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The effects of cryptolepine on a mouse model of thrombosis and on platelet aggregation
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The effects of cryptolepine on a mouse model of thrombosis
and on p|ate|et aggregation
Oyekan, A.O. DepasCn~nt of PhammcologF, New York Medical College, Valhalla,New York 10595, U.S.A.
Thrombosis is a multicomponent event involving platelet activity, vessel wall, blood flow and coagulation/fibrinolyric processes. An antithrombotic agent can therefore act by interfering with one or more of these events to exert protection against thrombosis. Previous studies on cryptolepine-the methylquindolanol alkaloid obtained from the roots of Cryptolepsis sanguinolenta (Dwuma-Badu et al., 1978) established its a2-blocking properties using the isolated vas deferens of the rat (Noamesi and Bamgbose, 1980). We opined that a possible blockade of a2 receptors by cryptolepine on another test system; the platelets, should elicit antiplatelet effects and therefore, an antithrombotic effect. We therefore evaluated the possible antithrombotic action of cryptolepine, alone and in combination with dipyridamole on a mouse model of arterial thrombosis (DiMinno and Silver, 1983). In this model, solutions (0,1 ml) of a mixture of collagen and adrenaline (made up in isotonic glucose solution, pH 2.7-2.9) containing 150/zg/ml collagen (Hormone-Chemie) and 18/zg/'ml of adrenaline (Parke-Davis) were injected into the tail veins of mice weighing between 25 and 30 gm. Within I rain aftex this thrombotic challenge, the mice became immobile, began gasping for breath and usually died within 1-3 rain. The ability of cryptolepine to prevent death from this thrombotic challenge was taken as a measure of antithrombotic action. Intraperitoneal premedication with cryptolcpine at doses of 0.5, 1.0, 2.0 and 5.0 mg/kg produced dose-dependent protection which reached a significant level (p < 0.05) at 1 mg/kg. The combination of dipyridamole (1.0 and 2.0 mg/kg) with each dose of cryptolepine produced far greater antithrombotic action (p < 0.01) than when both agents were gi~-n individually. The antithrombofie effects of cryptolepine seem to involve at least platelet activity as it dose-dependently ;.,'.~bited platelet a ~ e a t i o n in vitro in human, rabbit and rat platelet rich plasma (PRP). Platelet aggregation was studied in an aggregometer, aggrc~tion, being measured as percentage light transmission through a sample of PRP. The effects of cryptolepine on ag,~regation was determined by incubating it in graded doses with PRP 2 minutes before adding graded doses of ADP, AA and L~rombin. The EC50 v~ues (doses needed to reduce maximal aggregation to any platelet agonist by 50%) ranged between 8.1 x 10 -s M and 1.7 × 1 0 - : M for adenosine diphosphate (ADP), arachidonic acid (AA) and thrombin. In another aspect of the study, different concentrations of cryptolepine were added (in 10/tl volume) at t~e peak of agf,regatory response ~,~h near-maximal doses of ADP (7.5/tM), AA (25/zM) and ~hrombin (5 U/mi). Responses were compared with platelets to which 10 ttl of normal saline was added at the point of peak aggregation. Cryptolepine dissaggregated (dose-dependently), rat, rabbit and human platelets aggregated by near maximal doses of ADP, AA and thrombin. The latter effect suggests that cryptolepine possesses some profibrinolytic properties. These prelimina~ data show that cryptolepine is an antithrombotic agent possibly acting through combined effects on platelets as well as on the fibrinolytic process. Referem~es DiMinno, G~ ana_ Silver, M.I. (1983) J. Pharm. Exp. Ther. 225, 57-60. DwumaoBadu, D., Ayim, J.S.K., Fiagla, N.I.Y., Knapp, J.E., Schiff, Ph.D. and Slatkin, D.J. (1978) J. Pharm. Sci. 67, 433-434. N0mnesi, B.IC and Bamgbose, S.O.A. (1980) Planta Medica, 39, 51-56.
The effects of cryptolepine on a mouse model of thrombosis
and on p|ate|et aggregation
Oyekan, A.O. DepasCn~nt of PhammcologF, New York Medical College, Valhalla,New York 10595, U.S.A.
Thrombosis is a multicomponent event involving platelet activity, vessel wall, blood flow and coagulation/fibrinolyric processes. An antithrombotic agent can therefore act by interfering with one or more of these events to exert protection against thrombosis. Previous studies on cryptolepine-the methylquindolanol alkaloid obtained from the roots of Cryptolepsis sanguinolenta (Dwuma-Badu et al., 1978) established its a2-blocking properties using the isolated vas deferens of the rat (Noamesi and Bamgbose, 1980). We opined that a possible blockade of a2 receptors by cryptolepine on another test system; the platelets, should elicit antiplatelet effects and therefore, an antithrombotic effect. We therefore evaluated the possible antithrombotic action of cryptolepine, alone and in combination with dipyridamole on a mouse model of arterial thrombosis (DiMinno and Silver, 1983). In this model, solutions (0,1 ml) of a mixture of collagen and adrenaline (made up in isotonic glucose solution, pH 2.7-2.9) containing 150/zg/ml collagen (Hormone-Chemie) and 18/zg/'ml of adrenaline (Parke-Davis) were injected into the tail veins of mice weighing between 25 and 30 gm. Within I rain aftex this thrombotic challenge, the mice became immobile, began gasping for breath and usually died within 1-3 rain. The ability of cryptolepine to prevent death from this thrombotic challenge was taken as a measure of antithrombotic action. Intraperitoneal premedication with cryptolcpine at doses of 0.5, 1.0, 2.0 and 5.0 mg/kg produced dose-dependent protection which reached a significant level (p < 0.05) at 1 mg/kg. The combination of dipyridamole (1.0 and 2.0 mg/kg) with each dose of cryptolepine produced far greater antithrombotic action (p < 0.01) than when both agents were gi~-n individually. The antithrombofie effects of cryptolepine seem to involve at least platelet activity as it dose-dependently ;.,'.~bited platelet a ~ e a t i o n in vitro in human, rabbit and rat platelet rich plasma (PRP). Platelet aggregation was studied in an aggregometer, aggrc~tion, being measured as percentage light transmission through a sample of PRP. The effects of cryptolepine on ag,~regation was determined by incubating it in graded doses with PRP 2 minutes before adding graded doses of ADP, AA and L~rombin. The EC50 v~ues (doses needed to reduce maximal aggregation to any platelet agonist by 50%) ranged between 8.1 x 10 -s M and 1.7 × 1 0 - : M for adenosine diphosphate (ADP), arachidonic acid (AA) and thrombin. In another aspect of the study, different concentrations of cryptolepine were added (in 10/tl volume) at t~e peak of agf,regatory response ~,~h near-maximal doses of ADP (7.5/tM), AA (25/zM) and ~hrombin (5 U/mi). Responses were compared with platelets to which 10 ttl of normal saline was added at the point of peak aggregation. Cryptolepine dissaggregated (dose-dependently), rat, rabbit and human platelets aggregated by near maximal doses of ADP, AA and thrombin. The latter effect suggests that cryptolepine possesses some profibrinolytic properties. These prelimina~ data show that cryptolepine is an antithrombotic agent possibly acting through combined effects on platelets as well as on the fibrinolytic process. Referem~es DiMinno, G~ ana_ Silver, M.I. (1983) J. Pharm. Exp. Ther. 225, 57-60. DwumaoBadu, D., Ayim, J.S.K., Fiagla, N.I.Y., Knapp, J.E., Schiff, Ph.D. and Slatkin, D.J. (1978) J. Pharm. Sci. 67, 433-434. N0mnesi, B.IC and Bamgbose, S.O.A. (1980) Planta Medica, 39, 51-56.