| Popis: |
We have investigated the mechanism of Ca2+ entry in fura-2-loaded human platelets using the inhibitors of tyrosine kinases, genistein, and methyl-2,5-dihydroxycinnamate. Genistein (100 microM; 30 min) or methyl-2,5-dihydroxycinnamate (1 microgram/ml; 30 min) reduced ADP-evoked protein-tyrosine phosphorylation at specific bands as assessed by gel electrophoresis and Western blotting with a specific antiphosphotyrosine antibody. Both compounds also reduced ADP-evoked [Ca2+]i rises in the presence, but not the absence, of external Ca2+, suggesting a relatively selective inhibition of Ca2+ entry over internal release. The inactive analogue of genistein, daidzein, was without effect on protein-tyrosine phosphorylation or ADP-evoked Ca2+ elevation in the presence or absence of external Ca2+. Methyl-2,5-dihydroxycinnamate (1 microgram/ml; 5 min) significantly reduced the Ca2+ influx evoked by depletion of the intracellular Ca2+ stores using the inhibitor of the endomembranous Ca(2+)-ATPase, thapsigargin. These results with tyrosine kinase inhibitors are unlikely to be the result of the inhibition of other protein kinases since kinases A, C, and G all inhibit agonist-evoked rises in [Ca2+]i in platelets. These data support a role for tyrosine kinases in the control of Ca2+ entry in human platelets. |
