Tyrosine-kinase activity in rabbit platelets stimulated with platelet-activating factor. The effect of inhibiting tyrosine kinase with genistein on platelet-signal-molecule elevation and functional responses

Abstract

The temporal relationship of tyrosine phosphorylation of proteins in platelet-activating-factor-(PAF)-stimulated rabbit platelets was characterised by Western blotting using a monoclonal anti-phosphotyrosine antibody, demonstrated to be specific for detecting only tyrosine phosphorylated proteins. In addition, the protein tyrosine kinase (PTKase) inhibitor genistein, was used to investigate the role of endogenously activated PTKase(s) in the regulation of receptor-stimulated changes in both signal molecule production and in platelet functional responses. Several tyrosine phosphorylated protein bands (52-62 kDa) were observed in unstimulated platelets, however, within 5 s of PAF stimulation, two further groups of tyrosine phosphorylated protein bands were observed (35-45 kDa and 66-90 kDa) and within 30 s of PAF stimulation a further group was detected (90-150 kDa). Under conditions where intracellular Ca2+ was chelated with acetoxymethyl 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetate (BAPTA-AM) and extracellular Ca2+ was chelated with EGTA, the number of tyrosine-phosphorylated bands was greatly reduced. Tyrosine phosphorylation of the proteins induced by PAF stimulation were differentially inhibited by treatment with genistein. Genistein inhibited PAF-induced elevation of the signal molecule inositol 1,4,5-trisphosphate and also inhibited both mobilization of Ca2+ and the influx of Ca2+ through the plasma membrane. These results suggest a role for endogenously activated PTKase(s) in the early stages of signal transduction in PAF-stimulated platelets. Moreover, inhibition of genistein-sensitive PTKase(s) also caused an inhibition of PAF-induced thromboxane B2 generation, dense-granule release and platelet aggregation, indicating a role for PTKase(s) in the regulation of platelet functional responses. Platelets stimulated with alpha-thrombin, ionomycin and 12-O-tetradecanylphorbol 13-acetate gave a similar pattern of phosphorylated proteins to PAF-stimulated platelets, however, whereas genistein inhibited protein phosphorylation, it had no significant effect on functional responses in platelets stimulated with these agents, suggesting that an alternative signalling pathway exists.