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 Ib␣ (GPIb␣). Electron microscopy showed clusters of platelets in close contact. Binding of antibodies to the GPIb␣ N-terminal domain was decreased, whereas GPIX and GPV were normally detected. In Western blotting (WB), GPIb␣, ␣IIb, and ␤3 were normally present. Proteins involved in Ca 2؉ homeostasis were analyzed by quantitating platelet mRNA or by WB. Plasma membrane Ca 2؉ ATPase (PMCA)-4b and type III inositol trisphosphate receptor (InsP 3 -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. IntroductionVon Willebrand disease (VWD) is the most common inherited disorder of the platelet-vessel wall interaction and involves both quantitative and qualitative defects of von Willebrand factor (VWF), a crucial mediator of platelet function and carrier of the FVIII protein. In type 1 and type 3 VWD, deficiencies or absence of VWF protein are responsible for the bleeding syndrome, but in type 2 disease a functionally abnormal protein or the specific lack of large multimers account for the VWD phenotype. 1,2 In hemostasis, glycoprotein Ib␣ (GPIb␣) mediates platelet attachment to exposed subendothelium by binding through its N-terminus to the A1 domain of VWF exposed within the subendothelial matrix. 3,4 In healthy subjects, soluble VWF multimers in plasma fail to gain access to their binding site on GPIb␣, accessibility being controlled by a disulfide-linked double-loop region just below the leucine-rich repeats of GPIb␣. 5 In type 2B VWD, mutations giving rise to a selective number of amino acid substitutions in the A1 domain provide gain of function to the VWF multimers which then spontaneously bind to platelets in suspension through a direct interaction with GPIb␣. [6][7][8] This often results in the loss of the largest multimers from plasma, although these may be at least partially preserved in some cases. 9 Bleeding results from platelets having blocked GPIb function despite a heightened ristocetininduced platelet agglutination in platelet function testing, and perhaps through the relative hemostatic inefficiency of the remaining small multimers. The thrombocytopenia that accompanies this disorder in some, a...

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.

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. 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.Cell Ca 2ϩ signaling is a dynamic oscillatory process regulating a variety of important cellular functions such as secretion, contraction, metabolism, neuronal plasticity, and gene transcription (1). Accordingly, Ca 2ϩ signaling is strictly controlled in space, time, and amplitude. This very tight control is necessary to enable cells to extract relevant information from the Ca 2ϩ signal but also implies that disturbances in the intracellular Ca 2ϩ signaling can lead to a plethora of consequences.Understanding Ca 2ϩ signaling requires the basic knowledge of structures involved in this process. Among these structures are the Ca 2ϩ transport proteins inserted in the membranes of the endoplasmic reticulum (ER), 1 the intracellular reservoir of Ca 2ϩ ions. These include Ca 2ϩ channels (ryanodine and inositol 1,4,5-trisphosphate receptor channels) and Ca 2ϩ pumps (sarco/endoplasmic reticulum Ca 2ϩ -ATPases (SERCAs)). Although much data are available on the role of the channels in the elaboration of the Ca 2ϩ signal, little is known concerning how the signal may be modified by expression of SERCAs with different functional properties.In recent years, enormous advances in our understanding of SERCA structure-function relationships were made, including new insights regarding their three-dimensional structure (2, 3). Furthermore, relevant physiological functions of SERCAs have been defined through their potential role in various human diseases (4-6). However, some of the results contrast with those obtained in mice using SERCA1-3 knock ...