ALBERT

Description: Changes in glycans expression have been associated with defects in blood platelet counts. However, the role of posttranslational modifications on platelet production is poorly understood. Six genes encoding sialyltransferases (ST)3Gal-I to -VI that form a2-3 sialic acid linkage have been identified in the mammalian genome, and deletion of St3gal1 and St3gal4 genes has been associated with macrothrombocytopenia in mice. We and others have shown previously that St3gal4-null platelets are cleared by the hepatic Ashwell-Morell receptor. Loss of ST3Gal-I activity has been associated with core 1 O-glycan Galβ1-3GalNAcα1-Ser/Thr expression, also known as tumor-associated or Thomsen-Friedenreich antigen (T antigen). We here investigated the detailed mechanisms of macrothrombocytopenia associated with St3gal1 deficiency by generating St3gal1loxP/PF4+ mice that lack ST3Gal-I specifically in the megakaryocyte (MK) lineage. Blood platelet counts were reduced by ~50% in St3gal1loxP/PF4+ mice, compared to control mice. Other blood cell counts were normal in St3gal1loxP/PF4+ mice. The clearance rate of St3gal1-null platelets was increased by ~15%, as determined by in vivo platelet biotinylation. Bone marrow MK numbers were normal in St3gal1loxP/PF4+ mice, compared to control mice, indicating that mechanisms other than clearance regulate circulating platelet counts in St3gal1loxP/PF4+ mice. Both St3gal1loxP/PF4+ platelets and bone marrow MKs had increased T antigen expression, as evidenced by flow cytometry using peanut agglutinin (PNA) binding. St3gal1loxP/PF4+ mice had increased bone marrow macrophage numbers, as evidenced by immunohistochemistry and flow cytometry using the macrophage marker F4/80. Macrophages in St3gal1loxP/PF4+ mice had increased expression of CD68 (macrosialin), as determined by immunohistochemistry and flow cytometry, indicative of an activated macrophage state. Consistently, St3gal1loxP/PF4+ bone marrow smears stained with May-Grunwald/Giemsa revealed increased hemophagocytosis. Macrophage ablation by in vivo injection of clodronate-encapsulated liposomes normalized blood platelet counts and size, and significantly reduced the numbers of activated macrophages in St3gal1loxP/PF4+ mice. Together, our data indicates that platelet production in the bone marrow is reliant on correct glycosylation on MK surface proteins and that the intimate interaction between MKs and macrophages play an important role in regulating platelet production and bone marrow homeostasis. Disclosures No relevant conflicts of interest to declare.

Description: Dynamins are large and highly conserved GTPases involved in endocytosis and vesicle trafficking. Mutations K562E/del in the ubiquitous dynamin 2 (DNM2) have been associated with thrombocytopenia in humans. To determine the role of DNM2 in megakaryopoiesis we generated Dnm2fl/fl Pf4-Cre mice specifically lacking DNM2 in the megakaryocyte (MK) lineage. Dnm2fl/fl Pf4-Cre mice were viable, but had severe macrothrombocytopenia with moderately accelerated platelet clearance and prolonged bleeding due to poorly functional platelets. Dnm2-null bone marrow MKs had altered demarcation membrane system, appearing at times as a compact, narrow twisting membrane system of clathrin-coated vesicles. Fetal liver cell derived Dnm2-null MKs formed proplatelets poorly in vitro, showing that DNM2 plays a major role in MK membrane formation and thrombopoiesis. Both endogenous DNM2 and overexpressed DNM2 WT, but not DNM2 K562E/del mutants localized with the early endosome in bone marrow MKs. The endocytic pathway was disrupted in Dnm2-null MKs, as evidenced by severely reduced early endosome EEA1 and APPL1 staining and weak LysoTracker internalization. Endocytosis of the thrombopoietin (TPO) receptor Mpl was impaired in Dnm2-null platelets, causing constitutive phosphorylation of the tyrosine kinase JAK2 and elevated circulating TPO levels. MK-specific DNM2 deletion severely disrupted bone marrow homeostasis, as reflected by massive MK hyperplasia and myelofibrosis, and consequent extramedullary hematopoiesis and splenomegaly. However, additional Mpl genetic deletion failed to rescue the severe splenomegaly of Dnm2fl/fl Pf4-Cre mice, and Mpl-/- Dnm2fl/fl Pf4-Cre mice instead died at 4-5 weeks of age. Taken together, our data demonstrates that unrestrained MK growth and proliferation results in rapid myelofibrosis independently of Mpl expression and other bone marrow cell types, and establishes a previously unrecognized role for DNM2-dependent endocytosis in megakaryopoiesis, thrombopoiesis and bone marrow homeostasis. Disclosures No relevant conflicts of interest to declare.

Description: Platelet recovery following bone marrow transplant and radiochemotherapy is crucial to prevent bleeding complications. Defects in glycosylation have been associated with decrease in blood platelet counts. However, the role of posttranslational modification in platelet production remains elusive. We here investigated the role of β1,4 galactosyltransferase 1 (β4GalT1), the key glycosyltransferase regulating lactosaminoglycan (LacNAc or βGal1,4 GlcNAc) expression by adding galactose (Gal) to terminal N-acetylglucosamine (GlcNAc), in platelet production. Stromal cell-derived factor 1 (SDF-1), but not thrombopoietin promotes megakaryocyte (MK) migration towards the bone marrow sinusoids, thereby increasing platelet production. SDF-1 (CXCL12) upregulated LacNAc expression in fetal liver wild type (WT), but not β4galt1-null megakaryocytes (MKs) lacking β4GalT1, suggesting that SDF-1 promotes LacNAc expression in vivo to regulate MK migration and platelet production. In support of this hypothesis, β4galt1-null mice had severe macrothrombocytopenia with increased bone marrow MK numbers, but normal platelet clearance. Ploidy, expression of the major glycoproteins (GPIbα/V/IX and αIIbβ3) and the surface expression of the SDF-1 receptor CXCR4 were normal in β4galt1-null bone marrow MKs. However, β4galt1-null bone marrow MKs had increased surface and total β1 integrin expression, as determined by flow cytometry, immunoblot and immunofluorescence. Mature CD42b/CD41 positive β4galt1-null MKs co-localized poorly with endoglin positive bone marrow sinusoids (49.9 ± 2.1 %), compared to WT MKs (72.4 ± 0.6 %). Expression of laminin, the major β1 integrin ligand, was upregulated in β4galt1-null MKs, suggesting that loss of LacNAc expression on β1 integrin increased its function. To exclude an extrinsic contribution to the failure of β4galt1-null MKs to migrate, β4galt1-null fetal liver (E14.5) hematopoietic cells (FLHCs) were transplanted in lethally irradiated WT mice. Transplanted β4galt1-null FLHCs restored bone marrow MKs, but failed to migrate to sinusoids and produce circulating platelets. In marked contrast, production of β4galt1-null white blood cells was normal. Together, our results suggest that SDF-1 upregulates β4GalT1-dependent LacNAc expression to promote MK migration and interaction with BM sinusoids, likely regulating MK β1 integrin expression and interaction with components of the extracellular matrix, specifically laminin. Understanding of the mechanisms regulating posttranslational modifications induced by various hematopoietic stimulating chemokines and cytokines will contribute to better platelet recovery following bone marrow transplant and radiochemotherapy. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 192 Platelet turnover requires correct glycan presentation and expression. Deficiency in β-1,4-galactosyltransferase 1 (β4GalT1), the major enzyme that transfers galactose (Gal) from UDP-Gal to terminal N-acetylglucosamine (GlcNAc) to form type-2 lactosaminyl structures, is lethal in embryonic mice. However, E14.5 β4GalT1−/− embryos are grossly indistinguishable from wild type embryos. Here we sought to determine whether platelet formation and survival require β4GalT1. Hepatic stem cells were obtained from embryonic livers at E14.5 and megakaryoctes were differentiated in vitro in the presence of thrombopoietin. β4GalT1−/−megakaryocytes mature and differentiate normally in vitro as judged by their number, morphology and expression of main surface glycoproteins specific for the megakaryocyte lineage (GPIbα/β, GPIX, αIIb and β3). However, following differentiation, β4GalT1−/−megakaryocytes were unable to produce proplatelets and platelets in vitro. Addition of exogenous β4GalT1 and the donor substrate UDP-Galactose did not improve in vitro proplatelet and platelet formation. Importantly, platelet numbers were decreased by ∼ 75% in β4GalT1−/− E14.5 embryos when compared to wild type embryos. Platelet size was increased by ∼ 50% in β4GalT1−/− embryos, indicating that β4GalT1−/− mice have increased platelet clearance and/or that larger platelets are produced in vivo. Our data strongly support the notion that glycosylation mediated by β4GalT1 activity is crucial for platelet production in vitro and in vivo. Our findings demonstrate for the first time a role for posttranslational glycan modification in platelet production. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3257 The regulatory mechanisms of platelet homeostasis remain elusive. We investigated here the role of hepatic asialoglycoprotein receptor (a.k.a. Ashwell-Morell receptor) in platelet clearance. Mice lacking the hepatic asialoglycoprotein receptor Asgpr2 subunit had increased platelet survivals (T1/2 = 49.5±2h) when compared to wild type (WT, T1/2 = 31±4h) mice. Consequently, Asgpr2−/− mice had platelet counts increased by ∼20%, compared to WT, with increased terminal galactose exposure, as demonstrated using the galactose specific lectin RCA1. Bone marrow and spleen megakaryocyte numbers were reduced by ∼15% and ∼20% in Asgpr2−/− mice, compared to WT mice. Sialidase (NA, Clostidium perfringens, 50mU/mice) maximally desialylated circulating platelets when injected intravenously, as evidenced by increased RCA1 binding. Sialidase injection resulted in a ∼60% depletion of circulating platelets after 24h in Asgpr2−/− mice, compared to 〉90% in WT mice, indicating that desialylated platelets were partially removed by Asgpr1/2. In contrast to platelets, red blood cell counts were unaffected by sialidase treatment. Sialidase injection for 72h resulted in a 2.3-fold and 1.2-fold increase in megakaryocyte numbers in the spleen and bone marrow of WT mice, respectively, but not in Asgpr2−/− mice. In contrast to sialidase treatment, injections of rabbit anti-mouse platelet serum (RAMPS) depleted 〉95% of circulating platelets and increased by 70% bone marrow, but not spleen MK numbers in both WT and Asgpr2−/− mice. The data shows that removal of desialylated, i.e, senescent, platelets by the hepatic Ashwell-Morell receptor differs to that of antibody-mediated platelet clearance. Disclosures: No relevant conflicts of interest to declare.

Description: Platelet recovery following myelosuppressive/myeloablative chemotherapy is crucial to avoid bleeding complications of cancer treatment. Platelets are produced by bone marrow megakaryocytes (MKs), which develop and mature from hematopoietic stem cells (HSC). Mature MKs interact with sinusoidal bone marrow endothelial cells to form transendothelial pseudopods called proplatelets from which platelets are released into the bloodstream. Platelet survival is dependent on correct glycan expression. We here investigate the role of Type-2-Lactosaminoglycans (Type-2-LacNAc) in platelet production. beta1,4Galactosyltransferase 1 (beta4GalT1) is a major enzyme involved in Type-2-LacNAc synthesis which adds Galactose (Gal) to terminal N-Acetylglucosamine (GlcNAc) to form beta1,4Gal-GlcNAc (Type-2-LacNAc).beta4GalT1 deficient mice die in uthero between E15.5 and E16.5. A small percentage of beta4GalT1-/- mice survive until adulthood and they have severe macrothrombocytopenia but normal platelet clearance. Lethally irradiated wild type mice transplanted with beta4GalT1 deficient fetal liver cells failed to produce circulating beta4GalT1 deficient platelets, in marked contrast to beta4GalT1 deficient white blood cells, despite beta4GalT1 deficient MKs have been detected in the bone marrow of transplanted mice. beta4GalT1 deficient fetal liver MKs poorly produce proplatelets in vitro, following their normal maturation and differentiation, as judged by number, morphology, ploidy and expression of main surface glycoproteins. Our data strongly support the notion that glycosylation mediated by beta4GalT1 is crucial for platelet production in vitro and in vivo and demonstrate for the first time a role for post-translational glycan modification in platelet production. Disclosures: No relevant conflicts of interest to declare.