Antibody-mediated Thrombocytopenia Research Articles

Tacrolimus and Danazol Combination As a Second-Line Therapeutic Strategy for Steroid-Resistant or Relapsed Immune Thrombocytopenia: A Prospective, Randomized, Multicenter, Open-Label Study

Introduction: Immune thrombocytopenia (ITP) is an important health concern as a severe autoimmune bleeding disorder characterized by low platelet counts and an increased risk of bleeding. Traditionally, the first-line treatment for ITP involves glucocorticoids. However, a substantial number of patients develop resistance or relapse within the first year of treatment, presenting a formidable challenge for medical practitioners. The consequences of prolonged glucocorticoid therapy, such as severe adverse effects, further necessitate the exploration of alternative, more effective, and safer treatments. In recent years, tacrolimus, a potent calcineurin inhibitor, has emerged as a potential candidate for managing ITP. It has shown promising efficacy against anti-platelet antibody-mediated thrombocytopenia, thus expanding the therapeutic options for resistant or relapsed ITP patients. Our previous work ( BJH 2022) also highlighted the potential of tacrolimus in suppressing the activation of NLRP3 inflammasomes and the apoptosis of MSCs-C+ cells both in vivo and in vitro. This research formed a solid foundation for further investigation into the roles of tacrolimus in ITP. Building on this background, our current research focuses on evaluating the effectiveness and safety of combining tacrolimus and danazol as a second-line therapy for patients with steroid-resistant or relapsed ITP. This study was designed to provide a comprehensive understanding of this therapeutic combination, which could pave the way for optimized treatment regimens for ITP, ultimately improving patient outcomes and quality of life. Methods: This phase 2, randomized, open-label trial involved adult ITP patients demonstrating drug resistance or relapse from seven tertiary medical centers across China. Participants were randomly assigned to receive a 12-week treatment of either tacrolimus (initial dose 0.03 mg/kg/d, maintaining a blood concentration between 3-5 ng/mL) combined with danazol (200 mg bid) or monotherapy with danazol (200 mg bid). The primary endpoint entailed a 6-month sustained response, denoted by maintaining platelet counts > 50×10^9/L without additional ITP-modifying therapies at the 6-month follow-up. Secondary endpoints comprised an initial response, response duration, bleeding scores, and adverse events. This trial is registered with ClinicalTrials (NCT05471050). Results: The study enrolled 90 steroid-resistant or relapsed ITP patients, with 43 assigned to the tacrolimus plus danazol group and 47 to the danazol monotherapy group. The participants in the combination group had a median age of 48 years (range: 30-53), and in the monotherapy group, it was 40 years (range: 28-51). Both groups had a marginally higher proportion of female participants. The median primary ITP duration was two months for both groups. Baseline bleeding scores and bleeding or platelet transfusion instances at enrollment were similar across groups. The results showed a promising improvement in the patient response. The tacrolimus plus danazol group showed a significantly higher 6-month sustained response rate (53%, n=23) than the danazol group (30%, n=14) (odds ratio: 2.17, 95% CI: 1.16-6.13, p=0.032). While there was no significant difference in the response times across the groups, the combination group attained a significantly higher peak platelet count (90 ×10^9/L) than the monotherapy group (70 ×10^9/L) (p=0.031). This suggests that the addition of tacrolimus to the therapy can significantly boost the platelet count, potentially improving the clinical outcomes for the patients. Response durations were comparable in both groups, indicating that the addition of tacrolimus did not negatively impact the duration of response. Last, treatment was well-tolerated with no grade 3 or 4 events or treatment-related deaths reported in both groups. Conclusions: The tacrolimus and danazol combination emerges as a promising second-line treatment for steroid-resistant or relapsed ITP. This regimen demonstrated a higher 6-month sustained response rate and peak platelet count compared to danazol monotherapy, substantiating its potential role as a novel treatment strategy for adult ITP patients.

Real-World Study of the Use of Iguratimod to Treat Chronic/Refractory ITP

Introduction: Immune thrombocytopenia (ITP) is an acquired, organ-specific, autoimmune disease and one of the most common bleeding disorders that seriously endangers human health. Iguratimod is a small molecule compound that is widely used as a novel antirheumatic drug in the treatment of rheumatoid arthritis in China and Japan. Iguratimod inhibits the production of several inflammatory cytokines, including IL-1, IL-6, IL-8, and tumor necrosis factor (TNF), and reduces the production of immunoglobulins. Th1 and Th17 cell counts were significantly reduced in patients with rheumatoid arthritis after treatment with iguratimod. The low long-term remission rate of ITP makes the investigation of its pathogenesis and the search for new treatments important clinical problems to be solved. Abnormal T-cell function and anti-platelet antibody production play important roles in the pathogenesis of ITP, and iguratimod is a therapeutic agent that could potentially be used to treat ITP. Previous clinical studies have shown that iguratimod can increase platelet levels in patients with dry syndrome. The potential of iguratimod for treating anti-platelet antibody-mediated thrombocytopenia in mice with ITP by modulating T-cell differentiation suggests that iguratimod may be a new approach for the prevention and treatment of ITP. Recent retrospective clinical studies and case reports have also shown that the use of iguratimod in autoimmune diseases is associated with good safety and efficacy. In conclusion, the aim of this study was to investigate the effect of iguratimod on the treatment of ITP. Methods: The study began in December 2020 and is ongoing. A total of 76 patients who were treated with iguratimod for at least 3 months were selected. Of these, 51 patients were treated for more than 6 months, and 30 patients were treated for at least 12 months. Five patients withdrew from observation after 3 months: two patients due to NR, three patients due to epidemic and financial reasons, and no patients due to AE. Key secondary endpoints included initial response by Day 14 (OR, platelet count ≥30×109/L, at least a 2-fold increase in baseline platelet count, and absence of bleeding; and CR, platelet count ≥ 100×109/L), duration of response, bleeding scores, and adverse events (AEs). This study was registered with ClinicalTrials.gov (). Results: At baseline, 73.5% of the patients reported symptoms of grade 1 to 4 bleeding. After 3 months of treatment, the proportion of patients with any bleeding symptoms decreased to 61.8% and remained below baseline throughout the study period. At baseline, 63 patients reported the use of concomitant medications, which decreased by 36.5% between 3 and 6 months. The most frequently discontinued or reduced medications were glucocorticoids and rituximab. All the patients took the medication in strict accordance with the instructions and medical advice. The patients were monitored monthly to assess blood, liver, kidney function, and no abnormalities in laboratory tests, such as elevated transaminases or lymphocytopenia, were observed. Conclusions: Iguratimod has good efficacy and safety in the treatment of ITP and has the potential to become a new treatment for ITP.

Comments on Thrombosis After Vaccination: The Leader Sequence of the Spike Protein Might Be Responsible for Thrombosis and Antibody-Mediated Thrombocytopenia.

Comments on Thrombosis After Vaccination: The Leader Sequence of the Spike Protein Might Be Responsible for Thrombosis and Antibody-Mediated Thrombocytopenia.

Tacrolimus Plus High-Dose Dexamethasone Versus High-Dose Dexamethasone Alone As First-Line Treatment for Adult Immune Thrombocytopenia: The Phase 2, Open Label, Randomized Trial (TARGET 020)

▪IntroductionImmune thrombocytopenia (ITP) is an acquired, organ-specific, autoimmune disease and one of the most common bleeding disorders seriously endangering human health. Glucocorticoids and intravenous immunoglobulin are first-line treatments recommended by guidelines for patients with ITP. However, approximately 50%-85% of patients relapse during the first year of treatment. In addition, long-term use of glucocorticoids increases the risk for dose- and time-dependent glucocorticoid-related complications and serious side effects. Therefore, in-depth studies investigating new solutions for the first-line treatment of ITP are urgently needed. Tacrolimus is a calcineurin inhibitor, which forms a complex by binding to FK506-binding protein. It is currently widely used in the prevention of graft-versus-host disease for organ transplantation as well as for the treatment of autoimmune diseases. In addition to recent retrospective studies and case reports demonstrating its effectiveness in ITP, tacrolimus has been shown to improve anti-platelet antibody-mediated thrombocytopenia in mice, suggesting it may be a potential treatment for ITP. The aim of this study was to compare two first-line treatment options for ITP-a standard glucocorticoid-only regimen versus tacrolimus in combination with a standard glucocorticoid regimen-to determine which could help patients achieve stable platelet counts faster and experience a longer duration of remission.MethodsThis open-label, randomized, phase 2 trial, enrolled adult ITP patients from seven different tertiary medical centers in China. Elderly patients had confirmed, newly diagnosed, treatment-naive ITP, platelet counts <30×10 9/L, or < 50×10 9/L and significant bleeding symptoms (World Health Organization bleeding scale ≥ 2). Eligible patients were randomly assigned 1:1 with an interactive web-based response system to receive either oral tacrolimus (initial 0.03 mg/kg/day and maintain blood concentration at 3-5 ng/mL for 12 weeks) plus high-dose dexamethasone (HD-DXM) or HD-DXM monotherapy for 12 weeks. DXM (40 mg) was administered orally daily for 4 consecutive days to both study arms. The 4-day course of DXM was repeated on days 11-14 in patients who lacked response by day 10. The primary endpoint was 6-month sustained response (SR), defined as platelet count maintained >50×10 9/L without any additional ITP-modifying therapy at the 6-month follow-up. Key secondary endpoints included initial response by day 14 (OR, platelet count ≥30×10 9/L and at least 2-fold increase in baseline platelet count and absence of bleeding; and CR, platelet count ≥ 100×10 9/L), duration of response, bleeding scores, and adverse events (AEs). This trial was registered with ClinicalTrials.gov (NCT04747080).ResultsTotal 140 patients newly diagnosed with ITP were randomly assigned to either the tacrolimus plus HD-DXM (n=72) or HD-DXM monotherapy (n=68) groups. At the 6-month follow-up, the proportion of patients exhibiting SR was significantly higher in the tacrolimus plus HD-DXM group than in the HD-DXM monotherapy group (65.3% vs 42.6%, p= 0.007). Of the 140 patients with ITP (males accounted for 48.6%), the mean age was 32.8 years, the mean platelet count was 16.7×10 9/L. The combination group exhibited a higher 14-day early remission rate than the monotherapy group (76.4% vs 55.9%, P=0.001). Significantly fewer treatment failures occurred in patients randomly assigned to the combination group(19.4% vs 38.2%, P=0.0014). During the follow-up period, fewer patients in the combination group experienced relapse than in the monotherapy group; the median time to relapse was 77 days (Tacrolimus+HD-DXM) vs 36 days (HD-DXM). The combination group exhibited a lower proportion of bleeding events and a lower bleeding score. The incidence of serious AEs, rescue therapy, and treatment side effects were similar between the two groups, and treatment was well tolerated by all patients, with no grade 4 AEs or treatment-related deaths reported. There was no statistically significant difference in the incidence of treatment-related AEs between the two groups.ConclusionsLow-dose tacrolimus plus HD-DXM was an effective and safe treatment for ITP as first-line therapy and elicited a sustained prolonged response in adults. This therapy may be a new treatment option for adult patients with ITP. DisclosuresNo relevant conflicts of interest to declare. OffLabel Disclosure:It includes information or discussion of off-label drug use of tacrilimus.

Development of gates to measure the immature platelet fraction in C57BL/6J mice using the Sysmex XN-V series multispecies hematology analyzer.

The immature platelet fraction (IPF) is a measure of newly released platelets, which has been used as a marker of platelet production in multiple human studies but is not widely available in multispecies analyzers. We developed gates to measure the IPF in diluted and undiluted murine blood samples on the Sysmex XN-1000V multispecies hematology analyzer. IPF gates were created using undiluted and diluted (1/10) blood samples obtained from adult and newborn (postnatal day 10, P10) C57BL/6J wild-type (WT) mice, and from 3 murine models of thrombocytopenia: c-MPL-/- mice, which lack the thrombopoietin receptor (hyporegenerative); antibody-mediated thrombocytopenia; and acute inflammation-induced thrombocytopenia. P10 mice were chosen because, at their size, we could consistently obtain (by terminal phlebotomy) the blood volume needed to run an undiluted sample. The undiluted blood IPF gate successfully differentiated between mechanisms of thrombocytopenia in both adult and P10 mice. For diluted samples, 2 IPF gates were generated: a thrombocytopenic (T) gate, which performed well in samples with platelet counts (PCs) <800 × 109/L in adult mice and <500 × 109/L in newborn mice, and a non-thrombocytopenic (NT) gate, which performed well in samples with PCs above these thresholds. PCs and IPFs measured in diluted blood using these gates agreed well with those measured in undiluted blood and had good reproducibility. These diluted gates allow for the accurate measurement of PCs and IPFs in small (10 µL) blood volumes, which can be obtained easily from adult and newborn mice as small as P1 to assess platelet production serially.

The necroptotic cell death pathway operates in megakaryocytes, but not in platelet synthesis

Necroptosis is a pro-inflammatory cell death program executed by the terminal effector, mixed lineage kinase domain-like (MLKL). Previous studies suggested a role for the necroptotic machinery in platelets, where loss of MLKL or its upstream regulator, RIPK3 kinase, impacted thrombosis and haemostasis. However, it remains unknown whether necroptosis operates within megakaryocytes, the progenitors of platelets, and whether necroptotic cell death might contribute to or diminish platelet production. Here, we demonstrate that megakaryocytes possess a functional necroptosis signalling cascade. Necroptosis activation leads to phosphorylation of MLKL, loss of viability and cell swelling. Analyses at steady state and post antibody-mediated thrombocytopenia revealed that platelet production was normal in the absence of MLKL, however, platelet activation and haemostasis were impaired with prolonged tail re-bleeding times. We conclude that MLKL plays a role in regulating platelet function and haemostasis and that necroptosis signalling in megakaryocytes is dispensable for platelet production.

Tacrolimus ameliorates thrombocytopenia in an ITP mouse model.

Immune thrombocytopenia (ITP) is an autoimmune disease characterized by lower platelet count resulting from immune cells-mediated platelet clearance. Tacrolimus is an immunosuppressive agent which selectively inhibits T cell activation. Whether tacrolimus plays a role in ITP remains unclear. This study aimed to investigate the effect of tacrolimus on ITP in mice. An ITP mouse model was established by injection of rat anti-mouse integrin GPIIb/CD41 immunoglobulin and treated with tacrolimus followed by isolation of peripheral blood mononuclear cells and plasma. The mRNA expression of T-bet, GATA3, and Foxp3 was measured by RT-PCR, and level of IFN-γ, IL-12p70, IL-4, IL-13, and TGF-β in plasma was measured by ELISA. Tacrolimus inhibited antiplatelet antibody-mediated platelet clearance in ITP mouse model. Meanwhile, tacrolimus-treated ITP mice displayed a significant decrease in the mRNA expression of T-bet and plasma level of IFN-γ and IL-12p70 compared with ITP mice but without differences when compared with normal mice. Furthermore, the expression of GATA3, Foxp3, and plasma level of IL-4 and TGF-β were upregulated in tacrolimus-treated ITP mice without significant differences to normal mice (except TGF-β). Tacrolimus prevents antiplatelet antibody-mediated thrombocytopenia in ITP mice possibly through regulating T cell differentiations, suggesting it might be a novel approach for preventing ITP.

GPVI signaling is compromised in newly formed platelets after acute thrombocytopenia in mice

AbstractAt sites of vascular injury, exposed subendothelial collagens trigger platelet activation and thrombus formation by interacting with the immunoreceptor tyrosine-based activation motif (ITAM)–coupled glycoprotein VI (GPVI) on the platelet surface. Platelets are derived from the cytoplasm of megakaryocytes (MKs), which extend large proplatelets into bone marrow (BM) sinusoids that are then released into the bloodstream, where final platelet sizing and maturation occurs. The mechanisms that prevent activation of MKs and forming proplatelets in the collagen-rich BM environment remain largely elusive. Here, we demonstrate that newly formed young platelets (NFYPs) released after antibody-mediated thrombocytopenia in mice display a severe and highly selective signaling defect downstream of GPVI resulting in impaired collagen-dependent activation and thrombus formation in vitro and in vivo. The diminished GPVI signaling in NFYPs is linked to reduced phosphorylation of key downstream signaling proteins, including Syk, LAT, and phospholipase Cγ2, whereas the G protein–coupled receptor and C-type lectin-like receptor 2 signaling pathways remained unaffected. This GPVI signaling defect was overcome once the platelet counts were restored to normal in the circulation. Overall, these results indicate that the GPVI-ITAM signaling machinery in NFYPs after antibody-mediated thrombocytopenia only becomes fully functional in the blood circulation.

Zytopenien

Hematological alterations can often be observed during rheumatic diseases. The effects can be clinically severe, ranging from anemia of different grades of severity, through increased risk of hemorrhage due to thrombocytopenia up to severe infections as a result of high-grade leukocytopenia. The clinical sequelae for patients are predominantly determined by the extent of cytopenia. The underlying disease itself can initially be considered as the cause. Examples are anemia as a result of chronic inflammation, antibody-mediated thrombocytopenia as in systemic lupus erythematosus (SLE) or granulocytopenia within the framework of Felty's syndrome. Immunosuppressive treatment also often leads to alterations in the blood constituents. Although some substances, such as cyclophosphamide can suppress all three cell types, there are also selective effects, such as isolated thrombocytopenia under treatment with tocilizumab and JAK inhibitors. The differential diagnostic clarification of cytopenia can be difficult and necessitates a systematic work-up of the course of the disease and the subsequent treatment. The reviews of anemia, leukocytopenia and thrombocytopenia presented here summarize the most important components of the differentiation of hematological alterations in patients with rheumatic diseases.

TULA-2 (T-Cell Ubiquitin Ligand-2) Inhibits the Platelet Fc Receptor for IgG IIA (FcγRIIA) Signaling Pathway and Heparin-Induced Thrombocytopenia in Mice.

The objective of this study is to investigate the role of T-cell ubiquitin ligand-2 (TULA-2) in the platelet Fc receptor for IgG IIA (FcγRIIA) pathway and in the pathogenesis of heparin-induced thrombocytopenia (HIT). HIT is a life-threatening thrombotic disease in which IgG antibodies against the heparin-platelet factor 4 complex activate platelets via FcγRIIA. We reported previously differential expression of TULA-2 in human population was linked to FcγRIIA responsiveness. In this study, we investigated the role of TULA-2, a protein phosphatase, in the FcγRIIA pathway and HIT pathogenesis by crossing TULA-2-/- mice with transgenic FcγRIIA +/+ mice. Ablation of TULA-2 resulted in hyperphosphorylation of spleen tyrosine kinase, linker for the activation of T cells, and phospholipase Cγ2 in platelets via FcγRIIA activation. Platelet integrin activation, granule secretion, phosphatidylserine exposure, and aggregation were also enhanced in TULA-2-/- murine platelets. Compared with wild-type mice, TULA-2-/- mice showed aggravated antibody-mediated thrombocytopenia, augmented thrombin generation, and shortened tail bleeding time. In contrast, there was no significant difference between TULA-2-/- and TULA-2+/+ platelets in platelet spreading and clot retraction. Of note, heterozygous TULA-2+/- mice, whose platelets contained 50% as much protein as the TULA-2+/+ platelets, showed significantly increased platelet reactivity and more severe thrombocytopenia in vivo compared with TULA-2+/+ mice. Together, the data demonstrate that not only the absence of TULA-2 but also the relative level of TULA-2 expression modulates FcγRIIA-mediated platelet reactivity and HIT in vivo. TULA-2 expression could be a valuable marker for HIT and inhibiting TULA-2 may serve as a potential therapy to reverse the bleeding adverse effect of anticoagulants.

Platelet GPIba Is Important for Thrombopoietin Production and Thrombopoietin-Induced Platelet Generation

Background: Platelets are generated from megakaryocytes in the bone marrow and play a key role in hemostasis. GPIba is the major subunit in the GPIb-IX complex expressed on platelets and megakaryocytes (MKs). Deficiency of GPIba results in Bernard Soulier Syndrome (BSS), a severe bleeding phenotype characterized by thrombocytopenia and abnormal platelet morphology.The generation of megakaryocytes and platelets is regulated by the hematopoietic growth factor, thrombopoietin (TPO). In patients with thrombocytopenia, the TPO receptor agonists and recombinant TPO (rTPO) have been used to stimulate thrombopoiesis. TPO production primarily occurs in liver parenchymal cells with minor amounts synthesized in other tissues. The major clearance mechanism of TPO from circulation is via interaction between TPO and its receptor, c-Mpl, following which the c-Mpl-TPO complex undergoes internalization and degradation. However, whether it is the production or clearance of TPO the main regulator of circulating TPO levels has been debated but remains unclear.The prevailing theory was that hepatic TPO is generated constitutively, and the levels of circulating TPO are fine-tuned by its uptake and metabolism in platelets and megakaryocytes. Recently, this concept was modified by studies in Ashwell-Morell receptor (AMR) deficient mice, which highlighted the important role played by platelets in the regulation of hepatic TPO mRNA expression. As the most glycosylated platelet surface protein, GPIba contributes to chilled platelets clearance viahepatocytes, as well as antibody-mediated thrombocytopenia. However, the relationship between GPIba and plasma TPO has not been exploredMethods and Results: We observed a significant decrease (approx. 3 fold) of TPO levels in both plasma and sera of GPIba-/- mice compared to wild type (WT) controls. Given the enlarged size of GPIba-/- platelets compared to WT, we investigated whether the low circulating TPO levels could be due to increased TPO clearance. Interestingly, we found similar levels of intracellular TPO in WT and GPIba-/- platelets, and the capacity to adsorb TPO in vitro was not enhanced. Further, we observed GPIba-/- platelets, surprisingly, have less c-Mpl expression compared to WT. Therefore, the decreased circulating TPO levels in GPIba-/- mice is unlikely due to enhanced TPO clearance.We next quantified TPO mRNA from hepatic liver cells as they are known as the major source of TPO. Consistent with the low circulatory TPO levels, we found reduced hepatic TPO mRNA in GPIba-/- mice, suggesting a deficiency in hepatic TPO generation. To determine whether exogenous platelet transfusions could influence steady-state hepatic TPO production, we transfused WT or GPIba-/- mice with WT or GPIba-/- platelets. Post-transfusion of WT, but not GPIba-/- platelets elevated TPO levels to around 150% in GPIba-/- mice. Additionally, our preliminary data showed that co-culture of hepatocytes with WT, but not GPIba-/- platelets in vitro increased hepatic TPO mRNA expression, further supporting a role for GPIba in TPO generation.To test whether TPO can equally induced platelet generation in GPIba-/- and WT mice. We injected rTPO into the two groups of mice, and found that in contrast to WT controls, rTPO did not significantly increase the platelet number of GPIba-/- mice in the circulation.Conclusion: Our data demonstrated that GPIba is important for TPO generation in liver and is necessary for TPO-induced platelet generation. These discoveries may have significant implications in TPO therapy in ITP. DisclosuresNo relevant conflicts of interest to declare.

CD8+ T Cells Are Predominantly Protective In a Murine Model Of ITP and Is Required For Effective Steroid Therapy

BackgroundImmune thrombocytopenia (ITP) is a common bleeding disorder characterized by autoantibody mediated destruction of autologous platelets. The predominant autoantibody detected in ITP patients target the platelet Glycoprotein (GP)IIbIIIa, however autoantibodies can only be detected in 50-70% of patients. Cytotoxic CD8+ T-cells (CTL) may therefore also be a significant contributing factor for thrombocytopenia, either through direct cytotoxicity against platelets, or decreasing platelet production through interaction with megakaryocytes in the bone marrow. Nevertheless, whether CD8+ T cell mediated cytotoxicity significantly contributes to thrombocytopenia in ITP is controversial. Interestingly, CD8+ regulatory T cells have been demonstrated to play significant roles in other autoimmune diseases, but their function in the context of ITP has not been adequately examined. Methods and ResultsWe developed both passive and active murine models to investigate mechanisms of pathogenesis and steroid treatment of ITP. In the passive model, we injected anti-b3 or anti-GPIb antibodies to induce thrombocytopenia; this causes transient antibody-mediated thrombocytopenia. We found that a single intraperitoneal (IP) injection of steroids post-antibody injection was effective at rescuing platelet counts. We also adapted an active model of ITP whereby wild-type (WT) BALB/c mice were transfused with splenocytes from WT platelet immunized β3-/- mice to induce thrombocytopenia. This model encompasses both antibody and cell-mediated ITP and causes sustained thrombocytopenia in the mice. In this model, we found steroid treatment administered either orally or through IP-injection were equally efficacious at ameliorating thrombocytopenia. The successful use of steroids to treat thrombocytopenia in these animal models is representative of the therapeutic effects of steroid treatment seen in human ITP patients.To study the role of CD8+ T-cells in the response to steroid treatments in ITP, we depleted CD8+ T-cells from splenocytes prior to transfusion into WT mice. Unexpectedly, we found CD8+ T cell depleted splenocyte (lacking in CTL cells), engrafted mice had lower, but not higher, platelet counts. They were also less responsive to dexamethasone treatment compared to non-depleted engrafted mice. Furthermore, transfusion of splenocytes from immunized β3-/- mice in conjunction with antigen-primed CD8+ T-cells (isolated from immunized β3-/- splenocytes) was able to rescue platelet counts in WT mice. Co-transfusion of non-primed CD8+ T cells from β3-/- mice could not rescue platelet counts. These results indicate that platelet-antigen specific CD8+ Tregs play a dominant protective role in attenuating platelet clearance. In further support of our observations, we detected significantly increased CD8+CD25+Foxp3+ Treg percentages in the blood, thymus and spleen of immunized β3-/- mice. Further in vitro splenocyte cultures demonstrated putative regulatory mechanisms of CD8+CD25+ Tregs from immunized β3-/- splenocytes. We found CD8+CD25+ Tregs significantly inhibited CD4+ T and CD19+ B cell proliferation, platelet apoptosis, and platelet associated IgG production in the presence of platelet-antigens, but increased the secretion of the anti-inflammatory cytokine IL-10. ConclusionTo the best of our knowledge, these are the first reported animal models of steroid treatment of ITP. We demonstrated that steroid therapy was effective and CD8+ T cells are required for this efficacy. We further unveiled a population of CD8+ regulatory T-cells in the CD8+ T cell populations, which were able to rescue platelet counts. This suggests that CD8+CD25+Foxp3+Treg may impart a predominantly protective role, overcoming the cytotoxic function of CD8+ T-cells in ITP. These data provides significant insights into the understanding of immunopathogenesis of ITP, which may be important in designing effective therapy including the potential usage of CD8+ Tregs as a cellular therapeutic method against ITP. Disclosures:No relevant conflicts of interest to declare.

Gammaherpesvirus latency induces antibody-associated thrombocytopenia in mice

Human herpesviruses establish lifelong latency. Viral recrudescence can lead to the development of cancers, immunoproliferative disorders, transplantation complications, and thrombocytopenia. Although platelet-specific autoantibodies have been reported in patients infected with the Epstein–Barr virus (EBV), the mechanisms by which thrombocytopenia is induced remain unclear, as do the relative contributions of lytic viral replication and latent viral gene expression. The human gammaherpesviruses are tightly restricted in their ability to infect other mammals, so they are difficult to study in live animal models. Here we show that infection of mice with murine gammaherpesvirus-68 (γHV68), a rodent-specific pathogen closely related to EBV, induces the production of platelet-binding antibodies and causes thrombocytopenia. Infection of antibody-deficient mice does not lead to thrombocytopenia, indicating the platelet decrease is mediated by antibody. Additionally, infection with a latency-null recombinant γHV68 does not induce thrombocytopenia, suggesting factors associated with viral latency drive the infection-induced antibody-mediated thrombocytopenia. These studies describe an important animal model of gammaherpesvirus-induced autoimmune thrombocytopenia and demonstrate that this pathology is mediated by antibody and dependent on viral latency. This model will allow studies of the underlying mechanisms of disease progression and the testing of therapeutic strategies for the alleviation of virus-induced thrombocytopenia.

Antibody-Independent Thrombocytopenia in Lactate Dehydrogenase-Elevating Virus-Infected Mice

Previously we demonstrated that antibody-mediated thrombocytopenia is strongly enhanced by lactate dehydrogenase-elevating virus (LDV) infection. Here we report that mice infected with LDV develop a moderate thrombocytopenia, even in the absence of immunoglobulins or Fc receptors. A similar decrease of platelet counts was observed after mouse hepatitis virus infection. LDV-induced type I interferon-independent thrombocytopenia was partly suppressed by treatment with clodronate-containing liposomes. Therefore, we conclude that the thrombocytopenia results from increased phagocytosis of nonopsonized platelets by macrophages.

Papel de los anticuerpos antiplaquetarios en la infección viral: una revisión sistemática de la literatura

Introduction. Thrombocytopenia is a frequent phenomenon in viral infections. Peripheral platelet destruction mediated by anti-platelet antibodies has been one of the proposed causal mechanisms. Objective. Results were collected and analyzed from published studies on associations of human viral infections on anti-platelet antibodies and total platelet counts. Materials and methods. A PubMed search was conducted using the following terms: Viral infection (OR Virus diseases) AND antiplatelet antibody (OR thrombocytopenia) AND HIV (OR measles OR dengue OR chickenpox OR varicella OR Epistein Barr OR mumps OR rubella). Two hundred eighteen reference hits were obtained, 65 of which were relevant to this review. Results. Antiplatelet antibody-mediated thrombocytopenia has been documented in cases of HIV, measles, dengue, chickenpox, Epstein-Barr, mumps and rubella. Moreover, the presence of these antibodies has been associated with severity the disease and thrombocytopenia in viral infections. Conclusions. Although the presence of antiplatelet antibodies was not the only mechanism for explaining the thrombocytopenia developed in these viral infections, their presence was associated with severity of thrombocytopenia and with the clinical presentation of these patients.

Roll of antibodies antiplatelets in viral infection: a systematic review of literature

Thrombocytopenia is a frequent phenomenon in viral infections. Peripheral platelet destruction mediated by anti-platelet antibodies has been one of the proposed causal mechanisms. Results were collected and analyzed from published studies on associations of human viral infections on anti-platelet antibodies and total platelet counts. A PubMed search was conducted using the following terms: Viral infection (OR Virus diseases) AND antiplatelet antibody (OR thrombocytopenia) AND HIV (OR measles OR dengue OR chickenpox OR varicella OR Epistein Barr OR mumps OR rubella). Two hundred eighteen reference hits were obtained, 65 of which were relevant to this review. Antiplatelet antibody-mediated thrombocytopenia has been documented in cases of HIV, measles, dengue, chickenpox, Epstein-Barr, mumps and rubella. Moreover, the presence of these antibodies has been associated with severity the disease and thrombocytopenia in viral infections. Although the presence of antiplatelet antibodies was not the only mechanism for explaining the thrombocytopenia developed in these viral infections, their presence was associated with severity of thrombocytopenia and with the clinical presentation of these patients.

Serious Adverse Drug Reactions (sADRS) Associated with Hematopoietic Growth Factors: A Systematic Review From the Southern Network on Adverse Reactions (SONAR) Program

AbstractAbstract 2555Erythrpoiesis stimulating agents (ESAs) and the granulocyte hematopoietic growth factors granulocyte colony stimulating factor (G-CSF) and pegylated G-CSF revolutionized supportive care approaches to anemia and neutropenia, respectively. Thrombopoietin-receptor agonists improve antibody-mediated thrombocytopenia management. For these drugs, as with all agents, a comprehensive understanding of sADRs is important, however, often difficult to do. We reviewed sADRS described in meta-analyses and web-site materials maintained by regulatory agencies and manufacturers reports (1989 to July 2010; n=2,094). Overall, 28 systematic reviews, six package inserts, three medication guides, four FDA Risk Evaluation and Mitigation Systems (REMS) materials, 16 Summaries of Product Characteristics/Product Assessment Reports were included. For ESAs, common sADRs associated with increased relative risks (RRs) included: mortality among cancer patients with chemotherapy-associated anemia or cancer patients not receiving active treatment (RRs: 1.1–1.2); cerebrovascular and cardiovascular events among chronic kidney disease patients ((RRs: 1.3, 1.9), and venous thromboembolism in the cancer setting (RRs;16-1.7). For G-CSF/pegylated-GCSF, rare instances of myelodysplasia/acute myeloid leukemia (MDS/AML) associated with statistically increased RRs were reported among breast cancer patients receiving chemotherapy in trials reported from one NCI-sponsored clinical trials group, one meta-analysis, and one review of Medicare/SEER databases (RR: 1.9–2.1), although the absolute risks were very small (< 2 per 100,000 treated patients). Other rarely reported sADRs (total N < 20) included splenic rupture following G-CSF stimulation of healthy peripheral blood stem donors. Bone marrow fibrosis and collagen deposition were reported with thrombopoietin receptor agonists, although the implications of this finding is uncertain. Also, instances of VTEs were reported among persons who developed high platelet counts following administration of these thrombopoeitin receptor agonists. SADRs are commonly associated with ESA administration in a range of settings, are uncommon occurrences with G-CSF/pegylated G-CSF, and are not well characterized for thrombopoieitin receptor agonists. Formal pharmacovigilance initiatives are ongoing, including FDA mandated REMS with restricted distribution requirement for ESAs in the cancer setting (APPRISE) and the NEXUS and the PROMACTA CARES programs for thrombopoeitin receptor agonists.ESAs (Cancer)ESAs (CKD)G-CSF/Pegylated G-CSFVTEMortalityCerebrovascular and CVDAML/MDSRR1.57 and 1.671.10 to 1.171.34 and 1.922.14 and 1.9295% CI1.31–1.87 and 1.35–2.061.02–1.20 and 1.06–1.301.03–1.74 and 1.38–2.261.12–4.08 and 1.19–3.07Baseline RiskAbsolute RiskCommonCommonCommonRare (2/100,000) Disclosures:Bennett:Pfizer: Consultancy.

The Inhibitory Fc[gamma] Receptor is Unnecessary for IVIG Efficacy

AbstractIntravenous gamma globulin (IVIG) is used as an effective therapy for many different autoimmune and inflammatory diseases. The current paradigm for the mechanism underpinning anti-inflammatory effects links IVIG administration to increased expression and activity of the inhibitory Fc[gamma] receptor (Fc[gamma]RIIB) on splenic macrophages. This hypothesis has been disputed in the literature. Here we show that IVIG administered in the context of passive antibody-mediated thrombocytopenia to be therapeutically efficacious in both splenectomized and unmanipulated Balb/c mice despite no upregulation of Fc[gamma]RIIB mRNA in the spleen. Moreover, IVIG effectively ameliorated immune thrombocytopenia (ITP) in Fc[gamma]RIIB-deficient Balb/c mice, but not in Fc[gamma]RIIB-/- or Fc[gamma]RIIB+/+ control mice with a C57BL6/129S background. Our results demonstrate that Fc[gamma]RIIB is not relevant to the beneficial effects of IVIG. We anticipate that investigators will now shift their research focus away from Fc[gamma]RIIB and seek out other possible mechanism(s) to explain the plethora of diseases and conditions treatable with IVIG.

Antibody- and Cell-Mediated Immune Thrombocytopenia Are Differentially Sensitive to Intravenous Gammaglobulin Therapy

Immune thrombocytopenic purpura (ITP) is a bleeding disorder characterized by IgG autoantibody-opsonized platelets prematurely being destroyed in the spleen although recent evidence suggests that thrombocytopenia in perhaps as many as 40% of patients with ITP can be mediated by CD8+ T cells. Although several animal models of immune thrombocytopenia have been developed, few are induced by platelet-specific autoimmune mechanisms nor have any demonstrated cell-mediated platelet destruction. We developed a murine model of ITP by first immunizaing GPIIIa (CD61) knockout (KO) mice against wildtype (WT) CD61+ platelet transfusions and subsequently transferring their splenocytes (5×104 cells/transfer) intraperitonealy into irradiated SCID mouse recipients. The SCID mice developed significant bleeding mortality and thrombocytopenia within 2 weeks post-transfer. Lower doses (<104) of immune splenocytes transferred induced thrombocytopenia in the SCID mouse recipients with no mortality. Depletion of lymphocyte subsets in the splenocytes before transfer showed that both CD19+ B cell (antibody)- and CD8+ T cell (cell)-mediated thrombocytopenia existed and that bleeding mortality was particularly associated with antibody-mediated thrombocytopenia. Treatment of the SCID mouse recipients bi-weekly with 2g/kg intravenous gammaglobulins (IVIg) demonstrated that IVIg was beneficial in eliminating bleeding mortality and thrombocytopenia only in the recipients displaying the antibody-mediated form of ITP. Cell-mediated bleeding mortality and thombocytopenia was completely resistant to IVIg therapy. This model suggests a potential reason why some patients with ITP fail IVIg therapy and may aid in the development of new therapeutics for individuals inflicted with cell mediated thrombocytopenia.

A New Murine Model of Immune Thrombocytopenia: Evidence of Both Antibody- and CD8+ T Cell-Mediated Platelet Destruction.

Immune thrombocytopenic purpura (ITP) is an autoimmune-mediated bleeding disorder in which platelets are opsonized by autoantibodies and prematurely destroyed by Fc receptor (R)-mediated phagocytosis in the reticuloendothelial system (RES). Although the pathogenesis of the disorder has been considered to be primarily antibody mediated, recent reports have indicated that cell-mediated (e.g. CD8+ T cell) mechanisms can also lead to platelet destruction at least in those patients with ITP who have no detectible antibodies. To study these mechanisms, we developed a murine model of immune thrombocytopenia by transferring spleen cells (SC) from GPIIIa-knockout (KO) mice immunized against wild type (WT; GPIIIa-positive) platelets into syngeneic severe combined immunodeficient (SCID) mice. Results show that compared with naive SC, transfer of as few as 5x104 immune GPIIIa-KO SC caused significant thrombocytopenia and a bleeding diathesis and death in 60 percent of recipient SCID mice within 10 days post-transfer. Bleeding occurred primarily in the gut, lungs, subcutaneous tissues and brain. When the immune GPIIIa-KO SC were first depleted of CD4+T cells and transferred, thrombocytopenia or bleeding mortality occurred. In contrast, CD8+ T cell depletion of the GPIIIa-KO SC before transfer did not affect their ability to cause thombocytopenia nor bleeding. Interestingly, depletion of CD19+ B cells before transfer did not affect the ability of the SC to induce thrombocytopenia but prevented all bleeding. These results suggest that both antibody-mediated and CD8+ T cell-mediated platelet destruction occur in this animal model of immune thrombocytopenia, however, it appears that only antibody-mediated thrombocytopenia is associated with bleeding mortality. Because the immune thrombocytopenia is against a platelet-specific antigen e.g. GPIIIa (like in ITP), it will not only allow for the study of disease pathogenesis but may also be important for testing new immunospecific therapeutics to increase platelet counts in patients with ITP.