Understanding the Mechanisms Underlying Thrombocytopenia in Visceral Leishmaniasis

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

Blood ◽

10.1182/blood.v126.23.420.420 ◽

2015 ◽

Vol 126 (23) ◽

pp. 420-420

Author(s):

Melissa M. Lee-Sundlov ◽

Renata Grozovsky ◽

Silvia Giannini ◽

Martina McGrath ◽

Haley Elizabeth Ramsey ◽

...

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Flow Cytometric Analysis ◽

Antigen Expression ◽

Blood Platelet ◽

T Antigen ◽

Severe Thrombocytopenia ◽

Extrinsic Factors ◽

Platelet Production ◽

Platelet Counts

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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Mpl Expression on AML Blasts Predicts Cytopenia

Blood ◽

10.1182/blood.v126.23.1387.1387 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1387-1387

Author(s):

Philipp J Rauch ◽

Corinne Widmer ◽

Kristin Fritsch ◽

Jana M Ellegast ◽

Jeroen S Goede ◽

...

Keyword(s):

Platelet Count ◽

Negative Correlation ◽

Conflicts Of Interest ◽

Gene List ◽

Receptor Expression ◽

Severe Neutropenia ◽

Severe Thrombocytopenia ◽

Hematopoietic Stem ◽

Platelet Counts ◽

Blast Count

Abstract Acute myeloid leukemia (AML) induces profound impairment of healthy hematopoiesis. The production deficit in the bone marrow (BM) leads to development of peripheral anemia, thrombocytopenia and neutropenia, which is a major cause of AML-associated morbidity and mortality. Despite much progress in understanding of AML biology, the mechanisms by which AML blasts interact with elements of normal hematopoiesis to cause cytopenia are unclear. Conventional wisdom has it that blasts infiltrate the marrow and displace normal hematopoiesis. If this concept were to be true, there should be a strong correlation between BM blast count and peripheral cytopenia. Surprisingly, analysis of 223 patients with newly diagnosed AML at a tertiary referral center revealed lack of correlation between initial BM blast count [% of cellularity] and hemoglobin level (ρ=-0.11, P=0.12), platelet count (ρ=-0.00, P=0.53) and absolute neutrophil count (ρ=0.13, P=0.06). This indicates that mechanisms other than displacement of normal hematopoiesis dictate the severity of cytopenia in AML patients. Hematopoiesis is tightly regulated by cytokines. Among them, thrombopoietin (TPO) acts through its receptor c-Mpl as the master regulator of megakaryopoiesis, but also exerts upstream effects on hematopoietic stem and progenitor cells (HSPC). TPO levels are controlled by receptor-mediated scavenging by cells carrying c-Mpl on the surface, with platelets representing the lion's share in a healthy organism. This negative feedback loop results in strong negative correlation between serum TPO concentration and platelet count in the steady state. When we examined this relationship in our AML cohort, TPO levels did not follow the expected negative correlation with platelet counts (ρ=-0.10, P=0.59). Comparison with historic controls with thrombocytopenia induced by chemotherapy for non-hematopoietic malignancy revealed that the lack of correlation was driven by AML cases with severe thrombocytopenia that had lower than expected levels of TPO in the serum. As HSPC are known to express c-Mpl, we hypothesized that HSPC-derived AML blasts may also express the receptor and cause insufficiency of hematopoiesis by means of receptor-mediated TPO scavenging. To test this hypothesis, we compared c-Mpl expression on blasts in AML cases with severe thrombocytopenia and low TPO concentration (potential scavenger cases) to cases with TPO levels adequate for the degree of cytopenia. Both surface flow cytometry and qPCR demonstrated higher c-Mpl expression in potential scavenger cases (3.1-fold, P=0.02). To determine whether this difference in expression translates into increased serum TPO clearance, we incubated AML blasts with high (c-Mpl+) and low (c-Mpl-) receptor expression in serum containing recombinant human TPO at a concentration of 100 pg/mL. After 2h, TPO clearance reached 45 pg per 106 cells in wells with c-Mpl+ blasts, compared to only 4 pg per 106 cells in wells with c-Mpl- blasts (P=0.02). This confirms the hypothesis that AML blasts can lower TPO levels by virtue of their c-Mpl expression. Validation studies in an independent, multi-center Dutch-Belgian-Swiss cohort of 437 AML cases confirmed lack of correlation between initial BM blast count and cytopenia. Ranked gene list correlation analysis of whole genome microarray data proved significant enrichment of the MPL transcript in patients with severe thrombocytopenia when compared to patients with average platelet counts (rank 27/20'589, FDR<10-6). MPL enrichment could also be observed in patients with severe neutropenia (P<0.01), but there was no correlation between MPL transcript level and degree of anemia. Lastly, we asked if MPL expression was related to cytogenetic or molecular AML subtype: indeed, microarray analysis showed higher MPL expression in cases of AML with t(8;21) than in any other subtype (P<10-4). Concurrently, these patients displayed significantly lower platelet count (40 vs 83 x 109/L, P=0.02) when compared to all other AML cases. In summary, our study demonstrates that cytopenia in AML is independent of BM blast count, but strongly correlated with c-Mpl expression on blasts. We show that c-Mpl+ blasts clear TPO, causing insufficient TPO levels and contributing to development of thrombocytopenia and neutropenia. The work may have important ramifications for treatment of AML-induced cytopenia, especially in the relapsed or refractory setting. Disclosures No relevant conflicts of interest to declare.

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A Case of Clopidogrel Induced TTP Recurring After Four Years

Blood ◽

10.1182/blood.v116.21.4678.4678 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4678-4678

Author(s):

Nanda K. Methuku ◽

Abhinav B. Chandra ◽

Anuradha Belur ◽

Lech Dabrowski

Keyword(s):

Platelet Count ◽

Renal Insufficiency ◽

Peripheral Blood ◽

Blood Smear ◽

Reticulocyte Count ◽

Conflicts Of Interest ◽

Peripheral Blood Smear ◽

Severe Thrombocytopenia ◽

Platelet Counts ◽

Multiple Organs

Abstract Abstract 4678 Case description - A 61 year old woman was started on clopidogrel after having PTCA with stent placement in February 2006. Four weeks after starting clopidogrel she developed thrombocytopenia with platelet nadir of 17,000. Her LDH was 700 IU/L and she was anemic with hemoglobin of 7.4 gm/dl with elevated reticulocyte count. Peripheral blood smear showed schistocytes and diagnosis of TTP secondary to clopidogrel was made. She did not have renal insufficiency. Clopidogrel was discontinued and patient was started on plasmapheresis with recovery of platelet counts. Early attempts in weaning plasmapheresis resulted in drop in platelet count and Rituximab was given to the patient weekly for four weeks. Subsequently, patient was weaned off plasmapheresis. For four years patient was followed periodically with CBC showing platelet counts greater than 250,000. In May 2010, four years after initial event patient was admitted to hospital for abdominal pain and found to have splenic infarcts. Subsequently, she also developed bilateral cerebral infarcts. Platelet count had decreased to less than 100,000. Her LDH was elevated at 419 IU/L. Reticulocyte count was 2.3%. Peripheral blood smear revealed significant number of schistocytes. There was no renal insufficiency or fever. Trans-esophageal echocardiogram (TEE) was done that did not reveal any vegetations. Patient was diagnosed as having recurrence of TTP and started on plasmapheresis with recovery in platelet counts. Pt was also treated with Rituximab. Discussion- We describe a case of TTP initially occurring within weeks of starting clopidogrel. Patient was treated with plasmapheresis and Rituximab and clopidogrel was discontinued. Patient had recurrence after four years as manifested by infarcts in multiple organs, with mild thrombocytopenia, elevated LDH and significant number of schistocytes on peripheral blood smear. It is very uncommon for clopidogrel associated TTP to recur after such a prolonged period of 4 years. Most cases of clopidogrel associated TTP have mild thrombocytopenia. This patient had severe thrombocytopenia on first presentation of TTP but had mild thrombocytopenia on recurrence. This case illustrates the importance of extended follow up and high index of suspicion for TTP as delays in initiation of plasmapheresis has a poor clinical outcome. Disclosures: No relevant conflicts of interest to declare.

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Eltrombopag Can Stimulate Trilineage Hematopoiesis In Patients with Refractory Severe Aplastic Anemia

Blood ◽

10.1182/blood.v116.21.4427.4427 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4427-4427

Author(s):

Matthew J. Olnes ◽

Yong Tang ◽

Susan Soto ◽

Elaine M Sloand ◽

Philip Scheinberg ◽

...

Keyword(s):

Stem Cell ◽

Platelet Count ◽

Aplastic Anemia ◽

Severe Aplastic Anemia ◽

Successful Outcome ◽

Severe Thrombocytopenia ◽

Hematopoietic Stem ◽

Platelet Counts ◽

Transfusion Independence

Abstract Abstract 4427 Severe aplastic anemia (SAA) is characterized by trilineage marrow hypoplasia and a paucity of hematopoietic stem cell progenitors. SAA is treated with immunosuppression or allogeneic stem cell transplantation (SCT), with a successful outcome in a majority. However, 20–40% of patients without a suitable donor for SCT do not respond to immunosuppression and may have persistent severe thrombocytopenia. Thrombopoietin (TPO) is the principal regulator of platelet production, and it exerts its effects through binding the megakaryocyte progenitor TPO receptor mpl, which stimulates production of mature megakaryocytes and platelets. Eltrombopag, a small molecule TPO mimetic that binds to mpl, increases platelet counts in healthy subjects, and in patients with chronic immune thrombocytopenic purpura. Both TPO and eltrombopag stimulate more primitive multilineage progenitors and stem cells in vitro. Patients with SAA and thrombocytopenia have very elevated TPO levels; nevertheless, we asked whether pharmacologic doses of eltrombopag could stimulate hematopoiesis in these patients without other options. We are conducting a pilot phase II study of eltrombopag in SAA patients with severe thrombocytopenia refractory to immunosuppressive therapy. Consecutive eligible adult patients were treated with oral eltrombopag at an initial dose of 50 mg daily, with escalation to a maximum dose 150 mg daily, with the goal of maintaining a platelet count of >20,000/uL above baseline. Treatment response was measured after three months and was defined as platelet count increases to 20,000/uL above baseline, or stable platelet counts with transfusion-independence for a minimum of 8 weeks. Nine patients have been enrolled and six are evaluable for response to date. Two patients did not respond to treatment. Three patients achieved platelet responses by 12 weeks of treatment, and all have sustained their responses (median follow up 10 months). Four patients exhibited improved hemoglobin levels 12 weeks after starting treatment (median hemoglobin increase of 2.1 g/dL) and two patients who were previously dependent on packed red blood cell transfusions have achieved transfusion-independence. Three neutropenic patients exhibited increased neutrophil counts after treatment with eltrombopag (median increase 0.46K cells/uL). These results provide evidence that eltrombopag can improve platelet counts in patients with severe refractory thrombocytopenia, and perhaps more surprisingly, have a clinically relevant impact on erythropoiesis and myelopoiesis. Updated data will be presented at the Society's meeting. Disclosures: Off Label Use: Eltrombopag for thrombocytopenia in refractory severe aplastic anemia patients.

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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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Final Results from an International, Multi-Center, Single-Arm Study Evaluating the Safety and Efficacy of Romiplostim in Adults with Primary Immune Thrombocytopenia (ITP),

Blood ◽

10.1182/blood.v118.21.3279.3279 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3279-3279 ◽

Cited By ~ 6

Author(s):

Ann Janssens ◽

Michael D. Tarantino ◽

Robert Bird ◽

Maria Gabriella Mazzucconi ◽

Ralph Vincent V. Boccia ◽

...

Keyword(s):

Bone Marrow ◽

Platelet Count ◽

Board Of Directors ◽

Research Funding ◽

Platelet Response ◽

Employment Equity ◽

Advisory Committees ◽

Platelet Production ◽

Platelet Counts ◽

Equity Ownership

Abstract Abstract 3279 Background: ITP is an autoimmune disorder characterized by increased platelet destruction and suboptimal platelet production. Romiplostim stimulates platelet production via the TPO-receptor, and is recommended for second- and third-line treatment of chronic ITP in adults. We report final data from a large prospective study of romiplostim in adults with ITP of varying duration and severity. Methods: Eligibility criteria were broad: patients ≥18 years of age, who had received prior ITP therapies (final protocol amendment: ≥1, previous amendments: ≥3), with low platelet counts (final amendment: ≤ 30 × 109/L, previous amendments: ≤ 10, ≤ 20 × 109/L) or experiencing uncontrolled bleeding. The only excluded comorbidities were: hematological malignancy, myeloproliferative neoplasms, MDS and bone marrow stem cell disorder. Romiplostim was initiated at 1 (final amendment) or 3 (previous amendments) μg/kg/week, with dose adjustments allowed to maintain platelet counts ≥50 × 109/L. Patients could continue on study until they had access to commercially available romiplostim. Rescue medications were allowed at any time; concurrent ITP therapies could be reduced when platelet counts were > 50 × 109/L. Primary endpoint was incidence of adverse events (AEs) and antibody formation. Secondary endpoint was platelet response, defined as either (1) doubling of baseline count and ≥ 50 × 109/L or (2) ≥20 × 109/L increase from baseline. Results: A total of 407 patients received romiplostim, 60% of whom were female. Median (Q1, Q3) time since ITP diagnosis was 4.25 (1.20, 11.40) years (maximum 57.1 years), with 51% of patients splenectomised and 39% receiving baseline concurrent ITP therapies. Seventy-one percent of patients completed the study, with requirement for alternative therapy and withdrawn consent the most common reasons for discontinuation (5% each). Median (Q1, Q3) on-study treatment duration was 44.29 (20.43, 65.86) weeks (maximum 201 weeks), with a total of 20,201 subject-weeks on study. Incidence and type of AEs were consistent with previous studies. The most common serious treatment-related AEs were cerebrovascular accident, headache, bone marrow reticulin fibrosis (with no evidence of positive trichrome staining for collagen and no evidence suggesting primary idiopathic myelofibrosis), nausea, deep vein thrombosis, hemorrhage and pulmonary embolism, with each reported in 2 of 407 (0.5%) patients. All other serious treatment-related AEs were each reported in one patient. Eighteen patients died; 3 deaths (hemolysis, intestinal ischaema, aplastic anemia) were considered treatment-related. No neutralizing antibodies to romiplostim or TPO were reported. Approximately 90% of patients achieved each of the platelet response definitions, regardless of splenectomy status. Overall, median (Q1, Q3) time to response was 2 (1, 4) weeks for response definition 1, and 1 (1, 3) week for response definition 2. Median (Q1, Q3) baseline platelet count was 14 (8, 21) × 109/L. After 1 week of treatment median (Q1, Q3) platelet count had increased to 42 (18, 101) × 109/L. From week 8 onwards, and excluding counts within 8 weeks of rescue medication use, median platelet counts were consistently above 100 × 109/L (range 101.0–269.5 × 109/L). Median (Q1, Q3) average weekly romiplostim dose was 3.62 (1.99, 6.08) μg/kg. Summary/conclusions: This is the largest prospective study in adult ITP reported to date. The data reported here are similar to those reported for previous romiplostim studies, with romiplostim able to safely induce a rapid platelet response in adult ITP patients with low platelet counts or bleeding symptoms. Romiplostim is an important, well-tolerated, treatment option for adult ITP patients, which significantly increases and maintains platelet counts. Adverse Event Subject Incidence Platelet Response Disclosures: Janssens: Amgen: Consultancy; Roche: Speakers Bureau; GSK: Membership on an entity's Board of Directors or advisory committees. Tarantino:Cangene corporation: Research Funding; Baxter: Research Funding; Talecris: Honoraria, Speakers Bureau; Up-to-date: Patents & Royalties; The Bleeding and Clotting Disorders Institute: Board Member. Bird:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; GSK: Membership on an entity's Board of Directors or advisory committees. Boccia:Amgen: Equity Ownership, Honoraria, Speakers Bureau. Lopez-Fernandez:Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Kozak:Amgen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Steurer:Amgen: Honoraria. Dillingham:Amgen Limited: Employment, Equity Ownership. Lizambri:Amgen: Employment, Equity Ownership.

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Severe Thrombocytopenia Does Not Increase Bleeding Complications in Patients Undergoing Bronchoscopy

Blood ◽

10.1182/blood.v124.21.4293.4293 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4293-4293

Author(s):

Lakshminarayanan Nandagopal ◽

Muthu Veeraputhiran ◽

Tania Jain ◽

Ayman Soubani ◽

Charles A. Schiffer

Keyword(s):

Platelet Count ◽

Renal Dysfunction ◽

Platelet Transfusion ◽

Conflicts Of Interest ◽

Bleeding Complications ◽

British Thoracic Society ◽

Severe Thrombocytopenia ◽

Platelet Counts ◽

Number Of Patients ◽

Platelet Transfusions

Abstract Introduction Prophylactic platelet transfusions are often performed prior to bronchoscopy or broncho-alveolar lavage (BAL) to prevent bleeding in thrombocytopenic patients. There is a paucity of data to validate this approach, with a platelet transfusion threshold of <50,000/mm3 largely based on expert opinion. We conducted a retrospective study on the incidence of bleeding complications in thrombocytopenic patients undergoing bronchoscopy. Methods We identified 150 consecutive patients with platelet counts <100,000/mm3 who underwent bronchoscopy and/or BAL from January 2009 to May 2014 at our institution. Bronchoscopies performed in patients with frank hemoptysis and trans-bronchial lung biopsy procedures were excluded. Patient characteristics, underlying diagnosis, platelet count prior to bronchoscopy, administration of platelet transfusions and bronchoscopy details were recorded. Factors affecting bleeding risk including presence of renal dysfunction (defined as BUN >30 and/or Cr>2.0) and coagulation studies (PT, PTT, INR) were identified. The British Thoracic Society guidelines1 were used to categorize bleeding as a result of bronchoscopy. Data were analyzed using descriptive statistics. Results The median age was 59 years (range 27-90), with two-thirds of patients (63%) being male. One hundred and seventeen (78%) patients had underlying malignancy and 55 (37%) had thrombocytopenia related to malignancy. Fellows and residents under the supervision of a bronchoscopy certified attending performed all but 4 of the bronchoscopies. Infection (40%) was the primary indication for bronchoscopy with BAL performed in 127 (85%) patients. Fifty-eight of 89 (65%) patients with baseline platelet counts <50,000/mm3 received prophylactic transfusions compared to 8% of those with platelet counts >50,000/mm3. The platelet count did not rise to >50,000//mm3 in many transfused patients. Seventy patients (47%) had counts <50,000/mm3 and eighty patients (53%) had counts >50,000/mm3 at the time of bronchoscopy. 49% were receiving immunosuppressive medications, 45% had renal dysfunction and 8% had INR >1.5. Bloody lavage that resolved spontaneously without continuous suctioning (Grade 0) was observed in 9 (6%) patients. Bleeding that required continuous suctioning but then resolved spontaneously (Grade 1) was noted in 1 patient with a platelet count of 61,000/mm3. Of 10 total bleeding events, 7 occurred in patients who were intubated. Two additional patients with platelet counts of 30,000/mm3 and 53,000/mm3 had diffuse alveolar hemorrhage, which was present before bronchoscopy. “Old” blood and blood clots were observed in 6 patients. Discussion The low incidence of bleeding complications from bronchoscopy +/- BAL even in patients with platelet counts <30,000/mm3 (3 episodes in 31 patients, all grade 0) demonstrates that bronchoscopy can be safely done in severely thrombocytopenic patients. Adopting a lower threshold for prophylactic transfusions could save a considerable number of platelet units and translate into significant cost savings and decreased risk of transfusion-related complications. Table 1 Platelet count, transfusion history and bleeding complications during bronchoscopy Platelet count at the time of bronchoscopy Number (n) and percentage (%) of patients who underwent bronchoscopy Number of patients who received prior platelet transfusion Bleeding during bronchoscopy n % 0-15,000/mm3 9 6% (9/150) 5 Grade 0=1 pt 16-29 22 15% 16 Grade 0=2 pts 30-39 17 11% 9 Grade 0=1 pt 40-49 22 15% 9 Grade 0=3 pts 50-75 44 29% 14 Grade 1=1 pt 76-100 36 24% 10 Grade 0=2 pts Total 150 63 Grade 0=9 pts, Grade 1=1 pt. 1.Du Rand IA, Blaikley J, Booton R, et al. British Thoracic Society guideline for diagnostic flexible bronchoscopy in adults: accredited by NICE. Thorax. 2013:68 Suppl 1:i1-i44 Disclosures No relevant conflicts of interest to declare.

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Ectopic Expression of the Extracellular Domain of Mpl Is Sufficient to Induce a Hematopoietic Population Crisis

Blood ◽

10.1182/blood.v112.11.2886.2886 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2886-2886

Author(s):

Daniel C. Wicke ◽

Johann Meyer ◽

Guntram Büsche ◽

Hans Kreipe ◽

Zhixiong Li ◽

...

Keyword(s):

Gene Therapy ◽

Bone Marrow ◽

Ectopic Expression ◽

Extracellular Domain ◽

Target Cells ◽

Severe Thrombocytopenia ◽

Hematopoietic Stem ◽

Platelet Counts ◽

Cell Counts ◽

Population Crisis

Abstract The thrombopoietin receptor Mpl is required for regeneration of hematopoietic stem cells and governs megakaryocytic differentiation. Patients with inherited MPL deficiency suffer from severe thrombocytopenia which progresses to aplastic anemia, a disorder called congenital amegakaryocytic thrombocytopenia (CAMT). As a first step towards a potential gene therapy for MPL deficiency, we retrovirally expressed the receptor in a murine bone marrow transplantation model. An initial series of vectors used a strong enhancer-promoter derived from the spleen-focus forming virus (SFFV). Mice transplanted with hematopoietic cells modified by these constructs developed a profound yet transient elevation of multi-lineage hematopoiesis due to increased signaling of the Thpo receptor on target and non-target cells. Mice developed increased leukocyte, erythrocyte and platelet counts (2–3 times elevated) compared to GFP control animals. Histopathology revealed an elevated number of mature megakaryocytes with atypical features like numerous giant forms in the bone marrow and the spleen and atypical neoformation of bone in the spleen leading to the diagnosis of chronic myeloproliferative disorder (CMPD). A minority of mice (3/27) developed erythroleukemia with almost 100% CD71 and TER119 double positive cells as detected by flow cytometry. Histopathology presented infiltration of erythroblasts in all hematopoietic tissues like the BM, spleen and liver. Molecular analysis revealed retroviral vector insertions in sfpi1, fli1 and klf3 that seem to be the major driving force for the development of leukemia in these animals. Somewhat unexpectedly, in the majority of mice the CMPD converted into a progressive, potentially lethal pancytopenia. Animals had severely reduced blood cell counts with only 50% of leukocyte, 20% of erythrocyte and 10% of platelet counts compared to GFP control animals. This population crisis affected all major blood lineages and also involved co-existing unmodified hematopoiesis. Histopathology presented a dysmegakaryopoiesis with an increased number of atypical micro-megakaryocytes, histiocytes with erythrocytophagocytosis and atypical mast cell proliferation diagnosed as a myelodysplastic syndrome (MDS)-like disorder. In the bone marrow, pancytopenic mice had reduced cell numbers of the primitive cell fraction (LSK cells). To address the mechanism of pancytopenia, we expressed a dominant negative form of Mpl (dnMpl) consisting of the extracellular and transmembrane domain and lacking the intracellular signal transduction domain. Animals transplanted with dnMpl-modified cells failed to show the initial CMPD but developed the same pancytopenic, MDS-like end stage. A vector expressing Mpl under control of the PGK promoter or a fragment of the Mpl-promoter reduced or completely avoided the side effects (CMPD, MDS-like disorder) observed with vectors using stronger promoters. The induction of a hematopoietic population homeostasis thus depends upon Mpl expression levels, indicating the need for strictly regulated transgene expression in gene therapy for MPL deficiency. As ectopic expression of the extracellular domain is sufficient to cause MDS, sequestration of crucial niche factors like Thpo may contribute to the pathogenesis of this disorder. This study demonstrates that ectopic expression of a hematopoietic growth factor receptor may disturb organ homeostasis through interference with intra- and extracellular mechanisms of cell communication.

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Extreme Thrombocytosis Under Azacitidine in Patients with Myelodysplastic Syndrome

Blood ◽

10.1182/blood.v120.21.4961.4961 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4961-4961

Author(s):

Charikleia Kelaidi ◽

Dimitrios Kokkinidis ◽

Maria Protopappa ◽

Georgios Papaioannou ◽

Ioannis Batsis ◽

...

Keyword(s):

Platelet Count ◽

Myelodysplastic Syndrome ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasm ◽

Response To Treatment ◽

Jak2 Mutation ◽

Hematopoietic Stem ◽

Platelet Counts ◽

Hemorrhagic Event ◽

Count Response

Abstract Abstract 4961 Background: Platelet increase under azacitidine in patients with myelodysplastic syndrome (MDS) has been acknowledged as an early predictive factor of response to treatment. However, extreme thrombocytosis under azacitidine has not been reported. Methods: We studied consecutive patients with MDS or MDS/myeloproliferative neoplasm (MDS/MPN) who had platelet counts near or over 1, 000 G/L under azacitidine. Results: Four patients, sex ratio 1:1, with median age of 65 years, had extreme thrombocytosis under azacitidine. Baseline characteristics were: WHO classification RAEB-2/CMML-1/CMML-2 in 2/1/1 patients, median platelet count 248 G/L (<400 G/L in all), normal karyotype/+8, −9/−7 in 2/1/1 patients, IPSS low/int-2/high in 1/2/1 patients. None had reticulinic fibrosis or ring sideroblasts>15% at baseline. A median number of 8 cycles of azacitidine was administered. Individual platelet counts reached 2, 960 G/L, 800 G/L, 1, 188 G/L and 2, 740 G/L. Thrombocytosis occured early after treatment onset or resumption (Figure 1). Histologic findings under treatment were: Increased cellularity (N=4), micromegakaryocytes and other signs of megakaryocytic dysplasia (N=4), reticulinic fibrosis grade I and II in 1 and 2 patients, respectively. JAK2 V617F mutation was detected in 1 patient (with maximum platelet count of 2, 900 G/L) and was undetectable in the remaining patients. None had a thrombotic or hemorrhagic event. Two patients had a concomitant increase of WBC count. Response to azacitidine was CR, PR and stable disease in 1/1/2 patients. Three patients received hydroxyurea (HU) in addition to azacitidine and one patient underwent hematopoietic stem cell transplantation (HCT). AML transformation occurred in 1 patient 25 months after azacitidine onset. Median overall survival after azacitidine onset was 25 months. Conclusion: Extreme thrombocytosis of the range of essential thrombocytosis, with megakaryocytic dysplasia and hyperplasia, was noted under azacitidine in 4 patients with MDS-MDS/MPN and normal baseline platelet count. Hypothetically, azacitidine may induce the expression of critical genes of megakaryopoiesis or platelet release in patients with rare mutations. Notably, JAK2 mutation was detected in only one patient. Alternatively, demethylation could unmask an underlying unclassified MDS/MPN similar to RARS-T. 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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