[Recent topics in the pathophysiology and treatment of immune thrombocytopenic purpura].

Increased and impaired platelet productions via immunological abnormalities are the main pathophysiological mechanisms of primary immune thrombocytopenia (ITP). Recent studies have revealed that platelet removal from circulation involves not only Fc receptor-mediated phagocytosis of immunoglobulin G autoantibodies-bound platelets but also complement-dependent mechanism and platelet glycoprotein desialylation. Understanding the molecular mechanism of ITP pathophysiology has helped develop many novel molecular targeted drugs, and recent clinical trials have shown their effectiveness. In particular, fostamatinib, which is a Syk inhibitor, inhibits macrophage and B-cell activity and is already been approved in Europe for multidrug-resistant ITP. Recently, coronavirus disease-2019 (COVID-19) vaccine-associated newly-onset or ITP exacerbation has come to attention. Whether COVID-19 vaccines induce de novo ITP remains controversial. However, close attention is necessary after COVID-19 vaccination because a certain number of patients with ITP presented exacerbation after COVID-19 vaccination.

[Acquired hemophilia A following BNT162b2 mRNA COVID-19 vaccination].

Acquired hemophilia A (AHA) is a rare disease characteized by bleeding symptoms caused by decreased factor VIII activity due to the appearance of inhibitors to factor VIII triggered by malignancy or collagen disease. An 86-year-old woman developed purpura on her extremities after the first dose of the BNT162b2 mRNA COVID-19 vaccine. This symptom subsided after a few days. After the second dose of the BNT162b2 mRNA COVID-19 vaccine, purpura appeared again, and the patient was referred to our hospital Her APTT was remarkably prolonged to 110 seconds, and a cross-mixing test revealed an inhibitor pattern. Since FVIII activity was <1% and FVIII inhibitor was 51.6 BU, she was diagnosed with AHA. Prednisolone therapy was started, and coagulative complete remission was achieved. Because acquired hemophilia can develop after mRNA COVID-19 vaccination, as in this case, it is critical to monitor the appearance of bleeding symptom.

Reevaluation of platelet function in chronic immune thrombocytopenia: impacts of platelet size, platelet-associated anti-αIIbß3 antibodies and thrombopoietin receptor agonists.

Platelet function of immune thrombocytopenia (ITP) has been controversial because of methodological problems associated with low platelet counts. In this study, we evaluated platelet function in 21 patients with chronic ITP (cITP) using the recently developed flow cytometry (FCM)-based platelet aggregation assay (FCA) along with a PAC1/CD62P assay. Since ITP platelets are larger than controls, whole platelets (whole gating method) and size-adjusted platelets (size-adjusted method) were analysed in the PAC1/CD62P via FCM. We found that: (i) aggregation was equivalent [phorbol myristate acetate (PMA) or adenosine diphosphate (ADP)-induced] or enhanced [protease-activated receptor 1-activating peptide (PAR1AP)-induced] in cITP compared with control by FCA; (ii) PAC1 or CD62P was also equivalent or enhanced in cITP in the whole gating method; and (iii) in sharp contrast, the size-adjusted method revealed that ADP-, PAR1AP-, and collagen synthetic liquid reactive peptide (SRP)-induced PAC1 and ADP-induced CD62P were impaired in cITP. These data suggested that an increase in the number of larger-sized platelets may compensate for the impaired platelet function of cITP, leading to non-inferiority of overall platelet function in cITP. Furthermore, we revealed that ADP-induced aggregation was impaired in the patients with thrombopoietin receptor agonists (TPO-RAs) or platelet-associated anti-αIIbß3 antibodies compared with the control, suggesting that the presence of anti-αIIbß3 autoantibodies and/or administration of TPO-RAs may have a negative impact on platelet function.

[Reference guide for the treatment of adult idiopathic thrombocytopenic purpura].

The treatment strategy of adult idiopathic thrombocytopenic purpura (ITP) in Japan had consisted of corticosteroids as the first-line option, splenectomy as the second-line option, and others as the third-line option, respectively, for a long time. However, thrombopoietin receptor agonists (TPO-RAs) have been widely adopted recently for corticosteroid-resistant ITP, and indications for rituximab have been extended to adult ITP in Japan, suggesting that ITP treatment is dramatically changing. In the "Reference guide for adult ITP treatments in Japan" revised in 2019, TPO-RAs and rituximab are equally recommended as second-line treatments alongside splenectomy. It is suggested to select from among the second-line treatments with careful consideration of not only their advantages and disadvantages but also aspects of the condition and situation of each patient such as any comorbidities or lifestyle factors. Moreover, since multiple effective therapeutic options are now available as second-line options, it is preferable to consider an early transition to second-line treatments for corticosteroid-refractory/dependent ITP patients.

Human CalDAG-GEFI deficiency increases bleeding and delays αIIbß3 activation.

Affinity regulation of integrin αIIbß3 for fibrinogen by inside-out signaling plays a critical role in hemostasis. Calcium and diacylglycerol (DAG)-regulated guanine nucleotide exchange factor I (CalDAG-GEFI) was identified as a Rap1-activating molecule, and its role in inside-out αIIbß3 activation was established in CalDAG-GEFI-deficient mice. However, little information regarding CalDAG-GEFI in human platelets is available. Here, we report a 16-year-old girl with CalDAG-GEFI deficiency who has been suffering from severe bleeding tendency. Although talin and kindlin-3 were normally detected, CalDAG-GEFI was undetectable in her platelets by western blotting. Genetic analysis revealed compound heterozygous CalDAG-GEFI mutations, Lys309X and Leu360del, which were responsible for CalDAG-GEFI deficiency. The functional analysis demonstrated impaired αIIbß3 activation by various agonists except for phorbol myristate acetate, normal calcium mobilization, and impaired Rap1 activation, which were consistent with the phenotype of CalDAG-GEFI-deficient mice. Despite substantial αIIbß3 activation at high agonist concentrations, she had severe bleeding tendency. Further functional analysis demonstrated markedly delayed αIIbß3 activation velocity and decreased shear-induced thrombus formation. Contrary to CalDAG-GEFI-deficient mice, which showed integrin-dependent neutrophil functional abnormality, neutrophil ß2 integrin activation was not impaired in the patient. Our results demonstrate the critical role of CalDAG-GEFI in rapid αIIbß3 activation of human platelets.

Distinct effects of daratumumab on indirect and direct antiglobulin tests: a new method employing 0.01 mol/L dithiothreitol for negating the daratumumab interference with preserving K antigenicity (Osaka method).

BACKGROUND: There is an increasing demand for daratumumab (DARA), an immunoglobulin (Ig)G1κ monoclonal antibody (MoAb) that recognizes CD38, to manage relapsed or refractory multiple myeloma (MM) patients. However, DARA leads to positive and panreactive agglutination reactions in indirect antiglobulin tests (IATs) in vitro (the DARA interference). In addition, effects of DARA on red blood cells (RBCs) in vivo remains elusive. STUDY DESIGN AND METHODS: To develop a new method to negate the DARA interference, the effects of various concentrations of dithiothreitol (DTT) on RBC CD38 and Kell antigenicity in combination with an automatic blood cell washing centrifuge were compared with the AABB standard procedure in parallel. Moreover, direct antiglobulin tests (DATs) for RBCs in DARA-treated MM patients were examined. RESULTS: A quantity of 0.01 mol/L DTT as well as the AABB procedure (equivalent to 0.15 mol/L DTT in our procedure) markedly reduced the reactivity of phycoerythrin-mouse anti-CD38 MoAb and DARA with RBCs. In sharp contrast to the AABB procedure, 0.01 mol/L DTT partially preserved K antigenicity and allowed the determination of phenotype of K antigen even in the presence of the DARA interference. In contrast, DAT for RBCs obtained from MM patients showed a weak positive or negative reaction. Immunoblotting further indicated that DARA induced loss of CD38 in vivo. CONCLUSION: A simple and reliable method to negate the DARA interference with partially preserving Kell antigenicity is proposed (Osaka method). CD38 antigenicity is susceptible to 0.01 mol/L DTT treatment even in the presence of DARA. Our data also demonstrate distinct effects of DARA on IAT in vitro and DAT in vivo.

Autoimmune bullous disease and Hashimoto's disease complicated by acquired hemophilia A.

A 67-year-old man was admitted with a 1-month history of spontaneous hematoma in his right back and severe anemia. He had suffered from rashes with blisters involving both legs for 10 years, which had shown worsening and extended to his entire body concurrently with the hematoma. APTT was markedly prolonged to 119 seconds, and Factor VIII:C and FVIII inhibitor levels were less than 1% and 153.1 BU/ml, respectively, confirming the diagnosis of acquired hemophilia A (AHA). Skin biopsy revealed his rashes to be caused by autoimmune bullous disease (ABD), and laboratory and physical findings showed that he also had autoimmune hypothyroidism (Hashimoto's disease). Recombinant FVIIa was effective for management of his bleeding; in addition, FVIII inhibitor reduction and FVIII:C recovery, in parallel with improvement of the skin lesions, were achieved by administering prednisolone and cyclophosphamide. To our knowledge, this is the first report of AHA associated with ABD and Hashimoto's disease.

Expression levels of ABCG2 on cord red blood cells and study of fetal anemia associated with anti-Jr(a).

BACKGROUND: The Jr(a) antigen of JR blood group systems is located on ABCG2 and Jr(a-) subjects whose red blood cells (RBCs) lack ABCG2 have been identified mostly among the Japanese. Although anti-Jr(a) can cause fetal anemia, little is known regarding its mechanism. STUDY DESIGN AND METHODS: We reviewed clinical courses of all reported cases with fetal anemia due to anti-Jr(a) . We analyzed the ABCG2 expressions of cord RBCs at various gestational ages. We examined the effects of sera containing anti-Jr(a) from three pregnancies with fetal anemia or monoclonal anti-Jr(a) on erythropoiesis and phagocytosis. We also examined epitopes of anti-Jr(a) . RESULTS: Case series suggested that the majority of fetal anemia with anti-Jr(a) may not be progressive in the later gestational ages. ABCG2 expression levels of cord RBCs were significantly higher than those of adults and neonates with high individual variation and gradually decreased with advancing gestational ages. Anti-Jr(a) did not significantly impact erythroid colony formation, although we detected a tendency toward the suppression of erythroid burst-forming unit formation by anti-Jr(a) using feline marrow cells. Anti-Jr(a) did not induce phagocytosis of sensitized RBCs by monocytes. While many anti-Jr(a) recognized the same regions as a monoclonal anti-ABCG2, 5D3, epitopes of anti-Jr(a) did not correlate with the incidence of fetal anemia. CONCLUSION: ABCG2 expression levels in cord RBCs are higher than those of adults, and the change of ABCG2 expression in erythroid lineage cells may influence the clinical course of fetal anemia with anti-Jr(a) , although we could not detect significant effects of anti-Jr(a) on erythroid colony formation or phagocytosis.

[Recent advances in pathophysiology and treatment of immune thrombocytopenia].

Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by isolated thrombocytopenia caused by immune-mediated platelet destruction and impairment of platelet production. Recent studies have uncovered details involving the target regions of platelet-associated anti-GPIIb/IIIa antibodies, pathological differences depending on the specificity of target antigens, and cellular abnormalities, especially impairment of regulatory T cells contributing to the pathogenesis of ITP. Treatment of ITP has been changed dramatically by the application of thrombopoietin receptor agonists, TPO-RAs, in patients unresponsive to traditional steroids and splenectomy. Rituximab has also been used in Western countries for ITP patients and its long-term efficacy has become increasingly clear. Clinical problems awaiting solution in ITP management include improving the efficacy of treatments for newly-diagnosed ITP, confirmation of the long-term efficacy and safety of TPO-RAs, and determination of the positioning of rituximab in the treatment sequence of ITP.

Recognition of highly restricted regions in the ß-propeller domain of αIIb by platelet-associated anti-αIIbß3 autoantibodies in primary immune thrombocytopenia.

Platelet-associated (PA) IgG autoantibodies play an essential role in primary immune thrombocytopenia (ITP). However, little is known about the epitopes of these Abs. This study aimed to identify critical binding regions for PA anti-αIIbß3 Abs. Because PA anti-αIIbß3 Abs bound poorly to mouse αIIbß3, we created human-mouse chimera constructs. We first examined 76 platelet eluates obtained from patients with primary ITP. Of these, 26 harbored PA anti-αIIbß3 Abs (34%). Further analysis of 15 patients who provided sufficient materials showed that the epitopes of these Abs were mainly localized in the N-terminal half of the ß-propeller domain in αIIb (L1-W235). We could identify 3 main recognition sites in the region; 2 eluates recognized a conformation formed by the W1:1-2 and W2:3-4 loops, 5 recognized W1:2-3, and 4 recognized W3:4-1. The remaining 4 eluates could not be defined by the binding sites. Within these regions, we identified residues critical for binding, including S29 and R32 in W1:1-2; G44 and P45 in W1:2-3; and P135, E136, and R139 in W2:3-4. Of 11 eluates whose recognition sites were identified, 5 clearly showed restricted κ/λ-chain usage. These results suggested that PA anti-αIIbß3 Abs in primary ITP tended to recognize highly restricted regions of αIIb with clonality.

Reference guide for the diagnosis of adult primary immune thrombocytopenia, 2023 edition.

Primary immune thrombocytopenia (ITP) is an autoimmune disorder characterized by isolated thrombocytopenia due to accelerated platelet destruction and impaired platelet production. Diagnosis of ITP is still challenging because ITP has been diagnosed by exclusion. Exclusion of thrombocytopenia due to bone marrow failure is especially important in Japan because of high prevalence of aplastic anemia compared to Western countries. Hence, we propose a new diagnostic criteria involving the measurement of plasma thrombopoietin (TPO) levels and percentage of immature platelet fraction (RP% or IPF%); 1) isolated thrombocytopenia with no morphological evidence of dysplasia in any blood cell type in a blood smear, 2) normal or slightly increased plasma TPO level (< cutoff), 3) elevated RP% or IPF% (> upper limit of normal), and 4) absence of other conditions that potentially cause thrombocytopenia including secondary ITP. A diagnosis of ITP is made if conditions 1-4 are all met. Cases in which criterion 2 or 3 is not met or unavailable are defined as "possible ITP," and diagnosis of ITP can be made mainly by typical clinical course. These new criteria enable us to clearly differentiate ITP from aplastic anemia and other forms of hypoplastic thrombocytopenia and can be highly useful in clinical practice for avoiding unnecessary bone marrow examination as well as for appropriate selection of treatments.

A potential role for α-actinin in inside-out αIIbß3 signaling.

Many different biochemical signaling pathways regulate integrin activation through the integrin cytoplasmic tail. Here, we describe a new role for α-actinin in inside-out integrin activation. In resting human platelets, α-actinin was associated with αIIbß3, whereas inside-out signaling (αIIbß3 activation signals) from protease-activated receptors (PARs) dephosphorylated and dissociated α-actinin from αIIbß3. We evaluated the time-dependent changes of the αIIbß3 activation state by measuring PAC-1 binding velocity. The initial velocity analysis clearly showed that PAR1-activating peptide stimulation induced only transient αIIbß3 activation, whereas PAR4-activating peptide induced long-lasting αIIbß3 activation. When αIIbß3 activation signaling dwindled, α-actinin became rephosphorylated and reassociated with αIIbß3. Compared with control platelets, the dissociation of α-actinin from αIIbß3 was only transient in PAR4-stimulated P2Y(12)-deficient platelets in which the sustained αIIbß3 activation was markedly impaired. Overexpression of wild-type α-actinin, but not the mutant Y12F α-actinin, increased its binding to αIIbß3 and inhibited PAR1-induced initial αIIbß3 activation in the human megakaryoblastic cell line, CMK. In contrast, knockdown of α-actinin augmented PAR-induced αIIbß3 activation in CMK. These observations suggest that α-actinin might play a potential role in setting integrins to a default low-affinity ligand-binding state in resting platelets and regulating αIIbß3 activation by inside-out signaling.

Heterozygous ITGA2B R995W mutation inducing constitutive activation of the αIIbß3 receptor affects proplatelet formation and causes congenital macrothrombocytopenia.

Congenital macrothrombocytopenia is a genetically heterogeneous group of rare disorders. αIIbß3 has not been implicated in these conditions. We identified a novel, conserved heterozygous ITGA2B R995W mutation in 4 unrelated families. The surface expression of platelet αIIbß3 was decreased to 50% to 70% of control. There was spontaneous PAC-1 and fibrinogen binding to resting platelets without CD62p expression. The activation state of αIIbß3 in 293T cells was higher for αIIb-W995 than for ß3-H723 but was weaker than for ß3-N562. FAK was spontaneously phosphorylated in αIIb-W995/ß3-transfected 293T cells. These results indicate that αIIb-W995/ß3 has a constitutive, activated conformation but does not induce platelet activation. αIIb-W995/ß3-transfected CHO cells developed membrane ruffling and abnormal cytoplasmic protrusions. The increased size and decreased number of proplatelet tips in αIIb-W995/ß3-transduced mouse fetal liver-derived megakaryocytes indicate defective pro-platelet formation. We propose that activating mutations in ITGA2B and ITGB3 represent the etiology of a subset of congenital macrothrombocytopenias.

Progress in pathophysiological understanding and treatment of thrombocytopenia.

Immune thrombocytopenia (ITP), thrombotic thrombocytopenic purpura (TTP), and vaccine-induced immune thrombotic thrombocytopenia (VITT) all have "thrombocytopenia" in their name, and all but congenital TTP are caused by immune mechanisms, but these conditions are quite different in their clinical features and pathophysiology. This review series covers recent progress in pathophysiology and treatment of these diseases, as well as a recent epoch-making clinical trial of induced pluripotent stem cells (iPSC)-derived platelets for patients with thrombocytopenia.

FNAIT pathogenesis determined by serial analysis of three subsequent pregnancies of a woman with severe fetal and neonatal alloimmune thrombocytopenia (FNAIT) with anti-HPA-4b and anti-HPA-5b alloantibodies in the first sibling.

BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is caused by anti-HPA alloantibody, and anti-HPA-4b is the most common cause in Japanese. Anti-HPA-5b is frequently detected in pregnant women, but it is still controversial whether anti-HPA-5b causes severe FNAIT. CASE PRESENTATION: A Japanese woman with anti-HPA-4b and anti-HPA-5b alloantibodies delivered a baby with severe FNAIT who was both HPA-4b and HPA-5b incompatible. We carefully monitored the patient's following three pregnancies (the second and the fourth siblings were HPA-4b incompatible and HPA-5b compatible; the third sibling was both HPA-4b and HPA-5b compatible). FNAIT was not observed in all three siblings, although a modest decrease in cord blood platelet count was observed in the HPA-4b incompatible siblings compared to the HPA-4b compatible sibling. Serial monitoring of anti-HPA titer showed that anti-HPA-4b markedly decreased in late pregnancy and recovered after delivery of the HPA-4b incompatible siblings, but these decreases were not observed during the mother's pregnancy with the HPA-4b compatible sibling. In contrast, anti-HPA-5b remained at a high titer during pregnancy with all three siblings. CONCLUSION: Our data indicate that dynamic changes of anti-HPA-4b occur during pregnancy and strongly suggest that anti-HPA-5b was mainly responsible for severe FNAIT in this case.

Development of an Automated Chemiluminescent Enzyme Immunoassay for Measuring Thrombopoietin in Human Plasma.

Plasma thrombopoietin (TPO) measurements help distinguish between different types of thrombocytopenia but are not feasible in routine clinical practice. We developed a fully automated quantitative chemiluminescent enzyme immunoassay (CLEIA) for measuring TPO (TPO-CLEIA), which is a one-step sandwich-type assay. This assay utilizes a mouse monoclonal capture antibody, which has the neutralizing epitope of the interaction between TPO and the TPO receptor, and a newly generated rabbit monoclonal detector antibody. In analytical performance studies, this assay showed good linearity over the measuring range and high sensitivity. The limit of quantification (LoQ) of this assay was 3.4 pg/mL; low TPO concentration values of almost all healthy individuals exceeded the LoQ value. In clinical validation studies, TPO levels obtained from patients with aplastic anemia (AA) significantly increased, whereas those of patients with immune thrombocytopenia (ITP) were normal or slightly increased. The cutoff value for TPO-CLEIA corresponding to the previously reported values was useful for distinguishing between ITP and AA. These results suggest that TPO-CLEIA can quantify human plasma TPO levels with high accuracy and sensitivity and has the potential to facilitate routine clinical measurement of TPO in patients with various types of thrombocytopenia.

Increased cleavage of von Willebrand factor by ADAMTS13 may contribute strongly to acquired von Willebrand syndrome development in patients with essential thrombocythemia.

BACKGROUND: Patients with essential thrombocythemia (ET) often experience bleeding associated with acquired von Willebrand syndrome (AVWS) when the platelet count is markedly increased. OBJECTIVE: We investigated whether von Willebrand factor (VWF) degradation is enhanced in patients with ET. METHODS: Seventy patients with ET underwent VWF multimer (VWFM) analysis and measurement of VWF-related parameters. We calculated the VWFM index, defined as the ratio of intensities of a patient's molecular weight-categorized VWFMs, and those of a healthy subject's, using densitometric analysis. VWF degradation product (DP) was measured via ELISA using a monoclonal antibody that specifically recognizes Y1605 at the C-terminal boundary, which is exposed following ADAMTS13-mediated cleavage of the Y1605-M1606 bond of the VWF A2 domain. RESULTS: Patients with higher platelet counts had a significantly reduced high molecular weight (HMW)-VWFM index and an increased VWF-DP:VWF antigen (Ag) ratio compared to those with lower platelet counts. On multivariate analysis, the VWF-DP/VWF:Ag ratio was an independent predictor of the HMW-VWFM index. Patients who underwent cytoreductive therapy had a significantly higher HMW-VWFM index and lower VWF-DP/VWF:Ag ratio than those who did not. Among individual patients, there was also a significant increase in the HMW-VWFM index and a decrease in the VWF-DP/VWF:Ag ratio after cytoreductive therapy compared to pre-therapy values. CONCLUSION: In patients with ET, an increased platelet count is associated with enhanced cleavage of VWF at the Y1605-M1606 bond, primarily by ADAMTS13, leading to AVWS. Cytoreductive therapy reduces the platelet count, prevents excessive VWF cleavage, and improves VWFM distributions.

Autoimmune-mediated thrombocytopenia after allogeneic hematopoietic stem cell transplantation: significance of detecting reticulated platelets and glycoprotein-specific platelet autoantibodies.

Autoimmune hematological disorders are rare complications after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Diagnosis of immune thrombocytopenia (ITP) is challenging, especially after allo-HSCT, because various complications such as graft-versus-host disease, disease relapse, viral infection, thrombotic microangiopathy, and drug side effects can also cause thrombocytopenia. Assessment of reticulated platelets (RP) and plasma thrombopoietin (TPO) levels may be useful to distinguish between ITP and hypoplastic thrombocytopenia. ITP is generally characterized by an increased percentage of RP, and a normal or slightly increased plasma TPO level. We now report three cases of thrombocytopenia after allo-HSCT. RP% was elevated in these patients, as it is in primary ITP. However, in contrast to primary ITP, plasma TPO levels were high in two of three patients. Anti-αIIbß3 and anti-GPIb/IX-specific direct IgG antibodies were detected as well, suggesting occurrence of immune-mediated platelet destruction in addition to bone marrow suppression in two patients. All three patients were successfully treated with corticosteroids and/or thrombopoietin receptor agonists (TPO-RAs). These results suggest that increased RP% and detection of glycoprotein-specific platelet autoantibodies are useful for the diagnosis of ITP after HSCT.