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Macrothrombocytopenias are the most important subgroup of inherited thrombocytopenias. This subgroup is particularly heterogeneous because the affected genes are involved in various functions such as cell signaling, cytoskeleton organization, and gene expression. Herein we describe the clinical and hematological features of a consanguineous family with a severe autosomal recessive macrothrombocytopenia associated with a thrombocytopathy inducing a bleeding tendency in the homozygous mutated patients. Platelet activation and cytoskeleton reorganization were impaired in these homozygous patients. Exome sequencing identified a c.222C>G mutation (missense p.74Ile>Met) in PRKACG, a gene encoding the γ-catalytic subunit of the cyclic adenosine monophosphate-dependent protein kinase, the mutated allele cosegregating with the macrothrombocytopenia. We demonstrate that the p.74Ile>Met PRKACG mutation is associated with a marked defect in proplatelet formation and a low level in filamin A in megakaryocytes (MKs). The defect in proplatelet formation was rescued in vitro by lentiviral vector-mediated overexpression of wild-type PRKACG in patient MKs. We thus conclude that PRKACG is a new central actor in platelet biogenesis and a new gene involved in inherited thrombocytopenia with giant platelets associated with a thrombocytopathy., (© 2014 by The American Society of Hematology.)

Objective: Apoptotic-like phase is an essential step for the platelet formation from megakaryocytes. How controlled is this signaling pathway remained poorly understood. The aim of this study was to determine whether endoplasmic reticulum (ER) stress-induced apoptosis occurs during thrombopoiesis., Approach and Results: Investigation of ER stress and maturation markers in different models of human thrombopoiesis (CHRF, DAMI, MEG-01 cell lines, and hematopoietic stem cells: CD34(+)) as well as in immature pathological platelets clearly indicated that ER stress occurs transiently during thrombopoiesis. Direct ER stress induction by tunicamycin, an inhibitor of N-glycosylation, or by sarco/endoplasmic reticulum Ca(2+) ATPase type 3b overexpression, which interferes with reticular calcium, leads to some degree of maturation in megakaryocytic cell lines. On the contrary, exposure to salubrinal, a phosphatase inhibitor that prevents eukaryotic translation initiation factor 2α-P dephosphorylation and inhibits ER stress-induced apoptosis, decreased both expression of maturation markers in MEG-01 and CD34(+) cells as well as numbers of mature megakaryocytes and proplatelet formation in cultured CD34(+) cells., Conclusions: Taken as a whole, our research suggests that transient ER stress activation triggers the apoptotic-like phase of the thrombopoiesis process.

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 Ibalpha (GPIbalpha). Electron microscopy showed clusters of platelets in close contact. Binding of antibodies to the GPIbalpha N-terminal domain was decreased, whereas GPIX and GPV were normally detected. In Western blotting (WB), GPIbalpha, alphaIIb, and beta3 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.

Activation of the collagen receptor glycoprotein VI (GPVI) by a collagen-related peptide (CRP) induces stimulation of platelets and megakaryocytes through the phosphatidylinositol (PI) 3-kinase-dependent pathway leading to activation of Bruton tyrosine kinase (Btk) and phospholipase Cgamma2 (PLCgamma2). Here, we present evidence that both proteins undergo PI 3-kinase-dependent translocation to the plasma membrane on CRP stimulation that is markedly inhibited by wortmannin and LY294002. Translocation of PLCgamma2 but not Btk is also seen in megakaryocytes from X-linked immunodeficiency mice, which have a mutation that reduces the affinity of the pleckstrin homology (PH) domain of Btk for PI 3,4,5-trisphosphate (PI 3,4,5-P3). Activation of PC12 cells by epidermal growth factor (EGF) results in increased PI 3-kinase activity and high PI 3,4,5-P3 levels that trigger translocation of the green fluorescent protein (GFP)-labeled PH of Btk, but not the GFP-labeled PH and tandem Src homology 2 (SH2) domains of PLCgamma2. In contrast to the results with CRP, the G protein-coupled receptor agonist thrombin stimulates PI 3-kinase-independent translocation of Btk but not PLCgamma2. In conclusion, these results demonstrate that in mouse megakaryocytes, CRP leads to PI 3-kinase-dependent translocation of PLCgamma2 and Btk that are independent of one another, whereas thrombin only induces translocation of Btk through a pathway that is independent of PI 3-kinase activity.

This study examined the expression of the platelet collagen receptor glycoprotein VI (GPVI) in megakaryocyte cell lines and primary megakaryocytes by reverse transcriptase-polymerase chain reaction and by flow cytometry and ligand blotting using the snake venom toxin convulxin. Expression of GPVI is increased in the megakaryoblastic cell lines HEL and CMK on differentiation with the phorbol ester phorbol 12-myristate 13-acetate (PMA), along with the Fc receptor gamma-chain (FcR gamma-chain). The increase in GPVI expression is associated with marked potentiation of tyrosine phosphorylation and Ca(++) elevation in response to convulxin. Syk, linker for activated T cells, and phospholipase C gamma 2 (PLC gamma 2) are among the proteins tyrosine phosphorylated on convulxin stimulation in PMA-differentiated HEL cells. Studies on primary murine megakaryocytes grown in vitro confirmed that GPVI is up-regulated in parallel with functional activation, assessed by measurement of [Ca(++)](i), during differentiation. The results demonstrate that expression of GPVI is up-regulated along with the FcR gamma-chain during differentiation of megakaryocytes. (Blood. 2000;96:2740-2745)

The role of phosphatidylinositol 3,4,5-trisphosphate (PI3,4,5P(3)) and Btk in signalling by the collagen receptor glycoprotein VI was investigated. PI3,4,5P(3) was increased in platelets from mice deficient in the SH2 domain-containing inositol 5-phosphatase (SHIP), in response to collagen related peptide (CRP). Tyrosine phosphorylation and activation of phospholipase Cgamma2 (PLCgamma2) were unaltered in SHIP(-/-) platelets, whereas Btk was heavily tyrosine phosphorylated under basal conditions and maximally phosphorylated by low concentrations of CRP. There was an increase in basal Ca(2+), maximal expression of P-selectin, and potentiation of Ca(2+) and aminophospholipid exposure to CRP in SHIP(-/-) platelets in the presence of Ca(2+) (1 mM). Microinjection of PI3,4, 5P(3) into megakaryocytes caused a 3-fold increase in Ca(2+) in response to CRP, which was absent in X-linked immunodeficiency (Xid) mice, which have a mutation in the PH domain of Btk. There was a corresponding partial reduction in the sustained level of intracellular Ca(2+) in response to CRP in Xid mice but no change in PLC activity. These results demonstrate a novel pathway of Ca(2+) entry that involves PI3,4,5P(3) and Btk, and which is independent of increased PLC activity.

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. [ABSTRACT FROM AUTHOR]