ADP- and thapsigargin-evoked Ca2+ entry and protein-tyrosine phosphorylation are inhibited by the tyrosine kinase inhibitors genistein and methyl-2,5-dihydroxycinnamate in fura-2-loaded human platelets.
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.
Sargeant
P
P
Physiological Laboratory, University of Cambridge, United Kingdom.
Farndale
R W
RW
Sage
S O
SO
D000244:Adenosine Diphosphate / Q000494:pharmacology*; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D001798:Blood Proteins / Q000378:metabolism; D002118:Calcium / Q000097:blood*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002934:Cinnamates / Q000494:pharmacology*; D005456:Fluorescent Dyes; D016257:Fura-2; D019833:Genistein; D006801:Humans; D066298:In Vitro Techniques; D007529:Isoflavones / Q000494:pharmacology*; D007700:Kinetics; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000097:blood*; D013729:Terpenes / Q000494:pharmacology*; D019284:Thapsigargin; D013997:Time Factors; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism*
D001798:Blood Proteins; D002934:Cinnamates; D005456:Fluorescent Dyes; D007529:Isoflavones; D013729:Terpenes; D019000:Phosphotyrosine; D014443:Tyrosine; D000244:Adenosine Diphosphate; C004742:daidzein; C067472:methyl 2,5-dihydroxycinnamate; D019284:Thapsigargin; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D000252:Calcium-Transporting ATPases; D002118:Calcium; D016257:Fura-2