THE MECHANISM(S) OF THE ANTIAGGREGATORY EFFECTS OF CRYPTOLEPINE - THE ROLE OF CYCLIC ADENOSINE-MONOPHOSPHATE AND CELLULAR CA-2+

1. In this investigation, the properties and possible mechanisms of the antiaggregatory effects of cryptolepine were evaluated. 2. Cryptolepine had no effect on platelet shape change but inhibited aggregation in a time-dependent manner. The inhibition of aggregation lacks agonst specificity, the IC50 values ( x 10(-5) M) being 2.79 +/- 0.7 ADP 3.05 +/- 0.2 (U46619), 2.89 +/- 0.6 (A23187), 2.41 +/- 0.6 (thrombin), 4.05 +/- 0.9 (arachidonic acid) and 47.3 +/- 3.9 (PAF). 3. The antiaggregatory effects were fully reversible and surmountable at concentrations less-than-or-equal-to 75 muM but unsurmountable at concentrations greater-than-or-equal-to 100 muM. 4. The coincubation of cryptolepine (25 and 50 muM) with cpt-cAMP (50 muM) resulted in increased inhibition of aggregation from 24.2 +/- 2.1% (25 muM) and 45.1 +/- 3.4% (50 muM) to 69.5 +/- 5.8% and 84.2 +/- 6.4%, respectively. 5. Cryptolepine (10 muM) synergized with stimulants of platelet adenylate cyclase, prostacyclin (0.5 and 1 nM) and forskolin (2.5 and 5 muM) to inhibit aggregation induced by adenosine diphosphate (ADP). The inhibition of aggregation by cryptolepine (10 muM; 18.2 +/- 1.5%) or prostacyclin (0.5 nM; 23.4 +/- 2.0%) increased to 62.6 +/- 3.8% (P < 0.01) on combined administration. 6. Following pretreatment with IBMX (50 muM), a phosphodiesterase (PDE) inhibitor, the inhibitory effect of cryptolepine (25 muM) increased from 21.5 +/- 2. 1% to 42.3 +/- 3.7% (P < 0.01). In the presence of imidazole (2.5 mM), an activator of PDE, the inhibitory effects of cryptolepine reduced from 63.2 +/- 5.4% (50 muM) and 84.7 +/- 4.4% (75 muM) to 1.4 +/- 0.2% and 21.3 +/- 2.4%, respectively. 7. In Ca2+-free buffer (containing EGTA, 0.1 mM) or in the presence of extracellular Ca2+ (0.5 mM CaCl2), the IC50 of cryptolepine against thrombin-induced aggregation was not different (3.94 +/- 0.6 muM [0 mM Ca2+] vs 3.52 +/- 0.8 muM [0.5 mM Ca2+]). In platelets preincubated with ouabain (50 muM) to increase intracellular Ca2+, the inhibition of ADP-aggregation by cryptolepine (50 muM) was not different, the percentage inhibition of aggregation being 52.3 +/- 3.4% (+ouabain) and 59.6 +/- 3.4% (-ouabain). 8. The data suggest that cryptolepine inhibits platelet aggregation by increasing platelet cAMP levels, possibly through stimulation of platelet adenylate cyclase. The antiaggregatory effects do not seem to involve extracellular or intracellular Ca2+.