Recognition of Megakaryocyte-Specific T-Antigen By Macrophages Negatively Regulates Platelet Production in Bone Marrow

Abstract 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.

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T-Antigen Expression Causes Macrothrombocytopenia and Extensive Hemophagocytosis in Mice Lacking the Sialyltransferase ST3Gal-I Specifically in Megakaryocytes

Blood ◽

10.1182/blood.v124.21.94.94 ◽

2014 ◽

Vol 124 (21) ◽

pp. 94-94

Author(s):

Renata Grozovsky ◽

Silvia Giannini ◽

Haley Ramsey ◽

Martha Sola-Visner ◽

Karin M Hoffmeister

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Conflicts Of Interest ◽

Antigen Expression ◽

Blood Platelet ◽

T Antigen ◽

Platelet Production ◽

Platelet Counts ◽

Cell Counts

Abstract 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.

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Bone Marrow Macrophage Galectin-3 Regulates Platelet Production through Recognition of O-Glycans on Megakaryocytes

Blood ◽

10.1182/blood.v128.22.409.409 ◽

2016 ◽

Vol 128 (22) ◽

pp. 409-409

Author(s):

Melissa M Lee-Sundlov ◽

Renata Grozovsky ◽

Silvia Giannini ◽

Martina McGrath ◽

Haley E Ramsey ◽

...

Keyword(s):

Bone Marrow ◽

Antigen Expression ◽

Blood Platelet ◽

Activated Macrophages ◽

Bone Marrow Macrophage ◽

Platelet Production ◽

Platelet Counts ◽

Pathological Conditions ◽

Galectin 3

Abstract 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.

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Understanding the Mechanisms Underlying Thrombocytopenia in Visceral Leishmaniasis

Blood ◽

10.1182/blood-2019-128133 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2378-2378 ◽

Cited By ~ 1

Author(s):

Gulab Fatima Rani ◽

Olivier Preham ◽

Ian Hitchcock ◽

Paul Kaye

Keyword(s):

Bone Marrow ◽

Platelet Count ◽

Visceral Leishmaniasis ◽

Conflicts Of Interest ◽

Experimental Models ◽

Severe Thrombocytopenia ◽

Hematopoietic Stem ◽

Platelet Production ◽

Platelet Counts ◽

Splenic Macrophages

Visceral leishmaniasis (VL) is a neglected tropical parasitic disease caused by Leishmania parasites and only second to malaria in terms of worldwide morbidity and mortality. According to recent WHO report, there are 500,000 cases of VL worldwide leading to ~30,000 deaths per year. VL is endemic in 98 countries but the major disease burden is contributed by Brazil, India and Sudan. Disease manifestations include fever, weight loss, hepatosplenomegaly, immune dysregulations and extensive hematological complications. We have shown previously using experimental models of infection that the infiltration of CD4+ T cells results in disruption to the bone marrow environment, resulting in dysfunctional hematopoietic stem and progenitor cells self-renewal (Pinto et al, PLOS Pathogens, 2017) and aberrant medullary erythropoiesis causing pathological anemia (Preham et al, Frontiers in Immunology, 2018). Thrombocytopenia is also dominant hematological feature seen in both human and experimental models that may reflect either reduced platelet production or enhanced clearance. However, the mechanisms of VL-driven thrombocytopenia remain poorly understood. The aim of this study is to explore the possible underlying mechanisms from platelet production to phagocytic cells dependent clearance. Using a murine experimental model of VL, we demonstrate a steady decrease in the platelet count from d14 onwards in infected mice culminating in severe thrombocytopenia on d28 of infection (infected: 225.9 ±35.7 vs naïve: 1005 ±90.6, x 106/µl). Critically, thrombocytopenia is completely reversible after a single dose of liposomal amphotericin B (Ambisome @ 8mg/kg bodyweight, IV) which clears parasites by delivering the drug directly to parasite harbouring tissue macrophages, thereby improving parasite clearance and reducing toxicity. Despite significant thrombocytopenia, the number and gross morphology of bone marrow megakaryocytes (MKs) were not altered, but MK ultrastructure studies using transmission electron microscopy identified significantly reduced demarcation membranes in infected mice compared to naïve. Levels of plasma thrombopoietin (TPO), the key regulator of MK differentiation and platelet production, were decreased in infected vs naïve mice (1254 ± 95.49 vs 3249 ± 125.1 pg/ml) and administration of exogenous TPO resulted in complete recovery of platelet counts. Given that the majority of TPO is produced by the liver, reduction in the levels of circulating TPO during infection is likely due to destruction of liver architecture by parasite loaded hepatic granulomas. Together, these data suggest that despite some changes in MK cytoplasmic maturation, the bone marrow microenvironment remains supportive of MK differentiation capacity during VL. As platelet production is not significantly altered by VL, we next determined effects on platelet clearance. Large number of highly active splenic macrophages are common in VL and are known for their phagocytic properties. Experiments conducted on VL-infected splenectomised mice demonstrated a reduction in thrombocytopenia compared to sham-operated infected mice (685 ±32 vs 297± 16, x 106/µl) and showed a great response to exogenous TPO, implying splenic clearance may be involved in thrombocytopenia. Partial depletion of splenic macrophages in infected mice using clodronate liposomes did not alter platelet count, whereas neutrophil deletion (anti-Gr1 mAb @ 250ug/g IP) in infected mice resulted in a near 2-fold increase in platelet counts. Furthermore, circulating platelets in VL infected mice were IgG coated compared to naive which is likely to further enhance autoimmune platelet clearance. Severe thrombocytopenia and bleeding are important clinical manifestations of VL. Our findings clearly demonstrate that the mechanisms of thrombocytopenia in VL are multifactorial but do not cause permanent long term damage to the BM microenvironment. Critically, these changes could be reversed rapidly by clearing parasitemia, using TPO agonists to increase numbers of circulating platelets and/or by reducing platelet clearance. This highlights the possibility of re-evaluating the current treatment regimen in VL endemic countries by including therapeutic interventions aimed at reversing severe thrombocytopenia. Disclosures No relevant conflicts of interest to declare.

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Growth Factor Independence 1b (Gfi1b) Is An Essential Regulator of Late Stage Megakaryocyte Maturation and Platelet Production

Blood ◽

10.1182/blood.v118.21.2358.2358 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2358-2358

Author(s):

Lothar Vassen ◽

Tarik Moroy

Keyword(s):

Bone Marrow ◽

Late Stage ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Pcr Analysis ◽

Severe Thrombocytopenia ◽

Facs Analysis ◽

Platelet Counts ◽

Platelet Release ◽

Deficient Mice

Abstract Abstract 2358 Absence of Gfi1b in mice is embryonically lethal and causes failure to produce functional megakaryocytes and platelets. Thrombopoiesis, the production of platelets by megakaryocytes, is an essential process in hemostasis that needs to be well controlled. Too many platelets can cause thrombosis, too few cause excessive bleeding. How terminal megakaryocyte maturation and platelet release works is incompletely understood but requires many factors such as Fli1, Gata1, MyH9, p45-NFE2, or c-Myc. Expression array analysis of hematopoietic cells from conditionally Gfi1b deficient mice (Gfi1bfl/fl Mx-Cre) revealed an up-regulation of many factors important for megakaryocyte function like Itga2b, Itgb3, CD9, CD41, CD61, PF4 and Ppbp. Gfi1b ablation in adult Gfi1bfl/fl Mx-Cre mice leads to a severe drop in platelet counts to less than 20% of wt mice with an increase in mean platelet volume (MPV) by 40%. However, megakaryocyte numbers rise up to 100 fold over normal levels when Gfi1b is absent. FACS analysis of bone marrow cells of Gfi1b deficient mice showed a higher number of MEPs, a higher proportion of smaller megakaryocytes and an aberrant population of cKithiCD41hiCD9veryhi cells, which are not present in wt animals. Gfi1b−/− megakaryocytes can reach wt size and normal ploidy as shown by FACS analysis and immunofluorescence microscopy. Transmission electron microscopy (TEM) of pIpC induced Gfi1bfl/fl Mx-Cre megakaryocytes revealed an excess number and larger size of so called “demarcation membranes” in unusual parallel layers and a strongly reduced number of dense granula. Strikingly, both intact and fragmented megakaryocytes were frequently found within bone marrow blood vessels in Gfi1b−/− mice. In addition, a high percentage of megakaryocytes were found in a state of disintegration, without signs of proper platelet release. These features are rarely seen in wt mice. It is known that Gfi1b is required for erythropoiesis and Gfi1bfl/fl Mx-Cre mice show signs of anemia and stress erythropoiesis, which might explain high MEP numbers, which could explain the high numbers of megakaryocytes. To better define the function of Gfi1b in late stage megakaryocyte development, we decided to abrogate Gfi1b expression more specifically by using mice that express Cre recombinase under the megakaryocyte specific promoter of the PF4 gene (PF4-Cre). We observed that Gfi1bfl/fl PF4-Cre mice develop a very severe thrombocytopenia reaching only 2% of wt platelet counts in peripheral blood accompanied by an increase of MPV by 80% over wt levels. Most of Gfi1bfl/fl PF4-Cre mice died at 6–8 weeks of age from severe internal bleedings. Megakaryocyte numbers increase in these mice by 5 to 10 fold and they also reach high ploidy, but their morphology is highly disturbed. Gfi1bfl/fl PF4-Cre megakaryocytes contain less cytoplasm, few dense granula organized in a small patch and a lobulated, ring-shaped nucleus localized close to the cell membrane giving the cells an almost “inside-out” appearance and indicating a disturbed cytoskeleton organization. Gfi1bfl/flPF4-Cre mice show a stress induced splenic erythropoiesis and an increase in MEP numbers, probably a consequence of their substantial hemorrhaging owing to the low platelet counts. The high MEPs number might explain the increase in megakaryocytes in these mice compared to wt controls. Immunofluorescence analysis of Gfi1bfl/fl PF4-Cre megakaryocytes compared to wt counterparts showed less expression of van Willebrand factor (vWF), an important regulator of thrombopoiesis. Q-PCR analysis on mRNA from sorted Gfi1bfl/fl PF4-Cre wt megakaryocytes revealed a lower expression of vWF, but higher PF4, very high Mpl and high CCNE1 expression. Our data show that Gfi1b controls the production of platelets from megakaryocytes, but does not affect the maturation of megakaryocytes as such. However, Gfi1b is required to maintain the cellular organelle structure in megakaryocytes and, more specifically is required to control the formation of dense granula and demarcation membrane formation. Gfi1b ablation in megakaryocytes results in a phenotype with high similarities to Gata1-low mice and syndromes involving mutations in the Gata1 target Gpib, the receptor for vWF, causing Bernhard-Soulier Syndrome (BBS). It is thus possible that Gfi1b is another candidate gene involved in megakaryocyte related diseases. Disclosures: No relevant conflicts of interest to declare.

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Megakaryocyte Abnormalities Occur In The Absence Of Cell-Mediated Immunity In a Murine Model Of Passive Immune Thrombocytopenia (ITP)

Blood ◽

10.1182/blood.v122.21.3537.3537 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3537-3537

Author(s):

John W. Semple ◽

Kristin Hunt ◽

Yu Hou ◽

Rukhsana Aslam ◽

Edwin R. Speck ◽

...

Keyword(s):

Bone Marrow ◽

Immune Thrombocytopenia ◽

Conflicts Of Interest ◽

In Vivo Model ◽

Cell Mediated Immunity ◽

Severe Thrombocytopenia ◽

Platelet Destruction ◽

Platelet Counts ◽

Antiplatelet Antibodies ◽

Platelet Antibodies

Abstract Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder characterized by increased peripheral immune platelet destruction and megakaryocyte defects in the bone marrow. Although ITP was originally thought to be primarily due to humoral mediated autoimmunity it is now evident that T cells can also play a contributing role to the thrombocytopenia. In fact, the exact interplay between platelet destruction, megakaryocyte dysfunction, and the elements of both the humoral and cell mediated immune systems still remain incompletely defined. In murine passive models of ITP, the direct administration of anti-platelet antibodies can result in severe thrombocytopenia which is evident within 24 hours of injection. While most studies have focused on immune platelet destruction in the spleen, an additional possibility is that the anti-platelet antibody also has an effect on megakaryocytes. To unequivocally determine if antiplatelet antibodies have an effect on megakaryocytes in an in vivo model, BALB/c mice were intravenously administered 2 ug of an anti-GPIIbIIIa antibody (MReg30) or 50 uL of a high tittered anti-GPIIIa (anti-β3) serum from BALB/c GPIIIa (CD61) knockout mice immunized with wild type platelets. Platelet counts were assessed over time and the bone marrow and spleens were harvested for histological examination of megakaryocytes. Both preparations of antiplatelet antibodies significantly reduced platelet numbers within 1 day of antibody or serum administration. This thrombocytopenia could be rescued by administration of 2 g/kg of IVIg ip. Compared with naïve control mice, histological (H&E staining) examination of the bone marrow and spleens revealed that megakaryocytes were significantly increased in number and all exhibited abnormalities consistent with apoptosis e.g. pyknotic nuclei. IVIg administration completely prevented these megakaryocyte abnormalities. These results show that passively administered anti-platelet antibodies not only affect platelet counts but also significantly affect megakaryocyte physiology in the absence of cell mediated immunity. Disclosures: No relevant conflicts of interest to declare.

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Platelet Biogenesis in the Lung Circulation

Blood ◽

10.1182/blood-2018-99-109544 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. SCI-22-SCI-22

Author(s):

Mark Looney

Keyword(s):

Bone Marrow ◽

Lung Transplant ◽

Conflicts Of Interest ◽

Direct Proof ◽

Mouse Lung ◽

Mouse Strains ◽

Hematopoietic Progenitors ◽

Platelet Production ◽

Platelet Counts ◽

Transplant Procedure

Abstract Platelets are indispensable in hemostasis, thrombosis, and immune responses. In humans, billions of platelets are produced each day from megakaryocytes, however the mechanisms of mature platelet production are incompletely understood. Megakaryocytes are produced in the bone marrow and have been visualized to communicate with the bone marrow sinusoids to release proplatelet fragments. Megakaryocytes have also been found in other tissues, including the lung, but the function of megakaryocytes in these locations is unclear. Historical data indicate that the lung may be a site of platelet biogenesis. The concentration of megakaryocytes in the blood exiting the lung is much lower than the blood entering the lung (implying filtering) and conversely, platelet counts are higher in blood draining from the lungs. Additionally, when the lung circulation in entirely bypassed, megakaryocytes accumulate in the blood and there is a high incidence of thrombocytopenia. However, direct proof of platelet biogenesis in the lung is lacking. We used lung intravital microscopy combined with fluorescently labeled mouse strains and directly visualized intravascular megakaryocytes releasing platelets in the lung circulation. We also visualized megakaryocytes in the bone marrow and spleen releasing proplatelet fragments, and megakaryocyte migration in toto from the bone marrow, which are presumably the source material for lung platelet production. The megakaryocyte-releasing events in the lung were quantified and represent at least half of the total platelet production in mice, which can be increased by the application of thrombopoietin. We also observed a much larger extravascular pool of megakaryocytes in the lung that were not platelet generating as observed by lung intravital imaging. The function of these lung-resident megakaryocytes is unknown, but RNA-Seq data points to a potential role in lung immunity. Orthotopic, single-lung transplantation experiments into thrombocytopenic and hematopoietic progenitor-deficient animals (c-mpl-/-) revealed that peripheral blood platelet counts and bone marrow hematopoietic progenitors could be fully reconstituted by the lung transplant procedure implying the presence of hematopoietic progenitors in the mouse lung. Indeed, these progenitors were directly detected in the extravascular lung and purified populations of hematopoietic progenitors in the lung could correct thrombocytopenia in c-mpl-/-animals. Finally, the lung transplant procedure produced donor-derived chimerism of other hematopoietic lineages such as neutrophils and lymphocytes. We conclude that the lung has significant hematopoietic potential including being a major site of platelet biogenesis. Disclosures No relevant conflicts of interest to declare.

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Successful Treatment of Acquired Amegakaryocytic Thrombocytopenia with Rituximab: A Case Report.

Blood ◽

10.1182/blood.v114.22.4223.4223 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4223-4223

Author(s):

Laura R. Goldberg ◽

Lewis Glasser ◽

Tony Wu ◽

Ikue Shimizu ◽

Peter J. Quesenberry ◽

...

Keyword(s):

Bone Marrow ◽

Case Report ◽

Bone Marrow Biopsy ◽

Conflicts Of Interest ◽

Immunosuppressive Agents ◽

Peripheral Blood Smear ◽

Severe Thrombocytopenia ◽

Literature Report ◽

Platelet Counts ◽

Acquired Amegakaryocytic Thrombocytopenia

Abstract Abstract 4223 Acquired amegakaryocytic thrombocytopenia is a rare disorder in which there is a marked decrease in bone marrow megakaryocytes leading to severe thrombocytopenia with preserved hematopoeisis in the remaining lineages. The clinical course is variable and no standardized treatment exists. Multiple cases in the literature report treatment using immunosuppressive agents including cyclosporine and antithymocyte globulin. In this case report, we describe the first successful use of rituximab in a patient with amegakaryocytic thrombocytopenia in the absence of any underlying autoimmune disorders. Our patient, an 86- year-old woman, presented with epistaxis, ecchymoses, and blood-tinged sputum. She had thrombocytopenia (platelets 6 × 109/L, MPV 8.3) with a normal hemoglobin and white blood cell count. Peripheral blood smear showed no obvious cause for thrombocytopenia. Bone marrow biopsy revealed focal lymphocytic infiltrates and severe megakaryocytic hypoplasia with nearly absent megakaryocytes. Cytogenetic and molecular analyses were normal, revealing no evidence of a clonal myelo- or lymphoproliferative disorder. The diagnosis of amegakaryocytic thrombocytopenia was made. She was started on prednisone 50mg per day. There was no significant improvement in her platelet count and she was then given IVIG without response. Repeat bone marrow biopsy showed persistent severe bone marrow hypoplasia with near absence of megakaryocytes. After 6 weeks of platelet transfusion dependence and steroids, she was tapered off her steroids and was given rituximab 375mg/m2 weekly for 4 doses. Three weeks after starting rituximab, her platelet counts began to recover and by day 37, her platelet counts normalized and remained within normal limits 12 months after treatment. This case demonstrates the possible utility of rituximab in treating patients with isolated acquired amegakaryocytic thrombocytopenia. Disclosures: No relevant conflicts of interest to declare.

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Rapid In Vivo Consumption and Ex Vivo Phagocytosis of WASP(−) Platelets.

Blood ◽

10.1182/blood.v110.11.2103.2103 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2103-2103

Author(s):

Amanda Prislovsky ◽

Falk Nimmerjahn ◽

Jeffrey V. Ravetch ◽

Carl W. Jackson ◽

Ted S. Strom

Keyword(s):

Bone Marrow ◽

Ex Vivo ◽

Severe Thrombocytopenia ◽

Turnover Rates ◽

Platelet Production ◽

Platelet Counts ◽

Igg1 Antibody ◽

Reticulated Platelets ◽

Consumption Rates

Abstract Patients with the Wiskott-Aldrich Syndrome (WAS) have thrombocytopenia and increased platelet consumption rates, and may have reduced platelet production rates. WASP(−) mice have been reported to have only a mild thrombocytopenia. We find that when fully crossed onto the C57Bl/6J background, WASP(−) mice have a >50% reduction in platelet counts. Ex vivo labeled WASP(−)platelets are consumed 2 to 4-fold faster than are WT platelets in WT mice, and with exponential rather than linear kinetics. Clearance rates of WASP(−) platelets in WT mice, and vice versa, indicate that rapid consumption is due to factors both intrinsic and extrinsic to platelets. In vivo biotinylation demonstrates consumption rates comparable to those seen with ex vivo labeling, and shows a normal rate of consumption of WASP(−) reticulated (immature) platelets. Reticulated platelet counts are reduced, indicating that their production rate is reduced. Megakaryocytes are increased in spleen and bone marrow, and in the latter their ploidy distribution is normal, suggesting that impaired platelet production occurs at the level of thrombopoiesis. The absolute turnover rates of mature and reticulated platelets, however, indicate that maturation of the latter can account for only a fraction of the former’s turnover in either WT or WASP(−) mice. A subset of WASP(−) mice show an increased fraction of reticulated platelets and more severe thrombocytopenia, and some members of this subset also express serum anti-platelet antibodies. CMFDA-labeled WASP(−) platelets opsonized with anti-CD61(IgG1) antibody are more susceptible to ex vivo phagocytosis by bone marrow derived macrophages (BMDM) than WT platelets, and as susceptible as CD47(−/−) platelets. After opsonization with 6A6(IgG2b) antibody, WASP(−) platelets are also taken up more rapidly than WT platelets by BMDM, but less rapidly than are CD47(−/−) platelets. The in vivo consumption rate of WASP(−) platelets in WT recipients is more accelerated by opsonization with anti-CD61 antibody than is that of WT platelets. Increased phagocytosis is not due to (A) altered levels of the targeted antigens or of CD47 (B) increased exposure of phosphatidyl serine, or (C) antibody-induced activation as assayed by CD62P expression. Increased phagocytosis is not due to selective sensitivity to IgG1 vs IgG2b antibodies, as opsonization with an engineered 6A6(IgG1) antibody leads to reduced phagocytosis for both CD47(−/−) and WASP(−) platelets. Anti-CD61 opsonized platelets deficient in both WASP and CD47 show markedly increased ex vivo phagocytosis compared to platelets deficient in either protein, suggesting that platelet WASP does not function to amplify signals from platelet CD47 through macrophage SIRP-alpha. These results raise the possibility that the binding of low affinity or low titer antibodies that might have no effect on WT platelets could cause thrombocytopenia when the platelets lack WASP. Alternatively, rapid phagocytosis of opsonized WASP(−) platelets could promote a self-reinforcing cycle of increased host antigen presentation and increased immune responses to host antigens.

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Immature platelet values indicate impaired megakaryopoietic activity in neonatal early-onset thrombocytopenia

Thrombosis and Haemostasis ◽

10.1160/th09-03-0148 ◽

2010 ◽

Vol 103 (05) ◽

pp. 1016-1021 ◽

Cited By ~ 27

Author(s):

Hannes Hammer ◽

Christoph Bührer ◽

Christof Dame ◽

Malte Cremer ◽

Andreas Weimann

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Intensive Care ◽

Early Onset ◽

Absolute Number ◽

Severe Thrombocytopenia ◽

Platelet Counts ◽

Immature Platelet Fraction ◽

Neonatal Thrombocytopenia ◽

Immature Platelets

SummaryNewly released platelets, referred to as immature platelets, can be reliably quantified based on their RNA content by flow cytometry in an automated blood analyser. The absolute number of immature platelets (IPF#) and the immature platelet fraction (IPF%) reflect megakaryopoietic activity. We aimed to analyse the implication of these parameters in analysing the pathomechanism of early-onset neonatal thrombocytopenia. Platelet counts and IPF were determined at day 1 to 3 (d1 to d3) in 857 neonates admitted to intensive care. In thrombocytopenic patients (platelet counts<150 x 109/l, n=129), IPF# was significantly lower (8.5 ± 2.7 x 109/l), than in non-thrombocytopenic neonates (9.5 ± 3.6 x 109/l, n=682, p<0.05). IPF% was significantly higher in thrombocytopenic (9.3 ± 7.9%) vs. non-thrombocytopenic neonates (4.1 ± 1.8%, p<0.001). While neonates with early-onset infection (n=134) had lower platelet counts (199 ± 75 x 109/l) compared to controls (230 ± 68 x 109/l, n=574, p<0.01), there were no differences in IPF# or IPF%. Likewise, “small for gestational age” infants (SGA, n=149) had lower platelet counts at d1 (199 ± 75 x 109/l, p<0.001) than controls, but no differences in IPF. A trend towards lower IPF# was detected if SGA infants with platelet counts <100 x 109/l (5.4 ± 3.9 x 109/l, n=11) and thrombocytopenic neonates with infection (9.9 ± 7.3 x 109/l, n=10, p=0.11) were compared. The evaluation of IPF# indicates that thrombocytopenia in neonates is likely due to a combination of increased platelet consumption and inadequate megakaryopoietic response by the neonatal bone marrow. Furthermore, SGA neonates with moderate and severe thrombocytopenia might have a pronounced suppression of megakaryopoiesis compared to neonates with infection.

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Impact of Platelet Count on Bleeding in the Setting of Anti-Platelet Therapy

Blood ◽

10.1182/blood-2020-142893 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 18-18

Author(s):

Robert Hugh Lee ◽

Wolfgang Bergmeier

Keyword(s):

Real Time ◽

Conflicts Of Interest ◽

Thrombotic Event ◽

Platelet Inhibition ◽

Intravital Imaging ◽

Severe Thrombocytopenia ◽

Platelet Counts ◽

Anti Platelet Therapy ◽

The Impact

Anti-platelet therapy (APT) is used for secondary prevention of thrombosis. The most commonly prescribed anti-platelet drugs are aspirin and P2Y12 inhibitors, including clopidogrel, prasugrel and ticagrelor. Dual anti-platelet therapy (DAPT) consisting of aspirin and a P2Y12 inhibitor is often used in the first 1-12 months after an initial thrombotic event and has a greater anti-thrombotic effect than single agents, but is also associated with a higher risk of bleeding. Due to this risk of hemorrhage, the appropriate use of DAPT in patients requiring percutaneous coronary intervention (PCI) with baseline or periprocedural thrombocytopenia remains unclear. To study the impact of thrombocytopenia on bleeding with APT, we used intravital imaging in a murine hemostasis model and adoptive platelet transfer to generate mice with specific platelet counts with or without platelet inhibition. To generate experimental mice, we used transgenic mice in which platelets express a chimeric GPIb receptor with the extracellular domain replaced with a domain of the human IL-4R (hIL-4R/GPIb-Tg). Endogenous platelets were depleted by injection of anti-hIL-4R antibody, and the recipient mice were then transfused with wild-type (WT) platelets from donor mice treated, or not, with single or dual APT (aspirin 20 mg/kg; clopidogrel 25 mg/kg) to achieve specific platelet counts ranging from 50,000 to 400,000 platelets/μL. We also compared these mice with WT mice (with normal platelet counts, ~1,200,000 platelets/μL) treated with APT. Platelet inhibition was confirmed prior to performing in vivo experiments. Hemostasis was determined by intravital imaging in our saphenous vein laser injury model, in which a 50 μm injury was induced by laser ablation. Real-time top-down epifluorescence imaging was used to determine time to initial hemostasis, rebleeding events, and platelet and fibrin accumulation. In each mouse, 3-5 injuries were induced at different sites and each injury was visualized for 10 minutes. Following real-time imaging, spinning disk confocal Z-stacks of platelet plugs were obtained for 3D reconstruction to compare platelet plug volume. In untreated WT mice, hemostasis was achieved in ~20 seconds. In WT mice treated with DAPT, initial hemostasis was often rapidly achieved but this was followed by significant rebleeding events. Paradoxically, platelet accumulation was increased in WT + DAPT mice due to extravascular accumulation of platelets which occurred during bleeding. However, in plugs that stabilized, plug volume was reduced in WT + DAPT mice. In hIL-4R/GPIb-Tg mice with reduced platelet counts, untreated platelets were able to form a stable hemostatic plug even at 50,000/μL, although time to hemostasis was slightly prolonged. However, as platelet counts decreased in mice with DAPT-treated platelets, initial hemostasis became more prolonged and many injuries never achieved initial hemostasis. These results suggest that DAPT may not be safe in the setting of severe thrombocytopenia. Disclosures No relevant conflicts of interest to declare.

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