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Persistent or chronic immune thrombocytopenias (P/C-ITP) are acquired blood disorders lasting more than 3 months or 1 year, respectively. The pathogenesis of these disorders is thought to be immunological. We hypothesized that some patients with P/C-ITP might have an intrinsic megakaryopoiesis defect. We identified a group of P/C-ITP patients with acquired isolated mild thrombocytopenia (30-100 × 10(9) /l), undetectable anti-platelet antibodies, negative autoimmune investigations and no need for treatment. We examined in vitro megakaryocyte differentiation and compared these patients' results with those of acute-ITP patients and healthy controls. No difference in proliferation, ploidy or expression of surface markers was found. In contrast, P/C-ITP patients had significantly fewer proplatelet-forming megakaryocytes. This novel observation demonstrated that some patients diagnosed with P/C-ITP have an intrinsic megakaryopoiesis defect independent of the bone-marrow environment. Further investigations are needed to dissect mechanisms underlying this impaired proplatelet formation in these patients.

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Groupe d'Etude et de Recherches sur l'Hemostase (GEHT) Region Ile-de-France (CORDDIM) Leducq TransAtlantic Network of Excellence Thrombospondin-1 (TSP-1) gives rise to fragments that have both pro- and anti-angiogenic effects in vitro and in vivo. the TSP-HepI peptide (2.3 kDa), located in the N-terminal domain of TSP-1, has proangiogenic effects on endothelial cells. We have previously shown that TSP-1 itself exhibits a dual effect on endothelial colony-forming cells (ECFC) by enhancing their adhesion through its TSP-HepI fragment while reducing their proliferation and differentiation into vascular tubes (tubulogenesis) in vitro. This effect is likely mediated through CD47 binding to the TSP-1 C-terminal domain. Here we investigated the effect of TSP-HepI peptide on the angiogenic properties of ECFC in vitro and in vivo. TSP-HepI peptide potentiated FGF-2-induced neovascularisation by enhancing ECFC chemotaxis and tubulogenesis in a Matrigel plug assay. ECFC exposure to 20 mu g/mL of TSP-HepI peptide for 18 h enhanced cell migration (p < 0.001 versus VEGF exposure), upregulated alpha 6-integrin expression, and enhanced their cell adhesion to activated endothelium under physiological shear stress conditions at levels comparable to those of SDF-1 alpha. the adhesion enhancement appeared to be mediated by the heparan sulfate proteoglycan (HSPG) syndecan-4, as ECFC adhesion was significantly reduced by a syndecan-4-neutralising antibody. ECFC migration and tubulogenesis were stimulated neither by a TSP-HepI peptide with a modified heparin-binding site (S/TSP-HepI) nor when the glycosaminoglycans (GAGS) moieties were removed from the ECFC surface by enzymatic treatment. Ex vivo TSP-HepI priming could potentially serve to enhance the effectiveness of therapeutic neovascularisation with ECFC. (C) 2012 Elsevier Inc. All rights reserved. Univ Estado Rio de Janeiro, Dept Biol Celular, Lab Biol Celula Endotelial & Angiogenese LabAngio, Inst Biol Roberto Alcantara Gomes, BR-20550011 Rio de Janeiro, RJ, Brazil INSERM, U765, Paris, France Univ Paris 05, Paris, France Universidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, São Paulo, Brazil Univ Fed Rio de Janeiro, Inst Ciencias Biomed, Rio de Janeiro, RJ, Brazil Hop Europeen Georges Pompidou, AP HP, Dept Haematol, Paris, France INSERM, Paris Cardiovasc Res Ctr, U970, Paris, France Universidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, São Paulo, Brazil Leducq TransAtlantic Network of Excellence: 04CVD01-LENA Leducq TransAtlantic Network of Excellence: 04CVD02 -LINAT CNPq: E-26/110.780/2010 CAPES: 629/09 Web of Science

Background: Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell disorders, characterized by significant increase in one or more myeloid lineages. A mutation in the Janus kinase 2 (JAK2) gene, JAK2V617F, was identified in half of the patients with Philadelphia chromosome-negative (Ph-) MPNs. This activating mutation causes hypersensitivity to certain growth factors, which explain the increased proliferation of myeloid progenitors. It has been reported that patients with MPNs have an increased risk of thrombosis but also increased microvessel density that may reflect angiogenesis in the spleen and bone marrow (Medinger, Br J Haematol, 2009), with no clear physiopathological explanation. Several recent studies have demonstrated the presence of JAK2V617F not only in blood cells but also in endothelial cells (EC) in these patients (Sozer, Blood, 2009; Teofili, Blood, 2011; Rosti, Blood, 2012). Hypothesis: We hypothesized that the presence of JAK2V617F in EC could change their properties leading to an increased angiogenesis process in MPNs. Methods: To determine whether the presence of JAK2V617F in EC was responsible for increased angiogenesis, we used an in vitro approach with human JAK2V617F ECs and an in vivo mouse model. We first used HUVECs (human umbilical vein endothelial cells) transduced with GFP lentivirus encoding human JAK2V617F. An empty lentivirus encoding only for GFP was used as a negative control. Proliferation of HUVECs was quantified during 3 days culture in EGM-2 medium and tube formation after culture in Matrigel™ was assessed by microscopy. Cell migration was determined by microscopy after scratch assay. Proteins expression level in cell lysate and supernatant was determined using Proteome ProfilerTM Array (R&D). The intensity of dot blots was determined by imageJ. For the in vivo approach, we crossed JAK2Flex/WT mice with PDGFβcreERT2 mice to generate endothelial-specific JAK2V617F knock-in mouse line (PDGFβERT2-JAK2 V617F/WT). Our lab was pioneer in developing microCT vascular imaging in order to precisely measure arterial vessel density and organization in 3 dimensions. To analyze neoangiogenesis (in the setting of ischemia), we used the model of mouse hind limb ischemia (Oses, ATVB, 2009): 11 days after ligature of the femoral artery, mice are perfused with latex labeled with barium and the hind limb vascular network is visualized with microCT. Results: We first observed that JAK2V617F HUVECs proliferate more than controls (coefficient rate of 3,53+/-0,18 versus 1,98 +/-0,05), reminding the phenotype of JAK2V617F hematopoietic cells. We then showed that JAK2V617F HUVECs are able to form more tubular structures in Matrigel™. Using an in vitro cell migration assay, we observed that JAK2V617F HUVECs invaded 45% (+/-2.3%) of the total surface area versus 27% (+/-3.3%) for controls. To confirm these results obtained in vitro, we analyzed the vascular network after hind limb ischemia in mice expressing JAK2V617F mutation specifically in endothelial cells (PDGFβERT2-JAK2V617F/WT). Our first results show an increased neoangiogenesis and further results will be presented at the conference. In order to decipher the mechanism responsible for the increased angiogenesis, we then quantified the expression of proteins that regulate angiogenesis, either in transduced HUVECs or in the culture supernatant. Interestingly, we have demonstrated a greater secretion of angiogenin, PDGF-AA, Endostatin, IGFBP-1, MCP-1 and CXCL-16 by HUVEC JAK2V617F. Discussion: In summary, our data suggest that the presence of JAK2V617F mutation in EC modifies their properties toward a pro-angiogenic profile and can explain, at least in part, the reported increase of angiogenesis in MPN patients. Once the role of JAK2V617F mutation in modifying EC properties will be confirmed, further work will be required to identify the molecular mechanisms underlying these phenotypic changes. It will also be particularly important to investigate if ECs are involved in the pathogenesis of increased angiogenesis observed in other diseases. Thus, they could be a new target in the treatment of pathological neo-angiogenesis. Disclosures No relevant conflicts of interest to declare.

Abstract 624 Persistent/chronic immune thrombocytopenias (PC-ITP) are acquired thrombocytopenias characterized by a platelet count less than 100 G/L, lasting respectively more than 3 months/one year. It is a diagnosis of exclusion, and other causes of acquired thrombocytopenia such as neoplasms, infections, autoimmune diseases or drugs must have been ruled out. It is commonly admitted that the pathogenesis of ITP associates the presence of antiplatelet antibodies that bind to mature platelets, leading to their elimination by macrophages through Fc-mediated recognition mechanisms mainly in the spleen or liver, or both. These antibodies are also thought to bind to megakaryocytes impairing platelet production. Newly available TPO receptor agonists are highly effective in chronic ITP patients, with a dramatic increase in platelet count, which suggests a crucial role of altered megakaryopoiesis in this disorder. Because antiplatelet antibodies are only found in 20% of PC-ITP, we hypothesized that some of these patients might have an intrinsic defect in megakaryopoiesis rather than an immune-mediated dysmegakaryopoiesis. We thus analyzed in vitro megakaryocyte differentiation and proplatelet formation in 9 PC-ITP patients, 4 acute ITP patients (A-ITP), and 9 healthy controls (CTRL). All PC-ITP patients had ITP criteria regarding last international consensus (Neunert C et al, Blood 2011), with a slowly progressing thrombocytopenia (76 G/L, range: 29–97) lasting more than 3 months (median: 17 months; range: 7–36 months), no anti-platelet antibodies and normal medullar density and number of megakaryocytes. All A-ITP patients also matched ITP criteria. All samples were taken at the time of diagnosis, before any treatment was administered. They were compared with 9 controls including patients undergoing valvular replacement or healthy bone marrow donors with normal blood count. To analyze whether there was a defect of megakaryopoiesis that was cell-intrinsic, we isolated CD34 positive cells from the bone marrow and analyzed in vitro megakaryocytic differentiation with TPO-mimetic romiplostim. Proliferation was measured at days 3, 6 and 10 and compared in the 3 groups by a proliferation coefficient. Membrane maturation was assessed at day 6 and 10 by flow cytometry (FC) after CD41-FITC and CD42-PE staining. Megakaryocytic ploidy was measured at day 10 by FC after propidium iodure and CD41 staining. At day 8, large mature megakaryocytes were isolated after discontinuous HSA density gradient and proplatelets forming megakaryocytes were counted from between days 9 and 13. Late mature megakayocytes (day 12–13) were observed with a confocal microscope to qualitatively analyze proplatelet formation. We did not observe any difference between A-ITP or PC-ITP patients and controls in term of proliferation, ploidy, or expression of surface differentiation markers (CD41, CD42). In contrast, PC-ITP-derived megakaryocytes showed a defect in proplatelet formation, as only 12% of large, mature megakaryocytes were able to form proplatelets in liquid culture at day 11 vs 37% in CTRL (p=0,046) and 39% in A-ITP (p=0,03), 11% at day 12 vs 43,2% in CTRL (p=0,024) and 46% in A-ITP (p=0,0002), and 10% at day 13 vs 44,5% in CTRL (p=0,015) and 46% in A-ITP (p=0,03). Besides, we observed that proplatelet-forming megakaryocytes from ITP patients had less proplatelets per megakaryocyte and less bifurcation per proplatelet. In conclusion, our study shows that megakaryocytes from patients with persistent/chronic ITP have an intrinsic defect in megakaryocyte development that is independent from the medullar environment. This defect affects proplatelet formation and further investigations are now needed to better describe mechanisms underlying proplatelet alteration in this disease. Disclosures: Viallard: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees.