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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 × 10 4 /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., (© 2024. Japanese Society of Hematology.)

Thrombotic thrombocytopenic purpura (TTP) can rapidly become a life-threatening condition, and the importance of its appropriate diagnosis and treatment cannot be overstated. Until recently, TTP has mainly been diagnosed by clinical findings such as thrombocytopenia and hemolytic anemia. In addition to these clinical findings, however, reduced activity of a disintegrin-like and metalloprotease with thrombospondin type 1 motif 13 (ADAMTS13) below 10% has become internationally accepted as a diagnostic criterion for TTP. TTP is classified as immune-mediated TTP (iTTP) if the patient is positive for anti-ADAMTS13 autoantibodies, and as congenital TTP (cTTP) if ADAMTS13 gene abnormalities are detected. Fresh frozen plasma (FFP) transfusion is performed in patients with cTTP to supplement ADAMTS13. Plasma exchange therapy using FFP is conducted in patients with iTTP to supplement ADAMTS13 and to remove both anti-ADAMTS13 autoantibodies and unusually large von Willebrand factor (VWF) multimers. To suppress autoantibody production, corticosteroid therapy is administered in conjunction with plasma exchange. The monoclonal anti-CD-20 antibody rituximab is effective in patients with iTTP. In addition, caplacizumab, an anti-VWF A1 domain nanobody, has a novel mechanism of action, involving direct inhibition of platelet glycoprotein Ib-VWF binding. The recommended first-line treatments of iTTP in Japan are plasma exchange and corticosteroids, as well as caplacizumab., (© 2023. The Author(s).)

Hereditary antithrombin (AT) deficiency is an autosomal dominant inherited thrombophilia. In three pedigrees of hereditary type I AT deficiency, we identified novel variants c.126delC (p.Lys43Serfs * 7), c.165C > G (p.Tyr55 * ), and c.546delA (p.Lys182Asnfs * 102) in the open reading frame encoding AT in each patient. Each of these aberrant variants leads to premature termination of AT protein synthesis. To investigate whether these abnormal variants are involved in the pathogenesis of type I AT deficiency, we analyzed the function of these variants in HEK293 cells. Results of western blot analysis and immunofluorescence microscopy showed that all abnormal variants were expressed intracellularly, but p.Lys43Serfs * 7 and p.Tyr55 * protein were aggregated in the cells. These three variants were not detected in the spent culture medium, indicating that these novel variants affect protein secretion. In summary, we suggest that these variants in the AT-encoding gene are translated in the cell, but form abnormal proteins that form aggregates and/or inhibit secretion. These results provide insight into novel mechanisms of type I AT deficiency and potential therapies for the condition., (© 2022. Japanese Society of Hematology.)

This study investigated patients with thrombophilia and current peripartum management practices based on national surveillance in Japan. Between 2014 and 2018, antithrombin (AT), protein C (PC) and protein S (PS) deficiency were observed in 84, 67, and 443 pregnancies, respectively, with incidence rates among total deliveries at 0.012%, 0.009%, and 0.061%. The percentage of institutions that measured both antigens and AT, PC, and PS activity for the diagnosis of thrombophilia was 50.2%, and 46.9% of institutions did not perform gene analysis. Prophylactic anticoagulation therapy was used in the ante- and postpartum management of patients with AT deficiency at 67.1% and 66.3% of institutions, most commonly with 10,000 units of unfractionated heparin. Ante- and postpartum management of PC and PS deficiency was performed at 75.3% and 67.1% of institutions. Approximately half of the institutions performed peripartum prophylactic AT supplementation for AT deficiency. Low trough AT activity before supplementation was most commonly 50 ≤  < 70%, and the highest AT supplementation was 1500 ≤  < 3000 units. The number of pregnancies with AT, PC and PS deficiency might be as many as 29, 23 and 151 every year in Japan if complete answers were provided., (© 2022. Japanese Society of Hematology.)

Lipopolysaccharide (LPS) and tissue factor (TF) have frequently been used to induce disseminated intravascular coagulation (DIC) in experimental animal models. We have previously reported that the pathophysiology of DIC differs according to the inducing agents. However, inflammatory status and bleeding symptoms have not been fully compared between rat models of the two forms of DIC. We attempted to evaluate detailed characteristic features of LPS- and TF-induced DIC models, especially in regard to inflammatory status and bleeding symptoms, in addition to selected hemostatic parameters and pathologic findings in the kidneys. The degree of hemostatic activation in both types of experimental DIC was identical, based on the results of thrombin-antithrombin complex levels. Markedly elevated tumor necrosis factor, interleukin-6, and high-mobility group box-1 concentrations were observed with severe organ dysfunction and marked fibrin deposition in the kidney on administration of LPS, whereas markedly elevated D-dimer concentration and bleeding symptoms were observed with TF administration. Pathophysiology such as fibrinolytic activity, organ dysfunction, inflammation status, and bleeding symptom differed markedly between LPS- and TF-induced DIC models in rats. We, therefore, recommend that these disease models be assessed carefully as distinct entities to determine the implications of their experimental and clinical use., (© 2021. Japanese Society of Hematology.)

Patients with congenital protein S (PS) deficiency show a hereditary predisposition for thrombosis, and PS deficiency is prevalent among Japanese populations. Diagnosis is based on symptoms of thrombosis and reduced PS activity. Three reagents that use different measurement principles for determining PS activity are available in Japan. This study aimed to confirm the possibility of harmonization of these three reagents to establish a universal standard for PS activity in Japanese populations. Commercial normal plasma and plasma samples obtained from healthy individuals and healthy pregnant women were tested at three facilities using three reagents for measuring PS: STA-Staclot Protein S (STA-PS), HemosIL Protein S (Clotting) (IL-PS), and a total PS assay (SNT-PS). The within-run precision of each reagent was good, as each had a coefficient of variation of ≤ 3.8%. The dilution linearity for each reagent was also good. The correlation coefficient was 0.94 for STA-PS vs. IL-PS, 0.93 for SNT-PS vs. STA-PS, and 0.90 for SNT-PS vs. IL-PS, indicating a good correlation. Although the three reagents available in Japan for measuring PS activity use different measurement methods, each showed good performance, and large differences were not observed between the obtained values. Harmonization among them appears possible.

Abnormal bleeding is sometimes observed in patients with immunoglobulin light chain (AL) amyloidosis. Although several theories have been proposed regarding the pathological causes of the bleeding tendency in AL amyloidosis, many lacked sufficient evidence and full consensus. We conducted a retrospective survey at a single institution to assess bleeding manifestations, methods for evaluating hematological abnormalities, and treatments for bleeding in patients with systemic AL amyloidosis over the past 13 years. The participants were 10 men and 14 women, aged 39-84 years (mean 65 years). The prevalence of bleeding was 29%. Prolonged prothrombin time (PT), elevated plasmin-α2-antiplasmin complex, and factor X deficiency were distinctive to the bleeding group. Two case studies showed that tranexamic acid was effective for treating this hematological condition. However, two patients with normal PT and activated partial thromboplastin time (APTT) also had a bleeding manifestation. The rates of administration of coagulation and fibrinolytic tests were relatively low in the non-bleeding group. Therefore, a close investigation concerning coagulation and fibrinolysis should be performed in every patient with AL amyloidosis regardless of the PT/APTT values. A more careful, comprehensive, and large-scale study is required to reinforce these findings.

Congenital factor X (FX) deficiency is a rare bleeding disorder with an incidence of one in one million. The proband, a 2-year-old girl, exhibited easy bruising and a history of umbilical cord bleeding at birth. Prothrombin time (> 40 s) and activated partial thromboplastin time (65.0 s) were prolonged. Marked declines in FX activity (< 1%) and FX antigen levels (5%) were also observed. Genetic analysis of the proband identified two types of single-base substitutions, c.353G>A (p.Gly118Asp) and c.1303G>A (p.Gly435Ser), indicating compound heterozygous congenital FX deficiency. Genetic analysis of family members revealed that her father and older sister (5-year-old) were also heterozygous for p.Gly118Asp, and that her mother was heterozygous for p.Gly435Ser. To improve the bleeding tendency, the proband received regular replacement of 500 units of PPSB-HT, a prothrombin complex concentrate (PCC). Following continued regular replacement of 500 units of PPSB-HT once per week, the proband has exhibited no bleeding tendencies and no new bruises have been observed. There are no previous report of the use of PPSB-HT for regular FX replacement. Regular replacement therapy with PPSB-HT may be an effective method for preventative control of bleeding tendencies in FX deficiency.

Acquired coagulation factor inhibitors are rare. Among them, coagulation factor V (FV) inhibitor is particularly uncommon and presents with variable clinical manifestations. Certain acquired FV inhibitor patients have only mild bleeding or, in select cases, no symptoms at all, leading to spontaneous recovery. Others have life-threatening bleeding that requires medical attention. Thus, a prompt decision regarding diagnosis and clinical intervention is crucial for such patients. In five acquired FV inhibitor cases treated in our facility, each patient had a malignancy as an underlying disease and all unexpectedly showed prolongation of both prothrombin time (PT) and activated partial thromboplastin time (APTT). They all also displayed a discrepancy between PT and Normotest (Hepaplastintest, HPT) results. All but one patient experienced no bleeding at the time of diagnosis and achieved spontaneous recovery in 1-3 weeks. The patient with bleeding symptoms received plasma exchanges and a platelet transfusion. Useful markers in diagnosing the presence of an acquired FV inhibitor were a sudden prolongation of PT and APTT, and a discrepancy between the PT/APTT and HPT assays. Spontaneous recovery can be expected for patients with only minor bleeding.

Inherited antithrombin (AT) deficiency is one of the most clinically significant forms of congenital thrombophilia and follows an autosomal dominant mode of inheritance. We analyzed SERPINC1 in a patient who developed deep-vein thrombosis and low AT activity during pregnancy, and identified a novel missense mutation c.259A>G (p.Asn87Asp; N87D). Surprisingly, analysis of the parents' DNA showed that they did not possess this mutant, and thus, it may have been due to a de novo mutation. We also expressed this mutant AT protein in COS-1 cells and compared its intracellular localization and intracellular and extracellular antigen levels with that of wild-type AT. The expression experiment did not reveal a significant difference in the antigen levels of the mutant and wild-type AT in the cell lysate, but the mutant AT antigen level was markedly lower than that of its wild-type counterpart in the COS-1 cell supernatant. Immunofluorescence did not indicate any difference between the mutant and wild-type AT in terms of cytoplasmic localization of fluorescence signals. Our findings suggest that the patient's AT deficiency may have been caused by impaired extracellular secretion of mutant AT protein p.Asn87Asp.

We summarize causative genetic mutations for antithrombin (AT) deficiency and their clinical background in Japanese patients. A total of 19 mutations, including seven novel mutations, were identified. We also summarize clinical symptoms of thrombosis, age at onset, family history, and contributing factors for thrombosis, and review the use of prophylactic anticoagulation in pregnant women with heterozygous type II heparin binding site defects (HBS) AT deficiency. The prevalence of thrombosis in probands with type I AT deficiency (88%) was double that observed in those with type II AT deficiency (50%). The prevalence of thrombotic episodes among family members was also higher for type I AT deficiency subjects (82%) than for those with type II AT deficiency (0%). The most common contributing factor for thrombosis among women with type I AT deficiency was pregnancy. Forty-five percent of women with type I AT deficiency developed thrombotic events before the 20th week of gestation. In contrast, women with type II (HBS) AT deficiency appear to be at a lower risk of thrombosis during pregnancy. In conclusion, thrombotic risk varies among different subtypes. Risk assessments based on genetic/clinical backgrounds may contribute to appropriate diagnosis, treatment, and prophylaxis for patients with AT deficiency.

Pregnancy with paroxysmal nocturnal hemoglobinuria (PNH) is associated with significant risk of complications, such as life-threatening thrombosis. Recently, eculizumab has come into clinical use and revolutionized the treatment of PNH. However, clinical information regarding eculizumab use for PNH during pregnancy is limited. The present report describes pregnancies with PNH treated with eculizumab that were registered with the Japan PNH study group and reviews the literature. In case 1, the patient received eculizumab throughout pregnancy and delivered a healthy neonate at term, although breakthrough hemolysis occurred at 20 weeks of gestation. In case 2, the patient discontinued eculizumab before pregnancy and developed preeclampsia at 27 weeks of gestation. She received eculizumab and delivered a preterm, but healthy, neonate by cesarean section. In case 3, the patient received eculizumab from 18 weeks of gestation and delivered a healthy neonate at term without any complications. Reports of 11 pregnant women treated with eculizumab were identified in the literature. Of 14 pregnancies, including our own cases, breakthrough hemolysis and preeclampsia occurred in five and two cases, respectively. There were no thrombotic complications, maternal or neonatal deaths, or fetal structural abnormalities. Thus, eculizumab appears to be safe and effective for managing PNH during pregnancy.

Thrombomodulin, encoded by the THBD gene, is a critical regulator of coagulation and innate immunity. Its gene variant (rs3176123, 2729A>C) in the 3' untranslated region has been reported to be associated with vasculopathies. The present study analyzed the impact of THBD variation on transplant outcomes in a cohort of 317 patients who underwent unrelated HLA-matched bone marrow transplantation (BMT) for hematologic malignancies through the Japan Marrow Donor Program. The donor A/C or C/C genotype vs. the donor A/A genotype resulted in a lower incidence of grades II-IV acute graft-versus-host disease [GVHD; hazard ratio (HR) 0.66; 95 % confidence interval (CI) 0.44-0.99; P = 0.05] according to a multivariate analysis. In patients with grades II-IV acute GVHD, the donor A/C or C/C genotype vs. the donor A/A genotype was associated with significantly better overall survival rates (HR 0.45; 95 % CI 0.21-0.99, P = 0.05), while this effect was absent in other patients. A functional analysis using lymphocytes obtained from healthy individuals revealed that the 2729C allele has a higher level of THBD mRNA than the 2729A allele. These findings suggest the functional relevance of the rs3176123 variation and indicate that higher thrombomodulin expression by individuals with the 2729C allele likely accounts for their decreased risk for acute GVHD development and subsequent mortality.

Imatinib was the first BCR-ABL tyrosine kinase inhibitor to become clinically available. In this study, we retrospectively evaluated the long-term efficacy of low-dose imatinib (final maintenance dose <300 mg per day) due to intolerance, in comparison to optimal-dose imatinib (≥300 mg per day) in patients with Philadelphia chromosome-positive chronic myeloid leukemia in the chronic phase. The Kaplan-Meier estimates of the median time to complete cytogenetic response, major molecular response, and complete molecular response were longer for 31 patients receiving low-dose imatinib (360, 1360, and 1420 days, respectively) than 74 patients receiving optimal-dose imatinib (170, 420, and 720 days, respectively). However, the differences in response shrank over time and progression-free survival were comparable between the two groups. These findings suggest that long-term treatment with low-dose imatinib is an acceptable alternative for patients with intolerance to the optimal dose.

An inherited antithrombin deficiency is an autosomal dominant thrombotic disorder. We identified two pedigrees of inherited type I antithrombin deficiency and two responsible mutations in each. A novel 21-22delAA appeared to have caused a frameshift with a premature termination at amino acid +63 in one patient and a large deletion including all seven exons was identified by multiplex ligation-dependent probe amplification in the other. Some asymptomatic relatives of the second patient had the same mutation. The present findings support the value of using more than one method of gene analysis and of studying the families of probands with inherited thrombotic disorders.

Annexin II is a receptor of tissue-type plasminogen activator (t-PA). We have previously identified annexin II by immunolocalization in human atherosclerotic abdominal aortic aneurysms (AAAs). To investigate possible interactions between annexin II and AAA development, we examined annexin II mRNA and protein expression in a rat model of experimental AAA. AAAs were induced in rats by transient aortic infusion of elastase. The rats were divided into three groups: a saline-treated control group, a group with 15-min elastase infusion, and a group with 30-min elastase infusion. The 15-min elastase-infused group had smaller aneurysms and more preserved media than the 30-min elastase-infused group. Immunohistochemistry showed that annexin II expression was increased in the thickened intima and media of AAA rats as compared with the media of control rats. Furthermore, annexin II was colocalized with macrophages and smooth muscle cells. Quantitative real-time polymerase chain reaction showed that annexin II mRNA levels were up-regulated only in the smaller aneurysm group compared with the control group. In contrast, t-PA mRNA levels were increased in both the 15- and 30-min elastase-infused groups as compared with the control group. These results demonstrate various levels of annexin II expression within the aortic wall of rats with experimental AAAs. It has been suggested that alteration of fibrinolytic activity regulated by annexin II within the aortic wall may be associated with aneurysm formation.

Previous studies have found markedly elevated serum concentrations of proinflammatory cytokines in patients with Graves' disease (GD). We investigated the role of macrophage colony-stimulating factor (M-CSF) in GD. We assayed concentrations of M-CSF in sera from 32 patients with GD (25 untreated; 7 receiving thiamazole therapy). We also studied 32 age-matched healthy subjects as controls. Relationships between serum M-CSF and both thyroid state and serum lipids were examined. Moreover, to examine the effect of thyroid hormone alone on serum M-CSF, T3 was administered orally to normal subjects. Serum concentrations of M-CSF in GD patients who were hyperthyroid were significantly increased compared with GD patients who were euthyroid (P < 0.05) and control subjects (P < 0.0001). Serum M-CSF concentrations correlated closely with T3 levels in patients (r = 0.51, P < 0.005). Serial measurement of five individual patients revealed that serum concentrations of M-CSF were significantly decreased (P < 0.05), reaching normal control values upon attainment of euthyroidism. Furthermore, oral T3 administered to 15 volunteers for 7 days produced significant increases in serum levels of M-CSF (P < 0.05). The close correlation between serum M-CSF and serum thyroid hormone levels suggests that high circulating levels of thyroid hormones may directly or indirectly potentiate the production of M-CSF in patients with GD.

The patient, a 20-year-old male, was found to have a slightly prolonged prothrombin time (PT). No episodes of bleeding were noted. The measurement of coagulation factors revealed that the level of factor X (FX) activity was solely deficient, 51% (normal range: 70-130% ), and that of FX antigen was 100%. Analysis of the entire FX gene revealed the novel missense mutation of GTG to ATG, resulting in the substitution of the 196th amino acid valine --> methionine. The mother and younger brother had a normal PT time and expressed no episode of bleeding. The mother exhibited a normal level of FX activity and antigen; however the younger brother showed a slight decrease in both the parameters. This mutation was not observed in the mother and younger brother. Polymorphism is not observed at this point in healthy persons. The present novel FX mutation was named FX Hofu.

Plasma D-dimer (DD) is considered to be one of the most useful markers in the diagnosis and assessment of disseminated intravascular coagulation (DIC). The present study was performed to clarify the role of DD in a rat model of lipopolysaccharide (LPS)-induced DIC in which low-molecular-weight heparin (LMWH) and tranexamic acid (TA) were used. We investigated whether a relationship exists between plasma DD levels and severity of DIC. Experimental DIC was induced in rats by a sustained 4-hour infusion of 30 mg/kg LPS administered via the tail vein (LPS group). Rats received either LPS alone (LPS group) or LPS combined with 200 U/kg LMWH (LPS+LMWH group) or 50 mg/kg TA (LPS+TA group) from -30 minutes to 4 hours. Blood was drawn from each rat at 4, 8, and 12 hours. Plasma levels of thrombin-antithrombin complex (TAT) and creatinine were suppressed in the LPS+LMWH group, and less glomerular fibrin deposition was observed compared with the LPS group. On the other hand, an increased level of creatinine and increased glomerular fibrin deposition were observed in the LPS+TA group compared with the LPS group. LMWH demonstrated a protective effect against LPS-induced DIC, resulting in increased survival at 12 hours, whereas TA had the opposite effect. From these results, it appears that LMWH protects against LPS-induced DIC, but TA exacerbates LPS-induced DIC. It was interesting that plasma levels of DD were almost completely suppressed by concurrent administration of either TA or LMWH in this LPS-induced DIC model. This finding suggested that plasma levels of DD were suppressed by inhibition of coagulation (reduced deposition of fibrin) in the LPS+LMWH group and that DD levels were also suppressed by inhibition of fibrinolysis (reduced degradation of fibrin by plasmin) in the LPS+TA group. Thus care should be taken when evaluating the significance of plasma DD levels, because suppressed levels can occur with progressive fibrin deposition and worsening organ dysfunction or improvement in the course of DIC.

We investigated the molecular basis of factor X deficiency in a Japanese patient whose factor X activity and antigen level were 45% and 50% of normal control values, respectively. All exons and intron/exon junctions of the factor X gene were studied using a strategy combining polymerase chain reaction (PCR) amplification and nonradioactive single-strand conformational polymorphism (SSCP) analysis. Exon 5, containing the DNA fragment of the proband, showed aberrant migration by SSCP analysis. All exon-containing DNA fragments amplified by PCR were sequenced, and it was revealed that the proband was a heterozygote for a G --> A substitution in exon 5 of the factor X gene of the proband. This mutation predicts an amino acid replacement of arginine (Arg) for glycine (Gly) at codon 114 in the second EGF-like domain.