Tyrosine‐kinase activity in rabbit platelets stimulated with platelet‐activating factor

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 antiphosphotyrosine 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(Oaminophen0xy)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 B, generation, dense-granule release and platelet aggregation, indicating a role for PTKase(s) in the regulation of platelet functional responses. Platelets stimulated with a-thrombin, ionomycin and 12-0-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.Many phosphorylation events observed in platelets have been attributed to the role of protein kinase C (PKC) and myosin light chain kinase, both of which are serinelthreonine specific kinases (for review, see Siess, 1989). However, it is possible that the relatively recently discovered protein tyrosine kinases (PTKases) in platelets may also have a role in signal transduction.

Flavonoids inhibit platelet function through binding to the thromboxane A2 receptor

“…Moreover, the fact that the effect of flavonoids that exhibit higher affinity for the TxA 2 receptor in the inhibition of aggregation is stronger in collagen than in arachidonic acid and U46619‐stimulated platelets suggests that TxA 2 receptor blockade by flavonoids seems to be an additional mechanism to those that have been proposed, through which some of these compounds might inhibit the collagen signaling pathway. Hence, those flavonoids are also inhibitors of tyrosine kinases [16,34,35], an important pathway for collagen signaling leading to platelet aggregation [36], suggesting that this mechanism may also play a role in specificity of inhibition by collagen. Moreover, a methylated flavonoid, diosmetin, seems to specifically inhibit collagen‐induced aggregation and secretion, independently of any effect on the TxA 2 receptor blockade.…”

Phosphorylation of Threonine 558 in the Carboxyl-terminal Actin-binding Domain of Moesin by Thrombin Activation of Human Platelets

“…6. Calphostin C (54), genistein (55), and H-89 (56) are specific inhibitors of protein kinase C, proteintyrosine kinase, and cAMP-dependent protein kinase (protein kinase A), respectively, while staurosporine has broad specificity for inhibiting protein kinases (57)(58)(59). Genistein and staurosporine prevent the increased phosphorylation induced by thrombin in a dose-dependent manner, as well as reducing basal phosphorylation in unstimulated platelets.…”

An improved method for determination of isoflavones in soybean powder by liquid chromatography

“…The beneficial effects of isoflavones to human health have been well documented. For instance, isoflavones have been reported to possess antioxidant activity [1,2], inhibit growth of cancer cells [3,4] and prevent atherosclerosis [5]. However, isoflavones may undergo loss during processing of soybean foods, which may in turn decrease their biological activity [6,7].…”