Unrecognized blood clotting factors.

Most clotting factors were initially discovered as agents functionally deficient in the plasmas of rare patients with hereditary coagulation disorders. During 1940s to 1960s, many factors were named by different investigators after the name of the patient who lacked a new factor. Consequently, there were the same factors with different names. To avoid confusion, the International Committee on the nomenclature of clotting factors was founded and discussed the identity or non-identity of clotting factors by specialists. There remain, however, several factors that were not officially authorized. We attempt to review some of these factors that seem to be not identical with any known clotting factors.

Myh9 R702C is associated with erythroid abnormality with splenomegaly in mice.

MYH9 disorders are characterized by giant platelets, thrombocytopenia, and Döhle body-like cytoplasmic inclusion bodies in granulocytes. However, whether these disorders cause any changes in erythroid cells has yet to be determined. This study analyzed the influence of Myh9 R702C, as one of the most commonly detected MYH9 disorders, on erythroid cells in a mouse model. Knock-in mice expressing Myh9 R702C mutation either systemically or specific to hematological cells (R702C and R702C vav1 mice, respectively) were used in this study. Both displayed lower hemoglobin and higher erythropoietin levels than wild-type (WT) mice, along with significant splenomegaly. Flow cytometric analysis revealed erythroblasts present at a higher rate than WT mice in the spleen. However, no obvious abnormalities were seen in erythroid differentiation from megakaryocyte/erythroid progenitor to erythrocyte. Cell culture assay by fetal liver and colony assay also showed normal progression of erythroid differentiation from erythroid burst-forming unit to red blood cell. In conclusion, R702C and R702C vav1 mice displayed erythroid abnormality with splenomegaly. However, erythroid differentiation showed no obvious abnormality. Further research is required to elucidate the underlying mechanisms.

Effects of thrombomodulin alfa on hemostatic parameters in disseminated intravascular coagulation: Post hoc analysis of a phase 3 randomized controlled trial.

BACKGROUND: The efficacy and safety of thrombomodulin alfa (TM-α), a cofactor protein promoting thrombin-mediated protein C activation, have been examined in a phase 3 randomized, double-blinded, parallel-group trial in Japan. We have previously reported that TM-α is noninferior to heparin for the resolution of disseminated intravascular coagulation (DIC). OBJECTIVE: To investigate the basis for the efficacy of TM-α in the phase 3 clinical trial in Japan through post hoc analysis of coagulation and fibrinolysis parameters. PATIENTS/METHODS: The 227 patients of the full analysis set population described in the original phase 3 trial in Japan were included in this analysis. Changes in parameters between before and after TM-α or heparin administration in each of the two patient groups, with underlying diseases of either hematologic malignancy or infection, were studied separately and results were compared between TM-α and heparin treatment groups in a post hoc manner. RESULTS: TM-α administration did not prolong activated partial thromboplastin time but significantly decreased thrombin-antithrombin complex levels compared with heparin treatment. TM-α administration reduced consumption of endogenous anticoagulants such as antithrombin and protein C by DIC, compared with the heparin group. DIC scores were decreased in both TM-α and heparin groups during the 6-day treatment. CONCLUSION: TM-α can alleviate intravascular coagulation and consumption of anticoagulants without extending coagulation times. This may be associated with the relatively low risk of bleeding during TM-α treatment.

Effect of a Recombinant Human Soluble Thrombomodulin on Mortality in Patients With Sepsis-Associated Coagulopathy: The SCARLET Randomized Clinical Trial.

Importance: Previous research suggested that soluble human recombinant thrombomodulin may reduce mortality among patients with sepsis-associated coagulopathy. Objective: To determine the effect of human recombinant thrombomodulin vs placebo on 28-day all-cause mortality among patients with sepsis-associated coagulopathy. Design, Setting, and Participants: The SCARLET trial was a randomized, double-blind, placebo-controlled, multinational, multicenter phase 3 study conducted in intensive care units at 159 sites in 26 countries. All adult patients admitted to one of the participating intensive care units between October 2012 and March 2018 with sepsis-associated coagulopathy and concomitant cardiovascular and/or respiratory failure, defined as an international normalized ratio greater than 1.40 without other known etiology and a platelet count in the range of 30 to 150 × 109/L or a greater than 30% decrease in platelet count within 24 hours, were considered for inclusion. The final date of follow-up was February 28, 2019. Interventions: Patients with sepsis-associated coagulopathy were randomized and treated with an intravenous bolus or a 15-minute infusion of thrombomodulin (0.06 mg/kg/d [maximum, 6 mg/d]; n = 395) or matching placebo (n = 405) once daily for 6 days. Main Outcome and Measures: The primary end point was 28-day all-cause mortality. Results: Among 816 randomized patients, 800 (mean age, 60.7 years; 437 [54.6%] men) completed the study and were included in the full analysis set. In these patients, the 28-day all-cause mortality rate was not statistically significantly different between the thrombomodulin group and the placebo group (106 of 395 patients [26.8%] vs 119 of 405 patients [29.4%], respectively; P = .32). The absolute risk difference was 2.55% (95% CI, -3.68% to 8.77%). The incidence of serious major bleeding adverse events (defined as any intracranial hemorrhage; life-threatening bleeding; or bleeding event classified as serious by the investigator, with administration of at least 1440 mL [typically 6 units] of packed red blood cells over 2 consecutive days) was 23 of 396 patients (5.8%) in the thrombomodulin group and 16 of 404 (4.0%) in the placebo group. Conclusions and Relevance: Among patients with sepsis-associated coagulopathy, administration of a human recombinant thrombomodulin, compared with placebo, did not significantly reduce 28-day all-cause mortality. Trial Registration: ClinicalTrials.gov Identifier: NCT01598831.

Brief history of the Asiatic International Society of Hematology: our roots of international cooperation with Asian countries.

This year, 2018, marks the 60th anniversary of the Asiatic International Society of Hematology founded in 1958 and it seems to be a fitting occasion on which to reflect on our roots of the international cooperation with Asian countries. The Japanese Society of Hematology held the first meeting of the Asiatic International Society of Hematology in 1958 in Nagoya. Hematologists representing at least 10 Asian countries or districts, including Australia, Burma, Ceylon, Hong Kong, India, Indonesia, Korea, Philippines, Taiwan, and Thailand, participated. This meeting was perhaps the first International Congress of any kind in the medical field in Japan after World War II. In 1968, the Asian-Pacific Society of Hematology converged to join the International Society of Hematology (ISH) and became one of the three divisions of ISH (Asian-Pacific Division of ISH). The Annual Reports of the Society were published in Nagoya from 1969 to 1991. The meetings of the Asiatic International Society and the Asian-Pacific Division of ISH, and the distributions of the Annual Report to Asian countries, including China and Korea, seemed to help promote hematology in those countries, particularly in the early days.

The Japan Marrow Donor Program, 25 years of experience in achieving 20,000 bone marrow transplantations: organization structure, activity, and financial basis.

The Japan Marrow Donor Program (JMDP), established in 1991, has continued to grow in its capacity to facilitate unrelated bone marrow (BMT) and peripheral blood stem cell transplantation (PBSCT) for the past 25 years in Japan. The current donor pool is 463,465 (as of 31 December 2016) and 20,237 transplants were performed with the help of the Japanese Red Cross, government, and supporters. As JMDP introduced PBSCT in 2010, the vast majority of transplants are BMT. All donors are fully typed for HLA-A, B, C, and DR. The peak age of registered donors is around 40 years. The 8/8 HLA-matched donors are found in our registry for 96% of the patients and 54% of the patients receive a transplant. The median time between the initiation of donor search and the transplantation is approximately 122 days. The median interval between the initiation of donor search and identification of the first potential donor is 40 days. The most common diseases for which unrelated BMT/PBSCT is indicated are acute myelogenous leukemia (AML), acute lymphocytic leukemia (ALL), myelodysplastic syndrome (MDS), and malignant lymphoma. In recent years we have seen a marked increase in elderly patients who received BMT.

Missense mutations in the gene encoding prothrombin corresponding to Arg596 cause antithrombin resistance and thrombomodulin resistance.

Antithrombin (AT) and thrombomodulin (TM) play important roles in the process of natural anticoagulation in vivo. Recently, we reported that the prothrombin Yukuhashi mutation (p.Arg596Leu) was associated with AT and TM resistance-related thrombophilia. To assess the AT and TM resistances associated with other missense mutations by single base substitution in the Arg596 codon, we generated recombinant variants (596Gln, 596Trp, 596Gly, and 596Pro) and investigated the effects on AT and TM anticoagulant functions. All variants except 596Pro were secreted in amounts comparable to that of the wild-type but exhibited variable procoagulant activities. After a 30-minute inactivation by AT, the relative residual activity of wild-type thrombin decreased to 15 ± 4.0 %, in contrast to values of all variants were maintained at above 80 %. The thrombin-AT complex formation, as determined by enzyme-linked immunosorbent assay, was reduced with all tested variants in the presence and absence of heparin. In the presence of soluble TM (sTM), the relative fibrinogen clotting activity of wild-type thrombin decreased to 16 ± 0.12 %, whereas that of tested variants was 37 %-56 %. In a surface plasmon resonance assay, missense Arg596 mutations reduced thrombin-TM affinity to an extent similar to the reduction of fibrinogen clotting inhibition. In the presence of sTM or cultured endothelial-like cells, APC generation was enhanced differently by variant thrombins in a thrombin-TM affinity-dependent manner. These data indicate that prothrombin Arg596 missense mutations lead to AT and TM resistance in the variant thrombins and suggest that prothrombin Arg596 is important for AT- and TM-mediated anticoagulation.

Recombinant human soluble thrombomodulin (thrombomodulin alfa) to treat disseminated intravascular coagulation in solid tumors: results of a one-arm prospective trial.

BACKGROUND: Disseminated intravascular coagulation (DIC) associated with solid tumors (DIC-ST) is often encountered in clinical practice. Patients with DIC-ST are usually in poor condition and have bleeding diathesis due to advanced or metastatic diseases. Although some affected patients are treated with heparin, this strategy has not been prospectively studied. Recombinant human soluble thrombomodulin (thrombomodulin alfa, TM-α) is a new anticoagulant developed in Japan. We conducted a prospective study that evaluated the efficacy and safety of TM-α in patients with DIC-ST. METHODS: A prospective one-arm study with TM-α was conducted for DIC-ST. TM-α (380 U/kg) was given for 30 min intravenously once daily for 6-14 days. The primary efficacy endpoint was the DIC resolution rate. Change in DIC scores and improvement in bleeding symptoms and outcomes were also evaluated. Safety endpoints included the incidence of bleeding-related adverse events. RESULTS: A total of 101 patients were treated with TM-α. The three main underlying malignant diseases were lung, stomach, and breast cancer, which accounted for 60 % of all patients. The DIC resolution rate was 34.0 % at the end of TM-α treatment. Improvement in DIC scores was seen in 55.2 % of patients, while only 22.9 % of patients had worsening of DIC scores. The overall survival rate was 55.4 % on day 28. The incidence of hemorrhage related to TM-α was 12.9 % until day 28. Cases of severe hemorrhage related to TM-α did not occur. CONCLUSIONS: TM-α is effective and safe for DIC-ST. This agent is the treatment of choice for the management of DIC-ST.

Plasminogen activator inhibitor-1 in aging.

Plasminogen activator inhibitor-1 (PAI-1), a principal inhibitor of fibrinolysis, is induced in thrombotic, fibrotic, and cardiovascular diseases, which in turn primarily afflict the older population. This induction of PAI-1 may play an important role in the pathology of these diseases as PAI-1 can regulate the dissolution of fibrin and also inhibit the degradation of the extracellular matrix by reducing plasmin generation. PAI-1 expression is elevated in aged individuals and is significantly upregulated in a variety of pathologies associated with the process of aging, including myocardial and cerebral infarction, vascular (athero) sclerosis, cardiac and lung fibrosis, metabolic syndromes (e.g., hypertension, hyperlipidemia, and insulin resistance), cancer, and inflammatory/stress responses. Thus, PAI-1 may play a critical role in the development of aging-associated pathological changes. In addition, PAI-1 is recognized as a marker of senescence and a key member of a group of proteins collectively known as the senescence-messaging secretome. In this review, we highlight the role of PAI-1 in the pathophysiology of aging and aging-associated disorders.

Combined medialis pedis and medial plantar fasciocutaneous flaps for coverage of soft tissue defects of multiple adjacent fingers.

Soft tissue defects of adjacent multiple fingers covered by a single large flap require secondary separation of the flap into each finger. Such covering obstructs independent motion of injured fingers until the single large flap is separated. This report describes the technique of combined medialis pedis and medial plantar fasciocutaneous flaps for reconstructing soft tissue defects of multiple adjacent fingers. Three male patients (age range, 18-33 years) underwent soft tissue reconstructions of multiple adjacent fingers with combined flaps. Injuries involved three adjacent palmar fingers, two adjacent palmar fingers, and two adjacent dorsal fingers. Average sizes of the combined flaps were 4.2 × 4.0 cm for the medialis pedis flap and 3.0 × 1.8 cm for the medial plantar fasciocutaneous flap. All flaps survived without vascular complications, and donor sites healed uneventfully. All patients experienced excellent recovery of range of motion for the reconstructed fingers. In conclusion, combined flaps may offer an alternative for coverage of soft tissue defects that involve multiple adjacent fingers.

Development of a new laboratory test to evaluate antithrombin resistance in plasma.

INTRODUCTION: We recently reported a variant prothrombin (p.Arg596Leu: prothrombin Yukuhashi) that confers antithrombin resistance to patients with hereditary thrombosis. To detect antithrombin resistance in plasma, we devised a laboratory test analyzing the kinetics of thrombin inactivation using antithrombin. MATERIALS AND METHODS: After incubation with prothrombin activator components (phospholipids, CaCl2, and snake venom), samples were treated with excess antithrombin in the presence or absence of heparin for various time periods. Subsequently, H-D-Phe-Pip-Arg-p-nitoranilide was added and changes in absorbance/min (ΔA/min) were measured at 405 nm. RESULTS: After 1 min inactivation using antithrombin and heparin, the relative residual thrombin activity of recombinant mutant prothrombin (34.3% ± 2.2%) was higher than that of the wild-type (6.3% ± 1.2 %). After 30 min without heparin, the relative residual thrombin activity of recombinant mutant prothrombin (95.8% ± 0.4%) was higher than that of the wild-type (10.1% ± 1.7%), indicating that this assay could detect antithrombin resistance of the variant 596Leu prothrombin. Moreover, warfarinized plasmas from 2 heterozygous patients with prothrombin Yukuhashi mutation clearly showed higher values of the relative residual thrombin activity than those from 5 thrombosis patients lacking the mutation in the presence or absence of heparin. CONCLUSIONS: We have devised a laboratory test to detect antithrombin resistance in plasma by analyzing the kinetics of thrombin inactivation using antithrombin. This assay may be useful for detecting antithrombin resistance in plasma, even in warfarinized patients.

TUBB1 mutation disrupting microtubule assembly impairs proplatelet formation and results in congenital macrothrombocytopenia.

This report describes a family with TUBB1-associated macrothrombocytopenia diagnosed based on abnormal platelet ß1-tubulin distribution. A circumferential marginal microtubule band was undetectable, whereas microtubules were frayed and disorganized in every platelet from the affected individuals. Patients were heterozygous for novel TUBB1 p.F260S that locates at the α- and ß-tubulin intradimer interface. Mutant ß1-tubulin was not incorporated into microtubules with endogenous α-tubulin, and α-tubulin expression was decreased in transfected Chinese hamster ovary cells. Transduction of mutant ß1-tubulin into mouse fetal liver-derived megakaryocytes demonstrated no incorporation of mutant ß1-tubulin into microtubules with endogenous α-tubulin and diminished proplatelet formation, leading to the production of fewer, but larger, proplatelet tips. Furthermore, mutant ß1-tubulin was not associated with endogenous α-tubulin in the proplatelets. Deficient functional microtubules might lead to defective proplatelet formation and abnormal protrusion-like platelet release, resulting in congenital macrothrombocytopenia.

Establishment of mouse model of MYH9 disorders: heterozygous R702C mutation provokes macrothrombocytopenia with leukocyte inclusion bodies, renal glomerulosclerosis and hearing disability.

Nonmuscle myosin heavy chain IIA (NMMHCIIA) encoded by MYH9 is associated with autosomal dominantly inherited diseases called MYH9 disorders. MYH9 disorders are characterized by macrothrombocytopenia and very characteristic inclusion bodies in granulocytes. MYH9 disorders frequently cause nephritis, sensorineural hearing disability and cataracts. One of the most common and deleterious mutations causing these disorders is the R702C missense mutation. We generated knock-in mice expressing the Myh9 R702C mutation. R702C knock-in hetero mice (R702C+/- mice) showed macrothrombocytopenia. We studied megakaryopoiesis of cultured fetal liver cells of R702C+/- mice and found that proplatelet formation was impaired: the number of proplatelet tips was decreased, proplatelet size was increased, and proplatelet shafts were short and enlarged. Although granulocyte inclusion bodies were not visible by May-Grünwald Giemsa staining, immunofluorescence analysis indicated that NMMHCIIA proteins aggregated and accumulated in the granulocyte cytoplasm. In other organs, R702C+/- mice displayed albuminuria which increased with age. Renal pathology examination revealed glomerulosclerosis. Sensory hearing loss was indicated by lowered auditory brainstem response. These findings indicate that Myh9 R702C knock-in mice mirror features of human MYH9 disorders arising from the R702C mutation.

Molecular mechanisms of syndecan-4 upregulation by TNF-α in the endothelium-like EAhy926 cells.

Syndecan-4, a cell-surface heparan sulfate proteoglycan, can participate in inflammation and wound healing as a host defense molecule. Tumour necrosis factor (TNF)-α, one of the most potent proinflammatory cytokines, is known to upregulate syndecan-4 expression, but the precise mechanisms are unclear. To elucidate these mechanisms in detail, we examined syndecan-4 upregulation by TNF-α in the endothelium-like EAhy926 cell. Of the two putative nuclear factor kappa-B (NF-κB) binding sites in the syndecan-4 gene (SDC4) promoter, deletion or mutation of one or both sites significantly diminished the effects of TNF-α. Electrophoretic mobility shift assays showed that p65 and c-Rel, but not p50, bound to these NF-κB binding sites, whereas pull-down assays showed binding of all three NF-κB components. Chromatin immunoprecipitation assays clearly showed that p65 and phosphorylated p65, but not p50 or c-Rel, bound to the SDC4 promoter. An NF-κB inhibitor, p65 knockdown and a transcriptional elongation inhibitor completely blocked the effect of TNF-α on SDC4 promoter activity and significantly, but not completely, blocked that on SDC4 mRNA expression. These data suggest that NF-κB p65 could be a key mediator of syndecan-4 upregulation by TNF-α through two binding sites in the SDC4 promoter, but other NF-κB-p65 independent pathways might also be involved through transcriptional elongation.

Bernard-Soulier syndrome caused by a hemizygous GPIbß mutation and 22q11.2 deletion.

BACKGROUND: Bernard-Soulier syndrome (BSS) is a rare autosomal recessive bleeding disorder characterized by giant platelets, thrombocytopenia, and a prolonged bleeding time, which is caused by homozygous mutations in the GPIbα, GPIbß, or GPIX genes. The 22q11.2 deletion syndrome (22q11.2DS) is caused by a microdeletion on chromosome 22, which includes the GPIbß gene, and is characterized by abnormal development of the pharyngeal apparatus and heart. Thus, patients with 22q11.2DS are obligate carriers for BSS. METHODS: We evaluated two infants with BSS and performed the genetic analysis of the GPIbα, GPIbß, or GPIX genes, and investigated the segregation of the mutation within the families. The status of the 22q11.2 deletion was examined by fluorescence in situ hybridization and single-nucleotide polymorphism array copy number analysis. RESULTS: DNA sequencing analysis revealed that the infants were compound heterozygous for a hemizygous mutation in the GPIbß gene (p.Trp148X and p.Leu97Phe, respectively) and 22q11.2 deletion in the other chromosome. Both infants had the common 3Mb 22q11.2 deletion but did not show major phenotypic features of 22q11.2DS, such as developmental delay, cardiac defects, dysmorphic facial features, palatal anomalies, hypocalcemia, and immune deficiency. The 22q11.2DS would not have become clear if detailed molecular genetic analyses of BSS had not been performed. CONCLUSIONS: Our cases illustrate that a suspicion of 22q11.2 deletion is warranted in pediatric BSS patients with a mutation in the GPIbß gene, even without remarkable symptoms.

ACTN1 mutations cause congenital macrothrombocytopenia.

Congenital macrothrombocytopenia (CMTP) is a heterogeneous group of rare platelet disorders characterized by a congenital reduction of platelet counts and abnormally large platelets, for which CMTP-causing mutations are only found in approximately half the cases. We herein performed whole-exome sequencing and targeted Sanger sequencing to identify mutations that cause CMTP, in which a dominant mode of transmission had been suspected but for which no known responsible mutations have been documented. In 13 Japanese CMTP-affected pedigrees, we identified six (46%) affected by ACTN1 variants cosegregating with CMTP. In the entire cohort, ACNT1 variants accounted for 5.5% of the dominant forms of CMTP cases and represented the fourth most common cause in Japanese individuals. Individuals with ACTN1 variants presented with moderate macrothrombocytopenia with anisocytosis but were either asymptomatic or had only a modest bleeding tendency. ACTN1 encodes α-actinin-1, a member of the actin-crosslinking protein superfamily that participates in the organization of the cytoskeleton. In vitro transfection experiments in Chinese hamster ovary cells demonstrated that altered α-actinin-1 disrupted the normal actin-based cytoskeletal structure. Moreover, transduction of mouse fetal liver-derived megakaryocytes with disease-associated ACTN1 variants caused a disorganized actin-based cytoskeleton in megakaryocytes, resulting in the production of abnormally large proplatelet tips, which were reduced in number. Our findings provide an insight into the pathogenesis of CMTP.

Ribavirin-induced intracellular GTP depletion activates transcription elongation in coagulation factor VII gene expression.

Coagulation FVII (Factor VII) is a vitamin K-dependent glycoprotein synthesized in hepatocytes. It was reported previously that FVII gene (F7) expression was up-regulated by ribavirin treatment in hepatitis C virus-infected haemophilia patients; however, its precise mechanism is still unknown. In the present study, we investigated the molecular mechanism of ribavirin-induced up-regulation of F7 expression in HepG2 (human hepatoma cell line). We found that intracellular GTP depletion by ribavirin as well as other IMPDH (inosine-5'-monophosphate dehydrogenase) inhibitors, such as mycophenolic acid and 6-mercaptopurine, up-regulated F7 expression. FVII mRNA transcription was mainly enhanced by accelerated transcription elongation, which was mediated by the P-TEFb (positive-transcription elongation factor b) complex, rather than by promoter activation. Ribavirin unregulated ELL (eleven-nineteen lysine-rich leukaemia) 3 mRNA expression before F7 up-regulation. We observed that ribavirin enhanced ELL3 recruitment to F7, whereas knockdown of ELL3 diminished ribavirin-induced FVII mRNA up-regulation. Ribavirin also enhanced recruitment of CDK9 (cyclin-dependent kinase 9) and AFF4 to F7. These data suggest that ribavirin-induced intracellular GTP depletion recruits a super elongation complex containing P-TEFb, AFF4 and ELL3, to F7, and modulates FVII mRNA transcription elongation. Collectively, we have elucidated a basal mechanism for ribavirin-induced FVII mRNA up-regulation by acceleration of transcription elongation, which may be crucial in understanding its pleiotropic functions in vivo.