• equine;
  • platelets;
  • thrombin;
  • platelet-activating factor;
  • protein-tyrosine phosphorylation


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-dihydroxyciimamate (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 μmol/l MDHC and 300 μmol/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 μmol/l MDHC were similar, as were those in the presence of 300 μmol/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.