Anti-thrombopoietin Antibodies Research Articles

Detection of Anti-Thrombopoietin Antibodies in Patients with Immune Thrombocytopenia

Background: Immune thrombocytopenia (ITP) is an autoimmune disease characterized by the presence of autoantibodies against platelet membrane glycoproteins, which cause the autoantibody-mediated destruction of platelets and impaired platelet production. Thrombopoietin (TPO) binds to its receptor on the surface of hematopoietic stem cells and megakaryocytes and induces their maturation and proliferation. Patients with thrombocytopenia due to aplastic anemia have drastically elevated plasma levels of TPO, whereas patients with ITP have normal or slightly elevated plasma levels of TPO despite their low platelet count. Furthermore, based on the existence of a multitude of autoantibody reactivities in ITP, including antibodies against platelets and TPO receptors, the presence of anti-TPO antibodies in patients with ITP may be suspected.Objective: We developed assay systems to detect plasma anti-TPO antibodies and screen patients with ITP. We examined the clinical characteristics associated with anti-TPO antibodies and their pathogenic roles in patients with ITP.Methods: Plasma anti-TPO antibodies from 101 patients with ITP and 72 healthy controls were measured by enzyme-linked immunosorbent assay (ELISA) using recombinant human TPO (rhTPO) as an antigen. The specificity of anti-TPO antibody reactivity was confirmed by ELISA competition assay. The presence of anti-TPO antibodies was further examined using immunoprecipitation and immunoblotting using rhTPO. To investigate whether anti-TPO antibodies inhibited functional interactions between TPO and TPO receptors, we examined extracellular signal-regulated kinases (ERKs), downstream signals induced by TPO. The binding of TPO to TPO receptors induced the phosphorylation of ERK in TPO receptor-expressing UT-7/TPO cells.Results: The level of anti-TPO antibodies measured by ELISA was significantly greater in the samples from patients with ITP than in those from healthy controls (2.91 ± 3.64 units versus 1.45 ± 0.67 units, P < 0.001). Samples were classified as positive or negative for anti-TPO antibody, as determined by immunoprecipitation and immunoblotting. Thus, the ELISA positive-cutoff value was considered to be the mean plus 3.5 standard deviation (SD) of 72 healthy control plasma samples. Plasma anti-TPO antibodies were detected in twenty-four ITP patients (23.8%), but in none of the healthy controls. By ELISA competition assay, anti-TPO antibody reactivity was inhibited dose-dependently by preincubation of patient plasma with rhTPO. In addition, anti-TPO antibody-positive plasma samples inhibited the phosphorylation of ERK in UT-7/TPO cells. In contrast, healthy control plasma had no inhibitory effect. Furthermore, the number of megakaryocytes was decreased relatively in the anti-TPO antibody-positive ITP patients. There was no difference in the TPO levels in plasma between ITP patients with anti-TPO antibodies and patients without anti-TPO antibodies (63.6 ± 79.7 pg/ml versus 45.2 ± 49.3 pg/ml).Conclusion: Our results have thus demonstrated the presence of anti-TPO autoantibodies in patients with ITP. The ELISA using rhTPO was specific for the detection of anti-TPO antibodies and thus allows their easy and rapid measurement in clinical settings. These findings suggest that functional anti-TPO antibodies cause impaired megakaryocyte proliferation and platelet production in patients with ITP. DisclosuresHigashihara:Bristol-Myers Squibb: Research Funding; Baxter: Research Funding; Teijin: Research Funding; Pfizer: Research Funding; Astellas: Research Funding; Yakurt: Honoraria; KyowaHakkoKirin: Honoraria, Research Funding; Chugai: Honoraria, Research Funding; Eisai: Honoraria; GlaxoSmithKline: Honoraria, Research Funding; Nippon Shinyaku: Research Funding; Shionogi: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Celgene: Honoraria; Takeda: Honoraria; Janssen pharma: Honoraria, Research Funding; Alexion: Honoraria; Dainippon Sumitomo: Research Funding; Taisho Tomiyama: Research Funding.

“Almost Bleeding to Death”: The Conundrum of Acquired Amegakaryocytic Thrombocytopenia

Acquired amegakaryocytic thrombocytopenia (AAT) is a rare hematological disorder causing severe thrombocytopenia and bleeding. Previous in vitro studies postulated both cell-mediated suppression of megakaryocytopoiesis in early megakaryocytic progenitor cells and humoral-mediated suppression by anti-thrombopoietin antibodies as possible etiologies of AAT. Patients with AAT usually present with severe bleeding and thrombocytopenia that is unresponsive to steroids and intravenous immunoglobulin (IVIG). Although standard guidelines have not been established for management of AAT, a few case reports have indicated a response to immunosuppressive treatment. The prompt recognition of this disease entity is essential in view of the substantial risk of morbidity and mortality from excessive bleeding. We report a case of AAT successfully treated with equine antithymocyte globulin (ATG) and cyclosporine (CSP).

Anti-erythropoietin and anti-thrombopoietin antibodies induced after administration of recombinant human erythropoietin

Recombinant human erythropoietin (rhEPO) has been successfully used for correcting renal anemia. However, recent studies have raised some concerns about the safety of rhEPO treatment due to its immunogenic side effect — pure red cell aplasia (PRCA). We now report a case of development of anti-EPO neutralizing antibodies (Abs) implicated in thrombocytopenia as well as erythrocytopenia. A 35-year-old man had a history of administering rhEPO (epoetin alfa, epoetin beta and darbepoetin alfa) for 2years to treat renal anemia. The hematological parameters were collected. Anti-EPO, anti-platelet, and anti-thrombopoietin (TPO) Ab assays were performed to test the presence of autoreactive Abs. After performing antibody assays due to severe resistance to rhEPO treatment, a high titer of anti-EPO neutralizing Abs was detected. However, unexpectedly, this patient also showed thrombocytopenia rather than PRCA. We investigated the cause of the marked thrombocytopenia and found anti-TPO Abs in patient serum. To our best knowledge, this is the first report of the development of anti-TPO Abs during rhEPO treatment for anemia.

SLE Thrombocytopenia: From Peripheral Platelet Destruction to Central Hemopoietic Defect

Thrombocytopenia in Systemic Lupus Erythematosus is a common clinical manifestation affecting up to one third of patients in published cohorts. Antiplatelet antibodies, antithrombopoietin antibodies and faulty hemopoiesis have been implicated among other immunologic and non-immunologic causes. This mini review is an uptodate summary of these immunologic phenomena.

Anti-thrombopoietin antibodies suppress megakaryocytic colony formation in vitro in patients with systemic lupus erythaematosus

Autoantibodies against thrombopoietin (TPO) have been implicated in systemic lupus erythaematosus (SLE) thrombocytopoenia.1 2 To investigate their pathophysiological significance, we have evaluated the effect of SLE sera on megakaryocytic colony...

Current Concepts in SLE Thrombocytopenia: From Pathophysiology to Therapeutic Interventions

Thrombocytopenia in Systemic Lupus Erythematosus (SLE) is a common clinical manifestation affecting up to one third of patients in published cohorts. Anti-platelet antibodies have been implicated in its pathogenesis, as sensitized platelets interact with macrophages through the Fc receptor eliminating them from circulation. Non-specific, immunecomplex mediated platelet destruction is also implicated as are antiphospholipid syndrome, thrombotic microangiopathies and hemophagocytic syndrome. A few cases of thrombocytopenia with amegakaryocytic hypoplasia due to antibodies against c-mpl receptor have recently been identified. Pathophysiology has recently been intrigued by frequent findings of anti-thrombopoietin antibodies and faulty hemopoiesis in SLE patients with cytopenias. More specifically, histologic data has shown dysplastic changes and stromal alteration suggesting that bone marrow is a target organ in SLE. Although thrombocytopenia, per se, is a benign complication, with hemorrhagic manifestations being infrequent, it is associated with a more active disease and worse outcome: it marks a subgroup of SLE patients, having a higher risk of irreversible end-organ events throughout their disease course. These patients exhibit a predilection to a distinct pattern of damage, rendering thrombocytopenia a quantitive and qualitative marker of impending damage. Immunosuppressive therapy is required to restore normal platelet counts and treat concomitant organ involvement of other systems. Common therapeutic modalities include corticosteroids, intravenous immunoglobulin (IVIG), cytotoxic agents (mainly cyclophosphamide), immunomodulators (azathioprine) and androgens (Danazol). More recently, B-lymphocyte depletion (anti- CD20 immunotherapy) and mycofenolate mofetil have been successfully used in refractory cases with splenectomy as a last resort should other options fail.

An open‐label, unit dose‐finding study of AMG 531, a novel thrombopoiesis‐stimulating peptibody, in patients with immune thrombocytopenic purpura

Abstract The objective of this open label, phase 1-2, multicentre trial was to evaluate the safety of AMG 531, a novel thrombopoiesis-stimulating peptibody, and its effect on platelet counts in adults with immune thrombocytopenic purpura. Four patients were assigned to each of four unit-dose cohorts: 30, 100, 300 or 500 microg, administered subcutaneously on days 1 and 15 (or day 22 if the day 15 platelet count was >50 x 10(9)/l). Safety was assessed by adverse event (AE) monitoring, clinical laboratory studies and antibody assays. Platelet response was defined as a platelet count double the baseline value and between 50 and 450 x 10(9)/l. Sixteen patients (10 women) were enrolled. The 500-microg cohort was discontinued because the first patient's platelet count became unacceptably high. AEs were generally expected and mild or moderate; the most frequent was headache (eight of 16 patients). Two patients experienced serious AEs related to AMG 531 (severe headache and elevated serum lactic dehydrogenase; thrombocytopenia). Platelet responses occurred with all doses and with a dose equivalent to >/=1 microg/kg in eight of 11 patients. In summary, patients tolerated AMG 531 well at the doses tested. No anti-AMG or antithrombopoietin antibodies were detected. Doses equivalent to >/=1 microg/kg increased platelet counts.

Suspects in the tale of lupus-associated thrombocytopenia

Immunologically mediated thrombocytopenia is a frequent clinical manifestation in patients with systemic lupus erythematosus (SLE). Autoantibodies targeting platelet membrane glucoproteins have a central role in peripheral platelet destruction. Autoantibodies against thrombopoietin are also present in about one-third of patients, but their pathogenetic role is obscure. Thirty-eight serum samples from SLE patients were tested for anti-platelet antibodies, anti-thrombopoietin antibodies and levels of circulating thrombopoietin. Bone marrow histology was also assessed. Thirty-nine per cent of sera displayed anti-thrombopoietin antibodies and 29% had circulating anti-platelet antibodies. Anti-thrombopoietin antibodies were associated with lower thrombopoietin concentrations, and lower mean platelet values in long-term follow-up. Anti-platelet antibodies were present in about 40% of thrombocytopenic and non-thrombocytopenic individuals but were absent in patients who had recovered from thrombocytopenia, supporting their pathogenetic role. Both autoantibodies were absent in control sera from patients with rheumatoid arthritis and primary Sjögren's syndrome. Decreased bone marrow cellularity, normal or low number of hypolobulated, pyknotic megakaryocytes and stromal alterations were prominent findings in thrombocytopenic SLE patients, suggesting a defect in megakaryopoiesis. These findings were not evident in specimens from patients with idiopathic thrombocytopenic purpura who had increased megakaryocytes, normal cellularity and absence of stromal alterations. In conclusion, peripheral destruction due to platelet autoantibodies, anti-thrombopoetin antibodies, lower effective circulating thrombopoetin and impaired compensatory response due to bone marrow damage interact in SLE and thrombocytopenia ensues.

Monoclonal Anti-Thrombopoietin Antibodies Generated by Genetic Immunization

Thrombopoietin (TPO) is a megakaryocyte growth and differentiation factor that is currently being investigated as a therapeutic for cancer patients undergoing myelosuppressive chemotherapy. We generated monoclonal antibodies (MAbs) specific for human thrombopoietin (hTPO) by genetic immunization using an hTPO expression plasmid and an adjuvant plasmid that encodes mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). All genetically immunized mice exhibited a high humoral immune response. Splenocytes from these mice were used to generate hybridomas. Two MAbs, designated 2B9A10 and 4C16B15 (of IgG1 and IgG3 isotypes, respectively), were subsequently selected and produced. They specifically recognized and precipitated recombinant hTPO produced by mammalian cells and were effective in sandwich enzyme-linked immunosorbent assays (ELISAs) for hTPO quantitation. Our results demonstrate that these MAbs should be useful for purification and quantitation of hTPO in clinical and laboratory settings.

Prospective screening of 205 patients with ITP, including diagnosis, serological markers, and the relationship between platelet counts, endogenous thrombopoietin, and circulating antithrombopoietin antibodies.

Immune thrombocytopenia purpura (ITP) is characterized by destruction of circulating platelets and the presence of antiplatelet antibodies. Many of the current immunomodulatory therapies act by reducing platelet destruction and usually do not have a lasting effect. This prospective, exploratory study characterized patients with ITP by identifying their demographic and comorbid clinical factors, use of treatments, serologic markers of autoimmunity, and possible relationships between platelet counts, concentrations of endogenous thrombopoietin (eTPO), and the presence of circulating anti-TPO antibodies. Data including medical history and laboratory evaluations were collected at a single patient visit on 205 patients (19 children, 186 adults). Reported histories revealed a 5% rate of thrombotic/ischemic events. Autoimmune markers including direct antiglobulin test and antinuclear antibodies were found more frequently than in the normal population; antiplatelet antibody testing was not done. eTPO concentrations were comparable to concentrations found in healthy volunteers. Our study confirmed that no significant inverse correlation occurred between circulating concentrations of eTPO and platelet counts in patients with ITP (Spearman r = -0.15). Two of the 205 patients tested (1%) had neutralizing activity of recombinant human TPO in a biological assay; however, this activity was confirmed to be anti-TPO antibody in only 1 patient.

Serum thrombopoietin levels and anti-thrombopoietin antibodies in systemic lupus erythematosus.

Thrombocytopenia is a common phenomenon in patients suffering from systemic lupus erythematosus (SLE). The cause of thrombocytopenia in SLE, however, is poorly understood. In this study, 100 patients with SLE were evaluated for serum thrombopoietin levels, anti-thrombopoietin antibodies and routine laboratory parameters such as peripheral blood counts, parameters of blood chemistry and immunologic parameters of SLE. The median platelet count of SLE patients was 230 g/l and 19 were thrombocytopenic (range 8-148 g/l). Thrombopoietin levels in SLE patients were found to be significantly higher than in healthy controls (n = 96; median, 117 pg/ml vs 64 pg/ml, P < 0.01). When excluding thrombocytopenic SLE patients, thrombopoietin levels in SLE were still above controls (111 pg/ml, P < 0.01). The thrombopoietin levels were correlated to erythrocyte sedimentation rate and ECLAM score of disease activity, and inversely correlated to complement factor C4, but not to the platelet count. Anti-thrombopoietin antibody reactivity was found in 23% of SLE patients. Interestingly, these patients had lower platelet counts than SLE patients without anti-thrombopoietin antibodies (median 174 g/l and 253 g/l, respectively, P < 0.01), but thrombopoietin levels were not significantly different. Taken together, thrombopoietin levels are significantly higher in the sera of SLE patients than in healthy controls and anti-thrombopoietin antibodies are frequently found.