Na+/H+ exchange activity induced by thrombin is not inhibited by protein kinase inhibitors, staurosporine, K-252a, H-7 and sphingosine, in human platelets.

In human platelets stimulated with thrombin (40 mU/ml), Na+/H+ exchange activity [the ethylisopropylamiloride (EIPA)-sensitive increase of cytoplasmic pH (pHc)] and protein kinase C (PKC) activity [phosphorylation of 47 kDa protein (P47), a substrate for PKC] were determined in the presence of protein kinase inhibitors, staurosporine (0.05-1 microM), K-252a (0.5-10 microM), H-7 (100 microM) and sphingosine (20-40 microM). Staurosporine and K-252a completely blocked PKC activity. H-7 and sphingosine reduced the P47 phosphorylation to 64% and 35%, respectively. On the contrary, the thrombin-induced pHc increase was not inhibited by staurosporine, K-252a or H-7. Sphingosine elevated the resting pHc by 0.26-0.42 independently of the Na+/H+ exchanger and inhibited the thrombin-induced pHc increase. However, after the resting pHc elevated by sphingosine had been reduced to the initial level by adding sodium propionate, the thrombin-induced pHc increase was observed again. These results suggested that sphingosine inhibited the thrombin-induced pHc increase by elevating the resting pHc. Thus, we concluded that the Na+/H+ exchanger was activated by thrombin through a pathway independent of PKC.