Activation of a novel 33-kDa SER/THR kinase by tyrosine kinase and calpain in thrombin-stimulated platelets

Abstract number: P0717

Ido^* M., Hayashi† T., Suzuki† K.

†Mie University School of Medicine, Japan ^*Perinatal Center of Mie University Hospital, Japan;

Thrombin receptor/protease-activated receptor1 (PAR1) is a member of the G protein-coupled receptor family containing seven transmembrane domains. Thrombin cleaves extracellular region of PAR1 at the Arg41/Ser42 peptide bond and unmasks a tethered sequence beginning with Ser-Phe-Leu-Leu-Arg-Asn (thrombin receptor agonist peptide) (TRAP); subsequent binding of this ligand sequence to separate extracellular region of the receptor is coupled with a transmembrane signaling, leading to rapid activation of molecules that induces reorganization of the cytoskeletal signaling. Previously, we identified a 33-kDa Ser/Thr protein kinase (PK33) in platelets which activity is increased 10-fold by treatment with thrombin or TRAP. However, PK33-related signal transduction pathway and the role of PK33 in the platelet activation are still unclear. In the present study, we examined possible role of m-calpain and/or tyrosine kinase on thrombin- or TRAP-induced PK33 activation or platelet aggregation. PK33 activity was measured using in-gel renaturation protein kinase assay. Incubation of platelets with calpain inhibitor, calpeptin, or tyrosine kinase inhibitor, herbimycin A, inhibited thrombin- or TRAP-induced PK33 activation and platelet aggregation in a dose-dependent manner. Many signaling molecules including tyrosine kinases were found to be located in the specific membrane structure, lipid raft. Treatment of platelets with methyl-[fnof]À-cyclodextrin, which induces destruction of lipid raft, inhibited thrombin-induced PK33 activation. These findings suggest that m-calpain and tyrosine kinases are involved in the activation of PK33, which may play an important role in the platelet aggregation.