Activation of Human Prothrombin by Arginine-specific Cysteine Proteinases (Gingipains R) from Porphyromonas gingivalis *

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 ␣-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 ؋ 10 6 M ؊1 s ؊1 ) was 100-fold higher than that of RgpB (k cat /K m ‫؍‬ 1.2 ؋ 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.

Bacillolysin MA, a Novel Bacterial Metalloproteinase That Produces Angiostatin-like Fragments from Plasminogen and Activates Protease Zymogens in the Coagulation and Fibrinolysis Systems

“…Activation of pro-u-PA by a bacterial protease has not been reported previously, and the activation of factor X, prothrombin, and protein C has recently been demonstrated by arginine-specific cysteine proteases (gingipains) from Porphyromonas gingivalis, a causative bacterium of periodontitis (33)(34)(35). Proteases from foreign sources are thought to be virulence factors involved in inflammatory events occurring at infected sites, where fibrin deposition is a common feature.…”

“…7, A and B). The cleavage occurred at Asp 34 -Leu 35 and Arg 52 -Ile 53 of the heavy chain (Fig. 7, C and D).…”

Protease-Activated Receptor-2 Activation

“…As a matter of fact, studies have shown that P. gingivalis Arg-gingipains are able to activate prothrombin and coagulation factor X. 39,40 Interestingly, PAR 2 has been described as the endogenous receptor mediating the effects of factor Xa in endothelial cells. 41 Therefore, P. gingivalis Arg-gingipains could account for PAR 2 activation both directly (by cleaving the receptor) and indirectly (by inducing the release of PAR 2 -activating proteases).…”

Proteolysis of Non-phosphorylated and Phosphorylated Tau by Thrombin

“…Western blotting of human brain supernatant using anti-prothrombin (1:20,000; Dako Corp., Glostrup, Denmark) and anti-human thrombin (0.5 g/ml; American Diagnostica Inc., Stamford, CT) antibodies showed bands corresponding to prothrombin, prethrombin-1, prethrombin-2, the B chain of ␣-thrombin, and the B2 chain of ␤-thrombin ( Fig. 2) (22). Fig.…”