The endoplasmic reticulum (ER) plays a key role in Ca 2+ signalling through Ca 2+ release via inositol 1,4,5-trisphosphate receptors (Ins P 3 -Rs) and Ca 2+ uptake by sarco/endoplasmic reticulum Ca 2+ -ATPases (SERCAs). Here, we investigated the organization of platelet ER and its biogenesis during megakaryocytopoiesis. First, erythro/megakaryoblastic MEG 01, UT7, M-O7e and CHRF 288-11 cell lines, platelets and thrombopoietin-induced UT7-Mpl cells were selected for the study of SERCA2b and SERCA3 proteins by Western blotting using the antibodies IID8 and PL/IM430, respectively. As judged by platelet glycoprotein IIIa (GPIIIa) expression, an increase in SERCA3 proteins was observed while that of SERCA2b remained unchanged throughout maturation. Second, these studies were extended to the newly described alternatively spliced SERCA3a–c RNAs and Ins P 3 -Rs using the in vitro model of PMA-induced differentiation of MEG 01 cells. Time-course and dose–response studies showed a maximal approx. 4-fold up-regulation of SERCA3 proteins using 10 -8 M PMA for 3 days, which paralleled induction of GPIIIa expression. SERCA3 induction was found to occur at the level of mRNA. The modulation of the different SERCA3 species (i.e. 3a, 3b and 3c) was isoform-specific: while SERCA3a was slightly increased, an approx. 3-fold induction of SERCA3b, and a 4-fold induction of SERCA3c, was observed after 24h of PMA treatment. Isoform-specific Western blotting and/or reverse transcriptase PCR studies showed that Ins P 3 -R types I, II and III are expressed in MEG 01 cells, as well as in platelets. Study of the expression of these Ins P 3 -R types in PMA-induced MEG 01 cells revealed that: (i) Ins P 3 -RI protein and mRNA showed no changes; (ii) Ins P 3 -RII mRNA was up-regulated and peaked at hour 48 and (iii) Ins P 3 -RIII mRNA and protein showed a transitory maximal 3- and 2.3-fold increase at hours 6 and 30, respectively. Upon PMA treatment of CHRF 288-11 cells, in which GPIIIa is not induced upon treatment, a similar pattern of regulation of Ins P 3 -R types II and III was seen, but a distinct pattern of SERCA3 regulation was observed. These results suggest a profound reorganization of ER-protein patterns during megakaryocytopoiesis and underline the role of SERCA3 gene regulation in the control of Ca 2+ -dependent platelet functions.

Platelet Ca 2+ signalling involves intracellular Ca 2+ pools, whose content is controlled by sarco/endoplasmic reticulum Ca 2+ ATPases (SERCAs). Among these, a key role is played by the inositol trisphosphate-sensitive Ca 2+ pool, associated with the SERCA 3b isoform. We have investigated the control of this Ca 2+ pool through the cAMP-dependent phosphorylation of the GTP-binding protein, Rap (Ras-proximate) 1b. We first looked for this Ca 2+ pool target of regulation by studying the expression of the different SERCA and Rap 1 proteins in human platelets and various cell lines, by Western blotting and reverse transcription-PCR. Since co-expression of Rap 1b and SERCA 3b was obtained, we looked for their protein–protein interaction as a function of the cAMP-dependent phosphorylation of Rap 1b. Co-immunoprecipitations of SERCA 3b and Rap 1b proteins were found in the absence of phosphorylation, induced by the catalytic subunit of the cAMP-dependent protein kinase (csPKA). In contrast, upon pre-treatment of platelet membranes with csPKA, the SERCA 3b dissociated from the Rap 1b protein, in agreement with a role of its phosphorylated state in their interaction. Finally, we looked for adaptation of this complex in a platelet pathological model of hypertension. We investigated the expression of both proteins, as well as the cAMP-dependent phosphorylation of Rap 1b and SERCA 3b activity in platelets from control normotensive Wistar-Kyoto rats and from spontaneously hypertensive rats (SHRs). A decrease in SERCA 3b activity was associated with a decrease in Rap 1b endogenous phosphorylation in SHR platelets, consistent with a functional role in the regulation of the SERCA 3b-associated Ca 2+ pool.