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CONTEXT: Many hematological and non-hematological diseases can be hidden under the mask of isolated thrombocytopenia. The choice of therapeutic tactics is determinated by correct diagnosis. OBJECTIVE: to define the frequency of occurrence of primary immune thrombocytopenia (idiopathic thrombocytopenic purpura-ITP) in the group of patients with isolated thrombocytopenia. Materials and methods: We analysed clinical and laboratory data of 301 patients who applied to the outpatient department of National Research Center for hematology, Russian Federation with thrombocytopenia of unspecified origin. The first group is 183 patients who applied for the first time. The second group is 118 patients with long history of ITP. All patients were examined according to the extended differential diagnostic protocol used in isolated thrombocytopenia and based on international and National clinical recommendations for the diagnosis and treatment of ITP in adults. Results: Median age of patients in both groups was 36 years, male/female ratio in group 1 was 1:2, in group 2 - 1:4. In group 1, the count of platelets in the blood was more than 50*109/l in 87% of cases, while in the second group, in most cases (94%), there was a decrease in the count of platelets Conclusion: This study clearly presents a variety of hematological and non-hematological diseases occurring with isolated thrombocytopenia, which indicates the ambiguity of such concepts as the symptom of isolated thrombocytopenia and primary immune thrombocytopenia and requires a complete examination not only in the onset of the disease, but also in the recurrence of ITP. Disclosures No relevant conflicts of interest to declare.

Correction of murine models of β-thalassemia has been achieved through high-level globin lentiviral vector gene transfer into mouse hematopoietic stem cells (HSCs). However, transduction of human HSCs is less robust and may be inadequate to achieve therapeutic levels of genetically modified erythroid cells. We therefore developed a double gene lentiviral vector encoding both human γ-globin under the transcriptional control of erythroid regulatory elements and methylguanine methyltransferase (MGMT), driven by a constitutive cellular promoter. MGMT expression provides cellular resistance to alkylator drugs, which can be administered to kill residual untransduced, diseased HSCs, whereas transduced cells are protected. Mice transplanted with β-thalassemic HSCs transduced with a γ-globin/MGMT vector initially had subtherapeutic levels of red cells expressing γ-globin. To enrich γ-globin–expressing cells, transplanted mice were treated with the alkylator agent 1,3-bis-chloroethyl-1-nitrosourea. This resulted in significant increases in the number of γ-globin–expressing red cells and the amount of fetal hemoglobin, leading to resolution of anemia. Selection of transduced HSCs was also obtained when cells were drug-treated before transplantation. Mice that received these cells demonstrated reconstitution with therapeutic levels of γ-globin–expressing cells. These data suggest that MGMT-based drug selection holds promise as a modality to improve gene therapy for β-thalassemia.

Within the broad problem of host immune surveillance versus tumor immune evasion, a most intriguing question is how the cellular immunity can cope with cancerous cells that have gotten rid of the classical antigen-presenting machinery. One such option stems from (1) the fact that HLA loss is often attended with expression of Hsp70 on the tumor cell surface, and (2) our findings that human lymphocytes express a protein Tag7 (also known as PGRP-S) capable of tight and specific interaction with cognate Hsp70. Here we show that a subpopulation of human CD4+CD25+ lymphocytes, obtained either in culture as lymphokine-activated killers or directly from healthy donors, carry Tag7 and FasL on their surface and can indeed kill the HLA-negative tumor-derived cells K562 and MOLT-4 that expose Hsp70 and Fas. The primary binding of lymphocyte Tag7 to target-cell Hsp70 is very specific (eg, it is blocked by preincubating either cell with minimal peptides from the “partner” protein), and secures cell contact indispensable for subsequent FasL/Fas-triggered apoptosis. Unrelated to natural killer cell action or the putative role of Hsp as an antigen-presenting substitute, this novel mechanism is rather a backup analog of orthodox (CD8+) target recognition (Tag7 acting as built-in T-cell receptor and Hsp70 itself as ligand).

Binding of von Willebrand factor (VWF) to the platelet membrane glycoprotein (GP) Ib-IX-V complex initiates a signaling cascade that causes αIIbβ3 activation and platelet aggregation. Previous work demonstrated that botrocetin (bt)/VWF–mediated agglutination activates αIIbβ3 and elicits adenosine triphosphate (ATP) secretion in a thromboxane A2 (TxA2)– and Ca2+-dependent manner. This agglutination-elicited TxA2 production occurs in the absence of ATP secretion. However, the signaling components and signaling network or pathway activated by GPIb-mediated agglutination to cause TxA2 production have not been identified. Therefore, the focus of this study was to elucidate at least part of the signal transduction network or pathway activated by GPIb-mediated agglutination to cause TxA2 production. The phosphatidylinositol 3-kinase (PI3K) selective inhibitor wortmannin, and mouse platelets deficient in Lyn, Src, Syk, Src homology 2 (SH2) domain–containing leukocyte protein 76 (SLP-76), phospholipase Cγ2 (PLCγ2), linker for activation of T cells (LAT), or Fc receptor γ-chain (FcRγ-chain) were used for these studies. LAT and FcRγ-chain were found not to be required for agglutination-driven TxA2 production or activation of αIIbβ3, but were required for granule secretion and aggregation. The results also clearly demonstrate that bt/VWF-mediated agglutination-induced TxA2 production is dependent on signaling apparently initiated by Lyn, enhanced by Src, and propagated through Syk, SLP-76, PI3K, PLCγ2, and protein kinase C (PKC).

Members of the Src family of kinases are abundant in platelets. Although their localization is known, their role(s) in platelet function are not well understood. Lyn is a Src-family kinase that participates in signal transduction pathways elicited by collagen-related peptide; it has also been implicated through biochemical studies in the regulation of von Willebrand factor signaling. Here, we provide evidence that Lyn plays a role in γ-thrombin activation of platelets. Unlike the wild-type platelets, platelets from Lyn-deficient mice do not undergo irreversible aggregation, produce thromboxane A2, or secrete adenosine diphosphate in response to submaximal γ-thrombin concentrations that cause secretion-dependent irreversible aggregation. Phosphorylation of Akt, a downstream effector of phosphatidylinositol 3-kinase, also requires a higher concentration of γ-thrombin in Lyn-deficient platelets than in wild-type platelets. These findings demonstrate that Lyn signaling is required for thrombin induction of secretion-dependent platelet aggregation. Specifically, Lyn is required under these conditions to enable thrombin-induced TxA2 production and adenosine diphosphate secretion, necessary steps in secretion-dependent platelet aggregation.

Controlling the activation of platelets is a key strategy to mitigate cardiovascular disease. Previous studies have suggested that the ATP-binding cassette (ABC) transporter, ABCC4, functions in platelet-dense granules. Using plasma membrane biotinylation and super-resolution microscopy, we demonstrate that ABCC4 is primarily expressed on the plasma membrane of both mouse and human platelets. Platelets lacking ABCC4 have unchanged dense-granule function, number, and volume, but harbor a selective impairment in collagen-induced aggregation. Accordingly, Abcc4 knockout (KO) platelet attachment to a collagen substratum was also faulty and associated with elevated intracellular cyclic AMP (cAMP) and reduced plasma membrane localization of the major collagen receptor, GPVI. In the ferric-chloride vasculature injury model, Abcc4 KO mice exhibited markedly impaired thrombus formation. The attenuation of platelet aggregation by the phosphodiesterase inhibitor EHNA (a non-ABCC4 substrate), when combined with Abcc4 deficiency, illustrated a crucial functional interaction between phosphodiesterases and ABCC4. This was extended in vivo where EHNA dramatically prolonged the bleeding time, but only in Abcc4 KO mice. Further, we demonstrated in human platelets that ABCC4 inhibition, when coupled with phosphodiesterase inhibition, strongly impaired platelet aggregation. These findings have important clinical implications because they directly highlight an important relationship between ABCC4 transporter function and phosphodiesterases in accounting for the cAMP-directed activity of antithrombotic agents.

Introduction. Primary immune thrombocytopenia is a rare disease1. The incidence of ITP is not well estimated in Russia and worldwide. In adults it varies from 1,6 to 3,9/100 000 person-years2-3. The gender and age-associated results are discussed and differ in several investigations4-6. Study objectives: evaluation of the incidence of primary immune thrombocytopenia in adults in one region of Russia Patients and methods. The data source is the Registry of the patients with primary ITP in Russia. 272 adult patients: 77 males (28%) and 195 females (72%), age from 16 to 89 years (median 44 years) with ITP (ICD-10 code D69.3), newly diagnosed cases during the period from 12 Jan 2014 to 24 May 2016. Results. 221 (81%) cases were newly diagnosed in 12 regions of Russia in which registration was performed most actively - more than 5 cases for the duration of the study. But only one region was selected for the first evaluation of epidemiological characteristics because of the number of reasons. There is one hematological central clinic in this region in which diagnosis of ITP can be verified and patients with ITP are treated and monitored most properly. The early started and fully performed registration process can be regarded as covered most part of region population in this target region. 86 cases (27 male, 59 female) were registered in the target region. The gender-age distribution was following: male: age 60 = 10 (37%); female: age 60 = 15 (25%). The estimated incidence rate in the target region is shown in table 1. The estimated incidence rates in gender-age strata in the target region are demonstrated in table 2. Conclusion. Overall ITP incidence in one region of Russia is 3.20/100 000 person-years. It is compatible to the incidence in other European countries. Our data demonstrate the rise of incidence rate in males with age and its decrease with age in female population. Literature. 1) Rodeghiero F., Stasi R., Gernsheimer T., Michel M., Provan D., Arnold D.M., et al. Standardization of terminology, definitions and outcome criteria in immune thrombocytopenic purpura of adults and children: report from international working group. Blood. 2009; 113(11): 2386--93. doi: 10.1182/blood-2008-07-162503. 2) Terrell DR, Beebe LA, Vesely SK, Neas BR, Segal JB, George JN. The incidence of immune thrombocytopenic purpura in children and adults: A critical review of published reports. Am J Hematol. 2010; 85(3): 174-180. 3) Moulis G, Palmaro A, Montastruc J-L, Godeau B, Lapeyre-Mestre M, Sailler L. Epidemiology of incident immune thrombocytopenia: a natiowide population-based study in France. Blood. 2014; 124(22): 3308-3315. 4) Segal JB, Powe NR. Prevalence of immune thrombocytopenia: analyses of administrative data. J Thromb Haemost 2006; 4: 2377-83 5) Schoonen WM, Kucera G, Coelson J, et al. Epidemiology of immune thrombocytopenic purpura in the General Practise Research Database. Br J Haematol 2009; 145(2): 235-244. 6) Lisukov I.A., Maschan A.A., Shamardina A.V., Chagorova T.V., Davydkin I.L., Sycheva T.M., et al. Immune thrombocytopenia: clinical manifestations and response to therapy. Intermediate analysis of data of the Russian register of patients with primary immune thrombocytopenia and review of literature. Oncogematologiya. 2013; 2: 61--9]. Disclosures No relevant conflicts of interest to declare.

Hereditary macrothrombocytopenia is a hallmark of Wistar Furth (WF) rats. In addition, a platelet/megakaryocyte alpha granule defect, similar to that of patients with gray platelet syndrome, is present. Several observations indicate cytoskeletal abnormalities in WF platelets and megakaryocytes, suggesting the potential for functional defects in hemostatic processes requiring cytoskeletal reorganization, such as platelet adhesion and spreading. However, no bleeding abnormality has been noted. Here, we report a prolonged bleeding time (>30 minutes in 10 of 11 rats tested) with defective clot formation in the WF strain. Prolonged bleeding time can result from defects in platelet adhesion, aggregation, or the release reaction. Because aggregation to collagen and adenosine diphosphate were reported to be normal, we determined whether WF rat platelets are defective in their ability to adhere to substrates. Platelet adherence and spreading was evaluated from 30 seconds to 30 minutes on Formvar-coated, carbon-stabilized grids or poly-L-lysine–coated glass coverslips by transmission electron microscopy or immunofluorescence, respectively, and scanning electron microscopy. We classified the adhered platelets according to their pattern of spreading, ie, rounded, rounded or spreading with short filopodia, spindle-shaped, spreading with long filopodia, spreading with lamellipodia, and fully spread. Adherent normal rat platelets displayed all stages of spreading within 30 seconds to 2 minutes, including many spindle-shaped forms, and forms with multiple, long filopodia. In contrast, adhered WF platelets at these early time points rarely developed long filopodia or were spindle shaped. The majority of adherent WF platelets at these early time points were either round, spread with a few short filopodia, or extensively spread with wide lamellipodial skirts. By 15 to 30 minutes, most platelets in both Wistar and WF samples were fully spread. These data show abnormal WF platelet spreading. The paucity of spindle-shaped forms and forms with long filopodia may reflect an inability of WF platelets to undergo the early stages of spreading, or, alternatively, their more rapid than normal progression through these stages. We hypothesize that this failure to spread normally may relate to prolonged bleeding times in vivo and defective clot formation in WF rats.

Nonreceptor protein tyrosine kinases phosphorylate proteins, thereby activating many intracellular signaling pathways and mediating protein-protein interactions. Protein phosphorylation is regulated in large part by the subcellular localization of these kinases and their respective substrates. Src is the most studied of these kinases, although other members of the Src family have been shown to be important in the differentiation of specific cell types. Src and Src family members are reported to be membrane-associated, but detergent-extraction studies have demonstrated a major difference in the solubility of Src compared with other members of the Src family (Fgr, Fyn, Lck, Lyn, and Yes), suggesting that their subcellular distributions may be different. By immunoelectron microscopy, we demonstrate that, unlike Src, the Src-related kinases are associated with electron-dense cytoplasmic domains and plasma membrane domains that correspond in size and frequency to endocytotic vesicles and coated pits. Clusters of labeling for these kinases also were seen adjacent to granule membranes. These kinases colocalize with the coated vesicle protein, clathrin, confirming their association with this class of endocytotic vesicle. We hypothesize that this vesicular association of Src-related kinases indicates a role for them in the endocytotic vesicle–mediated uptake and trafficking of plasma proteins into platelet granules.

The roles of most cytoskeletal proteins in platelet formation and function remain largely undefined. We earlier detected megakaryocyte membrane blebbing and a unique antigenic determinant associated with a missense mutation in the cytoskeletal protein, talin, in an animal model of hereditary macrothrombocytopenia, the Wistar Furth (WF) rat, which led us to examine the distribution of talin and other cytoskeletal proteins in resting normal and WF rat platelets. In contrast to the conclusions of an earlier ultrastructural analysis, our biochemical and ultrastructural immunogold studies indicate a significant membrane-association of talin in both resting normal and WF rat platelets as found earlier for rat megakaryocytes. Talin was associated with plasma membranes, membranes of the surface-connected canalicular system, and with alpha-granule membranes of both normal and WF rat platelets, but as in WF megakaryocytes, talin was absent from the large membrane complexes of WF platelets. An even more striking difference was seen in the distribution of myosin in subcellular fractions of normal and WF rat platelets separated in density gradients, in which the proportion of myosin in the least dense WF rat platelet membrane fraction was one half that in the same normal platelet fraction. This difference was balanced by a fourfold increase in myosin in the most dense WF rat subcellular fraction, which is highly enriched for alpha-granules. These results support our hypothesis that the platelet abnormalities of the WF rat are related to defects in the megakaryocyte-platelet cytoskeleton.

Background: PV is a chronic myeloproliferative neoplasm (MPN) characterized by predominant proliferation of erythroid precursors, an elevated red blood cell mass, high risk of vascular and thrombotic complications, reduced quality of life due to a substantial symptom burden (pruritus, fatigue, constitutional symptoms, microvascular disturbances, and bleeding). Conventional therapeutic options aim at reducing vascular and thrombotic risk, with low-dose aspirin and phlebotomy as first-line recommendations for patients at low risk of thrombotic events and cytoreductive therapy (hydroxyurea or interferon alpha) recommended for high-risk patients. Long-term effective and well-tolerated treatments are still lacking. Few data are available concerning patients with this condition at Russian Federation. The aim of this study was to describe clinical and demographic characteristics of PV patients at diagnosis and review the current treatment landscape in PV. Methods: From 2004 to 2014 in the outpatient department of Hematology Research Center 1687 patients with MPN were observed. The proportion of patients with PV was 28% (470) PMF - 31% (523), ET - 23% (389), unclassified MPN - 18% (305). We present the results of observation of 100 patients with PV who treated in the outpatient department. Long-term follow up of patients ranged from 6 to 262 months. Median follow-up - 14 months. Results: The proportion of women was 67%, men - 33%. The age was from 23 to 80 years (median - 56 years). PV diagnosed on the classification of WHO 2008. Hemoglobin was from 149 to 260 g/L (median 181 g/L) in men, hemoglobin was from 136 to 247 g/L (median 177 g/L) in women. RBC was 4.4 - 10.0x1012/L (median 7,1x1012/L) in men, RBC 4.8 - 8.8 x1012/L (median 6,9x1012/L) in women. PLT 137 - 3934 hч109/L (median 551x109/L), WBC 4.0 - 69x109/L (median 10,5x109/L). Hematocrit 42 - 86% (median 53%). JAK2 V617F detected in 100%. Splenomegaly founded in 70%. All patients had headache, dizziness, 25% of patients - itching. All patients received symptomatic therapy, antiplatelet agents, preparations improving microcirculation and antihypoxants. 25% patients had thrombohemorrhagic complications in anamnesis. Treatment: 49% - hudroxiurea, 14% - INFα-2b, 14% - combination therapy (hydroxyurea and phlebotomy or INFα-2b and phlebotomy), 23% - only phlebotomy. Response to treatment was evaluated according to the criteria of the ELN 2009. In the whole group of patients without the therapy frequency of complete remission - 48% partial remission - 41%, with no effect - 11%. Change of therapy was carried out in case of failure, intolerance or treatment of complications. When switching treatment from one method to another complete remission was not achieved. Conclusion: Treatment of polycythemia is mainly symptomatic. The effectiveness of therapy I line (complete remission) from 14.5 to 71%. It is necessary to conduct clinical trials designed to evaluate the effectiveness and safety of new targeted therapies. Disclosures No relevant conflicts of interest to declare.

Sickle cell anemia is characterized by chronic hemolysis coupled with extensive vascular inflammation. This inflammatory state also mechanistically promotes a high risk of lethal, invasive pneumococcal infection. Current treatments to reduce vaso-occlusive complications include chronic hydroxyurea therapy to induce fetal hemoglobin. Because hydroxyurea also reduces leukocytosis, an understanding of the impact of this treatment on pneumococcal pathogenesis is needed. Using a sickle cell mouse model of pneumococcal pneumonia and sepsis, administration of hydroxyurea was found to significantly improve survival. Hydroxyurea treatment decreased neutrophil extravasation into the infected lung coincident with significantly reduced levels of E-selectin in serum and on pulmonary epithelia. The protective effect of hydroxyurea was abrogated in mice deficient in E-selectin. The decrease in E-selectin levels was also evident in human sickle cell patients receiving hydroxyurea therapy. These data indicate that in addition to induction of fetal hemoglobin, hydroxyurea attenuates leukocyte–endothelial interactions in sickle cell anemia, resulting in protection against lethal pneumococcal sepsis.

Collagen-induced activation of platelets in suspension leads to alpha(IIb)beta(3)-mediated outside-in signaling, granule release, thromboxane A2 (TxA2) production, and aggregation. Although much is known about collagen-induced platelet signaling, the roles of TxA2 production, adenosine diphosphate (ADP) and dense-granule secretion, and alpha(IIb)beta(3)-mediated outside-in signaling in this process are unclear. Here, we demonstrate that TxA2 and ADP are required for collagen-induced platelet activation in response to a low, but not a high, level of collagen and that alpha(IIb)beta(3)-mediated outside-in signaling is required, at least in part, for this TxA2 production and ADP secretion. A high level of collagen can activate platelets deficient in PLC gamma 2, G alpha q, or TxA2 receptors, as well as platelets treated with a protein kinase C inhibitor, Ro31-8220. Thus, activation of alpha(IIb)beta(3) in response to a high level of collagen does not require these signaling proteins. Furthermore, a high level of collagen can cause weak TxA2 and ADP-independent aggregation, but maximal aggregation induced by a high level of collagen requires TxA2 or secretion.

A single dose of Mpl ligand (Mpl-L) given immediately after lethal DNA-damaging regimens prevents the death of mice. However, the mechanism of this myeloprotection is unknown. The induction of p53-dependent apoptosis in response to DNA damage signals suggests that immediate administration of Mpl-L may inhibit p53-dependent apoptosis. This hypothesis was tested by administering a single injection of pegylated murine Megakaryocyte Growth and Development Factor (PEG-rmMGDF, a truncated recombinant Mpl-L) to p53(-/-) and wild-type mice immediately after carboplatin (80 mg/kg) and 7.5 Gy total body gamma-irradiation. PEG-rmMGDF was required to prevent the death of wild-type mice, whereas p53(-/-) mice survived with or without the exogenous cytokine. The degree of platelet depression and subsequent recovery was comparable in p53(-/-) mice to wild-type animals given PEG-rmMGDF. Hence, either Mpl-L administration or p53-deficiency protected multipotent hematopoietic progenitors and committed megakaryocyte precursors. The myelosuppressive regimen induced expression of p53 and the p53 target, p21(Cipl) in wild-type bone marrow, indicating that Mpl-L acts downstream of p53 to prevent apoptosis. Constitutive expression of the proapoptotic protein Bax, was not further increased. Bax(-/-) mice survived the lethal regimen only when given PEG-rmMGDF; however, these Bax(-/-) mice showed more rapid hematopoietic recovery than did identically-treated wild-type mice. Therefore, administration of Mpl-L immediately after myelosuppressive chemotherapy or preparatory regimens for autologous bone marrow transplantation should prevent p53-dependent apoptosis, decrease myelosuppression, and reduce the need for platelet transfusions.

Abstract 1063 Platelet activation is a highly regulated process, and cyclic nucleotide mediated signaling pathways are crucial to effective platelet activation. Vascular injury produces, exposed collagen which binds circulating platelets through the platelet's “collagen” receptor, GPVI, resulting in the activation of guanyly/adenlyl cyclases. These interactions result in the rapid alterations in the cyclic nucleotide concentration inside the platelets leading to activation of protein kinase A and G signaling pathways to modulate platelet function. While, ABCC4 functions as a plasma membrane transporter for cyclic nucleotides its contribution to platelet activation has been obscured because it was reportedly as primarily intracellular in the platelets dense granules. This original report (Jedlitschky, Tirschmann et al. 2004) evaluated ABCC4 localization by immune-fluorescence of platelets attached to collagen coated coverslips. However, attachment via collagen produces platelet activation leading to mobilization and fusion of alpha and dense granules to the plasma membrane, thus under these conditions distinguishing between plasma membrane and dense granules is not possible. We resolved this problem by labeling quiescent platelets with a cell impermeable biotinylating agent (EZ-Link Sulfo-NHS-LC-LC Biotin). Isolation of membrane and internal fraction demonstrated that of over ninety percent of Abcc4 localizes to the plasma membrane. Furthermore, confocal microscopy of platelets stained with specific antibodies against Abcc4 confirmed Abcc4 localization to the plasma membrane. We extended these studies to the Abcc4- knockout (KO) mouse model. The Abcc4- KO mouse does not have any change in the number of platelet or dense granules compared to the wild type mouse. Platelet activation in vivo can be initiated by interaction with collagen through the GPVI receptor that is expressed at the plasma membrane of the platelets. At the molecular level, the initiation of platelet activation by collagen results in an increase in the cyclic nucleotide concentration leading to activation of signaling cascade through protein kinase A or G. Expose of Abcc4-KO platelets to collagen and revealed impaired activation in response to collagen. However, Abcc4-KO platelets activated by either thrombin or ADP (which activate either G-coupled PAR receptors or P2Y12 receptor respectively) shows an aggregation profile almost identical to wildtype platelets, thus indicating the defect in Abcc4 -KO platelet aggregation is specific to the collagen pathway. To understand the basis for the impaired collagen aggregation of Abcc4-KO platelets, we investigated the collagen receptor (GPVI) signaling pathway in Abcc4-KO platelets. Interestingly, in the Abcc4-KO platelets after the platelet activation with collagen, cyclic nucleotide dependent phosphorylation of VASP through protein kinase A or G at Ser-157 or Ser-239 respectively is reduced compared to the wildtype. Notably, Abcc4-KO platelets had reduced GPVI surface expression that correlated with the reduced phosphorylation of VASP after collagen stimulation. The similar, protein levels of Syk and Plcg2, (downstream signaling molecules of GPVI signaling pathway), in the Abcc4 wildtype and KO platelets implies that GPVI expression is the primary defect in Abcc4 deficiency. These results suggest that Abcc4 plays a crucial role in regulating cyclic nucleotides in response to GPVI activation by collagen. These findings suggest ABCC4/Mrp4 loss of function or inhibition (by drugs) may disrupt platelet aggregation under conditions of vascular injury. As, many antiplatelet drugs are potent inhibitors of Abcc4 (e.g., Dipyridamole and Sildenafil) these conclusions have strong implications for not just the development of antiplatelet drugs, but also for further exploring the role of Abcc4 in regulating intracellular nucleotide levels and platelet biology. Disclosures: No relevant conflicts of interest to declare.

Abstract 1141 Cyclic nucleotides have an important role in platelet aggregation and the role of phosphodiesterases in regulating their concentration is well known. Currently it is unknown if plasma membrane cyclic nucleotide export proteins regulate cyclic nucleotide concentrations in platelets. The ATP-binding cassette transporter, ABCC4 functions as a cyclic nucleotide exporter that is highly expressed in platelets. However, its role as a cyclic nucleotide transporter in platelets is unknown, because it was reportedly localized intracellularly in the platelet dense granules. This original report (Jedlitschky, Tirschmann et al. 2004) evaluated ABCC4 localization by immune-fluorescence of platelets after attachment to collagen coated coverslips. However, collagen attachment activates platelets causing mobilization and fusion of alpha and dense granules to the plasma membrane, thus rendering conditions that distinguish between plasma membrane and dense granules almost impossible. To resolve this problem we isolated the platelets under conditions that minimize activation during isolation. Subsequently, these platelets membranes were labeled with the cell impermeable biotinylating agent (EZ-Link Sulfo-NHS-LC-LC Biotin). Analysis of total platelet lysate detected the dense granule marker, P-selectin and Abcc4. However, after precipitation of the plasma membrane with streptavidin-beads, we detected only Abcc4. This indicates Mrp4 is at the plasma membrane. We confirmed Abcc4 localization by confocal microscopy on platelets that were treated with a monoclonal antibody specific to Abcc4. Evidence that Abcc4 regulates cyclic nucleotide levels under basal conditions was then provided by the findings that Abcc4-null platelets have elevated cyclic nucleotides. We further used the Abcc4-null mouse model to explore the role of Abcc4 in platelet biology. The Abcc4-null mouse does not have any change in the platelet or dense granules number compared to the wild type mouse. Platelet activation in vivo can be initiated by interaction with collagen through the GPVI receptor that is expressed at the plasma membrane of the platelets. At the molecular level, the initiation of platelet activation by collagen results in an increase in the cyclic nucleotide concentration and phosphorylation of vasodilator-stimulated phosphoprotein (VASP) which can attenuate aggregation. To determine the Abcc4 role in this process we exposed Abcc4-null platelets to collagen and discovered that these platelets have impaired activation in response to collagen. However, Abcc4-null platelets activated by thrombin or ADP, which activate either G-coupled PAR receptors or P2Y12 receptor respectively, show an aggregation profile almost identical to wildtype platelets, thus indicating the defect in Abcc4-null platelet aggregation is specific to the collagen initiated pathway. To understand the basis for the impaired aggregation of Abcc4-null platelets, we examined VASP phosphorylation after collagen treatment, and discovered that the cyclic nucleotide dependent phosphorylation of VASP (Ser 157) is elevated in the Abcc4-null platelets. These results strongly suggest that Abcc4-null platelets have impaired GPVI activation by collagen due to elevated cyclic nucleotide concentrations. Based on these studies we conclude that Abcc4 plays a critical role in regulating platelet cyclic nucleotide concentrations and its absence or perhaps inhibition (by drugs) impairs the aggregation response to collagen. Because many antiplatelet drugs are potent inhibitors of Abcc4 (e.g., Dipyridamole and Sildenafil) these findings have strong implications for not just the development of antiplatelet drugs, but also for understanding the role of Abcc4 in regulating intracellular nucleotide levels. Jedlitschky, G., K. Tirschmann, et al. (2004). “The nucleotide transporter MRP4 (ABCC4) is highly expressed in human platelets and present in dense granules, indicating a role in mediator storage.” Blood 104(12): 3603–10. This work was supported by NIH and by the American Lebanese Syrian Associated Charities (ALSAC). Disclosures: No relevant conflicts of interest to declare.

Increased levels of red cell fetal hemogloblin (α2 γ2; HbF), whether due to hereditary persistence of HbF or from induction with hydroxyurea therapy, effectively ameliorate sickle cell disease (SCD). Therefore, we developed an erythroid-specific, γ-globin lentiviral vector for hematopoietic stem cell (HSC)-targeted gene therapy with the goal of permanent, high level expression of HbF in sickle red cells. The vector contained the γ-globin gene driven by 3.1 kb of transcriptional regulatory sequences from the β-globin LCR and a 130 bp β-globin promoter. Since adult erythroid cells have β-globin mRNA 3′UTR binding proteins that enhance β-globin mRNA stability, we replaced the native γ-globin 3′UTR with its β-globin counterpart. We tested the therapeutic efficacy of this vector using the BERK sickle cell mouse model. Five months following transplant, mice that received transduced lineage-depleted sickle steady-state bone marrow (BM) cells (n=10) expressed the g-globin transgene in 95% ± 2% of RBCs. We observed levels of HbF that equaled that of the endogenous HbS (HbF 48% ± 3% of total Hb). This was achieved with an average BM vector copy number of 1.7 ± 0.2 and led to correction of both the severe anemia and end-organ damage characterizing this SCD strain. Globin vector mice had a Hb level of 12.2 ± 0.2 g/dL, compared to 7.1 ± 0.3 g/dL of mice (n=16) transplanted with cells transduced with a control GFP vector. Urine concentrating ability was normal in globin vector mice, while severely impaired in control mice. At necropsy, minimal evidence of sickle-related organ damage was found in the globin vector recipient group. In contrast, severe renal, hepatic, splenic and pulmonary pathology was observed in control, mock-transduced animals. We then transplanted the BM from 6 primary recipients of globin vector-transduced cells into 23 secondary recipients. Five months after transplant, these animals maintained HbF levels similar to those of their primary donors, along with persistent resolution of anemia. This suggested that HSCs were transduced and that vector silencing was minimal. We then evaluated this vector using non-human primate CD34+ cells. Steady-state BM CD34+ cells from several different pigtail macaques were transduced with the globin lentiviral vector or with a GFP control vector. The GFP vector achieved an average transduction rate of 57% ± 6% (n=6) into CD34+ cells and 76% ± 9% into CFU, as judged by GFP expression. Similar high levels of gene transfer were obtained with the globin vector. Bulk CD34+ cells transduced with the globin vector and then cultured for 5 days demonstrated an average vector copy number of 0.6–1.0 as judged by Southern blot analysis and qPCR. High level transduction of CFU was also obtained as 12/16 and 16/16 colonies in two separate experiments were positive for the globin vector by PCR analysis of colony DNA. We are in the process of comparing globin gene transfer and expression with that of our standard GFP vector in the pigtail macaque autologous transplant model by transplanting a graft consisting of 50% globin lentiviral vector-transduced CD34+ cells and 50% GFP lentiviral vector-transduced cells.

Since increased levels of fetal hemoglobin can significantly ameliorate the severity of β-thalassemia, a strategy using autologous, stem cell-targeted transfer of a γ-globin gene capable of being expressed at high levels in adult erythroid cells may offer a potential cure. Previously, we and other groups have demonstrated correction of a murine model of severe β-thalassemia with an average globin lentiviral vector copy number of ∼1, meaning most, if not all, hematopoietic stem cells (HSCs) were genetically modified with the vector genome. Although mouse HSCs are easily modified with lentiviral vectors, evidence from non-human primate studies predicts much lower levels of lentiviral gene transfer into human HSCs. Therefore, we incorporated a drug resistance gene, methylguanine methyltransferase (MGMT), into our globin vector to allow in vivo selection of genetically modified cells following transplantation through the administration of drugs that kill the disease-causing, non-modified HSCs. We utilized a 3rd generation, self-inactivating vector design (SJ1) containing the γ-globin gene in reverse orientation under the transcriptional control of 3.1 kb of elements from the β-globin locus control region (LCR) coupled to a 130 bp minimal β-globin promoter. The MGMT cDNA was positioned in the opposite orientation from the globin cassette and was driven by an internal MSCV U3 promoter. Remarkably, this complex vector was produced at a mean unconcentrated titer of 7 × 105 units/ml; clinically relevant titers of >108 per ml were obtained by ultracentrifugation. We transplanted lethally irradiated mice with lineage negative β-thalassemic bone marrow cells transduced with the γ-globin/MGMT vector. Twelve weeks following transplant, animals were divided into 2 groups: a control group (CON; n=6) that received no further manipulation and was only observed, and a treatment group (RX; n=7) that received BCNU and benzylguanine every 6–8 weeks for 3 courses. The baseline percentage of F cells was similarly low in both groups (CON 4.6% ± 2.1 vs. RX 11.3% ± 4.3, p= 0.21) and total levels of HbF in the blood were virtually undetectable (< 5%). All animals were anemic with similar Hb values in both groups (CON 9.4 g/dL ± 0.3 vs. RX group 9.8 ± 0.3, p= 0.31) and blood smears showed typical RBC morphologic features of β-thalassemia. Although one CON mouse died during the experiment, RX mice tolerated the treatment well with no deaths. One month after the final BCNU/BG treatment, all mice were re-analyzed for their hematologic parameters. Five of 7 mice in the RX group demonstrated a significant increase in F cells (mean of 11% rising to 46%) and, importantly, total blood levels of HbF increased from a mean of less than 5% to 21% (range 13–30%). These increased levels of HbF resulted in resolution of anemia in the responding animals (Hb of 9.8 g/dL rising to 11.1). Blood smears of these animals showed normalization of RBC morphology. In contrast, the CON group of mice showed no improvement in any of these parameters compared to baseline and remained anemic (Hb 9.6). Secondary transplantation and clonal analysis studies of hematopoiesis in the responding RX mice is ongoing and will address both the durability of cure and the diversity of the stem cell population after vivo selection. Analysis of the effects of selection on the mean globin expression per cell will be presented. These data are the first to demonstrate the usefulness of in vivo selection drug selection in β-thalassemia using a globin lentiviral vector.

The AML1/CBFβ transcription complex, a critical regulator of the formation of definitive hematopoietic stem cells (HSC), is one of the most frequent targets of genetic alterations in acute leukemia. In addition to somatic alterations of AML1 and CBFβ in acute leukemia, germ-line loss-of-function mutations of AML1 are the underlying cause of an autosomal dominant familial platelet disorder with a predisposition to acute myeloid leukemia (FPD/AML). Importantly, a subset of the mutations identified in families with FPD/AML result in AML1 null allele, suggesting that AML1 haploinsufficiency is the underlying molecular abnormality. To explore the functional consequences of AML1 halpoinsufficiency on megakaryocyte development and platelet function, we analyzed the hematopoietic system of AML1+/- mice. Loss of a single AML1 allele resulted in a 15% reduction in the number of circulating platelets and a significant impairment in platelet function including a decrease in dense granule content and an impaired ability to aggregate in response to collagen stimulation. Further analysis indentified a left shift in the DNA ploidy of megakaryocytes and a reduction in GPV expression, consistent with impaired megakaryocyte maturation. In addition, electron microscopy indicated a reduction in platelet demarcation channels within the cytoplasm of megakaryocytes. Importantly, however, we did not observe a reduction in the total number of megakaryoctyes or a decrease in megakaryocyte colony forming units. These data suggest that the haploinsufficiency of AML1+/− does not alter the initial formation of megakaryocytes, but instead impairs the ability of these cells to efficiently mature and produce functional platelets. To explore the underlying mechanism responsible for the observed impairment in megakaryocyte maturation, we analyzed the pattern of expression of several putative AML1 transcriptional targets. Although AML1 binding sites have been identified within the promoter of c-mpl, the gene encoding the receptor for thrombopoietin (TPO), we did not observe any difference in c-mpl expression levels or in circulating TPO concentration between AML1+/− and +/+ mice. In addition, in vivo TPO stimulation induced a similar magnitude of megakaryocyte maturation and platelet production in both AML1+/+ and +/− mice. By contrast, analysis of members of the protein kinase C (PKC) family of gene, several which have been identified as transcriptional targets of AML1, revealed a reduction in PKCδ levels in platelets from AML1+/− mice. Taken together, our data suggest that AML1 haploinsufficiency leads to abnormalities in platelet that are identical to those observed in patients with FPD/AML. Thus, these mice should prove useful for exploring the molecular mechanisms through which AML regulates the normal maturation of megakaryocytes. Our early analysis suggests altered PKCδ signaling is a possible contributing factor to the observed phenotypic abnormalities.