Habutobin releases plasminogen activator (u-PA) from bovine pulmonary artery endothelial cells

Abstract

Habutobin is a thrombin-like enzyme, contained in the venom of Trimeresums flavoviridis, which has the strongest toxic effect in cases of habu bite. The present study was undertaken to examine the effect of habutobin on the release of plasminogen activators using cultured endothelial cells of the bovine pulmonary artery. The chemical characteristics of the plasminogen activators released into the conditioned medium were determined by fibrin autography and immunological analysis. A chromogenic substrate (S-2251) microassay was employed for quantitative estimation of the plasminogen activator activity in the conditioned medium and euglobulin fraction derived from the conditioned medium. The levels of plasminogen activator inhibitor released into the conditioned medium were determined by reverse fibrin autography. Fibrin autography revealed that cultured bovine pulmonary artery endothelial cells spontaneously released tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) into the conditioned medium with no stimulus. Exposure of confluent cultures to 50 nM habutobin, however, induced a time-dependent increase in the level of plasminogen activator activity in both the conditioned medium and euglobulin fraction, and the plasminogen activator activity in the euglobulin fraction at 24 hr was significantly higher than that in the control ( P < 0.05). Reverse fibrin autography demonstrated that the lysis-resistant zone of the supernatant of the euglobulin fraction (habutobin exposure) was wider than that in the case of no stimulus. These findings suggest that habutobin induced a time-dependent increase in the levels of plasminogen activator and plasminogen activator inhibitor concomitantly.