Evaluation of effects of various drugs on platelet functions using phorbol 12-myristate 13-acetate-induced megakaryocytic human erythroid leukemia cells

BACKGROUND: The hyperfunction and activation of platelets have been strongly implicated in the development and recurrence of arterial occlusive disease, and various antiplatelet drugs are used to treat and prevent such diseases. New antiplatelet drugs and many other drugs have been developed, but some drugs may have adverse effects on platelet functions. OBJECTIVE: The aim of this study was to establish an evaluation method for evaluating the effect and adverse effect of various drugs on platelet functions. MATERIALS AND METHODS: Human erythroid leukemia (HEL) cells were used after megakaryocytic differentiation with phorbol 12-myristate 13-acetate as an alternative to platelets. Drugs were evaluated by changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) mobilization in Fura2-loaded phorbol 12-myristate 13-acetate-induced HEL cells. Aspirin and cilostazol were selected as antiplatelet drugs and ibuprofen and sodium valproate as other drugs. RESULTS: There was a positive correlation between [Ca(2+)](i) and platelet aggregation induced by thrombin. Aspirin (5.6–560 µM) and cilostazol (5–10 µM) significantly inhibited thrombin-induced increases in [Ca(2+)](i) in a concentration-dependent manner. On the other hand, ibuprofen (8–200 µM) and sodium valproate (50–1,000 µg/mL) also significantly inhibited thrombin-induced increases in [Ca(2+)](i) in a concentration-dependent manner. Furthermore, the interaction effects of the simultaneous combined use of aspirin and ibuprofen or sodium valproate were evaluated. When the inhibitory effect of aspirin was higher than that of ibuprofen, the effect of aspirin was reduced, whereas when the inhibitory effect of aspirin was lower than that of ibuprofen, the effect of ibuprofen was reduced. The combination of aspirin and sodium valproate synergistically inhibited thrombin-induced [Ca(2+)](i). CONCLUSION: It is possible to induce HEL cells to differentiate into megakaryocytes, which are a useful model for the study of platelet functions, and the quantification of the inhibition of thrombin-induced increases in [Ca(2+)](i) is applicable to the evaluation of the effects of various drugs on platelets.