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Disruption of cell/ECM interactions resulting from uncontrolled pericellular proteolysis leads to detachment-induced cell apoptosis (anoikis), contributing to the morbid evolution of inflammatory vascular diseases. During cardiovascular infections, bacterial proteinases might induce vascular cells to enter a similar pathway. We focused on LasB, the predominant metalloproteinase secreted by the haematotropic pathogen Pseudomonas aeruginosa. While the exosecretome of the LasB-deficient pseudomonal strain PAO1lasBΔ had limited impact on human vascular cell adherence and viability, secretomes from the LasB-expressing reference strain, PAO1, or clinical isolates from patients with cardiac infection all induced anoikis, as did purified LasB. Immunofluorescence and/or immunoblotting analysis of heart valve myofibroblast cultures or whole tissue revealed an extensive, LasB-dependent degradation of ECM-associated fibronectin and vitronectin, that preceded cell de-adherence, whereas type I collagen showed limited degradation. Moreover, LasB produced a rapid endoproteolysis of the cell-associated urokinase receptor/uPAR, leaving a truncated receptor that is unable to support cell adherence and survival via interactions with vitronectin and integrins. Conversely, major myofibroblast integrins showed no or only minor alterations. Thus, among P. aeruginosa-secreted metalloproteinases, LasB can induce vascular cell anoikis through simultaneous proteolysis of ECM components and cell receptors, suggesting the uPAR-vitronectin axis as a major target in this process.

Cell activation, proliferation, differentiation and apoptosis are tidily regulated by calcium signals. Altered Ca2+ handling has been described in essential hypertension in different cells including platelets. In this review we will particularly focus on members of calcium pump family; the plasma membrane Ca2+ATPases (PMCAs) and the Sarco/ Endoplasmic Reticulum Ca2+ATPases (SERCAs) that are expressed in platelets. We will describe recently identified isoforms present in platelets and the regulation of their expressions along both human and rat essential hypertensions. Moreover, results suggesting that platelet Ca2+ATPase expressions in hypertensive patients can sign an abnormal megakaryocytopoiesis in this pathology will be presented and discussed.

Among the players involved in Ca(2+) homeostasis in heart tissue are SERCA (sarco/endoplasmic reticulum Ca(2+) ATPase)-type Ca(2+) pumps. Until recently, human heart was known to coexpress major SERCA2a and minor SERCA2b isoforms. Here, we will summarize data showing that nonfailing human heart is equipped with an increasing variety of SERCA isoforms comprised new SERCA2 (ATP2A2) and SERCA3 (ATP2A3) gene products. The novel 3'-ends of the human SERCA2 and -3 genes, the corresponding mRNAs and the carboxyl termini of the SERCA2a-2c and SERCA3a-3f isoforms will be presented. The intrinsic characteristics and effects on cellular Ca(2+) homeostasis of the SERCA2 and SERCA3 recombinant isoforms will be summarized. Evidence for the expression of SERCA2c and SERCA3a, -3d, and -3f mRNAs and/or endogenous proteins in the human heart will be summarized, the latter having being visualized thanks to newly generated isoform-specific antibodies. We will show how the strategic localization of the SERCA2c, SERCA3a, -3d, and -3f isoforms in cytoplasmic compartments, and the nucleus enables them to contribute to subsarcolemmal, cytoplasmic, and nuclear Ca(2+) signalling in the human heart and isolated cardiomyocytes. Comparative expressions of the additional SERCA isoforms in some failing hearts will also be summarized. Lastly, we will present what is known regarding the role the SERCA2c, SERCA3a, -3d, or -3f isoforms in cardiac muscle pathophysiology. To focus on up-to-date topics, this multi-SERCA system of human heart may sustain a distinct internal endoplasmic reticulum (ER) compartment in cardiomyocytes, as well as potential compensatory mechanisms and both SR/ER abnormalities in heart failure.

In type 2B von Willebrand disease, there is spontaneous binding of mutated von Willebrand factor (VWF) multimers to platelets. Here we report a family in which severe thrombocytopenia may also be linked to abnormal megakaryocytopoiesis. A heterozygous mutation in the VWF A1 domain gave a R1308P substitution in an interactive site for glycoprotein Ibα (GPIbα). Electron microscopy showed clusters of platelets in close contact. Binding of antibodies to the GPIbα N-terminal domain was decreased, whereas GPIX and GPV were normally detected. In Western blotting (WB), GPIbα, αIIb, and β3 were normally present. Proteins involved in Ca2+ homeostasis were analyzed by quantitating platelet mRNA or by WB. Plasma membrane Ca2+ ATPase (PMCA)-4b and type III inositol trisphosphate receptor (InsP3-R3) were selectively increased. The presence of degradation products of polyadenosine diphosphate (ADP)-ribose polymerase protein (PARP) suggested ongoing caspase-3 activity. These were findings typical of immature normal megakaryocytes cultured from peripheral blood CD34+ cells with TPO. Significantly, megakaryocytes from the patients in culture produced self-associated and interwoven proplatelets. Immunolocalization showed VWF not only associated with platelets, but already on the megakaryocyte surface and within internal channels. In this family, type 2B VWD is clearly associated with abnormal platelet production.

We recently documented the expression of a novel human mRNA variant encoding a yet uncharacterized SERCA [SR (sarcoplasmic reticulum)/ER (endoplasmic reticulum) Ca2+-ATPase] protein, SERCA2c [Gélébart, Martin, Enouf and Papp (2003) Biochem. Biophys. Res. Commun. 303, 676–684]. In the present study, we have analysed the expression and functional characteristics of SERCA2c relative to SERCA2a and SERCA2b isoforms upon their stable heterologous expression in HEK-293 cells (human embryonic kidney 293 cells). All SERCA2 proteins induced an increased Ca2+ content in the ER of intact transfected cells. In microsomes prepared from transfected cells, SERCA2c showed a lower apparent affinity for cytosolic Ca2+ than SERCA2a and a catalytic turnover rate similar to SERCA2b. We further demonstrated the expression of the endogenous SERCA2c protein in protein lysates isolated from heart left ventricles using a newly generated SERCA2c-specific antibody. Relative to the known uniform distribution of SERCA2a and SERCA2b in cardiomyocytes of the left ventricle tissue, SERCA2c was only detected in a confined area of cardiomyocytes, in close proximity to the sarcolemma. This finding led us to explore the expression of the presently known cardiac Ca2+-ATPase isoforms in heart failure. Comparative expression of SERCAs and PMCAs (plasma-membrane Ca2+-ATPases) was performed in four nonfailing hearts and five failing hearts displaying mixed cardiomyopathy and idiopathic dilated cardiomyopathies. Relative to normal subjects, cardiomyopathic patients express more PMCAs than SERCA2 proteins. Interestingly, SERCA2c expression was significantly increased (166±26%) in one patient. Taken together, these results demonstrate the expression of the novel SERCA2c isoform in the heart and may point to a still unrecognized role of PMCAs in cardiomyopathies.

The aetiology of adolescent idiopathic scoliosis (AIS), the most common form of scoliosis, is unclear. Previous studies showed controversial platelet abnormalities including intracellular calcium. Platelet Ca2+ homeostasis is controlled by a multi-Ca2+-ATPase system including SERCA (sarco/endoplasmic reticulum Ca2+-ATPase) and PMCA (plasma membrane Ca2+-ATPase) isoforms. Here, we first investigated the expression of PMCA4b, SERCA3a and SERCA2b isoforms in platelets of 17 patients with AIS. Patients presenting thoracic curves were found to present a higher PMCA4b expression coupled to a lower SERCA3a one in agreement with an abnormality in platelet maturation. Indeed, using PMA-treated MEG 01 cells, an in vitro model of megakaryocytopoiesis, we found an increase in SERCA3a expression, associated to a caspase-3 mediated C terminal proteolysis of PMCA4b. To look whether platelets reflect a basic defect in cell differentiation, we next identified osteoblast Ca2+-ATPases and studied their expressions in AIS. Major expressions of PMCA4b and SERCA2b were found in normal osteoblasts. Comparing platelets and osteoblasts in two additional patients with AIS, we found opposite and concerted regulations of the expressions of PMCA4b and caspase-3 substrate, PARP in both cell types. A systemic defect in cell differentiation involving caspase-3 can be proposed as a novel mechanism in the etiopathogenesis of the most frequent type of AIS. *R. Bredoux and E. Corvazier contributed equally to this work.

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.

Understanding of Ca(2+) signaling requires the knowledge of proteins involved in this process. Among these proteins are sarco/endoplasmic reticulum Ca(2+)-ATPases (SERCAs) that pump Ca(2+) into the endoplasmic reticulum (ER). Recently, the human SERCA3 gene was shown to give rise to five isoforms (SERCA3a-e (h3a-h3e)). Here we demonstrate the existence of an additional new member, termed SERCA3f (h3f). By reverse transcriptase-PCR using monocytic U937 cell RNA, h3f mRNA was found to exclude the antepenultimate exon 21. h3f mRNA expression appeared as a human-specific splice variant. It was not found in rats or mice. h3f mRNA gave rise to an h3f protein differing in its C terminus from h3a-h3e. Of particular interest, h3f diverged in the first amino acids after the first splice site but presented the same last 21 amino acids as h3b. Consequently, we further investigated the structure-function-location relationships of the h3b and h3f isoforms. Comparative functional study of h3b and h3f recombinant proteins in intact HEK-293 cells and in fractionated membranes showed the following distinct characteristics: (i) resting cytosolic Ca(2+) concentration ([Ca(2+)](c)) and (ii) ER Ca(2+) content ([Ca(2+)](er)); similar characteristics were shown for the following: (i) the effects of the SERCA inhibitor, thapsigargin, on Ca(2+) release ([Ca(2+)](Tg)) and subsequent Ca(2+) entry ([Ca(2+)](e)) and (ii) the low apparent Ca(2+) affinity and the enhanced rate of dephosphorylation of the E(2)P phosphoenzyme intermediate. Subcellular location of h3b and h3f by immunofluorescence and/or confocal microscopy using the h3b- and h3f-specific polyclonal and the pan-h3 monoclonal (PL/IM430) antibodies suggested overlapping but distinct ER location. The endogenous expression of h3f protein was also proved in U937 cells. Altogether these data suggest that the SERCA3 isoforms have a more widespread role in cellular Ca(2+) signaling than previously appreciated.

Sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs) pump Ca2+ into the endoplasmic reticulum. Recently, three human SERCA3 (h3a–c) proteins and a previously unknown rat SERCA3 (r3b/c) mRNA have been described. Here, we (i) document two novel human SERCA3 splice variants h3d and h3e, (ii) provide data for the expression and mechanisms regulating the expression of all known SERCA3 variants (r3a, r3b/c, and h3a–e), and (iii) show functional characteristics of the SERCA3 isoforms. h3d and h3e are issued from the insertion of an additional penultimate exon 22 resulting in different carboxyl termini for these variants. Distinct distribution patterns of the SERCA3 gene products were observed in a series of cell lines of hematopoietic, epithelial, embryonic origin, and several cancerous types, as well as in panels of rat and human tissues. Hypertension and protein kinase C, calcineurin, or retinoic acid receptor signaling pathways were found to differently control rat and human splice variant expression, respectively. Stable overexpression of each variant was performed in human embryonic kidney 293 cells, and the SERCA3 isoforms were fully characterized. All SERCA3 isoforms were found to pump Ca2+ with similar affinities. However, they modulated the cytosolic Ca2+ concentration ([Ca2+]c) and the endoplasmic reticulum Ca2+ content ([Ca2+]er) in different manners. A newly generated polyclonal antibody and a pan-SERCA3 antibody proved the endogenous expression of the three novel SERCA3 proteins, h3d, h3e, and r3b/c. All these data suggest that the SERCA3 gene products have a more widespread role in cellular Ca2+ signaling than previously appreciated.

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.

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.

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.

International audience; Within the vasculature, uncontrolled pericellular proteolysis can lead to disruption of cell-to-cell and cell-to-matrix interactions and subsequent detachment-induced cell apoptosis, or anoikis, contributing to inflammatory vascular diseases, with the endothelium as the major target. Most studies so far have focused on endogenous proteinases. However, during bloodstream infections, bacterial proteinases may also trigger endothelial anoikis. We thus investigated the potential apoptotic activity of the proteinases secreted by the haematotropic opportunistic pathogen, Pseudomonas aeruginosa, and particularly its predominant metalloproteinase, LasB. For this, we used the secretome of the LasB-expressing pseudomonal strain, PAO1, and compared it with that from the isogenic, LasB-deficient strain (PAO1?lasB), as well as with purified LasB. Secretomes were tested for apoptotic activity on cultured human endothelial cells derived from the umbilical vein or from the cerebral microvasculature. We found that the PAO1 secretome readily induced endothelial cell anoikis, as did secretomes of LasB-positive clinical pseudomonal isolates, while the PAO1?lasB secretome had only a limited impact on endothelial adherence and viability. Notably, purified LasB reproduced most of the effects of the LasB-containing secretomes, and these were drastically reduced in the presence of the LasB-selective inhibitor, phosphoramidon. A precocious and extensive LasB-dependent degradation of several proteins associated with the endothelial extracellular matrix, fibronectin and von Willebrand factor, was observed by immunofluorescence and/or immunoblotting analysis of cell cultures. Moreover, the PAO1 secretome, but not that from PAO1?lasB, specifically induced rapid endoproteolysis of two major interendothelial junction components, VE-cadherin and occludin, as well as of the anti-anoikis, integrin-associated urokinase receptor, uPAR. Taken as a prototype for exogenous haemorrhagic proteinases, pseudomonal LasB thus appears to induce endothelial anoikis not only via matrilysis, as observed for many pro-apoptotic proteinases, but also via cleavage of some essential cell-to-cell and cell-to-matrix adhesion receptors implicated in the maintenance of the endothelial barrier.

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.

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.

Disruption of cell/ECM interactions resulting from uncontrolled pericellular proteolysis leads to detachment-induced cell apoptosis (anoikis), contributing to the morbid evolution of inflammatory vascular diseases. During cardiovascular infections, bacterial proteinases might induce vascular cells to enter a similar pathway. We focused on LasB, the predominant metalloproteinase secreted by the haematotropic pathogen Pseudomonas aeruginosa. While the exosecretome of the LasB-deficient pseudomonal strain PAO1lasBΔ had limited impact on human vascular cell adherence and viability, secretomes from the LasB-expressing reference strain, PAO1, or clinical isolates from patients with cardiac infection all induced anoikis, as did purified LasB. Immunofluorescence and/or immunoblotting analysis of heart valve myofibroblast cultures or whole tissue revealed an extensive, LasB-dependent degradation of ECM-associated fibronectin and vitronectin, that preceded cell de-adherence, whereas type I collagen showed limited degradation. Moreover, LasB produced a rapid endoproteolysis of the cell-associated urokinase receptor/uPAR, leaving a truncated receptor that is unable to support cell adherence and survival via interactions with vitronectin and integrins. Conversely, major myofibroblast integrins showed no or only minor alterations. Thus, among P. aeruginosa-secreted metalloproteinases, LasB can induce vascular cell anoikis through simultaneous proteolysis of ECM components and cell receptors, suggesting the uPAR-vitronectin axis as a major target in this process.

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.

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.

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.

Platelet Ca(2+) homeostasis is controlled by a multi-Ca(2+)ATPase system including two PMCA (plasma membrane Ca(2+)ATPase) and seven SERCA (sarco/endoplasmic reticulum Ca(2+)ATPase) isoforms. Previous studies have shown similar platelet Ca(2+) abnormalities in diabetic and hypertensive patients, including an increase in intracellular [Ca(2+)](I), a possible modulation of PMCA activity and increased PMCA tyrosine phosphorylation. Very recently, we found that platelets from diabetic patients also exhibited increased PMCA4b expression. In the present study we looked for further similarities between diabetic and hypertensive patients. We first confirmed a decrease in Ca(2+)ATPase activity (mean 55 + 7%) in mixed platelet membranes isolated from 10 patients with hypertension compared with those from 10 healthy controls. In addition, the decreased Ca(2+)ATPase activity correlated with the DBP of the different patients, as expected for PMCA activity. Second, we performed a pilot study of six hypertensives to examine their expressions of PMCA and SERCA mRNA and proteins. Like the diabetic patients, 100% of hypertensives were found to present a major increase in PMCA4b expression (mean value of 218 +/- 21%). We thus determined that platelets from diabetic and hypertensive patients showed similar increased PMCA4b isoform. Since increased PMCA4b expression was recently found to be associated with a perturbation of megakaryocytopoiesis, these findings may also point to an abnormality in platelet maturation in hypertension.

Summary. Background: Previous studies have shown platelet Ca2+ abnormalities in diabetes mellitus and some reports suggest abnormal platelet production. Platelet Ca2+ homeostasis is controlled by a multi-Ca2+-ATPase system that includes two plasma membrane Ca2+-ATPase (PMCA) and seven sarco/endoplasmic reticulum Ca2+-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 Ca2+-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.

Sarco/endoplasmic reticulum Ca 2+ ATPases (SERCAs) pump free Ca 2+ from the cytosol into the endoplasmic reticulum. The human SERCA3 family counts six members named SERCA3a to 3f. However, the exact role of these different isoforms in cellular physiology remains undetermined. In this study, we compared some physiological consequences of SERCA3b and SERCA3f overexpression in HEK-293 cells. We observed that overexpression of SERCA3b affected cell adhesion capacity associated with a major disorganization of F-actin and a decrease in focal adhesion. Furthermore, we found that SERCA3f overexpression resulted in an increase in endoplasmic reticulum stress markers (including processing of X-box-binding protein-1 (XBP-1) mRNA and expression of chaperone glucose-regulated protein 78 (GRP78)). This was associated with the activation of caspase cascade and a higher spontaneous cell death. In conclusion, these data point for the first time to distinct physiological roles of SERCA3 isoforms in cell functions.

Sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs) pump Ca2+ into the endoplasmic reticulum. Recently, three human SERCA3 (h3a-c) proteins and a previously unknown rat SERCA3 (r3b/c) mRNA have been described. Here, we (i) document two novel human SERCA3 splice variants h3d and h3e, (ii) provide data for the expression and mechanisms regulating the expression of all known SERCA3 variants (r3a, r3b/c, and h3a-e), and (iii) show functional characteristics of the SERCA3 isoforms. h3d and h3e are issued from the insertion of an additional penultimate exon 22 resulting in different carboxyl termini for these variants. Distinct distribution patterns of the SERCA3 gene products were observed in a series of cell lines of hematopoietic, epithelial, embryonic origin, and several cancerous types, as well as in panels of rat and human tissues. Hypertension and protein kinase C, calcineurin, or retinoic acid receptor signaling pathways were found to differently control rat and human splice variant expression, respectively. Stable overexpression of each variant was performed in human embryonic kidney 293 cells, and the SERCA3 isoforms were fully characterized. All SERCA3 isoforms were found to pump Ca2+ with similar affinities. However, they modulated the cytosolic Ca2+ concentration ([Ca2+]c) and the endoplasmic reticulum Ca2+ content ([Ca2+]er) in different manners. A newly generated polyclonal antibody and a pan-SERCA3 antibody proved the endogenous expression of the three novel SERCA3 proteins, h3d, h3e, and r3b/c. All these data suggest that the SERCA3 gene products have a more widespread role in cellular Ca2+ signaling than previously appreciated.

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.

Abstract —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.

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.

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.

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.

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.

A sensitive and specific radioimmunoassay for prostaglandin D 2 has been developed using its stabilized 11-methoxime derivative, which was obtained after treatment of prostaglandin D 2 with methoxamine-HCl. The antiserum was obtained after injection of prostaglandin D 2 -methoxamine coupled to bovine serum albumin. A ( 125 I)-Histamide prostaglandin D 2 -methoxamine tracer was prepared by iodination of the corresponding histamide, followed by thin layer chromatography purification. The sensitivity of the assay was 280 femtomoles per ml at 50% displacement. The cross reactivities were 15% with prostaglandin D 1 -methoxamine and less than 0.20% with other prostaglandins. Determination of the half-life of prostaglandin D 2 in a solution containing albumin was also carried out, since it has been shown to catalyze prostaglandin D 2 destruction. The unstability of this prostaglandin is due to the presence of a β-hydroxy ketone group, and all prostaglandins possessing this labile moiety could be stabilized by such a derivatization before developing a radioimmunoassay.

The SERCA family includes 3 genes (SERCA1-3), each of which giving rise to various isoforms. To date, detailed structural data is only available for the SERCA1a isoform. Here, limited trypsinolysis of either human platelet membranes or recombinant SERCA3a in HEK-293 cells followed by Western blotting using antibodies covering different regions of the SERCA3(a) protein revealed two, kinetically distinct, Early (ETF) and Late (LTF) Tryptic Fragmentations. The ETF uses many tryptic sites while the LTF uses a unique tryptic site. Using site-directed mutagenesis: i) Arg(334), Arg(396) and Arg(638) were directly assigned to the ETF and ii) Arg(198) was assigned as the only tryptic site to the LTF. Arg(671), Lys(712)/Lys(713) and Lys(728) were also found to modulate the ETF. SERCA inhibitors Tg and tBHQ induced modest inhibition of the ETF. In contrast, the addition of CaCl(2), EGTA or AlF(4)(-) strikingly modified the ETF without any effect on the LTF. Trypsinolysis of the other recombinant SERCA3b-3f isoforms revealed: i) same ETF and LTF as SERCA3a, with variations of the length of the C-terminal fragments; ii) Arg(1002) as an additional tryptic site in SERCA3b-3e isoforms. Taken together, the two distinct SERCA3 fragmentation profiles sign the co-expression of SERCA3 proteins in two conformational states in cell membranes.

The effects of natural and synthetic sex hormones (10 −8 M) have been studied on human umbilical endothelial cell proliferation and prostacyclin production. 17 B estradiol and progesterone had no effect on cell multiplication. Ethinyl-estradiol increased proliferation when the initial plating density was 40,000 cells/ cm 2 . However, the production of 6-keto PGF 1 α induced by the Ca ++ ionophore A 23187 was not different between control, 17 B estradiol-or ethinyl estradiol-treated cultures. These results demonstrate an in vitro effect of synthetic estrogen, ethinyl estradiol on endothelial cell proliferation. At the present time it is however difficult to correlate these results with the clinical observation of an increase in thromboembolic complications in women under oral contraceptives.

The effect of ten flavonoids was studied on the stimulation of washed human platelets by either arachidonic acid or thrombin. The oxygenated metabolites released were analyzed by radioimmunoassay, glass-capillary-column gas chromatography and high-pressure liquid chromatography. No effect was evidenced for naringenin, rutinose and phloridzin up to 1000 microM. Thromboxane B2 and 12-hydroxyeicosatetraenoic acid production was depressed simultaneously by all other compounds at different IC50. When tested for their effect on reversibility, however, cyclooxygenase and lipoxygenase inhibition was found to be different depending upon the flavonoid used. All compounds, except morin and rutin, inhibited platelet aggregation and [14C]serotonin release with parallel inhibition of thromboxane synthesis when tested on arachidonic acid-induced platelet-rich plasma stimulation. Some flavonoids inhibited the metabolism of human neutrophils stimulated by ionophore A23187 as assessed by high-performance liquid chromatography. Our results show that flavonoids interfere with the different oxidative metabolisms of arachidonic acid. No clearcut specificity could be found between one compound and one metabolic pathway.

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.