Protein tyrosine phosphorylation in equine platelets: the effect of stimulation by thrombin and platelet-activating factor (PAF)
Protein tyrosine phosphorylation (PTP) in thrombin- and platelet-activating factor (PAF)-stimulated equine platelet activation was investigated in the absence and presence of 2 protein tyrosine kinase inhibitors (PTKIs), methyl 2,5-dihydroxycinnamate (MDHC) and genistein. Washed equine platelets aggregated irreversibly in response to thrombin or PAF in an agonist concentration dependent fashion. MDHC produced an MDHC concentration and time dependent inhibitory effect on rate and extent of thrombin- and PAF-induced aggregations, whereas the effect of genistein on the same parameters was only genistein concentration dependent. Western blotting demonstrated tyrosine phosphorylated proteins in resting platelets. Changes in the PTP pattern occurred both when platelets were stimulated with varying concentrations of thrombin or PAF for a standard time (3 min) or with a standard agonist concentration (0.17 u/ml thrombin or 10(-10) mol/l PAF) for varying times. Different patterns of PTP were produced by thrombin and PAF. 500 mumol/l MDHC and 300 mumol/l genistein each affected the PTP patterns produced in response to thrombin or PAF, but in different ways. PTP results with thrombin and PAF in the presence of 500 mumol/l MDHC were similar, as were those in the presence of 300 mumol/l genistein. However, there were many differences in the PTP results between thrombin (or PAF) in the presence of MDHC and between thrombin (or PAF) in the presence of genistein. Therefore, although both inhibitors are PTKIs, they have different effects on the PTP induced by either agonist. Our work has produced the first evidence of PTP in equine platelets. It is probable that the changes in PTP are related to events in the signal transduction pathway.
Dillon
A M
AM
University of Cambridge, Department of Clinical Veterinary Medicine, UK.
Heath
M F
MF
D000818:Animals; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D015153:Blotting, Western; D002934:Cinnamates / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D019833:Genistein; D006736:Horses / Q000097:blood* / Q000502:physiology; D007529:Isoflavones / Q000494:pharmacology; D008163:Luminescent Measurements; D010766:Phosphorylation; D010972:Platelet Activating Factor / Q000494:pharmacology*; D010974:Platelet Aggregation / Q000187:drug effects / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013917:Thrombin / Q000494:pharmacology*; D013997:Time Factors
D002934:Cinnamates; D007529:Isoflavones; D010972:Platelet Activating Factor; C067472:methyl 2,5-dihydroxycinnamate; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D013917:Thrombin