Thrombosis-related characteristics of pregnant women with antithrombin deficiency, protein C deficiency and protein S deficiency in Japan.

BACKGROUND:  We previously conducted a primary survey of pregnant women with hereditary thrombophilia based on national surveillance in Japan, but did not examine their thrombosis-related characteristics. Antithrombin (AT) deficiency, protein C (PC) deficiency and protein S (PS) deficiency are the major types of hereditary thrombophilia in Japan. METHODS: We examined their detailed information related to thrombosis, and evaluated peripartum outcomes in comparison with control data obtained from the Japan Society of Obstetrics and Gynecology. RESULTS: Definite or probable AT deficiency, PC deficiency and PS deficiency were observed in 80, 50, and 317 pregnancies, respectively, from 2014 to 2018 in Japan, with prevalence rates among total deliveries of 0.011%, 0.007%, 0.044%. The number of pregnancies with AT, PC and PS deficiency might have been as many as 27, 17 and 108 every year if complete answers had been provided. In the peripartum period of current pregnancies, 27.5% of women with AT deficiency, 28.0% with PC deficiency and 13.2% with PS deficiency developed thrombosis (p < 0.001 vs. control). Pregnant women with AT and PC deficiency were more susceptible to thrombosis than those with PS deficiency (P < 0.01). Of the thromboses, 92.3% occurred during pregnancy, 62.8% at less than 15 gestational weeks. The earliest onset of thrombosis was 5 gestational weeks. Prophylactic anticoagulation significantly prevented the onset of both antepartum and postpartum thrombosis (p < 0.0001). The rate of recurrent pregnancy loss in women with low PC or PS activities was significantly higher than in controls (p < 0.0001); however, it is unknown whether recurrent pregnancy loss is related to hereditary PS deficiency. There seem to have been few serious maternal or fetal/neonatal complications due to placental insufficiency related to a hypercoagulable state other than growth restriction. CONCLUSIONS: This survey revealed the thrombosis-related characteristics of pregnant women with hereditary thrombophilia in Japan. We suggest prophylactic anticoagulation to prevent maternal or fetal/neonatal complications.

The length of the sanitary napkins can be used as a handier index than pictorial blood loss assessment chart to predict the heavy menstrual bleeding.

AIM: Many women with inherited bleeding disorders are not diagnosed because of a lack of appropriate indicators. This study aimed to assess the predictability of the pictorial blood loss assessment chart (PBAC) as an indicator of menorrhagia and identify an easy indicator of menorrhagia resulting from bleeding disorders. METHODS: A multicenter study enrolled 9 patients with von Willebrand disease (VWD), 23 hemophilia carriers, and 71 controls aged 20-45 years who completed PBACs for two menstrual cycles as well as questionnaires. RESULTS: The PBAC scores of the VWD were significantly higher than those of other groups, even in multivariate analysis with age and sanitary item factors (p = 0.014). A PBAC score of 100 was not an appropriate cutoff because of its low specificity (VWD: sensitivity, 100; specificity, 29.5; hemophilia carriers: 74 and 29.5, respectively). In the ROC analysis, the cutoff of optimal PBAC for VWD was 171 (sensitivity, 66.7; specificity, 72.3; AUC, 0.7296). As the pad length increased, the total length of the pads used during one menstrual period could be a new and easy indicator. However, the cutoff for VWD was 735 cm (sensitivity, 42.9; specificity, 94.3; AUC 0.6837). A threshold could not be established for the hemophilia carrier. Therefore, we multiplied the coefficient by the length of thick pads, which caused a lower PBAC. For the VWD, the sensitivity increased to 85.7 (specificity, 77.1). For the hemophilia carrier, sensitivity (66.7) and specificity (88.6) could be separated from the control. CONCLUSIONS: The total length of the pads with a thick-pad adjustment can be a simple method to identify bleeding disorders.

Determination of diagnostic threshold in harmonization and comparison of clinical utility for five major antiphospholipid antibody assays used in Japan.

BACKGROUND: Anticardiolipin antibodies (aCL) and anti-ß2 -glycoprotein I antibodies (aß2 GPI) are essential in diagnosing antiphospholipid syndrome (APS) according to the international APS guideline. Five commercial assays for aCL and aß2 GPI are available in Japan, but their test results are quite discordant. For harmonization of diagnosing APS, upper reference limit (URL) and diagnostic accuracy of each assay were evaluated and compared by testing common sets of specimens across all assays. METHODS: We evaluated two manual and three automated assays for aCL and aß2 GPI of IgG- and IgM classes. 99%URL (the upper limit of reference interval: as per guideline) together with 97.5%URL were determined by testing sera from 198 to 400 well-defined healthy subjects. Both URLs were compared with the cutoff values, which were determined based on ROC analysis by testing 50 each of plasma specimens from patients with/without APS. Diagnostic accuracy was evaluated as area under curve (AUC) of the ROC curve. RESULTS: A variable degree of discrepancy between URLs and the cutoff values was observed, which was partly attributable to between-year assay variability. 97.5%URLs were set lower and closer to the cutoff values than 99%URLs. For all assays, diagnostic accuracies of both aß2 GPI-IgG and aCL-IgG were generally high (AUC: 0.84-0.93); whereas those for IgM-class assays were low (AUC: 0.57-0.67), implicating its utility is limited to rare IgG negative APS cases. CONCLUSION: To ensure harmonized APS diagnosis, the diagnostic thresholds of the five assays were evaluated by common procedures. Contrary to the guideline, 97.5%URL is rather recommended for diagnosing APS, which showed a closer match to the cutoff value.

Donor UNC-93 Homolog B1 genetic polymorphism predicts survival outcomes after unrelated bone marrow transplantation.

UNC-93 homolog B1 (UNC93B1) is a key regulator of toll-like receptors (TLRs), pattern recognition receptors that sense invading pathogens and manage the innate immune response and deliver them from the endoplasmic reticulum to their respective endosomal signaling compartments. Several types of TLRs are known to contribute to the inflammatory process after allogeneic hematopoietic stem cell transplantation (SCT), so UNC93B1 might play integral roles there. We investigated the influence of the UNC93B1 single-nucleotide polymorphism (SNP) rs308328 (T>C) on transplant outcomes in a cohort of 237 patients undergoing unrelated HLA-matched bone marrow transplantation (BMT) for hematologic malignancies through the Japan Marrow Donor Program. The donor UNC93B1 C/C genotype was associated with a better 3-year overall survival than the donor UNC93B1 C/T or T/T genotype. An analysis of the UNC93B1 rs308328 genotype may therefore be useful for selecting the donor, estimating the prognosis, and creating therapeutic strategies after allogeneic SCT.

Clinical guidance for peripartum management of patients with hereditary thrombophilia.

Hereditary thrombophilia is a condition in which individuals are susceptible to the formation of thrombi due to a hereditary deficiency in anticoagulant factors, antithrombin (AT), protein C (PC), or protein S (PS). Many Japanese thrombophilia patients have PS deficiency, especially PS p.K196E (also called as PS Tokushima), which is exclusive to the Japanese population, and thrombosis sometimes occurs during pregnancy. At present, no management guidelines for pregnancy and delivery in thrombophilia patients have been developed. The Study Group for Hereditary Thrombophilia, one of the research groups of blood coagulation abnormalities in the Research Program on Rare and Intractable Diseases supported with the Research Grants of the Ministry of Health, Labour and Welfare Science, has therefore developed this clinical guidance to provide healthcare workers with necessary information on safe pregnancy, parturition and neonatal management, adopting a format of responses to seven clinical questions (CQ). At the end of each answer, the corresponding Recommendation Level (A, B, C) is indicated.

Recurrent Cerebral Venous Thrombosis Treated with Direct Oral Anticoagulants in a Japanese Man with Hereditary Protein C Deficiency.

We herein report a case involving a 32-year-old Japanese man with recurrent cerebral venous thrombosis due to hereditary protein C deficiency. He was admitted to our hospital with impaired consciousness. Brain magnetic resonance imaging demonstrated high intensities diffusely along the bilateral sulci and magnetic resonance venography revealed left transverse sinus and superior sagittal sinus stenoses. His father had a history of cerebral infarction and venous thrombosis. The protein C activity level examined by chromogenic synthetic substrate assay was markedly reduced. He was diagnosed with protein C deficiency, and a genetic analysis revealed a heterozygous mutation at exon 3 c.199G>A,p.Glu67Lys on the protein C gene. Four months later, at his second admission, he had transient aphasia, and his protein C activity was under 10%. We switched warfarin to the direct oral anticoagulants edoxaban. He remains fully recovered with no adverse events after the administration of edoxaban for a year. Direct oral anticoagulants may be a new tool for treating cerebral venous thrombosis due to hereditary protein C deficiency.

[COVID-19-associated coagulopathy].

In 2020, infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) rapidly spread across the world to become a global pandemic. Coronavirus disease-2019 (COVID-19) is associated with a high rate of coagulopathy and thrombotic complications. The underlying mechanisms involved in these processes are complex. In addition to the low physical activity, blood coagulation activation accompanied by excessive immune/inflammatory reactions and vascular endothelialitis associated with the presence of intracellular SARS-CoV-2 and disrupted cell membranes contribute substantially to the complexity of the mechanisms. The types of thrombosis that occur include arterial thrombosis and venous thromboembolism. Microthrombi in alveolar capillaries are observed in COVID-19 patients. Considering the possible involvement of thrombosis in the worsening of COVID-19, prophylactic anticoagulant therapy, such as low-molecular-weight heparin or unfractionated heparin, is essential for patients with moderate and severe infections. Even with prophylactic anticoagulant therapy, the incidence of thrombosis remains high. Consequently, control of the underlying inflammation and vascular endothelial protection may be required in combination with anticoagulant therapy.

Potential therapeutics for antiphospholipid antibody associated thrombocytopenia: A systematic review and meta-analysis.

Objectives: Thrombocytopenia is frequently observed in antiphospholipid antibody (aPL) carriers. Due to the paradoxical risks of thrombosis and hemorrhage, the management of aPL-associated thrombocytopenia (APAT) is often deductive. We aimed to investigate the efficacy and safety of therapeutic approaches for APAT through a systematic review.Methods: Four therapeutic approaches for APAT, including antiplatelet agents, glucocorticoids, splenectomy and thrombopoietin receptor agonists, were selected. Clinical trials evaluating therapeutic outcomes including the remission, complications, mortality and relapse, were searched in MEDLINE, EMBASE and CENTRAL from the inception dates to 28 November 2016. A meta-analysis was performed to calculate risk ratios (RRs) and 95% confidence intervals (CIs) using random-effects models.Results: Out of 1407 papers, eight controlled clinical trials were included. In patients with APAT, the remission rates were higher in patients on glucocorticoids (RR 8.33 [95% CI 3.07-22.6]) or splenectomy (RR 8.37 [95% CI 1.61-43.7]) than in patients without those treatments. There was no significant association between glucocorticoids and thrombosis (RR 1.57 [95% CI, 0.17-14.9]) or between splenectomy and hemorrhage (RR 0.17 [95% CI 0.02-1.28]). The extracted data of mortality and relapse rate were not available for synthesis.Conclusion: Glucocorticoids or splenectomy seemed suitable therapeutic approaches for APAT.

Recipient ADAMTS13 Single-Nucleotide Polymorphism Predicts Relapse after Unrelated Bone Marrow Transplantation for Hematologic Malignancy.

Relapse remains a major obstacle to the survival of patients with hematologic malignancies after allogeneic hematopoietic stem cell transplantation. A disintegrin-like and metalloprotease with a thrombospondin type 1 motif (ADMATS13), which cleaves von Willebrand factor multimers into less active fragments, is encoded by the ADAMTS13 gene and has a functional single-nucleotide polymorphism (SNP) rs2285489 (C > T). We retrospectively examined whether ADAMTS13 rs2285489 affected the transplant outcomes in a cohort of 281 patients who underwent unrelated human leukocyte antigen (HLA)-matched bone marrow transplantation for hematologic malignancies. The recipient ADAMTS13 C/C genotype, which putatively has low inducibility, was associated with an increased relapse rate (hazard ratio [HR], 3.12; 95% confidence interval [CI], 1.25⁻7.77; P = 0.015), resulting in a lower disease-free survival rate in the patients with a recipient C/C genotype (HR, 1.64; 95% CI, 1.01⁻2.67; P = 0.045). Therefore, ADAMTS13 rs2285489 genotyping in transplant recipients may be a useful tool for evaluating pretransplantation risks.

Congenital coagulation factor X deficiency: Genetic analysis of five patients and functional characterization of mutant factor X proteins.

Congenital factor X (FX) deficiency is a rare bleeding disorder that is inherited as an autosomal recessive trait. In this study, a genetic analysis of the FX gene was performed in five families with this disorder. Four heterozygous mutations [p.Gly154Arg, p.Val236Met, p.Gly263Val and p.Arg387Cys] and one pair of compound heterozygous FX gene mutations consisting of p.Gly406Ser and p.Val424Phe were identified. Mutant FX proteins containing the identified amino acid substitutions were also expressed in cultured cells. These proteins were analysed by enzyme-linked immunosorbent assay and pulse-chase experiments. The results demonstrated normal intracellular synthesis and extracellular secretion of mutant FX proteins carrying the p.Val236Met, p.Arg387Cys and p.Gly406Ser amino acid substitutions. However, the results also showed that the p.Gly154Arg, p.Gly263Val and p.Val424Phe proteins were secreted less efficiently than the wild-type protein, although they were synthesized normally in the cell. Collectively, these observations suggest that the amino acid substitutions p.Gly154Arg, p.Gly263Val and p.Val424Phe induce protein misfolding, leading to the intracellular degradation of many FX proteins containing any of these mutations, and ultimately to the development of FX deficiency. On the other hand, for the p.Val236Met, p.Arg387Cys and p.Gly406Ser mutant proteins, we hypothesize that secreted FX proteins have impaired coagulant activities due to functional defects caused by these amino acid substitutions.

[Anticoagulants: current topics].

Despite the introduction of direct oral anticoagulants (DOAC), the need for more effective and safer antithrombotic strategies exists. Recently, the findings stating that the contact system is important for thrombus formation has identified factor XI as a potential target for new anticoagulants. Approximately 20-30% of patients who develop venous thromboembolism (VTE) also have cancer. To date, the drugs primarily used in the treatment of VTE are heparin in the acute phase and warfarin in the chronic phase. Recently, a large-scale international clinical trial, which examined the composite outcomes of VTE recurrence and major bleeding in cancer patients, found that edoxaban, a direct factor Xa inhibitor, is not inferior to low-molecular-weight heparin. The study also showed that DOACs have a promising potential to prove therapeutically effective in future studies. Anticoagulants are associated with a severe side effect, bleeding, which makes emergency neutralization an important concern. Four-factor prothrombin complex concentrate can be used to reverse the effect of warfarin and could also be effective as a neutralizing agent in patients having received DOACs. Moreover, more specific reversing agents include the approved human monoclonal antibody fragment idarucizumab for reversing the effects of dabigatran.

The first genome-wide association study identifying new susceptibility loci for obstetric antiphospholipid syndrome.

Antiphospholipid syndrome (APS) is the most important treatable cause of recurrent pregnancy loss. The live birth rate is limited to only 70-80% in patients with APS undergoing established anticoagulant therapy. Lupus anticoagulant (LA), but not anticardiolipin antibody (aCL), was found to predict adverse pregnancy outcome. Recent genome-wide association studies (GWAS) of APS focusing on aCL have shown that several molecules may be involved. This is the first GWAS for obstetric APS focusing on LA. A GWAS was performed to compare 115 Japanese patients with obstetric APS, diagnosed according to criteria of the International Congress on APS, and 419 healthy individuals. Allele or genotype frequencies were compared in a total of 426 344 single-nucleotide polymorphisms (SNPs). Imputation analyses were also performed for the candidate regions detected by the GWAS. One SNP (rs2288493) located on the 3'-UTR of TSHR showed an experiment-wide significant APS association (P=7.85E-08, OR=6.18) under a recessive model after Bonferroni correction considering the number of analyzed SNPs. Another SNP (rs79154414) located around the C1D showed a genome-wide significant APS association (P=4.84E-08, OR=6.20) under an allelic model after applying the SNP imputation. Our findings demonstrate that a specific genotype of TSHR and C1D genes can be a risk factor for obstetric APS.

Diagnosis and treatment of inherited thrombophilia.

Inherited thrombophilia is a thrombotic diathesis caused by a variety of genetic abnormalities in anticoagulant factors such as antithrombin (AT), protein C (PC), and protein S (PS), or coagulation factors such as prothrombin and factor V. Patients with inherited thrombophilia often present with unusual clinical episodes of venous thromboembolism (VTE), which manifests as deep vein thrombosis or pulmonary thromboembolism in young persons (younger than 40 years), often with a positive family history of thrombosis. Moreover, the thrombosis sometimes recurs, despite anticoagulant therapy. The genetic background is known to differ between Caucasians and East Asians. Starting this April, inherited thrombophilia, AT, PC, or PS deficiency, are officially designated as intractable diseases, and the treatment of these diseases is subsidized as public expense.

Familial discrepancy of clinical outcomes associated with fibrinogen Dorfen: A case of huge genital hematoma after episiotomy.

This study demonstrates a case of a huge genital hematoma after delivery, associated with fibrinogen Dorfen. Fibrinogen Dorfen is the mutation of a fibrinogen-coded exon gene, which has a single heterozygous GCC → GTC transition at codon 289 of the γ gene, predicting an Ala → Val substitution. Because Ala289 plays a crucial role in maintaining the structure of the polymerization site of hole 'a' via a hydrogen bond, it is speculated that the γ 289Ala → Val substitution can change not only the fibrinogen structure, but also the function of polymerization. In our case, although the patient's gene mutation was the same as that of her mother, there was a discrepancy in the clinical outcomes. Although the precise mechanism regarding this discrepancy remains unknown, it may cause different perinatal outcomes in terms of vaginal delivery, such as the severe bleeding in this patient and the absence of clinical symptoms in her mother. This is the first report suggesting the heterogeneity of fibrinogen functions of fibrinogen Dorfen, which may be critical to the clinical outcome.

[Diagnosis and treatment of microangiopathic hemolytic anemia].

"Microangiopathic hemolytic anemia (MAHA)" is now used to designate any hemolytic anemia related to RBC fragmentation, occurring in association with small vessel disease. In DIC, RBC fragmentation is thought to result from the deposition of fibrin or platelets within the microvasculature. The term "thrombotic microangiopathy (TMA)" is also used to describe syndromes characterized by MAHA, thrombocytopenia, and thrombotic lesions in small blood vessels. The most prominent diagnoses associated with TMA are thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS). Many different disorders, including preeclampsia, infections, adverse drug reactions, hematopoietic stem cell transplantation, autoimmune diseases, and malignancies, can cause TMA (i.e., secondary TMA). Recently, because the pathogeneses of TTP and HUS have been elucidated, great progress has been made in diagnosis and treatments. However, the pathogenesis of secondary TMA remains unclear. Clinical problems awaiting solution in TMA management include determination of the positioning of rituximab in the treatment sequence of primary TTP, management of Shiga-toxin producing Escherichia coli-HUS complicated by encephalopathy, confirmation of the efficacy and long-term safety of eculizumab in the treatment of atypical HUS, and elucidating the pathogenesis of secondary TMA as well as improving the efficacy of treatment.

[Genetic analysis].

Deficiencies of natural anticoagulant proteins including antithrombin (AT), protein C (PC) and protein S (PS) are important risk factors for venous thromboembolism (VTE) in Japanese people. The identification of deficiencies of these proteins is important for the prevention and treatment of VTE. Genetic analysis can help in making a definitive diagnosis of these inherited deficiencies, and can be useful both for the individual and potential thrombotic risk to family members. Mutations of AT, PC and PS are usually detected by direct DNA sequencing and multiple ligation-dependent probe amplification (MLPA). Large cohort studies have shown that AT and PC mutations are identified in 85% and 70% of patients, respectively. On the other hand, the detection rate in PS deficiency is lower in around 40% of patients.

Relapse of Acquired Hemophilia A after COVID-19 Infection.

Acquired hemophilia A (AHA) is a rare disease in which an autoantibody causes bleeding by interacting with and inhibiting the coagulation activity of endogenous factor VIII (FVIII). Most cases of AHA are idiopathic; known causes include autoimmune diseases, malignant tumors, pregnancy, drugs, and viral infections. An 86-year-old man was diagnosed with AHA based on the following results: an activated partial thromboplastin time (aPTT) extension of 130.7 seconds, presence of an inhibitor pattern in a mixing study, an endogenous factor VIII (FVIII) level of <1%, and an FVIII inhibitor titer of >5.1 Bethesda units (BU). The activity of von Willebrand factor (vWF) was diminished (<10%), which was considered a complication of acquired von Willebrand syndrome (AVWS). The patient was started on prednisolone, and the inhibitor level eventually became negative. vWF values also became normal. However, 1 year later, he was hospitalized for treatment of coronavirus disease 2019 (COVID-19). Blood testing showed an aPTT extension of 110.5 seconds, FVIII level of 4%, and FVIII inhibitor titer of 0.8 BU; thus, a relapse of AHA was diagnosed. After administration of corticosteroid and remdesivir, he recovered from COVID-19 and AHA. The inhibitor level became negative on the 9th day of admission. Several studies have implicated COVID-19 infection and vaccination in AHA. We recommend that aPTT be measured when patients with AHA are infected with SARS-CoV2, to confirm AHA relapse.

Platelet and large platelet ratios are useful in predicting severity of COVID-19.

The role of platelets in coronavirus disease (COVID-19) severity requires further exploration. To determine whether the platelet index is useful in predicting COVID-19 severity, we compared the platelet index in patients with higher and lower oxygen requirements (≥ 4 L/min vs. < 4 L/min) and patients without COVID-19. We also analyzed the time course of the platelet index in each group. A total of 285 patients with COVID-19 and 36 without COVID-19 who were hospitalized at Fussa Hospital were analyzed. After matching for oxygen requirement at admission, multivariate analysis was performed. Platelets (≤ 16.6 × 104/µL) and platelet-large cell ratio (P-LCR) (≥ 27.8%) were significant factors influencing severity. Based on these factors, we created the Fussa platelet score, and the group with a Fussa platelet score ≥ 2 was significantly more likely to reach the 4 L/min oxygen requirement (event-free survival: Fussa platelet score ≥ 2 versus < 2, P < 0.00000001). Analysis of platelet index by time period showed a significant increase from 6-10 days after onset. The Fussa platelet score can be measured quickly, easily, and inexpensively in a clinic and may be useful in determining need for transfer to a critical care hospital.