Thrombin, but not thromboxane, stimulates megakaryocytic differentiation in human megakaryoblastic leukemia cells

Physiologic stimuli for differentiation of bone marrow megakaryoblasts, a critical event leading to formation of platelets, have not been identified. Therefore, we examined the effects of two potent platelet agonists, thrombin and the thromboxane A2 mimetic U46619 [(15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z, 13E-dienoic acid], on the developmental regulation of cultured CHRF-288-11 megakaryoblast cells. Both thrombin and U46619 increased intracellular free calcium in fura-2 loaded, serum-deprived CHRF-288-11 cells with EC50 values of 0.07 +/- 0.01 U/ml and 194 +/- 27 nM, respectively (n = 4 each). Thrombin, but not U46619, increased intracellular pH from 7.44 +/- 0.03 to 7.55 +/- 0.06 (n = 4, P = .012) in 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein-loaded cells. Because increases in cytosolic free calcium and/or cytosolic pH are associated with mitogenic responses in many cell systems, we determined if thrombin or U46619 stimulated CHRF-288-11 proliferation or differentiation. U46619 (1 microM) inhibited cell growth but had no effect on cell size or morphology. In contrast, thrombin (0.2 U/ml) inhibited cell division and increased overall cell size to 116 +/- 2% of control (P less than .05). Microscopically, thrombin-treated cells had increased cytoplasmic content, nuclear lobulation and nucleolar content--hallmarks of megakaryocytic differentiation. Thrombin, but not U46619, increased levels of CHRF-288-11 c-fos and c-myc mRNA transcripts (protooncogene expression). We conclude that thrombin, but not thromboxane A2, induces terminal megakaryocytic differentiation of CHRF-288-11 cells. This suggests that thrombin may act as a physiologic regulator of platelet formation in vivo.