RgpB for 0, 1, 2, 5, 15, and 60 min, respectively; lanes o–r, α-thrombin (20 μm) incubated with RgpB (20 μm) for 0, 5, 15, and 60 min, respectively (2 μg loaded); lanes s andt, pure μ- and γ-thrombin, respectively (1.3 μg loaded). The positions of molecular mass standard markers are indicated to the right of the gel. The major fragments of prothrombin indicated by arrows were subjected to amino-terminal sequence analysis and identified as follows: 1, prethrombin 1; 2, fragment 1·2; 3, prethrombin 2;4, B-chain of α-thrombin; 5, fragment 1;6, B-2-chain of μ-thrombin; and 7, fragment 2.B, schematic diagram of prothrombin fragmentation with major cleavage sites for HRgpA and RgpB. The size of arrowheadsindicates the relative efficiency of cleavage of specific peptide bonds in the prothrombin polypeptide chain by the two proteinases tested. μ-Thrombin is generated by cleavage at the Arg70–Tyr71 or Arg73–Asn74 peptide bonds in the α-thrombin B-chain, giving rise to the B1 and B2 peptides. An additional cleavage at Lys154–Gly155 of the B2-chain generates γ-thrombin.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 2Time course of thrombin generation and degradation by gingipains R. Eighteen microliters of activated HRgpA (3.6 pmol) was added to 162 μl of prothrombin (3.68 nmol), and the mixture (20 nm and 20 μm final concentration of HRgpA and prothrombin, respectively) was incubated at 37 °C in 0.1 m Tris-HCl, 150 mm NaCl, 5 mm CaCl2, pH 7.6, containing 1 mmcysteine. At the given time intervals 10-μl aliquots were removed to 985 μl of the same buffer supplemented with 2 mmantipain, and thrombin activity released by HRgpA (○) and RgpB (▵) was measured using H-d-Phe-Pipecolyl-Arg-p-nitroanilide as substrate.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Due to autoproteolytic cleavage or fragmentation by other proteolytic enzymes, thrombin in vitro can occur in three major forms referred to as α-, β-, and γ-thrombin. All of these enzymes have amidolytic activity, but only α-thrombin is capable of clotting fibrinogen. Therefore, to determine if amidolytic activity released by gingipains from the zymogen is at least partially due to the presence of α-thrombin, which is the more important form of this proteinase, the samples of prothrombin incubated with gingipains were examined for clotting activity. As summarized in TableI both gingipains induced fibrinogen clotting activity from prothrombin in a time- and concentration-dependent manner. However, due to the apparent progressive cleavage of the α-thrombin B-chain and the creation of β- and/or γ-thrombin, a correlation between clotting and amidolytic activity was observed only at short preincubation times. Indeed, after prolonged incubation the former activity decreased much faster than the latter activity (data not shown). The same process of excessive α-thrombin cleavage in the presence of increased concentrations of gingipains most likely skewed a concentration-dependent release of fibrinogen clotting activity from prothrombin. This is particularly apparent in the case of RgpB where the doubling of enzyme concentration resulted only in the moderate shortening of the fibrinogen clotting time (Table I). In comparison to HRgpA, an ∼5-fold higher concentration of RgpB was necessary to induce clotting activity from prothrombin, and significantly, clotting times determined after the same preincubation time were 3–4 times longer. Taken together, these results indicate that HRgpA is about 20 times more efficient than RgpB in α-thrombin generation. This is in keeping with zymogen activation as measured with an amidolytic substrate in which HRgpA was shown to be 17-fold more efficient than RgpB (Fig. 1).Table IFibrinogen clotting by prothrombin incubated with gingipains RGingipain RConcentrationClotting time1 min 1-bPreincubation time of gingipain with prothrombin.2 min 1-bPreincubation time of gingipain with prothrombin.3 min 1-bPreincubation time of gingipain with prothrombin.nm 1-aConcentration during incubation with prothrombin.sssHRgpA5ND 1-cNot determined.41.1 ± 1.5ND1042.3 ± 1.225.7 ± 1.422.4 ± 1.415ND20.5 ± 1.1NDHRgpA-TLCK 1-dTreated with TLCK.15NDND>300 1-eNot clotted after a 300-s incubation.RgpB25NDND116.3 ± 6.150137.5 ± 8.1111.3 ± 8.493.7 ± 5.2RgpB-TLCK 1-dTreated with TLCK.50NDND>300 1-eNot clotted after a 300-s incubation.Ninety μl of prothrombin (90 μg/ml) was preincubated at 37 °C for 3 min before it was supplemented with 10 μl of a proteinase. One hundred μl of fibrinogen (3 mg/ml) prewarmed at 37 °C was then added to the mixture, and the clotting time was measured. Each value denotes the mean ± S.D. in triplicate assays.1-a Concentration during incubation with prothrombin.1-b Preincubation time of gingipain with prothrombin.1-c Not determined.1-d Treated with TLCK.1-e Not clotted after a 300-s incubation. Open table in a new tab Ninety μl of prothrombin (90 μg/ml) was preincubated at 37 °C for 3 min before it was supplemented with 10 μl of a proteinase. One hundred μl of fibrinogen (3 mg/ml) prewarmed at 37 °C was then added to the mixture, and the clotting time was measured. Each value denotes the mean ± S.D. in triplicate assays. The prothrombin activation assays in vitro based on generation of amidolytic and/or fibrinogen clotting activities do not reflect the complexity of reactions in blood plasma where a multitude of other proteins could hinder the interaction of gingipains R with prothrombin. Therefore, to determine if gingipains R can produce a significant amount of α-thrombin in plasma, we measured the clotting time of factor X-deficient plasma incubated with gingipains. In order to evaluate interference from any residual factor X, which may still exist in deficient plasma, the assay was performed in the presence of a factor Xa-specific inhibitor in c" @default.
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• W2087317222 date "2001-06-01" @default.
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• W2087317222 title "Activation of Human Prothrombin by Arginine-specific Cysteine Proteinases (Gingipains R) from Porphyromonas gingivalis *" @default.
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