Biogenesis of G-protein mediated calcium signaling in human megakaryocytes

To understand how platelet sig al transduction pathways develop during mega karyocytopoiesis we isolated human stem cells from umbilical cord blood and cultured the cells in the presence of thrombopoietin (TPO), Based on the e arly expression of CD61 and late expression of CD42b, immature (CD61(+)/CD4 2b(low)) and mature (CD61(+)/CD42b(high)) megakaryocytes were immunomagneti cally purified and, together with stem cells (CD34(+)), characterized for G alpha -protein expression and agonist-induced [Ca2+](i), increases. Megaka ryocytopoiesis was accompanied by down-regulation of the 43 kDa and 46 kDa variants of G(16)alpha, constant expression of G(s)alpha, and up-regulation of G(q)alpha and G(i)alpha1/2. The increase in G(q)alpha and G(i)alpha1/2 expression was accompanied by an increase in Ca2+ signaling triggered by th rombin and other agonists known to signal to Ca2+, via these G-proteins in platelets. The prostacyclin analog iloprost and TPO also induced [Ca2+](i), increases, and tile iloprost-induced Ca2+ response disappeared during matu ration. These data reveal sharp changes in Ca2+ regulation during megakaryo cytopoiesis.