Search

Immune thrombocytopenia (ITP) is a platelet disorder. Pediatric and adult ITP have been associated with sialic acid alterations, but the pathophysiology of ITP remains elusive, and ITP is often a diagnosis of exclusion. Our analysis of pediatric ITP plasma samples showed increased anti–Thomsen-Friedenreich antigen (TF antigen) antibody representation, suggesting increased exposure of the typically sialylated and cryptic TF antigen in these patients. The O-glycan sialyltransferase St3gal1 adds sialic acid specifically on the TF antigen. To understand if TF antigen exposure associates with thrombocytopenia, we generated a mouse model with targeted deletion of St3gal1 in megakaryocytes (MK) (St3gal1MK−/−). TF antigen exposure was restricted to MKs and resulted in thrombocytopenia. Deletion of Jak3 in St3gal1MK−/− mice normalized platelet counts implicating involvement of immune cells. Interferon-producing Siglec H–positive bone marrow (BM) immune cells engaged with O-glycan sialic acid moieties to regulate type I interferon secretion and platelet release (thrombopoiesis), as evidenced by partially normalized platelet count following inhibition of interferon and Siglec H receptors. Single-cell RNA-sequencing determined that TF antigen exposure by MKs primed St3gal1MK−/− BM immune cells to release type I interferon. Single-cell RNA-sequencing further revealed a new population of immune cells with a plasmacytoid dendritic cell–like signature and concomitant upregulation of the immunoglobulin rearrangement gene transcripts Igkc and Ighm, suggesting additional immune regulatory mechanisms. Thus, aberrant TF antigen moieties, often found in pathological conditions, regulate immune cells and thrombopoiesis in the BM, leading to reduced platelet count.

G protein–coupled receptors are critical mediators of platelet activation whose signaling can be modulated by members of the regulator of G protein signaling (RGS) family. The 2 most abundant RGS proteins in human and mouse platelets are RGS10 and RGS18. While each has been studied individually, critical questions remain about the overall impact of this mode of regulation in platelets. Here, we report that mice missing both proteins show reduced platelet survival and a 40% decrease in platelet count that can be partially reversed with aspirin and a P2Y12 antagonist. Their platelets have increased basal (TREM)-like transcript-1 expression, a leftward shift in the dose/response for a thrombin receptor–activating peptide, an increased maximum response to adenosine 5′-diphosphate and TxA2, and a greatly exaggerated response to penetrating injuries in vivo. Neither of the individual knockouts displays this constellation of findings. RGS10−/− platelets have an enhanced response to agonists in vitro, but platelet count and survival are normal. RGS18−/− mice have a 15% reduction in platelet count that is not affected by antiplatelet agents, nearly normal responses to platelet agonists, and normal platelet survival. Megakaryocyte number and ploidy are normal in all 3 mouse lines, but platelet recovery from severe acute thrombocytopenia is slower in RGS18−/− and RGS10−/−18−/− mice. Collectively, these results show that RGS10 and RGS18 have complementary roles in platelets. Removing both at the same time discloses the extent to which this regulatory mechanism normally controls platelet reactivity in vivo, modulates the hemostatic response to injury, promotes platelet production, and prolongs platelet survival.

The enzyme β-1-4 galactosyltransferase 1 (β4GalT1) plays a critical role in thrombopoiesis by modulating sialo-glycan (sialyl N-acetyl-lactosamine or LacNAc) content and function of the β1 integrin on megakaryocytes (MKs) (Nat. Commun. 2020;11(1):356). Recent data, however, point to a more complex role for β4GalT1 in hematopoiesis, as the promoter region of its conserved gene, B4galt1, is rich in enhancer sequences for transcription factors associated with cell identity and pro-oncogenic regulatory programs. Here, we investigated the homeostasis of hematopoietic stem and progenitor cells (HSPCs) in B4galt1-/- mice. We demonstrate that lack of Sialylated LacNAc synthesis perturbs HSPCs beyond the homing deficiency associated with lack of homing sialo-glycan motifs. Flow cytometry analysis showed that B4galt1-/- mouse bone marrows have increased numbers of Long-Term HSCs (LT-HSCs, defined as LineageNeg/Sca-1Pos/c-KitPos/CD150Pos/CD48Neg). The increase in HSC numbers led us to investigate their phenotypic and functional features further. While quiescence markers in B4galt1-/- LT-HSCs were indistinguishable relative to controls, LT-HSCs expressed more of the platelet marker CD41 on their surface, supporting a highly expanded CD41+ subset of LT-HSCs. Platelet-bias of LT-HSCs has been associated with inflammation and aging. However, our data do not support an increased cytokine inflammatory profile in the bone marrow. Instead, single-cell RNA sequencing (scRNA seq) of sorted β4galt1-/- LineageNeg/Sca-1Pos/c-KitPos (LSK) cells showed a significantly increased expression of the proto-oncogene Pim-1, its target, Myc, and the heavily O-glycosylated transmembrane receptor mucin 13 (MUC13), compared to control cells. Analysis of LT-HSC glycan expression using lectin microarray showed the expected decrease in N-glycosylation associated with B4galt1 deficiency, but also an increase in O-glycans, consistent with overexpression of MUC13, expression of which was enriched compared to other surface mucins. The data show that B4galt1 deletion leads to overexpression of the proto-oncogenes Pim-1, Myc, and MUC13 in HSPCs. The data suggest that MUC13-associated O-glycans and glyco-synthetic genes are potential therapeutic targets for hematologic malignancies since mucins have anti-inflammatory functions and alterations in mucin expression are with inflammation and cancer. Disclosures No relevant conflicts of interest to declare.

The thrombopoietin receptor Mpl plays a critical role in thrombopoiesis, as it is the primary signaling constituent in megakaryocyte (MK) differentiation from hematopoietic stem and progenitor cells (HSPCs) and maintains circulating blood thrombopoietin levels via clathrin-mediated endocytosis. Thus, thrombopoiesis is tightly controlled by Mpl cell-surface expression on HSPCs, MKs, and platelets. Previous work by our group has shown that Dnm2fl/flPF4-Cre (Dnm2Plt-/-) mice, lacking the large endocytic GTPase dynamin 2 (DNM2) specifically within MKs and platelets, develop a variety of clinical phenotypes that closely resemble myelofibrosis, such as bone marrow fibrosis, marked HSPC expansion, MK hyperplasia, extramedullary hematopoiesis, and severe splenomegaly (Bender, Giannini, et al. Blood. 2015;125(6):1014-1024). Dnm2Plt-/- mice also displayed elevated plasma TPO levels and constitutive JAK2 activation in platelets, due to defective Mpl endocytosis. Here, the role of Mpl endocytosis in the maintenance of normal hematopoiesis was assessed by generating Dnm2Plt-/- mice in the Mpl-/- background. At 3 weeks of age, Mpl-/- Dnm2Plt-/- mice displayed significantly reduced HSPCs, a near complete depletion of bone marrow MKs, similar to Mpl-/- mice, indicating that Mpl is the primary receptor contributing to the aberrant hyperproliferative phenotype of Dnm2Plt-/- mice. However, Mpl-/- Dnm2Plt-/- mice also showed severe anemia, defects in erythroblast maturation, grossly elevated plasma erythropoietin (EPO) levels, and splenomegaly, resulting in early fatality by 3 to 4 weeks of age, suggesting that Mpl contributes to normal erythropoiesis in young mice. Mpl-/- and Mpl+/- Dnm2Plt-/- mice displayed reduced erythroblast development at 3 weeks of age, which returned to normal with adulthood. Taken together, the data shows that DNM2-dependent Mpl endocytosis is required for steady-state hematopoiesis and provides novel insights into a developmentally-controlled role for Mpl in normal erythropoiesis. Disclosures Sola-Visner: Sysmex America, Inc.: Other: Laboratory equipment on loan, Research Funding.

Somatic mutations in the tyrosine kinase JAK2, the thrombopoietin (TPO) receptor Mpl and the chaperone calreticulin cause myelofibrosis due to constitutive TPO/Mpl signaling in abnormal hematopoietic stem cells (HSCs). Impaired Mpl-mediated endocytosis has been reported in myelofibrosis patients carrying the most frequent JAK2-V617F mutation. Mpl-mediated endocytosis is also impaired in Dnm2fl/fl Pf4-Cre (Dnm2Plt-/-) mice specifically lacking the highly conserved endocytic GTPase dynamin 2 (DNM2) in the megakaryocyte (MK) lineage. Consequently, Dnm2Plt-/-mice develop hallmarks of myelofibrosis such as elevated circulating TPO levels, constitutive JAK2 phosphorylation, marked expansion of HSCs, massive MK hyperplasia, bone marrow fibrosis, extramedullary hematopoiesis and splenomegaly. To determine whether the phenotype is due to unrestrained TPO/Mpl signaling in HSCs, Dnm2Plt-/- mice were crossed with Mpl-/-mice. Mpl-/- Dnm2Plt-/- mice were obtained with a normal Mendelian distribution at weaning, and bone marrow HSC and MK numbers were significantly reduced in Mpl-/- Dnm2Plt-/- mice, similar to those of Mpl-/- mice. Surprisingly, Mpl-/- Dnm2Plt-/- mice died at a median age of 26 days postnatal and presented a severe splenomegaly, similar to that of Dnm2Plt-/- mice. Complete blood counts were analyzed from birth to 3 weeks postnatal. Blood platelet counts increased over time in control mice, reaching maximal values at 3 weeks postnatal, and remained low in Mpl-/-, Dnm2Plt-/- and Mpl-/- Dnm2Plt-/- mice. Blood erythrocyte counts increased over time in control mice, were significantly slower in single Mpl-/- and Dnm2Plt-/- mice, and failed to increase in Mpl-/- Dnm2Plt-/-mice, resulting in severe anemia. Erythroid maturation in Mpl-/- Dnm2Plt-/- spleens was investigated by flow cytometry analysis using CD71 and Ter119 as erythroid markers, where immature erythroblasts are defined as CD71high/Ter119low and mature erythroblasts as CD71low/Ter119high. Approximately 75% of erythroid cells in control spleens were mature erythroblasts, and the distribution significantly decreased to 40% and 20% in single Mpl-/- and Dnm2Plt-/- spleens, respectively. In Mpl-/- Dnm2Plt-/- spleens, only 5% of erythroid cells were mature erythroblasts, consistent with severely impaired erythroid maturation. Flow cytometry phenotypic analysis of bone marrow MK and eryrthroid progenitors revealed a significant increase of the distribution of progenitors with both MK and erythroid potential (Pre-Meg-E) in Mpl-/- Dnm2Plt-/-mice. The data shows that TPO/Mpl signaling and endocytosis orchestrate erythropoiesis and thrombopoiesis to control the bone marrow environment. Mpl regulates both MK and erythroid progenitors, highlighting clinically relevant interactions between these two blood cell compartments in myelofibrosis development. Disclosures No relevant conflicts of interest to declare.

Bone marrow (BM) macrophages maintain both survival and retention of hematopoietic stem cells and regulate erythropoiesis. The role of macrophage lectins and glycans in thrombopoiesis remains unclear. We report a novel role for bone marrow macrophage galectin-3 in maintaining platelet counts, by phagocytosing megakaryocytes (MKs) expressing the Thomsen-Friedenreich (TF) antigen, which is often exposed under pathological conditions, such as cancer and malignancies. The TF antigen is a disaccharide presented in cryptic form on O-glycans and covered by a sialic acid moiety. The sialyltransferase ST3Gal1 transfers sialic acid onto the TF antigen. To investigate the role of O-glycans in thrombopoiesis, we generated mice with increased TF antigen in MKs by generating St3gal1loxP/PF4+ mice specifically lacking ST3Gal1 in the MK lineage. As expected, St3gal1loxP/PF4+ circulating platelets and BM MKs had increased TF antigen expression, compared to controls, as evidenced by peanut agglutinin (PNA) binding. Other blood cell lineages had no increase in TF antigen expression. St3gal1loxP/PF4+ mice developed mild thrombocytopenia, but surprisingly had virtually normal platelet clearance. BM MK colony forming units and in vitro proplatelet production were normal in St3gal1loxP/PF4+ mice, suggesting that extrinsic factors in the St3gal1loxP/PF4+BM environment affected platelet production. St3gal1loxP/PF4+ BM smears revealed increased hemophagocytosis, indicative of an increase in phagocytic macrophages. In vivo macrophage ablation by injection of clodronate-encapsulated liposomes significantly reduced the numbers of activated macrophages, thereby normalizing blood platelet counts and size. Flow cytometric phenotypic analysis of BM-derived macrophages showed an increased population of activated macrophages in St3gal1loxP/PF4+ mice, compared to controls, specifically macrophages with increased galectin-3 expression, a ligand for the TF antigen. Immunofluorescence staining of BM sections using a specific antibody towards the TF antigen showed that MK progenitors and pro-platelet-like structures expressed TF antigen in control BMs, which is significantly increased in St3gal1loxP/PF4+ mice and co-localized with galectin-3 expressing macrophages, supporting the notion that MK O-glycans and macrophage galectin-3 play a role in thrombopoiesis under steady state and pathological conditions. Consistent with this notion, galectin-3 deficient mice have slightly, but significantly increased blood platelet counts. We conclude that galactin-3 plays a minor role in normal thrombopoiesis. Activation of galectin-3 expressing macrophages by the MK TF antigen leads to MK phagocytosis, inhibition of platelet formation and thrombocytopenia. Disclosures No relevant conflicts of interest to declare.

The human body produces and removes 1011 platelets daily to maintain a normal steady-state platelet count. However, the regulatory mechanisms remain elusive. We have shown that platelets lacking sialic acid (desialylated platelets) are removed by the hepatic Ashwell-Morell receptor (AMR or asialoglycoprotein receptor type 2), thereby regulating platelet survival and hepatic TPO levels. Platelet counts and lifetime were increased in Asgr2-/- mice (AMR-null mice), compared to wild type (WT) mice. Platelet volume and immature platelet fraction (IPF) are decreased in AMR-null mice, consistent with the notion that platelets in AMR-null mice (AMR-null platelets) circulate longer and are older. By contrast, deficiency of the sialyltransferase St3gal4 gene induces a marked thrombocytopenia (St3gal4-null platelets), due to rapid platelet clearance by the hepatic AMR. Consistent with the rapid platelet clearance, platelet volume and IPF were increased in St3gal4-/- mice, reflecting high platelet turnover and younger platelets. While both AMR-null and St3gal4-null platelets are desialylated, they differ substantially in their time, i.e. age, in circulation. Here we investigated the effects of in vivo and in vitro aging on platelet function. Freshly isolated St3gal4-null platelets showed significantly increased integrin activation when stimulated with convulxin and thrombin, while AMR-null platelets showed a significantly lower response compared to WT platelets. Secretion of α-granule was significantly increased in St3gal4-null platelets. By contrast no significant difference was measured between WT and AMR-null platelets. Despite increased platelet counts, the tail-bleeding time was significantly prolonged in AMR-null mice, compared to WT mice, suggesting that increased circulatory time (age) negatively affects platelet function in vivo. We next performed in vitro storage for up to 72 hours at room temperature to stress platelet aging. Stored St3gal4-deficient platelets had increased integrin activation, α-granule secretion in response to convulxin and thrombin and showed increased phosphatidyl serine exposure as detected by Annexin V binding in response to calcium ionophore, compared to stored control platelets. By contrast, stored AMR-null platelets had significantly impaired integrin activation, α-granule secretion and Annexin V binding compared to controls. To further evaluate the propensity to undergo apoptosis, we tested caspase-3 activation and mitochondrial membrane potential. Surprisingly, we found that both St3gal4-null (young) and AMR-null (old) platelets showed a significantly lower caspase-3 activation in response to calcium ionophore and ABT-737 compared to WT platelets. Furthermore, the mitochondrial membrane potential was lower in both St3gal4-deficient and AMR-null platelets compared to WT platelets, indicating functionally impaired mitochondria. Taken together, our data indicate that younger St3gal4-null platelets have an increased baseline function while by contrast older AMR-null platelets have decreased function in vitro and in vivo. Interestingly, younger and older (longer circulating platelets) had reduced propensity to undergo apoptosis and impaired mitochondrial function. Overall, younger platelets represent a highly favorable profile during storage. Identification of donors with a larger fraction of younger platelets could result in safer and more efficacious platelet transfusions. Disclosures No relevant conflicts of interest to declare.

Glycosylation defects have been associated with low platelet counts. Six genes encoding sialyltransferases (ST), ST3gal1 to 6, that synthesize an α2,3 sialic acid (SA) linkage have been identified in the mammalian genome, and deletion of St3gal1 and St3gal4 genes has been associated with macrothrombocytopenia in mice. Despite the similarity in transferring SA in a α2,3-linkage to terminal galactose residues, St3gal1 and St3gal4 sialylate distinct glycans: St3gal1 is associated with core 1 O-glycan Galβ1,3GalNAcα1-Ser/Thr expression, also known as tumor-associated or Thomsen-Friedenreich antigen (T-antigen), whereas St3gal4 sialylates lactosaminyl Galβ1,4GlcNAc N-glycans. It has been previously shown that St3gal4-null platelets are cleared by the hepatic Ashwell-Morell receptor, causing severe thrombocytopenia in these mice. Herein, we generated St3gal1loxP/PF4+ mice specifically lacking ST3Gal1 in the megakaryocyte (MK) lineage to investigate the detailed mechanisms of macrothrombocytopenia associated with St3gal1 deficiency. Both St3gal1loxP/PF4+ circulating platelets and bone marrow (BM) MKs had increased T-antigen expression, compared to control, as evidenced by peanut agglutinin (PNA) binding. As expected, other blood cell lineages had no increase in T-antigen expression. Blood platelet counts were reduced by ~50% and platelets were enlarged in St3gal1loxP/PF4+ mice, compared to control, despite a virtually indistinguishable platelet clearance. BM MK numbers were normal despite the observed thrombocytopenia, BM MK colony forming units (CFUs) were reduced and in vitro proplatelet production was normal in St3gal1loxP/PF4+ mice, suggesting that extrinsic factors in the St3gal1loxP/PF4+ BM environment affected platelet production. We hypothesize that recognition of the T-antigen epitope on MKs mediate phagocytosis by macrophages. Macrophages in St3gal1loxP/PF4+ mice had increased expression of CD68 (macrosialin), indicative of an activated macrophage state. Flow cytometric analysis of BM derived macrophages of St3gal1loxP/PF4+ mice showed an increased population of resolving M2-type macrophages, which are normally involved in apoptotic cell clearance. Additionally, St3gal1loxP/PF4+ BM smears revealed increased hemophagocytosis, as evidenced by May-Grunwald/Giemsa, indicative of an unspecific increase in phagocytic macrophages. Macrophage ablation by in vivo injection of clodronate-encapsulated liposomes significantly reduced the numbers of activated macrophages in St3gal1loxP/PF4+ mice, thereby normalizing blood platelet counts and size. Taken together data show the contrasting effects of different SA loss on platelet homeostasis: Platelets lacking α2,3-linked SA on N-glycans have increased platelet clearance, whereas a lack of α2,3-linked on O-glycans do not affect platelet half-life, but cause defective thrombopoiesis in MKs. Disclosures No relevant conflicts of interest to declare.