Search

The dynamins are a family of ubiquitously expressed GTPase proteins, best known for their role in membrane remodeling. Their contribution to hematopoiesis is incompletely recognized. Individuals with Charcot-Marie-Tooth disease with dynamin-2 (DNM2) mutations often develop neutropenia. We previously reported that dynamin (DNM) inhibition impairs SDF1a-mediated migration in megakaryocytes. Here, we report on conditionally Dnm2 deleted mice in hematopoietic tissues using the Vav-Cre murine strain. Homozygous Dnm2 deletion in blood tissues is embryonic lethal. Dnm2het male mice only developed a slightly decreased hemoglobin level. Dnm2het female mice developed leukopenia by 40 weeks of age and neutropenia by 65 weeks of age. Flow cytometry revealed decreased lineage-negative cells and granulocyte-monocyte progenitors in Dnm2het female mice. Immunohistochemical staining of bone marrow (BM) for mature neutrophils with Ly6G was decreased and myelodysplastic features were present in the BM of Dnm2het female mice. A linear distribution of Ly6G+ BM cells along blood vessels was observed in fewer Dnm2het mice than in controls, suggesting that the migration pattern in the marrow is altered. Marrow neutrophils treated with dynamin inhibitor, dynasore, showed increased cell surface CXCR4, suggesting that abnormal migration results in marrow neutrophil retention. Dnm2het female mice also developed splenomegaly secondary to germinal center hyperplasia at younger ages, suggesting perturbed immunity. In summary, female mice with BM Dnm2 haploinsufficiency developed neutropenia as they aged with decreased granulocyte progenitor production and migration defects. Our studies indicate a potential mechanism for the development of chronic idiopathic neutropenia, a disease that predominantly presents in middle-aged women.

The dynamins are a family of ubiquitously expressed proteins with GTPase activity and are known for their role in membrane remodeling and intracellular trafficking. However, their exact role within various hematopoietic lineages is incompletely understood. In humans, most of the clinical cases with cytopenia in Charcot-Marie-Tooth (CMT) disease due to dynamin-2 mutations are associated with neutropenia, and CMT patients may suffer from impaired wound healing. Of interest, pregnancy notably worsens CMT disease, possibly due to hormonal changes. Antiprogesterone treatment was successfully given in a CMT rat model, and similar treatment is being considered for human CMT. We previously reported that inhibiting dynamin (DNM) activity impairs migration capability in mature megakaryocytes. We obtained the conditional deletion of Dnm2 and targeted its deletion in hematopoietic tissues with the vav-cre murine strain. Homozygous deletion of Dnm2 in blood tissues appears embryonic lethal. None of the pups born showed a Vav-cre/Dnm-2 fl/fl genotype, whereas a third of the pups born had a Vav-cre/Dnm-2 fl/wt (Dnm2 het) genotype. Bone marrow cells from the heterozygous female mice (Dnm2 het) had 35% to 50% decreased Dnm2 expression in comparison with age-matched controls (CTRL). Evaluation of the complete blood counts demonstrated that Dnm2 het female mice developed leukopenia which was detected from 40 weeks of age (average granulocyte-monocyte counts: CTRL 532/mm3 vs. Dnm2 het 300/mm3; p=0.0164). Neutropenia was unequivocal at 65 weeks of age (average neutrophil counts, CTRL 700/mm3 vs. Dnm2 het 343/mm3, p=0.016). Dnm2 het showed a trend for higher platelet counts than controls, but this was non-statistically significant. Further analysis of hematopoietic lineage maturation by flow cytometry indicated that lineage-negative cells and granulocyte-monocyte progenitors were decreased in Dnm2 het mice (average bone marrow lineage-negative cells: CTRL 2.8x10E6 vs. Dnm2 het 1.97x10E6, p =0.0056; average granulocyte-monocyte progenitors: CTRL 1.35x10E6 vs. Dnm2 het 0.85x10e6, p=0.01), along with a relative increase of common lymphoid progenitors and of megakaryocyte/erythrocyte progenitors in the bone marrow. Immunohistochemical staining for mature neutrophils with Ly6G showed an overall decreased number of mature granulocytes in the bone marrow of Dnm2 het mice (average Ly6G-positive cells: CTRL 20% vs. Dnm2 het 29%, p=0.0026). A linear pattern of distribution of Ly6G positive bone marrow cells along blood vessels was observed in fewer mice in the Dnm2 het group than in the CTRL group (25% vs. 59%, p=0.02), indicating that the migration pattern within the bone marrow is altered in the Dnm2 het group (see Figure). In addition, Dnm2 het mice developed splenomegaly (average spleen weight: Dnm2 het 146 mg vs. CTRL 99 mg, p=0.006), which was secondary to a marked florid reactive germinal center hyperplasia. Some of the Dnm2 het mice, including 5 mice whose pregnancy occurred in middle-age (p=0.005 when comparing with CTRL or young Dnm2 het mice) and 2 non-pregnant older mice, showed physical signs of distress with markedly reduced activity, poor grooming, ruffled furs, and hunched posture. Both non-pregnant sick mice showed a marked decrease in Ly6G positive mature neutrophils at 0.3% of total marrow cells (Figure), and the bone marrow from one mouse was completely effaced by immature myeloid precursors, consistent with the development of acute myeloid leukemia. In addition, a third of Dnm2 hetmice showed no distress but displayed morphological bone marrow abnormalities including megakaryocytic dysmorphology. In summary, female mice with loss of Dnm2 in the hematopoietic compartment develop persistent neutropenia as they age, with decreased granulocyte progenitor production and with migration defects. These abnormalities are associated with a risk for developing megakaryocytic dysplasia, and acute myeloid leukemia. These findings might also suggest a mechanism for chronic idiopathic neutropenia, which has a predominance in middle-aged women. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Megakaryocytes generate platelets through extensive reorganization of the cytoskeleton and plasma membrane. Cdc42 interacting protein 4 (CIP4) is an F-BAR protein that localizes to membrane phospholipids through its BAR domain and interacts with Wiskott-Aldrich Syndrome Protein (WASP) via its SRC homology 3 domain. F-BAR proteins promote actin polymerization and membrane tubulation. To study its function, we generated CIP4-null mice that displayed thrombocytopenia similar to that of WAS(-) mice. The number of megakaryocytes and their progenitors was not affected. However, the number of proplatelet protrusions was reduced in CIP4-null, but not WAS(-), megakaryocytes. Electron micrographs of CIP4-null megakaryocytes showed an altered demarcation membrane system. Silencing of CIP4, not WASP, expression resulted in fewer proplatelet-like extensions. Fluorescence anisotropy studies showed that loss of CIP4 resulted in a more rigid membrane. Micropipette aspiration demonstrated decreased cortical actin tension in megakaryocytic cells with reduced CIP4 or WASP protein. These studies support a new biophysical mechanism for platelet biogenesis whereby CIP4 enhances the complex, dynamic reorganization of the plasma membrane (WASP independent) and actin cortex network (as known for WASP and cortical actin) to reduce the work required for generating proplatelets. CIP4 is a new component in the highly coordinated system of megakaryocytic membrane and cytoskeletal remodeling affecting platelet production.

Abstract: Megakaryocytes (MKs) undergo directional migration from the proliferative osteoblastic niche within the bone marrow (BM) environment to the capillary-rich vascular niche for platelet production and release into the pulmonary circulation. This process is regulated, in part by dynamins, large GTPase proteins that regulate cellular functions such as endocytosis, vesicle transport and cell migration. Additional functions of dynamins include the formation of actin-rich structures, such as lamellipodia and dorsal membrane ruffles, invadopodia and podosomes. Previous studies have shown that mutations in Dynamin 2 (DNM2) cause thrombocytopenia in humans. To explore the function of dynamins in megakaryocyte migration and platelet production in more depth, we monitored the response of cells to chemotaxis SDF1α gradient signal by a microfluidic device-based approach. We observed an impaired directional migration by both human megakaryocytic cell lines and primary cells treated either with dynasore, a small molecule inhibitor of dynamins, or shRNA knockdown of Dynamin 2 and 3 (DNM2, DNM3). Since directional cell migration is tightly regulated by actin cytoskeletal rearrangements, we next measured actin polymerization and RhoA activity. We observed a profound decrease in the F-actin and Rho GTPase activity upon loss of DNM2 and DNM3 function. Next, since the response to chemoattractant signal is navigated by SDF1 through its receptor CXCR4, we explored the CXCR4 receptor response to ligand in dynamin defective megakaryocytes. Interestingly we observed an increase in CXCR4 expression in the dynasore treated primary human cells. Additionally, combined inhibition of DNM2 and DNM3 or over expression of dominant negative Dnm2-K44A or GTPase-defective DNM3 decreased the active β1- integrin (ITGB1) activity, which indicates a decrease in the integrin mediated endo/exocytic cycling during cell migration. Finally, to understand the role of dynamin in endosome recycling, we assayed the distribution of Rab11, a marker of recycling endosomes. We noticed an abnormal clustered staining pattern of Rab11 in dynasore-treated MKs which is indicative of a disruption in recycling pathways. This observation suggests decreased recruitment of the recycling pathway in dynasore-treated cells. Altogether, in this study we demonstrate that dynamins regulate MKs directional migration towards the SDF1α chemotaxis signal in the bone marrow and governs endocytosis and cell receptor trafficking. Disclosures Crispino: Scholar Rock: Research Funding; Forma Therapeutics: Research Funding.

Platelets are small, anucleated cells that participate in primary hemostasis by forming a hemostatic plug at the site of a blood vessel’s breach, preventing blood loss. However, hemostatic events can lead to excessive thrombosis, resulting in life-threatening strokes, emboli, or infarction. Development of multi-scale models coupling processes at several scales and running predictive model simulations on powerful computer clusters can help interdisciplinary groups of researchers to suggest and test new patient-specific treatment strategies.

Summary Background Mutations in the MYH9 gene cause autosomal dominant MYH9-related diseases (MYH9-RD) that associate macrothrombocytopenia with various other clinical conditions. The mechanisms giving rise to giant platelets remain poorly understood. Objectives/Patients To study the proplatelet formation (PPF) derived from megakaryocytes (MKs) generated in vitro from 11 patients with MYH9-RD with different mutations, compared with controls. Methods Proplatelet formation from cultured patients' MKs was evaluated with or without blebbistatin or the ROCK inhibitor Y27632. Myosin IIA and actin distribution were studied in spreading MKs on different surfaces by immunoconfocal analysis. Kinetic studies of contractility were performed on spreading MKs and the impact of blebbistatin on the maturation of the patients' MKs was evaluated by electron microscopy. Results and Conclusions We show that in vitro MKs of 11 patients formed significantly fewer proplatelets than controls. MKs from MYH9-RD displayed an abnormal spreading on polylysine, fibronectin and collagen, with a disorganized actin network and a marked increase in stress fiber formation. Traction force microscopy studies demonstrated an elevated level of contractile forces in adherent mutated MKs. The myosin II inhibitor blebbistatin and the ROCK inhibitor Y27632 both rescued the proplatelet formation defect and normalized the ultrastructural characteristics of MYH9-RD MKs. Altogether, our results show that in MYH9-RD, mutations modify the overall MYH9 function and provoke a proplatelet defect through an excess of actomyosin contractility in spreading MKs. These results may promote new therapeutic strategies aimed at reducing this actomyosin contractility.

Cdc42 interacting protein 4 (CIP4) is a membrane-associated BAR protein, which also forms a complex via its SH3 domain with the dynamins (DNMs) and Wiskott-Aldrich Syndrome (WAS) protein. Thus, CIP4 remodels the plasma membrane and cortical actin cytoskeleton. To determine its physiological function, we generated CIP4-null mice. They displayed thrombocytopenia similar to that of WAS-null mice and have abnormal megakaryocytes (MKs) with decreased proplatelet formation and underdeveloped demarcation membrane system (DMS) (Chen et al, Blood 2013). The DMS is an extensive network of membrane tubules which serves as a membrane reservoir for proplatelet formation. The membranes are enriched for polyphosphoinositides that are docking sites for BAR proteins and for pleckstrin homology domain-containing proteins such as the dynamins. Still, the formation of the DMS is poorly understood. Dynamins are cell vesicle trafficking proteins that possess a GTPase domain. They induce neck vesicle constriction and scission from the plasma membrane. When the GTPase activity is abrogated, vesicle scission does not occur; instead, the plasma membrane invagination induced by the BAR proteins results in deep plasma membrane tubulations. Of the three dynamin isoforms, DNM3 participates in MK development including DMS formation (Reems et al, Exp Hematol 2008; Wang et al, Stem Cells Dev 2011). Moreover, a recent genome-wide association study suggested that an MK-specific DNM3 isoform might play a role in human platelet size determination (Nürnberg et al, Blood 2012). However the exact mechanism for dynamin’s participation in DMS formation is unclear. A double knockout for dynamin 1 and dynamin 3 in neurons causes accumulation of long invaginations from the plasma membrane (Ferguson and De Camilli, Nat Rev Mol Cell Biol 2012). We initially hypothesized that CIP4’s association with DNM3 contributes to the DMS development during platelet biogenesis and wanted to test for functional redundancy with other dynamins present in MKs and platelets. To determine if CIP4 interacts with dynamin in the MK lineage, we found that following either phorbol ester (PMA) or fibronectin stimulation in the human MK cell line CHRF-288, CIP4 co-precipitated with DNM3 and colocalized by confocal microscopy. To determine dynamin’s effect on membrane biophysical properties, we measured the fluorescence anisotropy, which reflects the disorder of membrane lipids due to movement and indicate membrane rigidity. Compared with controls in CHRF-288 cells, shRNA-mediated knockdown (KD) of DNM2 or DNM3 resulted in higher membrane rigidity in response to PMA. The strongest effect was seen in double KD cells with decreased fluidity by 2.6 ± 0.3%, which is similar to what was observed with CIP4 KD and is physiologically significant (Chen et al Blood 2013). KD of DNM2 resulted in aberrant morphology, greater cell diameter, and electron microscopy (EM) showed formation of new multivesicular bodies (MVBs) which are sorting compartments during α- and dense granules formation. Single DNM3 KD cells had no observable phenotype. EM imaging of DNM2 and DNM3 double KD cells revealed plasma membrane tubulation that resembles the DMS. While control CHRF-288 cells, with high DNM3 protein expression, do not have a DMS at baseline, MK cell lines Meg-01 and L8057, with respectively lower or no dynamin-3 protein expression, both have a DMS (Battinelli et al PNAS 2001; Ishida Y et al, Exp Hematol 1993). Platelet microparticles (MPs) are known to mediate a prothrombotic state in patients. Having previously found that CIP4-null mice show reduced levels of platelet MPs, we measured MPs in dynamin knockdown cell supernatant by flow cytometry and CD41/Annexin V staining. Surprisingly, we found that microparticle levels were increased 2.9-fold in DNM2 KD cells and 3.8-fold in double DNM2 and DNM3 KD cells. Our findings suggest that: 1) there is only partial functional redundancy between DNM2 and DNM3 in platelet biogenesis, 2) DNM2 controls MVB formation and MP release in MK cells, and 3) the CIP4-dynamin pathway contributes to DMS formation. It is possible that CIP4’s interaction with dynamins restrains their spatial and temporal activity to allow for long invaginations to accumulate in the DMS. Dynamin depletion might also increase surface membrane availability for MP formation. Dynamins are thus potential targets to modulate thrombotic state and platelet biogenesis. Disclosures: No relevant conflicts of interest to declare.

Abstract 705 Using the Src kinase Lyn as bait in yeast two-hybrid screen, we isolated Cdc42-interacting protein 4 (CIP4). CIP4 is an F-BAR protein implicated in membrane curvature, membrane tubulation, and vesicle formation. CIP4 contains an SH3 domain that interacts with Wiskott-Aldrich Syndrome protein (WASP). We have reported thrombocytopenia in CIP4 knockout (KO) male mice, which was of similar severity as observed in WASP knockout male mice. Because a cardinal feature of Wiskott-Aldrich Syndrome is thrombocytopenia, we hypothesized that the CIP4-dependent thrombocytopenia occurs through the same mechanism as that in WASP-dependent thrombocytopenia. Interestingly, we have just generated a CIP4, WASP double knockout that displays a more severe thrombocytopenia. Neither CFU-MK progenitor studies nor ploidy studies showed a difference between CIP4 KO, WASP KO, double KO, and Wild Type (WT) mice. Thus, we reasoned that the defect lies in the structural production of platelets. We first studied where the proteins were localized and performed immunofluorescence staining and confocal microscopy analysis in cultured mouse megakaryocytes. We found a diffuse staining of CIP4 throughout the cytosol, the nucleus and proplatelets, with accumulation at the plasma membrane. In mouse megakaryocytes WASP staining was diffuse. In the human megakaryocytic CHRF-288-11 cell line, CIP4 staining was also diffuse. Because F-BAR proteins are also known to interact through their SH3 domain with members of the dynamin family, we investigated if CIP4 interacted with dynamin in the megakaryocytic lineage. While we saw diffuse staining for dynamin 3 in mouse megakaryocytes, we found co-localization of dynamin 3 with CIP4 at the cell periphery after exposure to known proplatelet-inducing agents such as phorbol ester (PMA) or fibronectin. We also performed functional assays in CIP4 KO mice. Since we did not find a difference in megakaryocytic progenitor numbers or in ploidy between the CIP4 knockout and the wild type mice, we studied later steps in platelet biogenesis. Preliminary studies were not indicative for decreased platelet lifespan in vivo in CIP4 KO mice. We counted the proplatelet formation in bone marrow-derived megakaryocytes in culture and found that CIP4 knockout megakaryocytes had decreased proplatelet formation compared with wild type (WT: 10.1 ± 1.6%, CIP4 KO: 4.5 ± 1%, p=0.04). We confirmed these findings in the CHRF-288-11 cell line with lentivirally mediated shRNA knockdown of CIP4 by counting the proplatelet-like protrusion formation in response to PMA (control: 15.4± 1.7%, CIP4 knockdown: 8.9 ± 1%, p=0.03). Because of the known direct interaction of CIP4 with the plasma membrane, we hypothesized that CIP4 deficit leads to changes in plasma membrane rigidity. By measuring fluorescence anisotropy, we confirmed decreased plasma membrane fluidity in CHRF-288-11 cells with CIP4 knockdown in response to agents that promote proplatelet-like protrusion formation, PMA and fibronectin. When using PMA, membrane fluidity increased by 4% or more in control samples (similar to the change obtained with known membrane perturbing agent ethanol 10 percent v/v), but did not increase in cells with CIP4 knockdown or N-WASP knockdown. Even more, plasma membrane rigidity increased by 3 ± 1.4% in CHRF-288-11 cells with CIP4 knockdown. Preliminary results showed the same loss of membrane fluidity in splenic B lymphocytes from CIP4 KO mice isolated (a 5% membrane fluidity increase in WT vs. none in CIP4 KO B lymphocytes in response to PMA). After exposure to fibronectin, membrane fluidity increased by 6.4 ± 1.3% in control CHRF-288-11 cells, whereas it did not increase in cells with CIP4 knockdown, and it increased in cells with N-WASP knockdown but only by 3 ± 0.9%. Interestingly, while CHRF-288-11 cells with knockdown of WASP still showed loss of plasma membrane fluidity in response to PMA (increased only by 1.8 ± 0.7%), this was not seen with fibronectin stimulation, where the membrane fluidity increase (5 ± 0.5%) did not appear statistically different from the response in control cells. Altogether our results point to decreased membrane fluidity in CIP4 deficient cells and they suggest that other pathways besides WASP pathways are involved in CIP4 dependent platelet biogenesis. Disclosures: No relevant conflicts of interest to declare.

Abstract 1153 Our lab isolated Cdc42 interacting protein 4 (CIP4) in a yeast two-hybrid screen using the Src kinase Lyn as bait. CIP4 is a Bin/Amphiphysin/Rvs (BAR) protein with a C-terminal SH3 domain that interacts with Wiskott Aldrich Syndrome protein (WASp) or dynamin. BAR proteins contain a domain that interacts with membrane lipids generating membrane curvature or tubulation. WASp and dynamin, on the other hand, function in actin polymerization and membrane vesicle scission respectively. WASp is defective in boys with the eponymous syndrome and is characterized by thrombocytopenia, which are small in size. Dynamin-3 has been shown to function during megakaryocyte development (Reems et al Exp Hematol. 36:12, 2008). We have observed that CIP4−/− mice displayed thrombocytopenia comparable to that observed in WAS-/ mice. Also, cultured megakaryocytes from CIP4 and WASp knockout mice showed decreased proplatelet extensions. As a result of those observations, we hypothesized that defects in membrane dynamics due to loss of CIP4 might decrease platelet production. To better understand the role of CIP4 in platelet biogenesis, CHRF-288-11 cell line, a megakaryoblastic cell line was used for studying CIP4's interaction with WASp and dynamin. CIP4's interaction with WASp and dynamin-3 was investigated by co-immunoprecipitation and microscopy in CHRF cells. The cells were induced to form proplatelets by 100 ng/ml phorbol myristate acetate (PMA) or fibronectin-coated plates. Activated cells showed CIP4/dynamin-3 co-precipitation in a time-dependent manner. Also, immunofluorescent images showed diffuse distribution of CIP4 and dynamin-3 prior to activation with PMA or fibronectin. However after activation, co-localization of both proteins were observed at the membrane. These findings suggest possible interaction of CIP4-dynamin in platelet biogenesis. Platelet microparticles may serve as a surrogate marker for membrane deformation, rigidity, and cleavability. PMP are formed during cellular activation or apoptosis from various cell types such as platelets. The process of PMP formation is incompletely understood. We hypothesized that circulating levels of platelet microparticles (PMP) would be affected by the loss of CIP4 and its affects on membrane remodeling. Using flow cytometric analysis we observed decreased circulating levels of CD41+/ AnnexinV+ PMP in 3–6 month old C57BL/6 male CIP4 −/− and CIP4−/−,WAS-/ mice when compared to controls. However, WAS-/ mice showed similar levels to control mice. We conclude that CIP4, a membrane deforming protein, promotes PMP formation. However, this is independent of its interaction with WASp. Because of dynamin's role in promoting membrane scission and its association with CIP4, we are currently investigating the role of dynamin in PMP formation. Disclosures: No relevant conflicts of interest to declare.

Abstract 4826 MYH9-related disorders (MYH9-RD) are characterized by an autosomal-dominant macrothrombocytopenia associated or not with glomerular impairment, hearing loss and/or cataracts. It is caused by mutations of the MYH9 gene (encoding for the nonmuscle myosin heavy chain IIA), present at the heterozygous state in patients. In knockout animal models, proplatelet formation (PPF) was found to be increased in certain conditions, but decreased in others (Chen et al, Blood 2007, Eckly et al, Blood 2007 and Thrombosis and Haemostasis 2010) and samples with heterozygosity did not show differences with the wild type. More recently, in a mouse model with a mutated MYH9 gene, mice with the mutation showed a phenotype similar to the one found in MYH9-RD, affecting multiple organs and, interestingly, mice heterozygous for the mutation showed an abnormal phenotype as well, although attenuated (Anderson et al, ASH Meeting 2010, Abstract 2527). However, opposite to the initial reports in mouse models with a knockout, proplatelet formation was found to be impaired in 4 patients (Pecci et al, Thrombosis and Haemostasis 2009). Therefore the question for the exact mechanism for thrombocytopenia still stands, so does the question whether the mutated protein causes disease through a dominant negative effect versus a functional haploinsufficiency. To address these questions, we performed studies on patient samples on one hand and seeked to knock down the gene expression in human megakaryocytes (MKs) on the other hand. To study PPF in these patients, we analyzed MK differentiation in CD34+ cells from 9 patients with mutations in exons encoding for the motor domain (2 patients) or for the coiled coil domain of myosin IIA (7 patients). Compared with cultured MKs derived from healthy donors, a 2 to 5 –fold decrease in PPF was observed in the patients (4.9 ± 1.2% for patients vs. 15.1 ± 1.8% for control) and proplatelet area was decreased in patients as well (threefold decrease, 6156 μm2 for patients versus 18064 μm2 for control). In confocal microscopy with immunofluorescence staining, we observed disorganization of the granules, abnormal spreading and stress fibers, meaning that actin-myosin network is unstable. We next used a shRNA strategy to knock down MYH9 expression during normal MK differentiation and compared shRNA-treated MKs (with 50% residual MYH9 expression) with those derived from patient CD34+ cells. Treatment with shRNA decreased in vitro PPF, as previously observed for cells from patients. Moreover, shRNA-treated MKs exhibited the same ultrastructural abnormalities as MKs from patients. Normal platelet production is dependent on the formation of branched long proplatelets. Surprisingly the defect in PPF formation seen in patients was rescued by blebbistatin, an inhibitor of class II myosin. In its presence the proplatelets increased by 2 to 5 fold, suggesting that the remaining myosin IIA might be hyperactivated and inhibiting PPF. Ultrastructural studies by electron microscopy performed on cells with blebbistatin treatment corrected abnormalities seen in the patients. Parallel to blebbistatin, ROCK (Y27632) or MLCK inhibitors (P18 or ML7) led to a threefold increase in proplatelet formation. We therefore compared by western blot the status of pMLC2 in patients and control MKs. In 3 out of 6 patients we found an increased pMLC2. In addition, we also found an increased pMLC2 in MKs treated with shRNA with 50% residual myosin expression. Altogether our results show that reducing the protein expression by half was sufficient to recreate features characteristic of the disease in megakaryocytes, while the increased PPF, relative to control, in response to rho/ROCK inhibitors and to myosin inhibitors, suggest that the defective PPF in patients may be related to an activated rho/ROCK pathway and/or increase of the contractile force. Disclosures: No relevant conflicts of interest to declare.

Abstract 4596 Normal platelet production is dependent on the formation of branched long cytoplasmic extensions, called proplatelets (PPT). Mutations of the Myh9 gene (encoding for the nonmuscle myosin heavy chain IIA) result in autosomal dominant disorders, where patients develop various degrees of macrothrombocytopenia, with sometimes glomerular impairment, hearing loss and cataracts. There has been questioning as to whether the mechanism for the macrothrombocytopenia is haploinsufficiency, or a dominant negative effect of the mutated gene. We performed an in vitro study to investigate PPF from patient megakaryocytes (MK). By this approach, a decrease in PPF from patient CD34 derived MKs was observed in comparison to normal cultured MK. Surprisingly this defect of PPF observed in patients was rescued by blebbistatin, an inhibitor of class II myosin. Immunofluorescence studies performed showed that besides clusterization of GPIb in patient's platelets, no major repartition abnormalities were seen in cultured MKs derived from patient's CD34 for myosin, actin, tubulin, vWF, and Rac (except in one patient where actin and Rac formed aggregates to some extend, in a small number of MKs). In order to better understand the role of myosin during normal and abnormal PPF, we used a shRNA strategy to disrupt the Myh9 expression during normal MK differentiation and compared shRNA-treated MKs with MKs derived from patient CD34. Megakaryocytes treated with a shRNA that knocks down the protein of about 50%, did not alter MK ploidization, but decreased in vitro PPF, as previously observed for cells issued from patients. Moreover, shRNA-treated MKs exhibited the same ultrastructural abnormalities as patient MKs. Addition of Blebbistatin to shRNA treated MKs led to an increase of PPF, suggesting that the remaining myosin II might be hyperactivated and inhibit PPF. Altogether this study strongly suggests that the thrombocytopenia of the Myh9 syndrome is essentially related to haploinsufficiency in myosin II. Disclosures: No relevant conflicts of interest to declare.

Cytomegalovirus (CMV) infection remains associated with increased mortality in immunocompromised populations, particularly in allogeneic hematopoietic stem cell transplant (HSCT) recipients. Risk factors for reactivation include serostatus and occurrence of graft-versus-host disease (GVHD).