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 alpha-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 x 10(6) m(-)1 s(-)1) was 100-fold higher than that of RgpB (k(cat)/K(m) = 1.2 x 10(4) m(-)1 s(-)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.
Highlights
Blood coagulation is an important defense system, protecting the body against blood loss from injured vessels
Endotoxin can induce fibrin accumulation in vivo through the Shwartzman reaction [14], presumably by activating monocytes to express tissue factor [15]. For this reason it is recognized as the component primarily responsible for blood coagulation associated with bacterial infections
Materials—Benzoyl-L-arginine-p-nitroanilide, tosyl-L-lysine chloromethyl-ketone (TLCK), leupeptin, and fibrinogen were purchased from Sigma
Summary
Materials—Benzoyl-L-arginine-p-nitroanilide, tosyl-L-lysine chloromethyl-ketone (TLCK), leupeptin, and fibrinogen were purchased from Sigma. Kinetic Analysis of Prothrombin Activation—Prothrombin, dissolved in 50 l 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 g/ml phospholipids at 37 °C for 30, 60, 90, or 120 s. SDS-Polyacrylamide Gel Electrophoresis and Western Blot Analysis—Eighteen microliters of activated HRgpA or RgpB (3.6 pmol) were added to 162 l of prothrombin (3.68 nmol in 0.1 M Tris-HCl, pH 7.6, 150 mM NaCl, 5 mM CaCl2, and 0.5 mM benzamidine), and the mixture (20 nM and 20 M final concentration of HRgpA or RgpB and prothrombin, respectively) was incubated at 37 °C. For Western blot analysis 2-l aliquots of the prothrombin/gingipain incubation mixture were transferred to 8 l of HEPES, pH 7.6, containing 10 M biotinylated Phe-Pro-Arg-chloromethyl ketone, incubated for 10 min at room temperature, and boiled in reducing treatment buffer. Excised bands were placed on a Polybrene-treated glass filter prior to sequence analysis
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