Response of a human megakaryocytic cell line to thrombin: increase in intracellular free calcium and mitogen release

Abstract

The CHRF-288-11 cell line has been previously shown to exhibit properties consistent with a megakaryocytic origin. The response of these cells to thrombin has now been investigated. Thrombin treatment of CHRF-288-11 cells results in both an increase in intracellular free calcium levels and secretion of mitogenic activity and beta-thromboglobulin. Cell viability is not affected. The mitogenic activity released from the cells is due primarily to the presence of basic fibroblast growth factor. Immunohistochemical data indicate a packaging of basic fibroblast growth factor into granular structures. Trypsin and phorbol 12-myristate 13-acetate also initiate release of mitogenic activity from this cell line, whereas under non-stirred conditions collagen and ADP do not. Through measurements of intracellular calcium levels it was determined that thrombin pretreatment of cells ablates a further response to thrombin, but does not block an increase in intracellular calcium levels due to trypsin. This suggests that these two agonists may act through different mechanisms. The thrombin-induced release reaction is inhibited almost completely by the reagents hirudin and dipyridamole, and only partially by indomethacin. These data indicate that the CHRF-288-11 cell line should provide an excellent model system in which to study the packaging of factors into granules which undergo regulated release.