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

Abstract

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 (0.05–10 μM), K-252a (0.5–10 μM), H-7 (100 μM) and sphingosine (20–40 μM). 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.