Risk factors for bleeding, including platelet count threshold, in newly diagnosed immune thrombocytopenia adults.

Essentials Risk factors of bleeding in adult immune thrombocytopenia are not known. This multicenter study assessed risk factors of bleeding at immune thrombocytopenia onset. Platelet count thresholds associated with bleeding were < 20 × 109 L-1 and < 10 × 109 L-1 . Exposure to anticoagulants was a major risk factor of severe bleeding. SUMMARY: Background The aim of this cross-sectional study was to assess risk factors for bleeding in immune thrombocytopenia (ITP) adults, including the determination of platelet count thresholds. Methods We selected all newly diagnosed ITP adults included in the Cytopénies Auto-immunes Registre Midi-PyrénéEN (CARMEN) register and at the French referral center for autoimmune cytopenias. The frequencies of any bleeding, mucosal bleeding and severe bleeding (gastrointestinal, intracranial, or macroscopic hematuria) at ITP onset were assessed. Platelet count thresholds were assessed by the use of receiver operating characteristic curves. All potential risk factors were included in logistic regression models. Results Among the 302 patients, the frequencies of any, mucosal and severe bleeding were 57.9%, 30.1%, and 6.6%, respectively. The best discriminant threshold of platelet count for any bleeding was 20 × 109 L-1 . In multivariate analysis, factors associated with any bleeding were platelet count (< 10 × 109 L-1 versus ≥ 20 × 109 L-1 , odds ratio [OR] 48.2, 95% confidence interval [CI] 20.0-116.3; between 10 × 109 L-1 and 19 × 109 L-1 versus ≥ 20 × 109 L-1 , OR 5.2, 95% CI 2.3-11.6), female sex (OR 2.6, 95% CI 1.3-5.0), and exposure to non-steroidal anti-inflammatory drugs (NSAIDs) (OR 4.8, 95% CI 1.1-20.7). A low platelet count was also the main risk factor for mucosal bleeding. Exposure to anticoagulant drugs was associated with severe bleeding (OR 4.3, 95% CI 1.3-14.1). Conclusions Platelet counts of < 20 × 109 L-1 and < 10 × 109 L-1 were thresholds for major increased risks of any and mucosal bleeding. Platelet count, female sex and exposure to NSAIDs should be considered for assessment of the risk of any bleeding. Exposure to anticoagulant drugs was a major risk factor for severe bleeding.

Acute coronary syndrome remodels the antiplatelet aggregation properties of HDL particle subclasses.

Essentials HDL subclasses were studied in acute coronary syndrome (ACS). HDL2 from ACS patients have better antiplatelet potency than HDL from non ACS subjects. ACS remodels the antiplatelet properties of HDL subclasses. Oxidized polyunsaturated fatty acids content of HDL is modified by ACS. SUMMARY: Background Although HDLs have antithrombotic effects by reducing platelet activation, the relationship between HDL levels and the risk of acute coronary syndrome (ACS) is unclear, as HDL particles are heterogeneous in composition and biological properties. Objective To characterize the effects of HDL2 and HDL3 subclasses from ACS patients and non-coronary artery disease (CAD) subjects on platelet activation. Methods We measured platelet aggregation and ex vivo thrombus formation, analyzed signaling pathways by flow cytometry, and performed a targeted lipidomics analysis on HDL subclasses. Results Analysis of human platelet aggregation in suspension, adhesion on von Willebrand factor and thrombus formation on collagen under arterial shear demonstrated that HDL2 from ACS patients had higher antiplatelet potency than HDL3 from ACS patients and HDL from non-CAD subjects. HDL binding to scavenger receptor class B type I was essential for this effect. A lipidomics analysis revealed that HDL2 from ACS patients had more oxidized polyunsaturated fatty acids (PUFAs). An inverse correlation between the concentrations of 9-hydroxyoctadecadienoic acid (9-HODE), 13-hydroxyoctadecadienoic acid (13-HODE), the eicosapentaenoic acid metabolite 18-hydroxyeicosapentaenoic acid (18-HEPE) and hydroxyeicosatetraenoic acid isomers in HDL2 and platelet aggregation was observed. This relationship was further demonstrated by the direct inhibitory effects of 18-HEPE, 9-HODE, 13-HODE, 17-hydroxydocosahexaenoic acid and 14-hydroxydocosahexaenoic acid on collagen-related peptide-induced platelet aggregation, indicating that oxidized PUFAs contribute to the antithrombotic effect of ACS HDL2. Conclusions Our data shed new light on the antiplatelet effects of HDL subclasses, and suggest physiological adaptation through the modulation of HDL properties in ACS patients that may limit their platelet-dependent thrombotic risk.

Mass Assays to Quantify Bioactive PtdIns3P and PtdIns5P During Autophagic Responses.

Autophagy is a cellular process whereby cytoplasmic substrates are targeted for degradation in the lysosome via the membrane structures autophagosomes. This process is initiated by specific phosphoinositides, PtdIns3P and PtdIns5P, which play a key role in autophagy by recruiting effectors such as Atg18/WIPI2. Therefore, quantifying those lipids is important to better understand the assembly of the complex autophagic machinery. Herein, we describe in detail methods to quantify PtdIns3P and PtdIns5P by specific mass assays feasible in most laboratories.

Cdc42-dependent F-actin dynamics drive structuration of the demarcation membrane system in megakaryocytes.

UNLABELLED: Essentials Information about the formation of the demarcation membrane system (DMS) is still lacking. We investigated the role of the cytoskeleton in DMS structuration in megakaryocytes. Cdc42/Pak-dependent F-actin remodeling regulates DMS organization for proper megakaryopoiesis. These data highlight the mandatory role of F-actin in platelet biogenesis. SUMMARY: Background Blood platelet biogenesis results from the maturation of megakaryocytes (MKs), which involves the development of a complex demarcation membrane system (DMS). Therefore, MK differentiation is an attractive model for studying membrane remodeling. Objectives We sought to investigate the mechanism of DMS structuration in relationship to the cytoskeleton. Results Using three-dimensional (3D) confocal imaging, we have identified consecutive stages of DMS organization that rely on F-actin dynamics to polarize membranes and nuclei territories. Interestingly, microtubules are not involved in this process. We found that the mechanism underlying F-actin-dependent DMS formation required the activation of the guanosine triphosphate hydrolase Cdc42 and its p21-activated kinase effectors (Pak1/2/3). Förster resonance energy transfer demonstrated that active Cdc42 was associated with endomembrane dynamics throughout terminal maturation. Inhibition of Cdc42 or Pak1/2/3 severely destructured the DMS and blocked proplatelet formation. Even though this process does not require containment within the hematopoietic niche, because DMS structuration was observed upon thrombopoietin-treatment in suspension, integrin outside-in signaling was required for Pak activation and probably resulted from secretion of extracellular matrix by MKs. Conclusions These data indicate a functional link, mandatory for MK differentiation, between actin dynamics, regulated by Cdc42/Pak1/2/3, and DMS maturation.

ATP-binding cassette transporter 1 (ABCA1) deficiency decreases platelet reactivity and reduces thromboxane A2 production independently of hematopoietic ABCA1.

UNLABELLED: ESSENTIALS: The role of ATP-binding cassette transporter 1 (ABCA1) in platelet functions is poorly characterized. We studied the impact of ABCA1 deficiency on platelet responses in a mouse model and two Tangier patients. ABCA1-deficient platelets exhibit reduced positive feedback loop mechanisms. This reduced reactivity is dependent on external environment and independent of hematopoietic ABCA1. BACKGROUND: The ATP-binding cassette transporter ABCA1 is required for the conversion of apolipoprotein A-1 to high-density lipoprotein (HDL), and its defect causes Tangier disease, a rare disorder characterized by an absence of HDL and accumulation of cholesterol in peripheral tissues. The role of ABCA1 in platelet functions remains poorly characterized. OBJECTIVE: To determine the role of ABCA1 in platelet functions and to clarify controversies concerning its implication in processes as fundamental as platelet phosphatidylserine exposure and control of platelet membrane lipid composition. METHODS AND RESULTS: We studied the impact of ABCA1 deficiency on platelet responses in a mouse model and in two Tangier patients. We show that platelets in ABCA1-deficient mice are slightly larger in size and exhibit aggregation and secretion defects in response to low concentrations of thrombin and collagen. These platelets have normal cholesterol and major phospholipid composition, granule morphology, or calcium-induced phosphatidylserine exposure. Interestingly, ABCA1-deficient platelets display a reduction in positive feedback loop mechanisms, particularly in thromboxane A2 (TXA2) production. Hematopoietic chimera mice demonstrated that defective eicosanoids production, particularly TXA2, was primarily dependent on external environment and not on the hematopoietic ABCA1. Decreased aggregation and production of TXA2 and eicosanoids were also observed in platelets from Tangier patients. CONCLUSIONS: Absence of ABCA1 and low HDL level induce reduction of platelet reactivity by decreasing positive feedback loops, particularly TXA2 production through a hematopoietic ABCA1-independent mechanism.

The short form of RON is expressed in acute myeloid leukemia and sensitizes leukemic cells to cMET inhibitors.

Several receptor tyrosine kinases (TKs) are involved in the pathogenesis of acute myeloid leukemia (AML). Here, we have assessed the expression of the Recepteur d'Origine Nantais (RON) in leukemic cell lines and samples from AML patients. In a series of 86 AML patients, we show that both the full length and/or the short form (sf) of RON are expressed in 51% and 43% of cases, respectively. Interestingly, sfRON is not expressed in normal CD34+ hematopoietic cells and induces part of its oncogenic signaling through interaction with the Src kinase Lyn. sfRON-mediated signaling in leukemic cells also involves mTORC1, the proapoptotic bcl2-family member, BAD, but not the phosphatidylinositol 3-kinase/Akt pathway. Furthermore, the expression of sfRON was specifically downregulated by 5-azacytidine (AZA). Conversely, AZA could induce the expression of sfRON in sfRON-negative leukemic cells suggesting that the activity of this drug in AML and myelodysplastic syndromes could involve modulation of TKs. cMET/RON inhibitors exhibited an antileukemic activity exclusively in AML samples and cell lines expressing sfRON. These results might support clinical trials evaluating cMET/RON inhibitors in AML patients expressing sfRON.

Sex differences in the GSK3ß-mediated survival of adherent leukemic progenitors.

Therapeutic resistance of acute myeloid leukemia stem cells, enriched in the CD34(+)38(-)123(+) progenitor population, is supported by extrinsic factors such as the bone marrow niche. Here, we report that when adherent onto fibronectin or osteoblast components, CD34(+)38(-)123(+) progenitors survive through an integrin-dependent activation of glycogen synthase kinase 3ß (GSK3ß) by serine 9-dephosphorylation. Strikingly, GSK3ß-mediated survival was restricted to leukemic progenitors from female patients. GSK3ß inhibition restored sensitivity to etoposide, and impaired the clonogenic capacities of adherent leukemic progenitors from female patients. In leukemic progenitors from female but not male patients, the scaffolding protein RACK1, activated downstream of α(5)ß(1)-integrin engagement, was specifically upregulated and controlled GSK3ß activation through the phosphatase protein phosphatase 2A (PP2A). In a mirrored manner, survival of adherent progenitors (CD34(+)38(-)) from male but not female healthy donors was partially dependent on this pathway. We conclude that the GSK3ß-dependent survival pathway might be sex-specific in normal immature population and flip-flopped upon leukemogenesis. Taken together, our results strengthen GSK3ß as a promising target for leukemic stem cell therapy and reveal gender differences as a new parameter in anti-leukemia therapy.

Platelet membrane phospholipid asymmetry: from the characterization of a scramblase activity to the identification of an essential protein mutated in Scott syndrome.

Like all eukaryotic cells, platelets maintain plasma membrane phospholipid asymmetry in normal blood circulation via lipid transporters, which control transbilayer movement. Upon platelet activation, the asymmetric orientation of membrane phospholipids is rapidly disrupted, resulting in a calcium-dependent exposure of the anionic phospholipid, phosphatidylserine (PS), at the outer platelet surface. This newly-exposed PS surface is a major component of normal hemostasis because it supports platelet procoagulant function. Binding of blood clotting enzyme complexes to this negatively-charged membrane surface allows a dramatic increase in the rate of conversion of zymogens to active serine proteases, which in turn produce a burst of thrombin leading to the formation of a fibrin clot and further platelet activation. Cells have the capacity to catalyze transbilayer phospholipid exchange via ATP-requiring translocase enzymes (flippases and floppases), which control unidirectional phospholipid transport against a concentration gradient. They also use an energy-independent, calcium-dependent scramblase activity to govern the bidirectional exchange of phospholipids between the two leaflets of the bilayer; this activity is essential for PS exposure during platelet activation. Scramblase activity, biochemically characterized in the 1980s, is deficient in patients with Scott syndrome, a rare inherited bleeding disorder with defective platelet procoagulant activity. Despite considerable efforts, the platelet scramblase protein remained elusive for years but a significant advance has recently been made with the identification of TMEM16F, a membrane protein essential for calcium-dependent PS exposure whose loss of function mutations are found in Scott syndrome. This review recalls historical aspects of platelet membrane asymmetry characterization, summarizes the mechanisms and roles of PS exposure following platelet activation and discusses the recent identification of TMEM16F and its significance in the scrambling process.

Targeted drug therapy: the platelet side.

Targeted therapy is certainly considered the future of cancer treatment. Several new molecules targeting critical intracellular signaling actors, particularly kinases, are arriving in clinics and many other are under development. However, proteins targeted by these drugs are common to many cell types and are particularly implicated in the highly dynamic processes of platelet activation. Therefore, the effects of targeted drugs, including kinase inhibitors, on platelet activation have to be considered in clinical practice. Moreover, their analysis also represents an opportunity to increase our knowledge in platelet biology and physiology and to develop novel antiplatelet strategies. In this review we briefly describe the major platelet signaling pathways that may be affected by these new drugs and discuss some clinical implications of their use.

The Syk-kinase inhibitor R406 impairs platelet activation and monocyte tissue factor expression triggered by heparin-PF4 complex directed antibodies.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a rare but severe complication of heparin therapy in which immunoglobulin G (IgG) antibodies against the platelet factor 4-heparin complex activate platelets through the FcγRIIA receptor. Clustering of FcγRIIA initiates signaling cascades involving tyrosine kinases including the spleen tyrosine kinase (Syk). Moreover, besides the critical role of platelets, the expression of tissue factor (TF) by human monocytes triggered by HIT antibodies has been shown to contribute to the hypercoagulability and the thrombotic complications in HIT patients. OBJECTIVES: We investigated the effect of R406, a small molecule inhibitor of Syk developed as a potential treatment of autoimmune diseases, allergic disorders and B-cell related hematological malignancies, on FcγRIIA-mediated platelet activation. To further assess the potential activity of Syk inhibitors in HIT treatment, the effect of R406 was also evaluated on HIT antibodies-induced expression of TF and procoagulant activity of monocytic cells. RESULTS: We show that R406 is a potent inhibitor of platelet signaling and functions initiated by FcγRIIA cross-linking by specific antibodies or by sera from HIT patients. Syk inhibition efficiently prevents FcγRIIA-induced LAT phosphorylation and activation of phosphoinositide 3-kinase, Akt, phospholipase Cγ2 and p38 MAP-kinase. As a consequence, FcγRIIA-induced platelet aggregation, granule secretion and microparticles production are strongly inhibited by R406. Moreover, the Syk inhibitor efficiently impairs the expression of TF and the procoagulant activity of human monocytes triggered by HIT antibodies. CONCLUSION: Syk inhibitors may be of therapeutic interest in the treatment of HIT by reducing HIT antibodies-mediated platelet activation and monocyte procoagulant activity.

Inhibition of Rac controls NPM-ALK-dependent lymphoma development and dissemination.

Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) is a tyrosine kinase oncogene responsible for the pathogenesis of the majority of human ALK-positive lymphomas. We recently reported that it activated the Rac1 GTPase in anaplastic large-cell lymphoma (ALCL), leading to Rac-dependent formation of active invadopodia required for invasiveness. Herein, we went further into the study of this pathway and used the inhibitor of Rac, NSC23766, to validate its potential as a molecular target in ALCL in vitro and in vivo in a xenograft model and in a conditional model of NPM-ALK transgenic mice. Our data demonstrate that Rac regulates important effectors of NPM-ALK-induced transformation such as Erk1/2, p38 and Akt. Moreover, inhibition of Rac signaling abrogates NPM-ALK-elicited disease progression and metastasis in mice, highlighting the potential of small GTPases and their regulators as additional therapic targets in lymphomas.

Internalization of microparticles by endothelial cells promotes platelet/endothelial cell interaction under flow.

BACKGROUND: Microparticles (MPs) released by activated or apoptotic cells increase in number in the blood of subjects with vascular or metabolic diseases and may contribute to thrombotic complications. OBJECTIVES: In this study, we investigated whether MPs promoted platelet recruitment to endothelial cells in flow conditions, and by which mechanism. METHODS: Human umbilical vein endothelial cells (HUVECs) grown in microslide perfusion chambers were exposed to MPs prepared in vitro from HUVECs, monocytes or platelets. RESULTS: Videomicroscopy of DIOC-labelled blood perfused at arterial rate on human umbilical vein ECs demonstrated that, irrespective of their cell origin, MPs promoted the formation of platelet strings at the surface of HUVECs. This platelet/endothelial cell interaction was dependent on von Willebrand factor (VWF) expression at the HUVEC surface and involved Glycoprotein Ib and P-selectin. Interestingly, HUVECs internalized MPs within a few hours through a process involving anionic phospholipids, lactadherin and αvß3 integrin. This uptake generated the production of reactive oxygen species via the xanthine/xanthine oxidase system (inhibited by allopurinol and the ROCK inhibitor Y-27632) and the NADPH oxidase (inhibited by SOD). Reactive oxygen species appeared essential for VWF expression at the endothelial cell surface and subsequent platelet/endothelial cell interaction under flow. The pathophysiological relevance of this process is underlined by the fact that circulating MPs from Type I diabetic patients induced platelet/endothelial cell interaction under flow, with an intensity correlated with the severity of the vasculopathy.

Reactive oxygen species and lipoxygenases regulate the oncogenicity of NPM-ALK-positive anaplastic large cell lymphomas.

The chimera nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), the tyrosine kinase activity of which is constitutively upregulated, is the causative agent of 75% of the anaplastic large-cell lymphomas (ALCLs). We have demonstrated that NPM-ALK induces the production of reactive oxygen species (ROS) by a pathway involving the arachidonic acid-metabolizing enzymes of the lipoxygenase (LOX) family. The use of the LOX inhibitor nordihydroguaiaretic acid (NDGA) and of the anti-oxidant N-acetylcysteine (NAC) demonstrated that ROS are important in maintaining the ALK kinase active. Consistent with this, NDGA treatment resulted in the inhibition of key pathways, such as Akt, signal transducer and activator of transcription factor 3 (STAT3) and extracellular signal-regulated kinase (ERK), which are involved in NPM-ALK antiapoptotic and pro-mitogenic functions. Conversely, the stress-activated kinase p38, described in some instances as a mediator of apoptosis, was activated. Interestingly, 5-LOX, an isoform involved in many cancers, was found to be activated in NPM-ALK(+) cells. Functional studies have shown that transforming properties, namely proliferation and resistance to apoptosis, were abrogated by treatment with either NDGA or the 5-LOX inhibitor (N-(3-phenoxycinnamyl)-acetohydroxamic acid) (BW A4C). Together, these data point to the ROS/LOX pathway as a potential new target for therapy in NPM-ALK-positive tumors.

The level of AKT phosphorylation on threonine 308 but not on serine 473 is associated with high-risk cytogenetics and predicts poor overall survival in acute myeloid leukaemia.

The phosphoinositide 3-kinase/Akt pathway is an important signalling pathway governing cell survival and proliferation in acute myeloid leukaemia (AML). As full activation of Akt requires phosphorylation on both threonine 308 (Thr308) and serine 473 (Ser473) residues, we studied the level of phosphorylation on the both sites in 58 AML samples by flow cytometry. The ratio of the mean fluorescence intensity of Thr308 and Ser473 represented a continuum ranging from 0.3 to 5.6 and from 0.4 to 2.87, respectively. There were no significant correlations between age, gender, French-American-British classification, leukocytosis, FLT3-ITD and Akt phosphorylation. However, the level of phosphorylation on Thr308, but not on Ser473, was significantly correlated with high-risk karyotype. Thr308(high) patients had significantly shorter overall survival (11 vs 47 months; P=0.01), event-free survival (9 vs 26 months; P=0.005) and relapse-free survival (10 months vs not reached; P=0.02) than Thr308(low) patients. Neither screening for AKT1 E17K mutation nor changes in the level of PTEN expression and phosphorylation could be linked to increased phosphorylation on Thr308 in high-risk cytogenetic AML cells. However, PP2A activity was significantly reduced in high-risk samples compared with intermediate-risk samples. Moreover, the specific Akt inhibitor, Akti-1/2, inhibited cell proliferation and clonogenic properties, and induced apoptosis in AML cells with high-risk cytogenetics, suggesting that Akt may represent a therapeutic target in high-risk AML.

A caspase-dependent cleavage of CDC25A generates an active fragment activating cyclin-dependent kinase 2 during apoptosis.

The cellular level of the CDC25A phosphatase is tightly regulated during both the normal and genotoxic-perturbed cell cycle. Here, we describe a caspase-dependent cleavage of this protein at residue D223 in non-genotoxic apoptotic conditions. This specific proteolysis generates a catalytically active C-terminal fragment that localizes to the nuclear compartment. Accumulation of this active CDC25A fragment leads to reduced inhibitory phosphorylation of the CDC25A substrate cyclin-dependent kinase 2 (CDK2) on Tyr15. Moreover, CDK2 was found stably associated with this fragment, as well as with an ectopically expressed CDC25A224-525 truncation mutant that mimicks the cleavage product. Ectopic expression of this mutant induced CDK2 Tyr15 dephosphorylation, whereas its catalytically inactive version did not. Finally, this 224-525 mutant initiated apoptosis when transfected into HeLa cells, whereas its catalytic inactive form did not. Altogether, this study demonstrates for the first time that caspase-dependent cleavage of CDC25A is a central step linking CDK2 activation with non-genotoxic apoptotic induction.

Elevated levels of PtdIns5P in NPM-ALK transformed cells: implication of PIKfyve.

Phosphatidylinositol 5-monophosphate (PtdIns5P), one of the latest phosphoinositides discovered, has been suggested to play important cellular functions. Here, we report the presence of higher levels of this lipid in cells expressing the oncogenic tyrosine kinase nucleophosmin anaplastic lymphoma kinase (NPM-ALK), a chimeric protein found in the large majority of anaplastic large cell lymphomas (ALCLs). In addition, we describe that a pool of PtdIns5P is located in the membrane extensions characteristic of NPM-ALK-transformed cells. Finally, we show that the increase of PtdIns5P is controlled by the kinase PIKfyve, which is known for its role in vesicular trafficking. These data suggest for the first time a role of PtdIns5P and PIKfyve in oncogenesis, potentially linking intracellular trafficking to cancer.

Activation of Rac1 and the exchange factor Vav3 are involved in NPM-ALK signaling in anaplastic large cell lymphomas.

The majority of anaplastic large cell lymphomas (ALCLs) express the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) fusion protein, which is oncogenic due to its constitutive tyrosine kinase activity. Transformation by NPM-ALK not only increases proliferation, but also modifies cell shape and motility in both lymphoid and fibroblastic cells. We report that the Rac1 GTPase, a known cytoskeletal regulator, is activated by NPM-ALK in ALCL cell lines (Karpas 299 and Cost) and transfected cells (lymphoid Ba/F3 cells, NIH-3T3 fibroblasts). We have identified Vav3 as one of the exchange factors involved in Rac1 activation. Stimulation of Vav3 and Rac1 by NPM-ALK is under the control of Src kinases. It involves formation of a signaling complex between NPM-ALK, pp60(c-src), Lyn and Vav3, in which Vav3 associates with tyrosine 343 of NPM-ALK via its SH2 domain. Moreover, Vav3 is phosphorylated in NPM-ALK positive biopsies from patients suffering from ALCL, demonstrating the pathological relevance of this observation. The use of Vav3-specific shRNA and a dominant negative Rac1 mutant demonstrates the central role of GTPases in NPM-ALK elicited motility and invasion.