The biogenesis of trimeric G proteins was investigated by measurement of the expression of alpha-subunits in the megakaryoblastic cell lines MEG-01, DAMI, and CHRF-288-11, representing stages of increasing maturation, and compared with platelets. Megakaryoblasts and platelets contained approximately equal amounts of Gi alpha-1/2, Gi alpha-3, Gq alpha, and G12 alpha protein. Maturation was accompanied by (1) downregulation of mRNA for Gs alpha and disappearance of iloprost-induced Ca2+ mobilization, (2) upregulation of the long form of Gs alpha protein (Gs alpha-L) and an increase in iloprost-induced cAMP formation, and (3) upregulation of G16 alpha mRNA and G16 alpha protein and appearance of thromboxane A2-induced signaling (Ca2+ mobilization and stimulation of prostaglandin I2-induced cAMP formation). Gz alpha protein was absent in the megakaryoblasts despite weak expression of Gz alpha mRNA in DAMI and relatively high levels of Gz alpha mRNA and Gz alpha protein in platelets. These findings reveal major changes in G protein-mediated signal transduction during megakaryocytopoiesis and indicate that G16 alpha couples the thromboxane receptor to phospholipase C beta.

Differentiation of CD34(+) stem/progenitor cells into megakaryocytes is thought to be a uniform, unidirectional process, in which cells transform step by step from less differentiated precursor stages to more differentiated megakaryocytes. Here we propose the concept and present evidence based on single-cell analysis that differentiation occurs along multiple, partially asynchronous routes. In all CD34(+) cells cultured with thrombopoietin, surface appearance of glycoprotein IIIa (GPIIIa) preceded that of GPIb, indicating that the expression of these glycoproteins occurs in a timely ordered manner. Cellular F-actin content increased in parallel with GPIb expression. Only cells that expressed GPIb were polyploid, pointing to co-regulation of GPIb expression, actin cytoskeleton formation and polyploidization during megakaryocytopoiesis. On the other hand, most progenitor cells responded to thrombin but not to thromboxane A(2) analogue by rises in cytosolic [Ca(2+)](i). The appearance of thromboxane-induced responses during megakaryocytopoiesis was not strictly linked to glycoprotein expression, because cells showed responsiveness either before or after GPIb expression. The same non-strictly sequential pattern was observed for disappearance of the Ca(2+) response by prostacyclin mimetic; in some megakaryocytes it occurred before and in others after GPIb expression. Thus, megakaryocytic differentiation follows along independent routes that are either strictly sequential (GPIIIa and GPIb expression) or proceed at different velocities (Ca(2+) signal regulation).

Abstract-The immature megakaryoblastic cell line MEG-01 responds to iloprost with an increase in cytosolic Ca 2ϩ and cAMP. The Ca 2ϩ response is almost absent in CHRF-288-11 cells, but cAMP formation is preserved in this more mature megakaryoblastic cell line. Also, in human hematopoietic stem cells, iloprost induces a Ca 2ϩ response and cAMP formation. The Ca 2ϩ response is downregulated during megakaryocytopoiesis, but cAMP formation remains unchanged. The Ca 2ϩ increase may be caused by cAMP-mediated inhibition of Ca 2ϩ sequestration, because it is (1) independent of Ca 2ϩ entry; (2) mimicked by forskolin, an activator of adenylyl cyclase, and isobutylmethylxanthine, an inhibitor of phosphodiesterases; and (3) preserved in the presence of inhibitors of protein kinase A and inositol-1,4,5-triphosphate receptors. The small GTPase Rap1 has been implicated in the control of Ca 2ϩ sequestration. Indeed, Rap1 activation parallels the iloprost-and forskolin-induced Ca 2ϩ increase and is unaffected by the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ,-tetraacetic acid-AM. These findings reveal a novel mechanism for elevating cytosolic M egakaryocytopoiesis is accompanied by downregulation of stem cell properties and upregulation of properties that later determine platelet functions. One of the first characteristics of megakaryocyte differentiation is the appearance of the fibrinogen receptor, integrin ␣ IIb ␤ 3 (glycoprotein IIb/IIIa, or CD41/CD61), together with the disappearance of the stem cell marker CD34. 1 A second early event is the synthesis of von Willebrand factor (vWF), which starts in immature, both CD61 ϩ and CD34 ϩ megakaryocytes. At a later stage, the vWF receptor, glycoprotein Ib␣ (CD42b), is expressed, which marks the beginning of polyploidization. 1 The transition from proliferating to differentiating megakaryocytes is accompanied by loss of nuclear-associated acetylcholinesterase activity. 2 The megakaryoblastic cell lines MEG-01, DAMI, and CHRF-288-11 have properties in common with normal megakaryocytes at different stages of maturation. 3 The immature MEG-01 cells already show an increase in cytosolic Ca 2ϩ concentration, [Ca 2ϩ ] i , on stimulation by thrombin and platelet-activating factor. The more mature DAMI and CHRF-288-11 cells upregulate this property and, in addition, become sensitive to thromboxane A 2 . These cells respond to the prostacyclin analogue iloprost with an increase in cAMP, a response that is also upregulated in the more mature cell lines. Hence, the immature megakaryoblast already has the capacity to regulate Ca 2ϩ and cAMP via mechanisms also seen in platelets. An interesting exception is that in MEG-01 cells, iloprostinduced cAMP formation is accompanied by a rise in [Ca 2ϩ ] i . This is in sharp contrast with platelets, which do not raise [Ca 2ϩ ] i when treated with prostacyclin and show Ca 2ϩ responses by thrombin and other platelet-activating agents that are completely blocked by an increase in cAMP. 4 In the present study, we aimed to clarify ...