|Budget Amount *help
¥2,400,000 (Direct Cost : ¥2,400,000)
Fiscal Year 1996 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1995 : ¥1,500,000 (Direct Cost : ¥1,500,000)
It has been known that the platelet integrin alpha_<IIb>beta_3 is involved bi-directional signaling across the plasma membrane. When platelets are stimulated by agonists, intracellular signal transduction can be activated, leading to a conformational change in the extracellular domains of integrin alpha_<IIb>beta_3 that increases its affinity for adhesive ligands, for example fibrinogen, resulting in platelet aggregation. Conversely, ligand binding to integrin alpha_<IIb>beta_3 is involved in outside-in signals ; its association with cytoskeleton causes biochemical changes including tyrosine phosphorylation of several proteins, increase in intracellular calcium level. Ourprevious study indicated that thrombin activation of human platelets causes cleavage of PLCbeta3 by calpain leading to its enhanced activity by Gbetagamma subunits.
To investigate a role of phospholipase C (PLC) isozymes in the integrin alpha_<IIb>beta_3-mediated signaling, their location was examined in thrombin-activa
ted human platelets, revealing different regulation of their translocation to the cytoskeleton (CSK). In resting platelets, the major PLCs such as PLCbeta2, PLCbeta3a (155KDa) and PLCgamma2, and the minor PLCs (PLCbeta1 and PLCgamma1) were located in the Triton X-100-soluble (Tx.Sol) fraction and the membrane skeleton (MSK) , while PLCbeta3b (140KDa) was present only in Tx.Sol fraction. Thrombinstimulation caused a rapid and transient translocation of PLCbeta3 (a, b) and a slower accumulation of PLCbeta2 and PLCgamma2 in the reorganized CSK.The translocation to CSK of both PLCbeta3 (a, b) , but not PLCbeta2, was dependent on integrin alpha_<IIb>beta_3-mediated aggregation. Furthermore, an actin polymerization inhibitor, cytochalasin D or a protein tyrosine kinase inhibitor, genistein abolished the CSK association of alpha_<IIb>beta_3, PLCbeta3 and Gq, but not PLCbeta2 or Gi2. The late diacylglycerol generation induced by thrombin stimulation was reduced by the genistein treatment. These results suggest that the integrin alpha_<IIb>beta_3-mediated cytoskeletal association of PLCbeta3 is regulated by protein tyrosine kinase and also that the activation of the relocated PLC may play a role in the late platelet-to-platelet aggregation in thrombin-stimulated human platelets. Less