Platelet PMCA- and SERCA-type Ca2+ -ATPase expression in diabetes: a novel signature of abnormal megakaryocytopoiesis.

BACKGROUND: Previous studies have shown platelet Ca(2+) abnormalities in diabetes mellitus and some reports suggest abnormal platelet production. Platelet Ca(2+) homeostasis is controlled by a multi-Ca(2+)-ATPase system that includes two plasma membrane Ca(2+)-ATPase (PMCA) and seven sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) isoforms. In addition, we recently found that the expression of PMCA4b and SERCA3 isoforms may serve as new markers of abnormal megakaryocytopoiesis [Nurden P et al. Impaired megakaryocytopoiesis in type 2B von Willebrand disease with severe thrombocytopenia. Blood 2006; 108: 2587-95]. AIM: To analyze the expression of major platelet Ca(2+)-ATPases in 27 patients with type 1 or type 2 diabetes (T1D or T2D) compared with normal donors. METHODS: Investigation of protein and mRNA expressions of PMCA1b and PMCA4b, and SERCA2b, SERCA3a and SERCA3b, using specific Western blotting and reverse transcriptase-polymerase chain reaction, respectively. RESULTS: Remarkably, all patients with T1D were found to present a higher expression of PMCA4b protein (212% +/- 28%; n = 10) and PMCA4b mRNA (155% +/- 16%; n = 17), coupled with a higher expression of SERCA3b mRNA (165% +/- 9%) in some cases. Patients with T2D (n = 10) were also studied for protein expression and were found to present similar major upregulation of the expression of PMCA4b protein (180% +/- 28%; n = 10). Lastly, five of 10 patients with T1D were studied for PMCA4b expression after insulin treatment, with four of five recovering normal expression (96% +/- 15%; n = 5). CONCLUSIONS: Compared with the expression of PMCA4b upon platelet maturation, platelets from diabetic patients exhibit similarities with immature megakaryocytes. Thus, this study reinforces the idea that abnormal megakaryocytopoiesis can provide additional insights into diabetes and could represent a novel therapeutic target for antithrombotic drugs.

How many Ca(2)+ATPase isoforms are expressed in a cell type? A growing family of membrane proteins illustrated by studies in platelets.

Ca(2+) signaling plays a key role in normal and abnormal platelet functions. Understanding platelet Ca(2+) signaling requires the knowledge of proteins involved in this process. Among these proteins are Ca(2+)ATPases or Ca(2+) pumps that deplete the cytosol of Ca(2+) ions. Here, we will particularly focus on two Ca(2+) pump families: the plasma membrane Ca(2+)ATPases (PMCAs) that extrude cytosolic Ca(2+) towards the extracellular medium and the sarco/endoplasmic reticulum Ca(2+)ATPases (SERCAs) that pump Ca(2+) into the endoplasmic reticulum (ER). In the present review, we will summarize data on platelet Ca(2+)ATPases including their identification and biogenesis. First of all, we will present the Ca(2+)ATPase genes and their isoforms expressed in platelets. We will especially focus on a member of the SERCA family, SERCA3, recently found to give rise to a number of species-specific isoforms. Next, we will describe the differences in Ca(2+)ATPase patterns observed in human and rat platelets. Last, we will analyze how the expression of Ca(2+)ATPase isoforms changes during megakaryocytic maturation and show that megakaryocytopoiesis is associated with a profound reorganization of the expression and/or activity of Ca(2+)ATPases. Taken together, these data provide new aspects of investigations to better understand normal and abnormal platelet Ca(2+) signaling.

Biogenesis of endoplasmic reticulum proteins involved in Ca2+ signalling during megakaryocytic differentiation: an in vitro study.

The endoplasmic reticulum (ER) plays a key role in Ca(2+) signalling through Ca(2+) release via inositol 1,4,5-trisphosphate receptors (InsP(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 InsP(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 InsP(3)-R types I, II and III are expressed in MEG 01 cells, as well as in platelets. Study of the expression of these InsP(3)-R types in PMA-induced MEG 01 cells revealed that: (i) InsP(3)-RI protein and mRNA showed no changes; (ii) InsP(3)-RII mRNA was up-regulated and peaked at hour 48 and (iii) InsP(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 InsP(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+)ATPases : a plural, species-specific, and multiple hypertension-regulated expression system.

Gaining insight into nonmuscle Ca(2+) signaling requires basic knowledge of the major structures involved. We investigated the expression of platelet Ca(2+)ATPases in normal and hypertension-associated abnormal Ca(2+) signaling. First, overall identification of normotensive Wistar-Kyoto rat Ca(2+)ATPases was attempted by looking for newly described human platelet 3'-end alternatively spliced sarco/endoplasmic reticulum Ca(2+)ATPases (SERCA) 3b mRNA and plasma membrane Ca(2+)ATPase (PMCA) 1b and 4b proteins, in addition to SERCA2b and SERCA3a isoforms. For SERCAs, comparative analyses of human and Wistar-Kyoto rat SERCA3 platelet mRNA by reverse transcription-polymerase chain reaction (RT-PCR) followed by sequencing established that human platelets coexpressed SERCA3b and a third SERCA3c, while rat cells were devoid of them but expressed a still unknown splice variant that we termed rSERCA3b/3c. Its identification using 3'-end SERCA3 gene and rapid amplification of cDNA ends (RACE)-PCR studies showed that it results from an additional SERCA3 alternative splicing process, which uses a second alternative polyadenylation site located in the last intron. For PMCAs, with the use of gene-specific RT-PCR followed by sequencing and Western blotting using 5F10 monoclonal antibody, expression of human and rat platelet PMCA1b and PMCA4b was similar. Second, comparative analysis of these newly identified Ca(2+)ATPases and SERCA3a in age-matched spontaneously hypertensive rat platelets demonstrated (1) a marked downregulation of rSERCA3b/3c, which became null, and a 1.71-fold increase in SERCA3a and (2) an opposite regulation of the 2 PMCAs, namely, a 3.3-fold decrease in PMCA1b mRNA and a 3.7-fold increase in PMCA4b mRNA. Hence, platelets coexpress multiple, diverse, and species-specific Ca(2+)ATPases, including a novel fourth SERCA3. Moreover, expression of PMCA (1b and 4b), SERCA3a, and rSERCA3b/3c was modulated in rat hypertension. Hence, Ca(2+)ATPases should be regarded as constituting a new rational basis for the understanding of nonmuscle cell Ca(2+) signaling.

Platelet sarco/endoplasmic reticulum Ca2+ATPase isoform 3b and Rap 1b: interrelation and regulation in physiopathology.

Platelet Ca2+ signalling involves intracellular Ca2+ pools, whose content is controlled by sarco/endoplasmic reticulum Ca2+ATPases (SERCAs). Among these, a key role is played by the inositol trisphosphate-sensitive Ca2+ pool, associated with the SERCA 3b isoform. We have investigated the control of this Ca2+ pool through the cAMP-dependent phosphorylation of the GTP-binding protein, Rap (Ras-proximate) 1b. We first looked for this Ca2+ 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 Ca2+ pool.

Expression of two isoforms of the third sarco/endoplasmic reticulum Ca2+ ATPase (SERCA3) in platelets. Possible recognition of the SERCA3b isoform by the PL/IM430 monoclonal antibody.

Human platelets express several sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) isoenzymes: SERCA2b of 100 kDa apparent molecular mass and two distinct enzymes of 97 kDa, one of them identified as being the SERCA3a isoform. The molecular identity of the third enzyme specifically recognized by the PL/IM430 monoclonal antibody has remained elusive. First, the study of the 3'-end part of platelet SERCA3 mRNA, by means of RT-PCR amplification using sets of primers covering the N-3 to N (ultimate) exons of the human SERCA3 sequence, revealed the presence of two distinct mRNA sequences, SERCA3a and a longer variant. Second, this additional sequence was identified as SERCA3b and found to refer to the insertion of a new exon of 73 bp, located at bp 349 from the beginning of the intronic sequence, linking the penultimate (N-1) exon to the last exon (N) of the human SERCA3 gene. Third, a relationship between the expression of this SERCA3b mRNA and the PL/ IM430 recognizable SERCA protein was observed. SERCA3b mRNA was found to be absent in epithelial HeLa cells not recognized by the PL/IM430 antibody and the expression of this SERCA3b RNA species correlated with that of the SERCA protein recognized by PL/IM430 which was down-modulated in the platelet precursor megakaryocytic CHRF 288-11 cell line as well as upon in vitro lymphocyte activation. Taken together, these results strongly support the notion of the presence of the SERCA3b protein in human cells by showing SERCA3b mRNA in platelets and the fact that the protein corresponding to this mRNA species is very likely the 97 kDa protein recognized by the PL/IM430 antibody.

Immunolocalization of the multi-sarco/endoplasmic reticulum Ca2+ ATPase system in human platelets.

We recently identified a multi-SERCA (sarco/endoplasmic reticulum Ca2+ ATPase) system in haemopoietic cells comprising the SERCA 2b, SERCA 3 and a new monoclonal anti-Ca2+ ATPase antibody (PL/IM 430) recognizable SERCA isoforms. We have now investigated the subcellular localization of these enzymes in human platelets by Western blotting of subcellular membrane fractions and by immunoelectron microscopy. We precisely defined the recognition specificity of the polyclonal anti-SERCA 2b, anti-SERCA 3, anti-SERCA 1 antibodies as well as of the monoclonal antibody PL/IM 430 by testing their recognition of the tryptic fragments of the SERCA isoforms. The analysis of fragmented membranes enriched in plasma membrane and intracellular membrane components by Western blotting showed that the SERCA 2b and the SERCA 3 isoforms were found in both the plasma membrane and the intracellular membrane fractions, whereas the PL/IM 430 recognizable SERCA isoform was restricted to membranes associated with the plasma membrane fraction. The immunoelectron microscopical study of the SERCA isoforms in resting platelets showed that: (i) the SERCA 2b isoform was expressed in membranes associated with the plasma membrane and open canalicular system, some alpha-granules and in unidentified membranes; (ii) the SERCA 3 isoform was found associated with plasma and intracellular membranes; and (iii) the PL/IM 430 recognizable SERCA isoform was observed only in structures associated with the cytoplasmic face of the plasma membranes, as confirmed by flow cytometry. Finally, since the PL/IM 430 antibody was raised against intracellular membranes, we looked for a potential membrane redistribution during the isolation procedure used for the preparation of the immunizing membranes. Neuraminidase treatment indeed induced a translocation of the PL/IM 430 recognizable SERCA isoform from plasma to intracellular membranes. Thus, the multi-SERCA system in platelets: (i) is distributed over different platelet membranes, (ii) presents a sub-compartmental organization with some overlapping, and (iii) is partly associated with motile membranes, reflecting an unrecognized level of complexity of Ca2+ stores in these cells.

The platelet Ca2+ transport ATPase system.

The Ca2+ signal accompanying cell function involves the activities of plasma membrane Ca2+ transport ATPases (PMCA) which transport Ca2+ ions out of the cell and those of sarco/endoplasmic reticulum Ca2+ transport ATPases (SERCA), which pump Ca2+ ions into intracellular Ca2+ pools. Although a platelet Ca2+ transport ATPase was described three decades ago, for a long time it remained poorly understood in terms of its cellular localization and identity. By integrating data obtained during recent years, including newly available information in the literature for the PMCAs and aspects of our work concerning the SERCAs, the present review will show how the overall view of the platelet Ca2+ATPase system has to be modified due to the presence of a number of Ca2+ATPases in these cells. These Ca2+ATPases include a typical 144 kDa PMCA protein, although its molecular identity still remains to be established, expressed together with a multi-SERCA system constituted by the ubiquitous 100 kDa SERCA 2b isoform, the 97 kDa SERCA 3 isoform and a new 97 kDa SERCA isoform recognized by the monoclonal antibody termed PL/IM 430 which also remains to be identified. The new paradigm of the platelet multi-Ca2+ATPase system will be discussed including: (i) the problems solved, as it has now become possible to reconciliate previous contradictory observations and (ii) those which still remain due to the fact that the platelet Ca2+ATPase system is more complex than previously assumed. Finally, to put this complexity of the platelet Ca2+ transport ATPase system into perspective, the biological significance of the multi-SERCA system in the context of Ca2+ signalling will be tentatively discussed in an attempt to produce a model of the organization of the intracellular Ca2+ pools in platelets.

Smooth muscle cell cycle and proliferation. Relationship between calcium influx and sarco-endoplasmic reticulum Ca2+ATPase regulation.

The role of Ca2+ influx in the regulation of the sarco-endoplasmic reticulum Ca2+ATPases (SERCA) associated with intracellular Ca2+ pools was investigated during smooth muscle cell (SMC) proliferation induced by platelet-derived growth factor (PDGF). We first defined that the previously described up-regulation of the SERCA2a isoform found in vascular SMC after a 24-h stimulation with PDGF (Magnier, C. , Papp, B., Corvazier, E., Bredoux, R., Wuytack, F., Eggermont, F., Maclouf, J., and Enouf, J. (1992) J. Biol. Chem. 267, 15808-15815) was precisely associated with SMC entry into S phase as it appeared linked with [3H]thymidine incorporation. This was further confirmed by testing the effect of transforming growth factor-beta1, which inhibited both aortic SMC proliferation associated with G1 cell cycle arrest and PDGF-induced SERCA2a up-stimulation. Then, we tested the role of Ca2+ influx by using SR 33805, a new Ca2+ channel blocker, which was characterized with regard to the voltage Ca2+ channel blocker nifedipine and the capacitative entry Ca2+ blocker SKF 96365. SR 33805 was found to be the most potent inhibitor of both PDGF-induced SMC proliferation and the associated rise in intracellular Ca2+ concentration with IC50 values of 0.2 +/- 0.1 and 0.31 +/- 0. 04 microM, respectively. Finally, by examining in parallel both SERCA2a and SERCA2b isoforms, in terms of activity and expression, we could determine that PDGF-induced stimulation of total SERCA activity (detected by formation of the phosphorylated intermediate, E approximately P) and of SERCA2a expression (Western blotting) were abolished when extracellular Ca2+ entry was prevented by SR 33805. This study demonstrates that SERCA2a up-regulation is: 1) related to the G1/S transition step of cell cycle and 2) dependent on Ca2+ entry during PDGF-induced SMC proliferation.

Relationship between Rap1 protein phosphorylation and regulation of Ca2+ transport in platelets: a new approach.

Although the interrelationship between the two messengers Ca2+ and cyclic AMP in platelet function is well documented, its mechanism of action still remains to be established. We investigated here the question of the regulation of platelet Ca(2+)-ATPases by cyclic AMP through the phosphorylation of the Rap1 protein using a pathological model. We first found experimental conditions where Ca(2+)-transport by platelet membrane vesicles appeared to be dependent on the phosphorylation of the Rap1 protein. Then, we studied platelets of patients with congestive heart failure for their expression of the potential 97 kDa Ca(2+)-ATPase target of regulation through the Rap1 protein as well as the phosphorylation of the Rap1 protein using the catalytic subunit of the cyclic AMP-dependent protein kinase (C. Sub.). In the first patients studied, we found no significant modification in the expression of the 97 kDa Ca(2+)-ATPase by Western blotting using the PL/IM 430 monoclonal antibody which specifically recognized this isoform. In contrast, the Rap1 protein was differentially phosphorylated when using 15 micrograms/ml of the C. Sub. These results allowed us to use these pathological platelets to study the relationship between the expression of Rap1 protein and the regulation of Ca2+ transport by selecting a patient with severe heart failure. We could show a decrease in the expression as well as in the phosphorylation of Rap1 protein and demonstrate a lower effect of C. Sub. on Ca2+ transport. Finally, by studying a further series of patients, we could confirm that the decrease in Rap1 protein expression in heart failure, whatever its extent, was variable, and could strictly correlate the expression of Rap1 protein with the stimulatory effect of C. Sub. on Ca2+ transport. Besides the evidence for regulation of the expression of the Rap1 protein in platelets from patients with heart failure, these findings constitute a new approach in favour of the regulation of platelet Ca2+ transport through the phosphorylation of the Rap1 protein.

Evidence for Rap1 in vascular smooth muscle cells. Regulation of their expression by platelet-derived growth factor BB.

The effect of platelet-derived growth factor (PDGF) on Rap1 expression was investigated in rat vascular smooth muscle cells (SMC). First, evidence for Rap1 proteins was shown by their: (i) detection in membranes using a specific anti-Rap1 antibody, (ii) typical shift in electrophoretic mobility as a consequence of reduction, and (iii) cAMP-induced phosphorylation and immunoprecipitation. Then, the mitogenic activity of 10 ng/ml PDGF AA and BB for 48 h, resulting in a 2- and 5-fold increase in [3H]thymidine incorporation, was correlated with that of total Rap1 protein expression which was found to be 99% +/- 36% and 260% +/- 70%, respectively. Further time-course studies established that this up-regulation of Rap1 proteins was only observed after 48 h of PDGF BB treatment. Lastly, comparative RT-PCR of both rap1a and rap1b mRNAs showed that PDGF BB also up-regulated the rap1a mRNA species, which was 1.5-fold increased in contrast with the rap1b mRNA species. It is concluded that the PDGF BB-induced SMC proliferation is associated with an up-regulation of Rap1a protein.

Controlled proteolysis of Ca(2+)-ATPases in human platelet and non-muscle cell membrane vesicles. Evidence for a multi-sarco/endoplasmic reticulum Ca(2+)-ATPase system.

Two sarco/endoplasmic reticulum Ca(2+)-ATPases (SERCAs) have been previously identified in platelets: the 100-kDa SERCA2b and the 97-kDa SERCA3 isoforms. Analysis of the acylphosphate intermediate (E-P) formation and the immunoreactivity of the platelet Ca(2+)-ATPases and their proteolytic fragments upon controlled trypsinolysis revealed the presence of an additional 97-kDa Ca(2+)-ATPase that comigrates with SERCA3 on SDS-polyacrylamide gels. At a trypsin/membrane protein ratio of 0.025 at 4 degrees C, tryptic fragments of 73-, 68- and 40-kDa, previously unknown in the SERCA family, could be detected by using the PL/IM 430 anti-Ca(2+)-ATPase antibody that had been shown to recognize a 97-kDa Ca(2+)-ATPase. The 73- and 68-kDa fragments were precursors of the 40-kDa one. Ca(2+)-dependent phospholabeling of the 73-kDa fragment and immunostaining of all these proteolytic products by another antibody raised against SERCA1 established the SERCA nature of the 97-kDa parent enzyme. The SERCA3-related E-P-forming 80-kDa tryptic fragment appeared during trypsinolysis with a different time course from that of the 73-, 68-, and 40-kDa ones. At a trypsin/membrane protein ratio of 0.125 at 37 degrees C, it reached its maximum level at 5 min of digestion, while the 73-, 68-, and 40-kDa fragments were fully degraded at 2 min of trypsinization. This 80-kDa species was immunostained neither with the PL/IM 430, nor with the anti-SERCA1 antibodies. Similar results were found in some megakaryoblastoid and lymphoblastoid cell lines. All these data indicate the presence of two distinct tryptic fragmentation patterns attributed to two 97-kDa SERCA isoforms and point to the existence of a multi-SERCA system in different human non-muscle cells.

Abnormal cAMP-induced phosphorylation of rap 1 protein in grey platelet syndrome platelets.

We previously demonstrated abnormal Ca2+ transport by microsomes in platelets from a grey platelet syndrome patient. Here, we investigated the platelet Ca2+ ATPases that mediate this transport, as well as its possible regulation by rap 1 protein. We showed that grey platelet syndrome platelets expressed the same two distinct Ca2+ ATPases as those recently described in normal platelets; the 100 kD SERCA2-b isoform (Sarco/Endoplasmic Reticulum Ca2+ATPase) and a new 97 kD SERCA isoform. The two Ca2+ATPases formed similar amounts of transient phosphorylated intermediates. The expression of these two Ca2+ATPases was compared by Western blotting using specific antibodies, which again emerged in similar amounts in normal and grey platelet syndrome platelets. As regards the protein phosphorylated by cAMP, it was found to be identical to rap 1 protein when it was immunoprecipitated with an antibody raised against a synthetic peptide specific for rap 1 protein. Although the expression of rap 1 protein was similar in membranes isolated from grey platelet syndrome and normal platelets, its exogenous phosphorylation by cAMP was abnormal, with a concentration (10 micrograms/ml) of the catalytic subunits of the cAMP-dependent protein kinase (C.Sub.), as it decreased to half the control level. It is concluded that the abnormal Ca2+ transport found in grey platelet syndrome platelets is not due to the abnormal expression of the Ca2+ATPases, but is associated with an abnormality of rap 1 protein phosphorylation by cAMP.

Correlated expression of the 97 kDa sarcoendoplasmic reticulum Ca(2+)-ATPase and Rap1B in platelets and various cell lines.

Evidence has accumulated that cyclic AMP (cAMP)-induced phosphorylation of a Ras-related protein (Rap1) regulates platelet Ca2+ transport. As this transport was recently found to be controlled by two isoforms of sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA), the 100 kDa SERCA2b and the newly identified 97 kDa SERCA, we attempted to establish which isoform is involved in this regulation. For this purpose, we studied the expression and regulation of both the SERCA and Rap1 isoforms in platelets, haemopoietic cells and various cancer cell lines. SERCA2b was shown to be equally expressed in all the cell lines tested, as determined by detection of its phosphoenzyme formation and by Western blotting using an isoform-specific antibody. In contrast, the expression of the 97 kDa SERCA, studied by the same methods, varied from total absence in the cancer cells to high levels in the megakaryocytic cell lines. With regard to the potential regulatory Rap1 proteins, Western blotting showed different expression of total Rap1 isoforms among the cell lineages, thus ruling out any possible relationship between Rap1 and SERCA2b. However, the expression of Rap1 proteins correlated with that of the 97 kDa SERCA isoform. More refined analysis of the rap1A and rap1B isoforms by reverse transcription PCR and by determining cAMP-induced phosphorylation of Rap1B, i.e. its functional mechanism, confirmed the correlation between Rap1B and the 97 kDa SERCA expression. This relationship was also established by the concerted up-regulation of these two proteins demonstrated in the pathological model of platelets from hypertensive rats. It is concluded that the expressions of 97 KDa SERCA and Rap1B are related, suggesting that regulation of the platelet Ca(2+)-ATPase system by cAMP-induced phosphorylation of Rap1B specifically involves the 97 kDa SERCA.

The rat platelet 97-kDa Ca2+ATPase isoform is the sarcoendoplasmic reticulum Ca2+ATPase 3 protein.

We recently showed that human and rat platelets express two types of SERCAs (Sarco Endoplasmic Reticulum Ca2+ATPases): a 100-kDa SERCA2b isoform and a 97-kDa SERCA isoform. Here, we explored the possibility that the rat 97-kDa isoform is identical to the SERCA3 protein. For this purpose, we first attempted to detect SERCA3 mRNA in rat platelet total RNA by reverse transcription-polymerase chain reaction using SERCA3-specific primers, and demonstrated the presence of this mRNA species by sequencing the amplification product. We then searched for a relationship between the expression of the SERCA3 mRNA and of the 97-kDa protein using either rat aortic smooth muscle cells, previously found not to express the 97-kDa SERCA isoform (negative model), or platelets of spontaneously hypertensive rats (SHR), which overexpress this isoform (overexpression model) but express the 100-kDa SERCA2b isoform normally. No expression of SERCA3 mRNA was detectable by analysis of smooth muscle cell RNA, but comparison by reverse transcription-polymerase chain reaction of the SERCA2b and SERCA3 mRNAs from the platelets of normotensive (Wistar-Kyoto, WKY) rats and SHR clearly demonstrated a 238 +/- 43% increase in the expression of the SERCA3 mRNA in SHR platelets only. Last, by comparative Western blotting of WKY rat and SHR platelet membranes using a recently developed polyclonal anti-SERCA3 antibody, we established that the 97-kDa SERCA and the SERCA3 protein are identical, as immunostaining of the 97-kDa protein revealed a 230 +/- 25% increase in the expression of this protein in SHR versus WKY rat platelets. It is concluded that the 97-kDa platelet SERCA isoform, which is up-regulated in SHR, is the SERCA3 protein. As far as we know, this constitutes the first demonstration of the actual presence of this Ca2+ATPase isoform in normal cells, in addition to the artificial transfection systems.

Spontaneously hypertensive rats and platelet Ca(2+)-ATPases: specific up-regulation of the 97 kDa isoform.

The use of platelets instead of smooth muscle cells (SMC) to study the abnormal Ca2+ handling found in hypertension was investigated using spontaneously hypertensive rats (SHR). We studied the regulation of platelet Ca(2+)-ATPases, as we have recently demonstrated that human platelets, like SMC, contain the Ca(2+)-ATPase isoform termed SERCA2-b (sarco-endoplasmic reticulum Ca(2+)-ATPase). In mixed membranes isolated from platelets of normotensive Wistar-Kyoto (WKY) rats and SHR, total Ca(2+)-ATPase activity was found to be 43% higher in SHR than in WKY rats. By the use of autophosphorylation of rat platelet Ca(2+)-ATPases with [gamma-32P]ATP, followed by SDS/PAGE and Western blotting, we found that rat platelets express two distinct Ca(2+)-ATPases: a 100 kDa isoform, recognized by a SERCA2-b-specific anti-peptide antibody, and a 97 kDa isoform, specifically recognized by a polyclonal anti-SERCA antibody. Comparative analysis of platelet membrane Ca(2+)-ATPases from WKY rats and SHR demonstrated that the expression of the SERCA2-b isoform did not change significantly (128 +/- 22%), whereas that of the 97 kDa isoform reached 300 +/- 35% in SHR when compared with WKY rats. We concluded that the upregulation of total platelet Ca(2+)-ATPases in SHR is not due to the 100 kDa SERCA2-b isoform found in SMC, but is specific to the 97 kDa Ca(2+)-ATPase isoform which is not present in SMC. Therefore platelets should be used with extreme caution as a surrogate model of vascular smooth muscle Ca2+ homeostasis.

Regulation of sarco-endoplasmic reticulum Ca(2+)-ATPases during platelet-derived growth factor-induced smooth muscle cell proliferation.

The role of the sarco-endoplasmic reticulum Ca(2+)-ATPases (SERCA) in the regulation of cell proliferation by Ca2+ was investigated by testing the effect of platelet-derived growth factor (PDGF) on cultured pig aorta smooth muscle cells. For this purpose, the PDGF-mediated rise in the Ca2+ concentration was first examined for its ability to induce the formation of prostaglandins from the specific membrane enzyme, cyclooxygenase. In parallel experiments, similar conditions (10 ng/ml PDGF for 24 h) were used to investigate the smooth muscle cell membrane SERCA2 isoforms. Total SERCA2 activity rose by 472% as reflected by their specific formation of phosphorylated intermediate (E approximately P). This rise correlated with an increase in the amount of SERCA2 proteins (100 kDa) as shown by Western blotting. With isoform-specific anti-SERCA2-a and anti-SERCA2-b antibodies, we demonstrated that the increase in total SERCA2 proteins concerned the minor isoform SERCA2-a, which rose 10-fold, whereas SERCA2-b proteins were not affected. Lastly, Northern blotting using riboprobes showed that PDGF treatment increased the SERCA2-a mRNA species by 82%, and concomitantly decreased the SERCA2-b mRNA by 28%, as a result of isoform switching. We conclude that up-regulation of the SERCA2-type Ca(2+)-ATPases occurs in PDGF-treated smooth muscle cells, which suggests that this enzymatic system plays an essential part in cell proliferation.

Evidence for a role of rap1 protein in the regulation of human platelet Ca2+ fluxes.

The relationship between the 22-24 kDa cyclic AMP (cAMP)-dependent phosphoprotein previously described as being involved in the regulation of human platelet membrane Ca2+ transport and a GTP-binding protein of low molecular mass (ras-like protein) was investigated. After isolation of plasma membranes and intracellular membranes, it was found that guanosine 5'-[gamma-thio]triphosphate (GTP[S]) bound to plasma membrane proteins ranging in molecular mass from 22 to 29 kDa, but not to intracellular membranes. The major GTP-binding protein appeared as a 24 kDa protein under reduced conditions and a 22 kDa protein under non-reduced conditions. A similar membrane location and electrophoretic mobility were found for both the cAMP phosphoprotein and the protein recognized by a specific anti-rap1 antibody. The identity between the cAMP phosphoprotein and the rap1 GTP-binding protein was further examined by studying the functional effect of GTP on plasma membrane Ca2+ transport. A maximal GTP[S] concentration of 40 microM was found to: (1) inhibit to the same degree (40%) both Ca(2+)-ATPase activity and the Ca2+ transport function mediated by the Ca(2+)-ATPase; (2) inhibit the phosphorylation of the 22-24 kDa protein by the catalytic subunit of the cAMP-dependent protein kinase (C.Sub.); and (3) abolish the stimulation of Ca2+ uptake induced by C.Sub. It is concluded that the platelet cAMP phosphoprotein is indeed the rap1 GTP-binding protein, and that it regulates plasma membrane Ca2+ transport, thus providing evidence for a new role of a ras-related protein.

Characterization of GTP-gamma-S binding to isolated human platelet plasma membranes and its relationship with the stimulation of a phospholipase C activity.

Binding parameters for the interaction of GTP-gamma-[35S] with isolated platelet plasma membranes have been studied. Analysis of the data by a non-linear curve fitting program indicates that the interaction can be satisfactory described by a model with a single, high affinity binding site (Kd = 0.3 +/- 0.07 microM and Bm = 0.4 +/- 0.2 nmoles of GTP-gamma-S/mg of membrane protein). Binding is selectively inhibited by GDP-beta-S and GMP-PNP (1 microM), but not affected by ATP, CTP, ITP, or UTP, even at mM concentration. Optimal conditions for the interaction were 30 degrees C and pH 8.0. Incubation of the isolated membranes with GTP-gamma-S results in a measurable phospholipase C activity (as detected both by a breakdown of phosphoinositides and an increase of inositide phosphates) which under our experimental conditions is only slightly enhanced by addition of cytosolic proteins. Our results indicate that platelet plasma membranes contain all the necessary elements for signal transduction through the diacylglycerol/inositolphosphates pathway.