Vaccine-Induced Immune Thrombotic Thrombocytopenia (VITT): Targeting Pathomechanisms with Bruton Tyrosine Kinase Inhibitors.

A series of cases with rare thromboembolic incidents including cerebral sinus vein thrombosis (some of them fatal) and concomitant thrombocytopenia occurring shortly after vaccination with the coronavirus disease 2019 (COVID-19) vaccine AZD1222 (Vaxzevria) have caused significant concern and led to its temporary suspension in many countries. Immediate laboratory efforts in four of these patients have identified a tentative pathomechanism underlying this syndrome termed initially vaccine-induced prothrombotic immune thrombocytopenia (VIPIT) and renamed recently vaccine-induced immune thrombotic thrombocytopenia (VITT). It encompasses the presence of platelet-activating antibodies to platelet factor-4/heparin complexes, possibly emulated by polyanionic constituents of AZD1222, and thus resembles heparin-induced thrombocytopenia (HIT). Because these immune complexes bind and activate platelets via Fcγ receptor IIA (FcγRIIA), high-dose intravenous immunoglobulin G has been suggested for treatment of VITT in addition to non-heparin anticoagulants. Here we propose inhibitors of Bruton tyrosine kinase (Btk) approved for B cell malignancies (e.g., ibrutinib) as another therapeutic option in VITT, as they are expected to pleiotropically target multiple pathways downstream of FcγRIIA-mediated Btk activation, for example, as demonstrated for the effective inhibition of platelet aggregation, dense granule secretion, P-selectin expression and platelet-neutrophil aggregate formation stimulated by FcγRIIA cross-linking. Moreover, C-type lectin-like receptor CLEC-2- and GPIb-mediated platelet activation, the interactions and activation of monocytes and the release of neutrophil extracellular traps, as encountered in HIT, could be attenuated by Btk inhibitors. As a paradigm for emergency repurposing of approved drugs in COVID-19, off-label use of Btk inhibitors in a low-dose range not affecting haemostatic functions could thus be considered a sufficiently safe option to treat VITT.

Co-Inhibition of the Immunoproteasome Subunits LMP2 and LMP7 Ameliorates Immune Thrombocytopenia.

The immunoproteasome, a special isoform of the 20S proteasome, is expressed when the cells receive an inflammatory signal. Immunoproteasome inhibition proved efficacy in the treatment of autoimmune diseases. However, the role of the immunoproteasome in the pathogenesis of immune thrombocytopenia (ITP) remains unknown. We found that the expression of the immunoproteasome catalytic subunit, large multifunctional protease 2 (LMP2), was significantly upregulated in peripheral blood mononuclear cells of active ITP patients compared to those of healthy controls. No significant differences in LMP7 expression were observed between patients and controls. ML604440, an specific LMP2 inhibitor, had no significant impact on the platelet count of ITP mice, while ONX-0914 (an inhibitor of both LMP2 and LMP7) increased the number of platelets. In vitro assays revealed that ONX-0914 decreased the expression of FcγRI in ITP mice and decreased that of FcγRIII in ITP patients, inhibited the activation of CD4+ T cells, and affected the differentiation of Th1 cells in patients with ITP. These results suggest that the inhibition of immunoproteasome is a potential therapeutic approach for ITP patients.

The abnormal function of CD39+ regulatory T cells could be corrected by high-dose dexamethasone in patients with primary immune thrombocytopenia.

Primary immune thrombocytopenia is an autoimmune disease, characterized with decreased platelet and increased risk of bleeding. Recent studies have shown the reduction and dysfunction of regulatory T (Treg) cells in ITP patients. CD39 is highly expressed on the surface of Treg cells. It degrades ATP to AMP and CD73 dephosphorylates AMP into adenosine. Then adenosine binds with adenosine receptor and suppresses immune response by activating Treg cells and inhibiting the release of inflammatory cytokines from effector T (Teff) cells. Adenosine receptor has several subtypes and adenosine A2A receptor (A2AR) plays a crucial role especially within lymphocytes. The CD39+ Treg cells and the expression of A2AR showed abnormality in some autoimmune disease. But knowledge of CD39+ Treg cells and A2AR which are crucial in the adenosine immunosuppressive pathway is still limited in ITP. Thirty-one adult patients with newly diagnosed ITP were enrolled in this study. CD39 and A2AR expression was measured by flow cytometry and RT-PCR. The function of CD39 was reflected by the change of ATP concentration detected by CellTiter-Glo Luminescent Cell Viability Assay. CD39 expression within CD4+CD25+ Treg cells in ITP patients was decreased compared to normal controls. After high-dose dexamethasone therapy, response (R) group showed increased CD39 expression within Treg cells while non-response (NR) group did not show any difference in contrast to those before treatment. The expression of A2AR in CD4+CD25- Teff and CD4+CD25+ Treg cells was both lower in ITP patients than that of normal controls. After therapy, CD4+CD25- Teff cells had higher A2AR expression while CD4+CD25+ Treg cells did not show any difference in comparison to that before treatment. The enzymatic activity of CD39 was damaged in ITP patients and improved after high-dose dexamethasone therapy. In ITP, there was not only numerical decrease but also impaired enzymatic activity in CD39+ Treg cells. After high-dose dexamethasone treatment, these two defects could be reversed. Our results also suggested that ITP patients had reduced A2AR expression in both CD4+CD25+ Treg cells and CD4+CD25- Teff cells. CD4+CD25- Teff cells had increased A2AR expression after treatment.

Akt-mediated platelet apoptosis and its therapeutic implications in immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by low platelet count which can cause fatal hemorrhage. ITP patients with antiplatelet glycoprotein (GP) Ib-IX autoantibodies appear refractory to conventional treatments, and the mechanism remains elusive. Here we show that the platelets undergo apoptosis in ITP patients with anti-GPIbα autoantibodies. Consistent with these findings, the anti-GPIbα monoclonal antibodies AN51 and SZ2 induce platelet apoptosis in vitro. We demonstrate that anti-GPIbα antibody binding activates Akt, which elicits platelet apoptosis through activation of phosphodiesterase (PDE3A) and PDE3A-mediated PKA inhibition. Genetic ablation or chemical inhibition of Akt or blocking of Akt signaling abolishes anti-GPIbα antibody-induced platelet apoptosis. We further demonstrate that the antibody-bound platelets are removed in vivo through an apoptosis-dependent manner. Phosphatidylserine (PS) exposure on apoptotic platelets results in phagocytosis of platelets by macrophages in the liver. Notably, inhibition or genetic ablation of Akt or Akt-regulated apoptotic signaling or blockage of PS exposure protects the platelets from clearance. Therefore, our findings reveal pathogenic mechanisms of ITP with anti-GPIbα autoantibodies and, more importantly, suggest therapeutic strategies for thrombocytopenia caused by autoantibodies or other pathogenic factors.

Protein kinase A determines platelet life span and survival by regulating apoptosis.

Apoptosis delimits platelet life span in the circulation and leads to storage lesion, which severely limits the shelf life of stored platelets. Moreover, accumulating evidence indicates that platelet apoptosis provoked by various pathological stimuli results in thrombocytopenia in many common diseases. However, little is known about how platelet apoptosis is initiated or regulated. Here, we show that PKA activity is markedly reduced in platelets aged in vitro, stored platelets, and platelets from patients with immune thrombocytopenia (ITP), diabetes, and bacterial infections. Inhibition or genetic ablation of PKA provoked intrinsic programmed platelet apoptosis in vitro and rapid platelet clearance in vivo. PKA inhibition resulted in dephosphorylation of the proapoptotic protein BAD at Ser155, resulting in sequestration of prosurvival protein BCL-XL in mitochondria and subsequent apoptosis. Notably, PKA activation protected platelets from apoptosis induced by storage or pathological stimuli and elevated peripheral platelet levels in normal mice and in a murine model of ITP. Therefore, these findings identify PKA as a homeostatic regulator of platelet apoptosis that determines platelet life span and survival. Furthermore, these results suggest that regulation of PKA activity represents a promising strategy for extending platelet shelf life and has profound implications for the treatment of platelet number-related diseases and disorders.

Tpl2 kinase regulates FcγR signaling and immune thrombocytopenia in mice.

The MAPK3 Tpl2 controls innate and adaptive immunity by regulating TLR, TNF-α, and GPCR signaling in a variety of cell types. Its ablation gives rise to an anti-inflammatory phenotype characterized by resistance to LPS-induced endotoxin shock, DSS-induced colitis, and TNF-α-induced IBD. Here, we address the role of Tpl2 in autoimmunity. Our data show that the ablation and the pharmacological inhibition of Tpl2 protect mice from antiplatelet antibody-induced thrombocytopenia, a model of ITP. Thrombocytopenia in this model and in ITP is caused by phagocytosis of platelets opsonized with antiplatelet antibodies and depends on FcγR activation in splenic and hepatic myeloid cells. Further studies explained how Tpl2 inhibition protects from antibody-induced thrombocytopenia, by showing that Tpl2 is activated by FcγR signals in macrophages and that its activation by these signals is required for ERK activation, cytoplasmic Ca(2+) influx, the induction of cytokine and coreceptor gene expression, and phagocytosis.

Decreased expression of indoleamine 2,3-dioxygenase 1 in dendritic cells contributes to impaired regulatory T cell development in immune thrombocytopenia.

Along with their immunogenic role, dendritic cells (DCs) are also critical in maintaining tolerance to self-antigens by inducing regulatory T cells (Tregs) via the expression of the immunomodulatory enzyme indoleamine 2,3-dioxygenase 1 (IDO1). In turn, Tregs modulate the maturation and/or function of DCs. In immune thrombocytopenia (ITP), the interaction between DCs and Tregs has never been investigated although decreased number/function of Tregs as well as altered DCs have been described. Here, we ask whether, in ITP: (1) IDO1 expression/activity is decreased in mature DCs; (2) IDO1-mediated Treg generation is impaired; and (3) DC maturation is abnormally modulated by Tregs. We found that in ITP, DCs show reduced capability of upregulating the expression/activity of IDO1. This finding results in the reduced ability of mature DCs of converting T cells into Tregs. In turn, Tregs are characterized by decreased interleukin-10 production and show lower ability of inhibiting DC maturation. In conclusion, these data point out the role of IDO1 in the impaired regulatory T cell development of ITP patients and suggest that the cross-talk between Tregs and DCs is hampered and plays a pathogenetic role.

[Characterization of unusual macro CK1 of IgG3kappa-cK-MM type detected in the serum of a patient with idiopathic thrombocytopenic purpura].

An unusual creatine kinase (CK) band detected in the serum of a patient with idiopathic thrombocytopenic purpura (ITP) was studied. The number of platelets of the patient decreased to 2.3 x 10(4)/microL, and serum CK and CK-MB activities increased to 899 and 62.7 U/L, respectively. The unusual CK band with a molecular size of 320,000 migrated to the cathodal side of CK-MM through electrophoresis, and the position was similar to that of octameric mitochondrial CK(Mi-CK). However, the cathodal band did not react with anti ubiquitous or anti-sarcomeric Mi-CK antibodies; instead, a reaction of the band occurred with anti-CK-M subunit antibodies and CK activity was inhibited. In addition to the cathodal band of CK-MM, an atypical CK band was simultaneously detected on the anodal side of CK-MM. It was found that the cathodal and anodal bands were macro CK1 of IgG3kappa-CK-MM and IgG1kappa-CK-MM, respectively, by immunofixation electrophoresis with anti-IgG subclass antibodies. The number of platelets began to increase one week after the elimination of Helicobacter pylori. The cathodal band activity showed an opposite migration one to two weeks following the platelet count change. Change of the anodal band began one month or longer following the platelet count change. One year following the first medical examination, the level of IgG3 in the serum of the patient decreased to 45%, and the percentage was lower than those of the other IgG subclasses. These results suggest that changes in the cathodal band are associated with those of the number of platelets and reflect the disease state of the patient. In conclusion, the cathodal band of CK-MM in the serum of an ITP affected patient was macro CK1 of IgG3kappa-CK-MM type, it migrated to the opposite sides of the platelet count change, and reflected the disease state of the patient.

DNA methyltransferase 3B (DNMT3B -579G>T) promotor polymorphism and the susceptibility to pediatric immune thrombocytopenic purpura in Egypt.

Idiopathic thrombocytopenic purpura (ITP) is an autoimmune disease characterized by increased platelet destruction. Although the etiology of ITP remains unclear, it is accepted that both environmental and genetic factors play an important role in the development of the disease. The present study aimed at exploring a novel molecular determinant that may influence the susceptibility and course of ITP in Egyptian children. To achieve our aim, genotyping of DNMT3B -579G>T promotor polymorphism by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assay. The current study was conducted on 140 ITP patients and 150 age and gender matched healthy controls. The results obtained revealed that DNMT3B -579 TT homotype was significantly higher in ITP patients and conferred almost three fold increased risk of ITP (OR=3.16, 95%CI=1.73-5.79). There was no statistically significant difference between ITP patients with wild or mutant genotypes as regards their clinical or laboratory data. Furthermore, there was no statistical difference in the distribution of DNMT3B -579G>T genotypes between acute and chronic ITP patients. In conclusion, DNMT3B -579G>T promotor polymorphism represents a novel genetic risk factor for ITP but not a predictor for tendency to chronicity in pediatric ITP in Egypt.

Role of DNA methyltransferase 3A mRNA expression in Egyptian patients with idiopathic thrombocytopenic purpura.

INTRODUCTION: Idiopathic thrombocytopenic purpura (ITP) is an organ-specific autoimmune hemorrhagic disease characterized by breakdown of self-tolerance and triggering autoreactive lymphocytes' response against platelets. The underlying etiology of ITP remains largely unknown. DNA methylation plays an essential role in maintaining T-cell function, and impaired methylation can lead to inappropriate gene expression and contribute to T-cell autoreactivity and autoimmunity. The aim of this study was to evaluate the role of DNA methyltransferase 3A gene expression in the pathogenesis of ITP. METHODS: This study included 60 subjects: 20 healthy volunteers as a control group, 20 patients with acute ITP, and 20 patients with chronic ITP. DNA methyltransferase 3A (DNMT3A) mRNA expression in peripheral blood mononuclear cells was measured by real-time quantitative polymerase chain reaction. Plasma S-adenosylhomocysteine (SAH) levels were assayed with reversed-phase high-performance liquid chromatography. RESULTS: DNMT3A mRNA expression was significantly decreased in patients with ITP as compared with that of the control group. Plasma SAH level was significantly elevated in patients with ITP than in healthy controls. However, no significant difference was found in DNMT3A mRNA expression or plasma SAH level between patients with acute and chronic ITP. CONCLUSIONS: Aberrant DNA methylation status reflected by decreased mRNA expression of DNMT3A and increased plasma SAH level may play an important role in the pathogenesis of ITP, although the precise underlying mechanisms still await further investigations, and extensive work in this field is clearly needed to provide novel therapeutic targets for ITP.

Residues Arg568 and Phe592 contribute to an antigenic surface for anti-ADAMTS13 antibodies in the spacer domain.

BACKGROUND: The majority of patients diagnosed with thrombotic thrombocytopenic purpura have autoantibodies directed towards the spacer domain of ADAMTS13. DESIGN AND METHODS: In this study we explored the epitope specificity and immunoglobulin class and immunoglobulin G subclass distribution of anti-ADAMTS13 antibodies. The epitope specificity of anti-spacer domain antibodies was examined using plasma from 48 patients with acute acquired thrombotic thrombocytopenic purpura by means of immunoprecipitation of ADAMTS13 variants containing single or multiple alanine substitutions. Using similar methods, we also determined the presence of anti-TSP2-8 and CUB1-2 domain antibodies in this cohort of patients. RESULTS: Antibody profiling revealed that anti-ADAMTS13 immunoglobulin G1 and immunoglobulin G4 predominate in plasma of patients with acquired thrombotic thrombocytopenic purpura. Analysis of anti-spacer domain antibodies revealed that Arg568 and Phe592, in addition to residues Arg660, Tyr661, and Tyr665, also contribute to an antigenic surface in the spacer domain. The majority of patients (90%) lost reactivity towards the spacer domain following introduction of multiple alanine substitutions at Arg568, Phe592, Arg660, Tyr661 and Tyr665. Anti-TSP2-8 and anti-CUB1-2 domain-directed antibodies were present in, respectively, 17% and 35% of the patients' samples analyzed. CONCLUSIONS: Immunoglobulin G directed towards a single antigenic surface comprising residues Arg568, Phe592, Arg660, Tyr661 and Tyr665 predominates in the plasma of patients with acquired thrombotic thrombocytopenic purpura.

Heterogeneous pathogenic processes of thrombotic microangiopathies in patients with connective tissue diseases.

To clarify the pathogenic processes of thrombotic microangiopathies (TMAs) in patients with connective tissue disease (CTD), we analysed clinical characteristics and plasma ADAMTS13 levels in 127 patients with CTD-TMAs, including patients with systemic lupus erythematosus (SLE), systemic sclerosis, polymyositis/dermatomyositis, and rheumatoid arthritis (RA), and 64 patients with acquired idiopathic thrombotic thrombocytopenic purpura (ai-TTP). Plasma levels of ADAMTS13 activity, antigen, and inhibitors were determined by enzyme immunoassays. IgG type anti-ADAMTS13 antibodies were also detected by immunoblots using purified ADAMTS13. ADAMTS13 activity was significantly decreased in CTD-TMAs, regardless of the underlying disease, but the frequency of severe deficiency (defined as <0.5% of normal) was lower in CTD-TMA patients than in ai-TTP patients (16.5% vs. 70.3%, p < 0.01). Severe deficiency of ADAMTS13 activity was predominantly detected in patients with RA- and SLE-TMAs, and was closely associated with the presence of anti-ADAMTS13 IgG antibodies. CTD-TMA patients with severe deficiency of ADAMTS13 activity appeared to have lower platelet counts and better therapeutic outcomes. At least two phenotypic TMAs occur in patients with CTDs: a minor population with deficient ADAMTS13 activity caused by neutralising autoantibodies, and a major population with normal or moderately reduced activity. Classifying CTD-TMAs by ADAMTS13 activity may be useful in predicting the clinical course and therapeutic outcomes, as patients with moderately reduced activity are likely to have more prominent renal impairment and poor prognoses.

Fostamatinib, a Syk inhibitor prodrug for the treatment of inflammatory diseases.

Rigel Pharmaceuticals Inc is developing fostamatinib, a prodrug of the spleen tyrosine kinase (Syk) inhibitor R-406, for the potential treatment of autoimmune diseases such as rheumatoid arthritis (RA), idiopathic thrombocytopenic purpura (ITP) and B-cell lymphomas. Syk is a key mediator of Fc and B-cell receptor signaling in inflammatory cells, such as B-cells, mast cells, macrophages and neutrophils. Preclinical studies of R-406 or fostamatinib demonstrated a significant reduction in major inflammatory mediators such as TNFalpha, IL-1, IL-6 and IL-18, leading to reduced inflammation and bone degradation in models of RA. In a phase II clinical trial, fostamatinib treatment effectively improved American College of Rheumatology response rates in patients with RA. Preclinical studies and phase II trials also suggested the potential of using fostamatinib for the treatment of ITP and B-cell lymphomas, by increasing platelet counts and inducing response rates, respectively. Fostamatinib is orally bioavailable and was well tolerated in phase I and II trials, with the most common side effect being gastrointestinal symptoms. At the time of publication, phase II trials for fostamatinib were ongoing in patients with RA, ITP and B-cell lymphomas. The Syk inhibitor appears to be a promising therapeutic for immunological diseases, but further data are required to establish the efficacy and long-term safety of the drug in humans.

Effect of protein kinase C on T lymphocyte proliferation and apoptosis in acute idiopathic thrombocytopenic purpura.

Previous studies have shown that T lymphocyte proliferation and apoptosis are abnormal in idiopathic thrombocytopenic purpura (ITP) children; however, the underlying regulated mechanisms in signal transductions remain unknown. In this paper, we investigated the changes of protein kinase C (PKC) activity in peripheral blood T lymphocytes and the effect of PKC on the peripheral blood T lymphocyte proliferation and apoptosis in ITP children. We demonstrated that T lymphocytes from ITP children were more susceptible to the activator (phorbol myristate acetate) and the inhibitor (H-7) of PKC. In ITP children, phorbol myristate acetate and H-7 dramatically affected T lymphocyte proliferation and apoptosis, but altered little in healthy children. Compared with healthy children, PKC activity was significantly enhanced in ITP children, increasing the expressions of Fas ligand on CD3+, CD4+ and CD8+ T cells, indicating positive correlations between PKC activity and the expressions of Fas ligand on T cells, while the relations between PKC activity and platelet count showed negative correlations. Taken together, our findings suggest that PKC signal transductions may participate in the procedure of T lymphocyte proliferation and apoptosis in ITP children. PKC activation may enhance T lymphocyte activity, suppress T cell apoptosis, and be involved in thrombocyte damage, which can be related to the immunity pathogenesis of ITP.

[Analysis of protein kinase C activity of peripheral blood T lymphocytes in children with acute idiopathic thrombocytopenic purpura].

OBJECTIVE: Acute idiopathic thrombocytopenic purpura (AITP) is a common autoimmune disease in children. Thrombocytes decrease extremely in serious patients, its pathogenesis involves abnormal activation of T lymphocytes and T cell-dependent production of autoantibody. The aim of the present study was to investigate changes of protein kinase C (PKC) activity in peripheral blood T lymphocytes in children with AITP and the relationships between PKC activity and T lymphocytes activation and thrombocytopenia. METHODS: Peripheral blood specimens were collected from children with acute ITP (n = 35) and healthy children (n = 30), and T lymphocytes were isolated and purified by using T cells Segregation Enrichment Column. PKC activity was detected by using PepTag Assay, a non-radioactive detection method. The reaction mixture, in a final volume of 25 microl, consisted of 5 microl reaction buffer, PepTag C1 5 microl (0.4 microg/microl), PKC activator solution (DG) 5 microl, peptide protection solution 1 microl and sample 9 microl. Phosphorylation reaction was allowed to continue for 30 minutes, then 25 microl reaction mixture was subjected to electrophoresis on a 0.8% agarose gel at 100 V for about 20 minutes. After electrophoresis, the PepTag C1 peptides which were phosphorylated and non-phosphorylated were separated, phosphorylated PepTag C1 peptide with negative electricity migrated toward the anode (+), but nonphosphorylated PepTag C1 peptide with positive electricity migrated toward cathode (-), the gel was photographed. Electrophoresis bands on anode represented PKC activity and were analyzed quantitatively. FasL, which is T cell activation marker, was determined by flow cytometer and platelet was counted by cell counting meter. RESULTS: Compared with healthy children, children with AITP had significantly higher PKC total activity [(0.97 +/- 0.21) nmol/(min.ml) vs. (0.55 +/- 0.13) nmol/(min.ml), (P < 0.05)]. Expression of FasL on T cell subpopulation in children with AITP was significantly higher [Th FasL: (32.7 +/- 3.4) vs. (14.7 +/- 4.2); Tc FasL: (17.3 +/- 9.7) vs. (11.6 +/- 8.5)%, (P < 0.05)]. Besides, relationships between the changes of PKC activity, Th FasL and Tc FasL had positive correlation (r(1) = 0.68, r(2) = 0.53, P < 0.05). However, PKC activity and platelet count had a significantly negative correlation (r = -0.75, P < 0.05). CONCLUSION: Increased PKC activity was seen in children with AITP, which can cause damage to thrombocytes and reduction of thrombocytes. PKC signal transduction pathway might play an important role in the immunopathogenesis of AITP.