title: NP-313, 2-acetylamino-3-chloro-1,4-naphthoquinone, a novel antithrombotic agent with dual inhibition of thromboxane A(2) synthesis and calcium entry.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  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
  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.
authors.affiliation: Graduate Institute of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan.
authors.forename: Heng-Lan
authors.initials: HL
authors.lastname: Kuo
authors.forename: Jin-Cherng
authors.initials: JC
authors.lastname: Lien
authors.forename: Chien-Hsin
authors.initials: CH
authors.lastname: Chang
authors.forename: Ching-Hu
authors.initials: CH
authors.lastname: Chung
authors.forename: Sheng-Chu
authors.initials: SC
authors.lastname: Kuo
authors.forename: Chun-Chieh
authors.initials: CC
authors.lastname: Hsu
authors.forename: Hui-Chin
authors.initials: HC
authors.lastname: Peng
authors.forename: Tur-Fu
authors.initials: TF
authors.lastname: Huang
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005343:Fibrinolytic Agents / Q000008:administration & dosage / Q000494:pharmacology*; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D009285:Naphthoquinones / Q000008:administration & dosage / Q000494:pharmacology*; D019007:P-Selectin / Q000235:genetics; D010974:Platelet Aggregation / Q000187:drug effects*; D010975:Platelet Aggregation Inhibitors / Q000008:administration & dosage / Q000494:pharmacology*; D013928:Thromboxane A2 / Q000096:biosynthesis; D013929:Thromboxane B2 / Q000096:biosynthesis
chemical_list: C559199:2-acetylamino-3-chloro-1,4-naphthoquinone; D015220:Calcium Channels; D005343:Fibrinolytic Agents; D009285:Naphthoquinones; D019007:P-Selectin; D010975:Platelet Aggregation Inhibitors; D013929:Thromboxane B2; D013928:Thromboxane A2; D002118:Calcium