Inhibition of Na+/H+ exchange reduces Ca2+ mobilization without affecting the initial cleavage of phosphatidylinositol 4,5-bisphosphate in thrombin-stimulated platelets

AbstractStimulation of human platelets increases cytoplasmic pH (pHi) via activation of Na+/H+ exchange. We have determined the effect of inhibiting Na+/H+ exchange on (i) thrombin-induced Ca2+ mobilization and (ii) turnover of 32P-labelled phospholipids. Blocking Na+/H+ exchange by removal of extracellular Na+ or by ethylisopropylamiloride (EIPA) inhibited Ca2+ mobilization induced by 0.2 Uml thrombin, whereas increasing pHi by NH4C1 enhanced the thrombin-induced increase in cytosolic free Ca2+. The effect of EIPA was bypassed after increasing pHi by moneasin. The thrombin-induced cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2) was unaffected by treatments that blocked Na+/H+ exchange or increased pHi. It is concluded that activation of Na+/H+ exchange is a prerequisite for Ca2+ mobilization in human platelets but not for the stimulus-induced hydrolysis of PIP2

Медико-психологическая характеристика и дифференциальная диагностика дезадаптивных состояний у военнослужащих

Діагностична і експертна оцінка дезадаптивних станів є актуальною проблемою сучасної психіатрії. У цієї роботі розглядаються дезадаптивні стани з погляду девіантної поведінки у акцентуйованих осіб. Результати дослідження підтверджувалися психологічними, нейрофізіологічними методами.Diagnostics and expert estimation of deadaptation states is a topical problem of modern psychiatry. This article represents an examination of deadaptation states from the point of view of deviant behavior of accentuated personalities. The research results were confirmed by psychological and neurophysiological methods

Biogenesis of G-protein mediated calcium signaling in human megakaryocytes

Biogenesis of G-protein mediated calcium signaling in human megakaryocytes. den Dekker E, Gorter G, van der Vuurst H, Heemskerk JW, Akkerman JW. Department of Haematology, University Medical Center Utrecht, The Netherlands. [email protected] To understand how platelet signal transduction pathways develop during megakaryocytopoiesis, we isolated human stem cells from umbilical cord blood and cultured the cells in the presence of thrombopoietin (TPO). Based on the early expression of CD61 and late expression of CD42b, immature (CD61+/CD42b(low)) and mature (CD61+/ CD42b(high)) megakaryocytes were immunomagnetically purified and, together with stem cells (CD34+), characterized for Galpha-protein expression and agonist-induced [Ca2+]i increases. Megakaryocytopoiesis was accompanied by down-regulation of the 43 kDa and 46 kDa variants of G16alpha, constant expression of Gsalpha, and up-regulation of Gqalpha and Gialpha1/2. The increase in Gqalpha and Gialpha1/2 expression was accompanied by an increase in Ca2+ signaling triggered by thrombin and other agonists known to signal to Ca2+ via these G-proteins in platelets. The prostacyclin analog iloprost and TPO also induced [Ca2+]i increases, and the iloprost-induced Ca2+ response disappeared during maturation. These data reveal sharp changes in Ca2+ regulation during megakaryocytopoiesis

Improved platelet survival after cold storage by prevention of glycoprotein Ibα clustering in lipid rafts

ABSTRACT Background Room temperature storage of platelets for transfusion increases the risk of microbial infection and decreases platelet functionality, leading to out-date discard rates of up to 20%. Cold storage may be a better alternative, but this treatment leads to rapid platelet clearance after transfusion, initiated by changes in glycoprotein Ibα, the receptor for von Willebrand factor. Design and Methods We examined the change in glycoprotein Ibα distribution using Förster Resonance Energy Transfer by time-gated Fluorescence Lifetime Imaging Microscopy. Results Cold storage induced deglycosylation of glycoprotein Ibα ectodomain, exposing N-acetyl-Dglucosamine residues, which sequestered with GM1 gangliosides in lipid rafts. Raft-associated glycoprotein Ibα formed clusters upon binding of 14-3-3ζ adaptor proteins to its cytoplasmic tail, a process accompanied by mitochondrial injury and phosphatidyl serine exposure. Cold storage left glycoprotein Ibα surface expression unchanged and although glycoprotein V decreased, the fall did not affect glycoprotein Ibα clustering. Prevention of glycoprotein Ibα clustering by blockade of deglycosylation and 14-3-3ζ translocation raised the survival of cold-stored platelets above levels of room temperature platelets without compromising hemostatic functions. Conclusions We conclude that glycoprotein Ibα translocates to lipid rafts upon cold-induced deglycosylation and forms clusters by associating with 14-3-3ζ. Interference with these steps provides a means to enable cold storage of platelet concentrates in the near future

Expression of transient receptor potential mRNA isoforms and Ca(2+) influx in differentiating human stem cells and platelets.

AbstractStore-regulated Ca2+ entry (SOCE) is an important mechanism of elevating cytosolic [Ca2+]i in platelets, though the Ca2+ influx channels involved are still unclear. We screened human platelets and their precursor cells (human stem cells and megakaryocytes) for the presence of candidate influx channels, i.e., isoforms of the Trp family of proteins. Primary stem cells were cultured with thrombopoietin to allow differentiation into megakaryocytes. The undifferentiated stem cells (CD34+) showed mRNA expression of only a spliced variant Trp1A. Immature (CD61+/CD42blow) and mature (CD61+/CD42bhigh) megakaryocytes as well as platelets expressed in addition unspliced Trp1 as well as Trp4 (less abundant) and Trp6 isoforms. This unspliced isoform appeared to be specific for cells of the megakaryocyte/platelet lineage, since immature (CD14+/CD61−/CD42b−) and mature monocytes expressed only the Trp1A isoform. This conclusion was confirmed by the presence of Trp1A, 3, 4 and 6 transcripts in the immature megakaryocytic Dami cell line, and of Trp1, 1A, 4 and 6 transcripts in the more mature CHRF-288 cell line. The up-regulation of Trp1, 4 and 6 in the lineage from primary stem cells to mature megakaryocytes and platelets was accompanied by increased influx of extracellular Ca2+ after pretreatment of the cells with thapsigargin or thrombin. Expression of new Trp isoforms in the differentiated cells is thus accompanied by increased SOCE