Dysfunction of the PI3 kinase/Rap1/integrin αIIbβ3 pathway underlies ex vivo platelet hypoactivity in essential thrombocythemia.
ABSTRACT Patients with myeloproliferative disorders (MPD) such as essential thrombocythemia (ET) have increased risk of thrombosis and bleeding, which are major sources of morbidity and mortality. Most MPD patients have a gain of function mutation in Janus kinase 2 (JAK2V617F), but little is known how JAK2V617F affects platelet function. Here, we demonstrate that platelets from ET patients have impaired SFLLRN-mediated fibrinogen binding and have lost the potentiating effect of thrombopoietin (which couples to JAK2) on this pathway. In contrast, SFLLRN-mediated P-selectin expression, ATP secretion, phosphorylation of the PKC substrate pleckstrin and Ca(2+) mobilisation were unaffected in JAK2V617F positive platelets. In addition, thrombopoietin-mediated JAK2 phosphorylation was unchanged, suggesting that signalling pathways activated downstream of JAK2 are impaired. Indeed, we found that platelets from JAK2V617F positive ET patients have significantly reduced phosphorylation of the PI3 kinase substrate Akt, and have reduced activation of Rap1 in response to thrombopoietin, IGF-1, ADP, SFLLRN and thrombin. This effect was independent of Giα P2Y12 purinergic receptor function as ADP-mediated inhibition of VASP phosphorylation was unchanged. These results demonstrate that the PI3 kinase/Rap1 pathway is intrinsically impaired in platelets from JAK2V617F positive ET patients, resulting in diminished thrombin and thrombopoietin-mediated integrin α(IIb)β(3) activation.
SourceAvailable from: Stephane Schurmans[Show abstract] [Hide abstract]
ABSTRACT: Rasa3 is a GTPase activating protein of the GAP1 family which targets Ras and Rap1. Ubiquitous Rasa3 catalytic inactivation in mouse results in early embryonic lethality. Here, we show that Rasa3 catalytic inactivation in mouse hematopoietic cells results in a lethal syndrome characterized by severe defects during megakaryopoiesis, thrombocytopenia and a predisposition to develop preleukemia. The main objective of this study was to define the cellular and the molecular mechanisms of terminal megakaryopoiesis alterations. We found that Rasa3 catalytic inactivation altered megakaryocyte development, adherence, migration, actin cytoskeleton organization and differentiation into proplatelet forming megakaryocytes. These megakaryocyte alterations were associated with an increased active Rap1 level and a constitutive integrin activation. Thus, these mice presented a severe thrombocytopenia, bleeding and anemia associated with an increased percentage of megakaryocytes in the bone marrow, bone marrow fibrosis, extramedular hematopoiesis, splenomegaly and premature death. Altogether, our results indicate that Rasa3 catalytic activity controls Rap1 activation and integrin signaling during megakaryocyte differentiation in mouse.PLoS Genetics 06/2014; 10(6):e1004420. DOI:10.1371/journal.pgen.1004420 · 8.52 Impact Factor
Article: Is JAK2V617F finally off the hook?[Show abstract] [Hide abstract]
ABSTRACT: In this issue of Blood, Lamrani et al use various mouse models to demonstrate that the disease phenotype, rather than the JAK2(V617F) mutation, is responsible for the increased risk of thrombosis and bleeding in mouse models of myeloproliferative neoplasms (MPNs).Blood 08/2014; 124(7):992-3. DOI:10.1182/blood-2014-07-583856 · 9.78 Impact Factor
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ABSTRACT: Background The current study was to evaluate the anti-thrombotic effect of alpha-linolenic acid (ALA) which was isolated and purified from Jiaomu in vivo. Methods The seeds were crushed and subsequently subjected to saponification, acid hydrolysis, gradient freezing, urea inclusion and complexation of silver nitrate to obtain the unsaturated fatty acids. The chemical characteristics of isolated ALA were validated by 1HNMR, 13CNMR and mass spectrometry, and then the anti-thrombotic effect of ALA and its mixture with linoleic acid (1:1) were evaluated in the following experiments. Results The alpha-linolenic acid was isolated and purified from Jiaomu through our newly established methods. ALA and its mixture with linoleic acid can prolong the hemorrhage and coagulation time as well as enhanced the survival rate of mice subjected to collagen-adrenaline induced thrombosis. In addition, the thrombosis on A-V bypass and platelet aggregation of rats will be reduced after treated with ALA or its mixture, and the expression level of Akt and PI3K protein decreased 26% and 31%, respectively. Conclusions We designed and optimized a very simple and high-yield procedure to isolate ALA and linoleic acid mixture from seeds of Zanthoxylum bungeanum Maxim and demonstrated that such mixture can obtain a good anti-thrombotic effect through the modulation of PI3K/Akt signaling.BMC Complementary and Alternative Medicine 09/2014; 14(1):348. DOI:10.1186/1472-6882-14-348 · 1.88 Impact Factor