Molecular mechanism of thrombin receptor pathway that mediated apoptosis in neural cells

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Thrombin is a multifunctional serine protease that plays an important role in hemostasis and wound healings. High concentrations of thrombin in the central nervous system is often associated with neural cell death. Protease-activated receptor 1 (PAR1/ thrombin receptor) is a member of the G protein-coupled receptor containing seven transmembrane domains. Thrombin cleaves the receptor at the Arg41/Ser42 peptide bond and unmasks a tethered amino-terminal sequence beginning with the Ser-Phe-Leu-Leu-Arg-Asn (thrombin receptor agonist peptide/TRAP) ; subsequent binding of this ligand sequence to the body of the receptor is coupled with a transmembrane signaling mediated by G proteins. Neural cell death by thrombin is mediated by the activation of PAR1. Thus, regulation is coupled with internalization and PAR1. Phosphorylation of Ser and/or Thr in this region by Ala residue or truncation of this region results in resistance to desensitization and internalization. However, protein kinase capable of phosphorylating PAR1 in human platelet is unknown. In the present study, we identified the 33-kDa Ser/Thr protein kinase, which was activated by thrombin, TRAP, but not by hirudin-treated thrombin or diisopropylfluoro- phosphate-inactivated thrombin, suggesting that it is activated through PAR1. Furthermore, treatment of cells with thromboxane AィイD22ィエD2 analog, STAィイD22ィエD2, led to an activation of this protein kinase and phosphorylation of PAR1. In conclusion, the present study provides evidence of homologous and heterologous activation of a novel 33-kDa Ser/Thr kinase that phosphorylates the cytoplasmic tail of PAR1.