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, Ca(2+) 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 B(2) formation and [Ca(2+) ](i) elevation in platelets stimulated by Thrombin and collagen. NP-313 at 10 µM inhibited cyclooxygenase, thromboxane A(2) synthase, and protein kinase Cα, whereas it did not affect Phospholipase A(2) or Phospholipase C activity. In the presence of indomethacin and an adenosine 5-diphosphate scavenger, NP-313 concentration-dependently inhibited thrombin- and A23187-induced [Ca(2+)](i) increase through its inhibitory effects on Ca(2+) influx, rather than blocking Ca(2+) release from intracellular stores. NP-313 also inhibited thapsigargin-mediated Ca(2+) influx through store-operated Calcium Channel but had no effect on Ca(2+) 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 A(2) synthesis and Ca(2+) influx through SOCC.