Exploration of the antiplatelet activity profile of betulinic acid on human platelets

Abstract

Betulinic acid, a natural pentacyclic triterpene acid, presents a diverse mode of biological actions including antiretroviral, antibacterial, antimalarial, and anti-inflammatory activities. The potency of betulinic acid as an inhibitor of human platelet activation was evaluated, and its antiplatelet profile against in vitro platelet aggregation, induced by several platelet agonists (adenosine diphosphate, thrombin receptor activator peptide-14, and arachidonic acid), was explored. Flow cytometric analysis was performed to examine the effect of betulinic acid on P-selectin membrane expression and PAC-1 binding to activated platelets. Betulinic acid potently inhibits platelet aggregation and also reduced PAC-1 binding and the membrane expression of P-selectin. Principal component analysis was used to screen, on the chemical property space, for potential common pharmacophores of betulinic acid with approved antithrombotic drugs. A common pharmacophore was defined between the NMR-derived structure of betulinic acid and prostacyclin agonists (PGI2), and the importance of its carboxylate group in its antiplatelet activity was determined. The present results indicate that betulinic acid has potential use as an antithrombotic compound and suggest that the mechanism underlying the antiplatelet effects of betulinic acid is similar to that of the PGI2 receptor agonists, a hypothesis that deserves further investigation.

Figure 1

Dose-response curves for betulinic acid demonstrating the inhibition of platelet aggregation induced by ADP (A), Arachidonic acid (AA) (B) and TRAP (C).

Figure 2

Representative histograms, obtained by flow cytometry analysis, illustrating the effect of betulinic acid (A), (C) and betulin (B), (D) on PAC-1-FITC binding, and CD62P-PE membrane expression on activated with TRAP platelets, respectively.

Figure 3

Principal component analysis (PCA) comparison of five families of 18 approved antithrombotic drugs representative of five mechanistic groups: adenosine diphosphate receptor inhibitors (orange), prostanoid prostacyclin receptor agonists (blue), COX inhibitors (purple), thromboxane inhibitors (green) and phosphodiesterase inhibitors (red). The first two principal components account for 84.1% of the original information. Clusters of antithrombotic drugs were identified by considering the hierarchical clustering approach (single linkage method) (25). The 4 prostacyclin receptor agonists cluster largerly in one region of the plot (grey area).

Figure 4

(A) NMR derived 3D structure of betulinic acid. The two chemotypes of betulinic acid, the carboxylate group and the hydroxyl group, are highlighted and their van der Waals surfaces are colored in red. (B) Superposition of the NMR derived structure of betulinic acid to the synthetic PGI2 analogue iloprost. Iloprost is shown in its bound form to the hIP receptor as was previously determined (34). Betulinic acid is colored in blue and iloprost in orange.

Scheme 1

2D structures of betulinic acid and betulin. The 2D structure of prostacyclin and its synthetic analogues (iloprost, beraprost, treprostinil) show common features: the C1-COOH, the α-chain, the C11-OH, the C15-OH and the ω-chain.