Biogenesis of G-protein mediated calcium signaling in human megakaryocytes.
To understand how platelet signal transduction pathways develop during megakaryocytopoiesis, we isolated human stem cells from umbilical cord blood and cultured the cells in the presence of thrombopoietin (TPO). Based on the early expression of CD61 and late expression of CD42b, immature (CD61+/CD42b(low)) and mature (CD61+/ CD42b(high)) megakaryocytes were immunomagnetically purified and, together with stem cells (CD34+), characterized for Galpha-protein expression and agonist-induced [Ca2+]i increases. Megakaryocytopoiesis was accompanied by down-regulation of the 43 kDa and 46 kDa variants of G16alpha, constant expression of Gsalpha, and up-regulation of Gqalpha and Gialpha1/2. The increase in Gqalpha and Gialpha1/2 expression was accompanied by an increase in Ca2+ signaling triggered by thrombin 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 the iloprost-induced Ca2+ response disappeared during maturation. These data reveal sharp changes in Ca2+ regulation during megakaryocytopoiesis.
den Dekker
E
E
Department of Haematology, University Medical Center Utrecht, The Netherlands. e.dendekker@lab.azu.nl
Gorter
G
G
van der Vuurst
H
H
Heemskerk
J W
JW
Akkerman
J W
JW
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid / Q000494:pharmacology; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D001792:Blood Platelets / Q000166:cytology / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology*; D002417:Cattle; D002454:Cell Differentiation / Q000235:genetics; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D005312:Fetal Blood / Q000166:cytology; D005434:Flow Cytometry; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D018507:Gene Expression Regulation, Developmental; D020962:Heterotrimeric GTP-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D006801:Humans; D016285:Iloprost / Q000494:pharmacology; D008533:Megakaryocytes / Q000187:drug effects / Q000378:metabolism*; D010972:Platelet Activating Factor / Q000494:pharmacology; D020033:Protein Isoforms / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology; D013926:Thrombopoietin / Q000494:pharmacology
D010972:Platelet Activating Factor; D020033:Protein Isoforms; D011518:Proto-Oncogene Proteins; D000244:Adenosine Diphosphate; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D013926:Thrombopoietin; D013917:Thrombin; C112631:G protein alpha 16; C498015:GNAI2 protein, human; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D019205:GTP-Binding Protein alpha Subunits, Gs; D020962:Heterotrimeric GTP-Binding Proteins; D016285:Iloprost; D002118:Calcium