ALBERT

Description: Key Points CIP4 affects the remodeling of both plasma membrane and cortical cytoskeleton in megakaryocytes. CIP4 in platelet biogenesis involves cortical tension, as does WASP, and WASP-independent plasma membrane reorganization.

Description: Key Points Nbeal2 −/− mice are a model of human GPS, characterized by macrothrombocytopenia and α-granule-deficient platelets. NBEAL2 is required for normal platelet function and megakaryocyte development.

Description: Platelet P-selectin plays important roles in inflammation and contributes to thrombosis and hemostasis. Although it has been reported that von Willebrand factor (VWF) affects P-selectin expression on endothelial cells, little information is available regarding regulation of platelet P-selectin expression. Here, we first observed that P-selectin expression was significantly decreased on platelets of fibrinogen and VWF double-deficient mice. Subsequently, we identified this was due to fibrinogen deficiency. Impaired P-selectin expression on fibrinogen-deficient platelets was further confirmed in human hypofibrinogenemic patients. We demonstrated that this impairment is unlikely due to excessive P-selectin shedding, deficient fibrinogen-mediated cell surface P-selectin binding, or impaired platelet granule release, but rather is due to decreased platelet P-selectin content. Fibrinogen transfusion completely recovered this impairment in fibrinogen-deficient (Fg−/−) mice, and engagement of the C-terminus of the fibrinogen γ chain with β3 integrin was required for this process. Furthermore, Fg−/− platelets significantly increased P-selectin expression following transfusion into β3 integrin–deficient mice and when cultured with fibrinogen. These data suggest fibrinogen may play important roles in inflammation, thrombosis, and hemostasis via enhancement of platelet P-selectin expression. Since human fibrinogen levels vary significantly in normal and diseased populations, P-selectin as an activation marker on platelets should be used with caution.

Description: Emerging data indicate that germline mutations in transcription factors involved in hematopoiesis can lead to a cascade of downstream molecular alterations that modify the function of megakaryocytes (MK) and platelets. Our group and others have found that mutations in ETV6 lead to mild thrombocytopenia with a bleeding diathesis, red cell macrocytosis, and predisposition to lymphoblastic leukemia. The mechanisms responsible for thrombocytopenia and propensity for bleeding in patients with ETV6 mutations are unknown. We described families with missense mutations in the central domain (p.Pro214Leu) and the ETS DNA binding domain (p.Arg418Gly) of ETV6 that result in aberrant cellular localization of ETV6, decreased transcriptional repression, and impaired MK maturation. Deep sequencing of the platelet transcriptome revealed significant differences in mRNA expression levels between patients with the ETV6 p.Pro214Leu mutation and non-affected family members, indicating that ETV6 is critically involved in defining the molecular phenotype and function of platelets. We hypothesize that normal regulation and function of ETV6 is essential for the transcriptional machinery that controls megakaryocyte differentiation and formation of platelets that function normally under homeostatic conditions. We have successfully generated a CRISPR-Cas9 model to edit the genome of ETV6-expressing iPSC derived megakaryocyte cell line (imMKCL) to characterize the role of wild-type ETV6 in megakaryocyte development and elucidate the molecular mechanism driving mutant ETV6 mislocalization, transcriptional dysregulation, and subsequent dysmegakaryopoiesis and thrombocytopenia. In this imMKCL model, we have genetically engineered the cells to express wild-type, P214L, and the DNA binding domain mutations R418G and R369Q ETV6 fused to HALOtag, a reporter protein that can react with ligands carrying a variety of functionalities, including fluorescent labels, affinity handles, and attachment to solid phase, making this novel reporter conducive to immunofluorescence imaging, biochemical pulldown, and ChIPSeq. This system allows us to express wild type and mutant forms of ETV6 in appropriate allele ratios in imMKCL cells and various hematopoietic-relevant cell lines. Using this approach, we detected nuclear localization of wild-type ETV6 and altered cytoplasmic localization of both P214L and R418G ETV6 mutants. We have also demonstrated dimerization between both wild-type and mutant ETV6 in this cell model. Importantly, we have used HALOtag protein immunoprecipitation to demonstrate ETV6 binding to FLI1, another ETS family member and key transcriptional regulator of megakaryocyte development, suggesting that ETV6 and FLI1 cooperate to regulate megakaryopoiesis under homeostatic conditions. Altogether, these data suggest that mutant ETV6 functions as a dominant negative, sequestering wild type ETV6 in the cytoplasm, de-regulating key transcriptional targets for homeostatic megakaryocyte development. Ongoing studies will define the full repertoire of protein interactions and transcriptional targets of wild-type and mutant ETV6. Discoveries from this novel tool will further advance our understanding of normal megakaryocyte and platelet biology, and will provide potential therapeutic targets for disorders of platelet number and function to optimize the clinical approach to these patients. Disclosures Callaghan: Bayer: Consultancy, Speakers Bureau; Alnylum: Equity Ownership; Biomarin, Bioverativ, Grifols, Kedrion, Pfizer, Roche/Genentech, Shire, and Spark Therapeutics: Consultancy; Takeda: Consultancy, Research Funding; Sanofi: Consultancy; Global Blood Therapeutics: Consultancy; Novonordisk: Consultancy, Speakers Bureau; Octapharma: Consultancy; Pfizer: Research Funding; Roche: Research Funding; Shire/Takeda: Speakers Bureau; Roche/Genentech: Speakers Bureau.

Description: Key Points DNM2-dependent endocytosis in MKs regulates megakaryopoiesis, thrombopoiesis, and bone marrow homeostasis.

Description: 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.

Description: Autosomal dominant macrothrombocytopenias such as the May-Hegglin anomaly, Sebastian syndrome, Fechtner syndrome and Epstein syndrome are all characterized by mutations in the MYH9 gene. It has been proposed that these MYH9-related diseases are not distinct entities but represent a variable expression of a single disorder with a continuous clinical spectrum varying from mild macrothrombocytopenia with leukocyte inclusions to more severe conditions encompassing hearing loss, cataracts and renal failure. Mutations in the MYH9 gene cause abnormal expression and/or function of the 224 kDa nonmuscle myosin heavy chain IIA (NMMHC-IIA) protein. Class II myosins are hexameric complexes composed of 2 heavy chains and 2 pairs of light chains forming a structure containing 2 N-terminal globular domains and an elongated α-helical C-terminal tail. Since identical mutations in the MYH9 gene cause variable clinical presentations in different patients, other factors likely modulate the mutant phenotype. We describe a patient with congenital macrothrombocytopenia, mild bleeding problems, Döhle-like leukocyte inclusion bodies, normal hearing, normal renal function and absent cataracts. The patient has macrothrombocytes with an average manual platelet count of 100 x 109/L. Immunofluorescence confocal microscopy using an antibody specific for NMMHC-IIA revealed unique localization of NMMHC-IIA in the patient’s leukocytes. Platelets and leukocytes from both parents and one sister are normal. Megakaryocytes cultured from the patient’s and one parent’s peripheral blood CD34+ cells also demonstrated abnormal distribution of NMMHC-IIA in only the patient’s megakaryocytes. Ultrastructural analysis using electron microscopy revealed distinct inclusion bodies in the patient’s leukocytes and megakaryocytes that were not observed in either parent. The patient’s macrothrombocytopenia together with the abnormal distribution of NMMHC-IIA within leukocytes is highly suggestive of a MYH9-related disorder. The absence of these findings in either parent suggested a de novo MYH9 mutation. We therefore sought to identify the mutation via amplification and DNA sequencing of all MYH9 exons and intron/exon boundaries. Exons 1, 10, 16, 24, 25, 26, 30, 38 and 40 representing all of the previously described mutations in MYH9-related disorders were found to be normal. Surprisingly, the remaining MYH9 gene exons and intron/exon boundaries were also normal. This unexpected finding suggests that another factor is involved in the normal assembly and/or localization of NMMHC-IIA in human leukocytes and megakaryocytes. It also suggests that a deficiency of this factor may lead to congenital macrothrombocytopenia with features indistinguishable from those of MYH9-related disorders. Identification of this factor could enhance our understanding of NMMHC-IIA-related congenital macrothrombocytopenias and would allow us to gain insights into the normal assembly and/or function of NMMHC-IIA in human megakaryocytes and platelets.

Description: Abstract SCI-34 Platelet secretory granules develop within maturing bone marrow-resident megakaryocytes, where α-granules, δ-granules, and lysosomes are transported to extending proplatelets (1) and undergo further maturation after platelets are released into the circulation. Mature platelets contain 50 to 80 membrane-enclosed α-granules, three to eight dense (δ-) granules, and a few lysosomes. δ-granules store calcium, phosphate, ADP, ATP, and serotonin, which play important roles during platelet activation. α-granules store numerous soluble and membrane-bound proteins, including adhesion molecules, cytokines, chemokines, coagulation and fibrinolytic proteins, immunologic modulators, and an assortment of complement, growth, and pro- and antiangiogenic factors. These play important roles in clotting, angiogenesis, inflammation, wound healing, and bone remodeling, and provide defenses against infections. Insights into megakaryocyte and platelet δ-granule development have come from studying inherited δ-granule deficiencies such as Hermansky-Pudlak syndrome (HPS) and Chediak-Higashi syndrome (CHS; MIM214500), for which mouse models also exist. Several genes/proteins linked to the regulation of vesicle trafficking have been implicated in δ-granule formation. These include components of BLOC (biogenesis of lysosome-related organelles complex) protein complexes (BLOC-1, −2, and −3), known vesicle-trafficking proteins (VPS33A and the β3A and δ subunit of AP-3), and the BEACH domain, containing protein LYST. Less is known about α-granule development, in which two inherited disorders result in platelets lacking α-granules: ARC syndrome (Arthrogryposis, Renal dysfunction, and Cholestasis; MIM208085) and gray platelet syndrome (GPS; MIM139090). GPS is characterized by variable thrombocytopenia and large, gray-appearing platelets on blood smears, with α-granules and α-granule proteins markedly decreased or absent. We and others recently determined that GPS is caused by mutations in NBEAL2, encoding a BEACH protein (2, 3, 4). Our work has also shown that the large α-granule-deficient platelets in ARC syndrome can arise due to mutations in VPS33B, encoding the Sec1/Munc18 (SM) protein VPS33B involved in vesicular trafficking (5). SM proteins are known to interact with membrane-associated soluble N-ethylmaleimide-sensitive fusion (NSF)-attachment protein receptors (SNAREs) of the syntaxin subfamily. Recently we have also identified VPS16B as a VPS33B-binding protein. A patient with homozygous missense mutations in C14orf133, encoding VPS16B, has ARC syndrome, with platelets lacking α-granules and stored α-granule proteins. Thus VPS16B is also required for megakaryocyte and platelet α-granule formation, and, in contrast to GPS, in which platelets have α-granule membrane proteins such as P-selectin, VPS16 null platelets lack P-selectin. The observation that GPS and ARC platelets lack α-granules but contain δ-granules, while HPS platelets are devoid of δ-granules but contain α-granules, suggests there are distinct pathways for δ-granule and α-granule biogenesis in maturing megakaryocytes. Immunofluorescence microscopy suggests that VPS16B and VPS33B act along the trans-Golgi network/late endosome/α-granule vesicular trafficking pathway during formation of α-granules in megakaryocytes. It is predicted that complexes containing VPS33B and VPS16B facilitate docking and fusion of intracellular vesicles during α-granule formation, while NBEAL2 promotes the maturation of nascent α-granule vesicles. Disclosures: No relevant conflicts of interest to declare.