NP-313, 2-acetylamino-3-chloro-1,4-naphthoquinone, a novel antithrombotic agent with dual inhibition of thromboxane A(2) synthesis and calcium entry

BACKGROUND AND PURPOSE

1,4-Naphthoquinones exhibit antiplatelet activity both in vivo and in vitro. In the present study, we investigated the antiplatelet effect of a novel naphthoquinone derivative NP-313, 2-acetylamino-3-chloro-1,4-naphthoquinone and its mechanism of action.

EXPERIMENTAL APPROACH

We measured platelet aggregation, Ca²⁺ mobilization, thromboxane B2 formation and P-selectin expression and examined several enzymatic activities. Furthermore, we used the irradiated mesenteric venules in fluorescein sodium–treated mice to monitor the antithrombotic effect of NP-313 in vivo.

KEY RESULTS

NP-313 concentration-dependently inhibited human platelet aggregation induced by collagen, arachidonic acid, thapsigargin, thrombin and A23187. NP-313 also inhibited P-selectin expression, thromboxane B₂ formation and Ca²⁺]_i elevation in platelets stimulated by thrombin and collagen. NP-313 at 10 µM inhibited cyclooxygenase, thromboxane A₂ synthase, and protein kinase Cα, whereas it did not affect phospholipase A₂ or phospholipase C activity. In the presence of indomethacin and an adenosine 5-diphosphate scavenger, NP-313 concentration-dependently inhibited thrombin- and A23187-induced Ca²⁺]_i increase through its inhibitory effects on Ca²⁺ influx, rather than blocking Ca²⁺ release from intracellular stores. NP-313 also inhibited thapsigargin-mediated Ca²⁺ influx through store-operated calcium channel but had no effect on Ca²⁺ influx through store-independent calcium channel evoked by the diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol. Nevertheless, it had little effect on cyclic AMP and cyclic GMP levels. Also, intravenously administered NP-313 dose-dependently inhibited the thrombus occlusion of the irradiated mesenteric vessels of fluorescein-pretreated mice.

CONCLUSIONS AND IMPLICATIONS

Taken together, these results indicate that NP-313 exerts its antithrombotic activity through dual inhibition of thromboxane A₂ synthesis and Ca²⁺ influx through SOCC.