Abstract
Fibrinogen-clotting enzymes purified from snake venoms, ancrod, batroxobin and crotalase induce a fibrinolytic state in the circulation of humans and animals. In vivo, but not in vitro, the enzymes indirectly convert fibrinogen into plasmin-derived degradation products. To explain this effect, we investigated the secretion of plasminogen activators by cultured human umbilical vein endothelial cells in response to purified snake venom enzymes. The activity of plasminogen activators was measured by a solid-phase 125I-fibrin lysis assay and the type demonstrated by fibrin zymography. Unstimulated primary or passaged cultures secreted activators at a mean rate of 0.183 or 46.6 milliunits/10 6 cells, respectively. Ancrod, batroxobin and crotalase did not affect biosynthesis and secretion of tissue plasminogen activator and did not induce pro-urokinase from primary cell cultures after 4 h. However, in passaged cells treated for 4 h, ancrod and batroxobin stimulated the secretion of plasminogen activators in a dose-dependent manner. Maximum stimulation by the two enzymes at 3 and 2 u/ml, respectively, was twofold higher than control values. In contrast, crotalase (1 u/ml), and thrombin (1 u/ml) decreased activator secretion to 32 and 11% of control values, respectively. The use of specific antibodies showed that the secreted plasminogen activators were inhibited by 90% using anti-urokinase antibodies and by 10% with anti-tissue plasminogen activator antibodies. Fibrin zymography supported the data and demonstrated that fibrinogen-clotting enzymes affected both extracellular and intracellular pro-urokinase levels. Intracellular plasminogen activators increased in cells treated with crotalase to 130% but decreased with ancrod to 40%, batroxobin to 80% and thrombin to 23%. The data indicated that the effects of the four enzymes on the secretion of plasminogen activators from passaged endothelial cells were independent of their action on fibrinogen. It is concluded that ancrod or batroxobin may activate the fibrinolytic system in vivo by stimulation of vascular endothelial cells to selectively increase pro-urokinase secretion without alteration of tissue plasminogen activator output.
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