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Among the acquired coagulation factor deficiencies, autoimmune coagulation factor deficiencies (AiCFD) are rare and result from autoantibody production against coagulation factors. In Japan, a nationwide survey on AiCFD has been conducted since 2009. Autoimmune factor XIII, factor VIII, von Willebrand factor, factor V, and factor X deficiencies (AiF13D, AiF8D, AiVWFD, AiF5D, and AiF10D, respectively) have been enacted as "designated intractable disease-282." The incidence of AiF8D, AiF13D, and AiF5D was 1.83, 0.044, and 0.038 per million people/year, respectively, whereas that of AiVWFD and AiF10D was not calculable owing to the small number of patients. AiF13D and AiF8D were often idiopathic, whereas AiVWFD was often associated with plasma cell neoplasms. Epistaxis was a characteristic symptom of AiVWFD, intramuscular bleeding was frequent in AiF13D and AiF8D, and subcutaneous bleeding (purpura) was frequent in AiF13D and AiF10D, although none were specific to any one disease. Differential diagnosis cannot be made based on bleeding symptoms alone; therefore, rapid and accurate testing is mandatory. Definitive diagnosis of AiCFD necessitates identifying the presence of coagulation factor "inhibitors" and/or "autoantibodies." Therefore, these tests should be performed upon unexplained severe acquired coagulation factor deficiencies. The mainstay of treatment for AiCFD was hemostatic therapy and autoantibody eradication therapy, which included the replacement of coagulation factors or "bypass" agents and administration of immunosuppressants. The rate of hemorrhagic death was high in AiF13D (13%), followed by AiF5D (7%) and Ai10D (5%); therefore, early diagnosis and optimal treatment are essential for AiCFDs. Given the unknown long-term prognosis, "intractable disease platform registries" have begun to accumulate in Japan., Competing Interests: None declared., (Thieme. All rights reserved.)

Background:  Coagulation factor XIII (FXIII) is a proenzyme of plasma transglutaminase. It comprises two catalytic A subunits (FXIII-A) and two carrier B subunits (FXIII-B). We previously reported that alloantibodies against FXIII-B could promote FXIII clearance in a patient with congenital FXIII-B deficiency who had received infusions of plasma-derived human FXIII (A 2 B 2 heterotetramer)., Objectives:  We aimed to investigate whether anti-FXIII-B antibodies affect the catalytic function of FXIII., Methods:  FXIII activation and fibrin crosslinking were examined in the presence of patient plasma, isolated patient IgG, or rat anti-FXIII-B monoclonal antibodies., Results:  Alloantibody levels were increased by repeated infusions of plasma-derived A 2 B 2 heterotetramer, which enhanced binding to the functionally important FXIII-B sushi domains. The patient plasma strongly inhibited cleavage of the FXIII-A activation peptide, amine incorporation, and fibrin crosslinking in normal plasma. Furthermore, anti-FXIII-B alloantibodies blocked the formation of the complex of FXIII-B with FXIII-A, and fibrinogen. Rat monoclonal antibodies against the 10th sushi domain of FXIII-B inhibited the incorporation of FXIII-B to fibrin, FXIII activation (i.e., cleavage of FXIII-A activation peptide), and ultimately fibrin crosslinking in normal plasma, independent of their effect on heterotetramer assembly with FXIII-A. Alloantibody binding to the A 2 B 2 heterotetramer blocked the access of thrombin to the FXIII-A cleavage site, as indicated by the reaction of the alloantibodies to the A 2 B 2 heterotetramer and FXIII-B, but not to FXIII-A., Conclusion:  Anti-FXIII-B antibodies binding to the A 2 B 2 heterotetramer and FXIII-B inhibited FXIII activation and its crosslinking function despite being directed against its noncatalytic subunit (FXIII-B)., Competing Interests: None declared., (Thieme. All rights reserved.)

Autoimmune factor XIII (FXIII) deficiency (AiF13D) is an acquired life-threatening bleeding disorder due to anti-FXIII autoantibodies (autoAbs). We previously established an immunochromatographic test (ICT) for detection of anti-FXIII-A subunit (FXIII-A) autoAbs. Conversely, the detection of anti-FXIII-B subunit (FXIII-B) autoAbs is currently performed in a limited number of medical facilities through time-consuming and expensive laboratory tests, such as dot-blotting analysis and enzyme-linked immunosorbent assay (ELISA). Accordingly, in this study, we generated eight rat monoclonal antibodies (mAbs) against human FXIII-B using the rat lymph node method. By employing an ELISA, two mAbs, 2G12B10 and 8H12B9, were selected considering the distance between the recognition regions of each mAb (the 6th and 9th-10th Sushi domain, respectively) and the strength of their reactivity. Using this mAb combination, we prototyped an ICT to detect anti-FXIII-B autoAbs and distinguish between AiF13D and "nonimmune" acquired FXIII deficiency (acF13D), and tested it with 22 healthy controls, 23 acF13D patients, 15 AiF13D patients without anti-FXIII-B autoAbs, and 8 AiF13D patients with anti-FXIII-B autoAbs. Receiver operating characteristic curve analyses of ICTs for anti-FXIII-B autoAbs were performed and revealed a precision similar to dot-blot analysis. Human anti-FXIII-A mAbs were also generated from a single patient with AiF13D using a new cDNA cloning method, and their binding properties were characterized. Consequently, anti-FXIII-A immunoglobulin G preparations were established as potentially permanent positive controls of ICT for anti-FXIII-A antibodies. Combining the previously developed ICT for anti-FXIII-A autoAbs and the novel ICT for anti-FXIII-B autoAbs may reduce false negatives and lead to appropriate diagnosis and treatment., Competing Interests: None declared., (Thieme. All rights reserved.)

Competing Interests: None declared.

von Willebrand factor (VWF) forms high-molecular-weight multimers and plays an essential role in hemostasis, and thus its deficiency leads to bleeding symptoms. Acquired von Willebrand syndrome (AVWS) is rare, but potentially underdiagnosed, and develops in various underlying disorders. AVWS caused by anti-VWF autoantibodies is a rare subcategory of AVWS that can also be referred to as autoimmune VWF deficiency (AiVWFD). We performed a search of patients with autoimmune coagulation factor deficiencies in our nationwide survey in Japan. Among these, suspected cases of AiVWFD were extremely few, with only 11 case consultations in the last 10 years. Of these, three and five were respectively positive for anti-VWF autoantibodies (anti-VWF-Ab) and VWF inhibitor (VWF-inh). We also performed an extensive literature search of other cases from Japan, and in total, 40 cases were finally identified to have AiVWFD, with mean age of 55.0 years. Most underlying disorders were lympho- or myeloproliferative diseases, followed by autoimmune diseases. The major bleeding sites were subcutaneous and mucosal, the bleeding severity was moderate, and there were no hemorrhagic deaths. Bleeding time was prolonged; factor VIII activity, VWF antigen, and VWF activity were decreased, and high-molecular-weight VWF multimers were absent or decreased. These are similar to the common abnormal laboratory findings observed among general AVWS cases. Hemostatic therapy often involved VWF concentrates and vasopressin, and antibody eradication therapy often included corticosteroids and achieved remission. Notably, of all cases, 68% had anti-VWF-Abs, and 83% of anti-VWF-Ab-positive patients were also VWF-inh positive. To accumulate precise clinical information on AiVWFD, it is necessary to verify and improve the measurement methods for both anti-VWF-Ab and anti-VWF-inh. These findings from Japan should be confirmed in other geographic localities., Competing Interests: A.I. reports grants from the Japanese Ministry of Health, Labor, and Welfare, during the conduct of the study. The remaining authors do not report any conflicts of interests., (Thieme. All rights reserved.)

Competing Interests: None declared.

Coagulation factor V (or FV for the purpose of medical safety) is an essential cofactor of coagulation factor X in the common pathway of coagulation; severe FV deficiency leads to a bleeding tendency. Although both congenital and acquired FV deficiencies are widely recognized, FV deficiency also presents as an autoimmune disorder. A nationwide survey on autoimmune coagulation factor deficiencies (AiCFDs) conducted in Japan by our Japanese Collaborative Research Group identified 24 new patients with autoimmune FV deficiency (AiFVD) in the past 5 years. Furthermore, our extensive literature search confirmed that 177 AiFVD cases have been reported in previous articles published from Japan. Patients with AiFVD in Japan were predominantly men, with age similar to those with other AiCFDs. AiFVD was confirmed as a relatively mild type of bleeding diathesis, associated with lower mortality rate than that for AiFVD and other AiCFDs reported in previous studies. Patients with AiFVD had variable FV inhibitor titers and both neutralizing anti-FV autoantibodies and nonneutralizing counterparts. Although spontaneous resolution occurs in some patients, timely initiation of hemostatic and immunosuppressive therapies helps arrest the bleeding and eliminate anti-FV antibodies, resulting in a high cumulative recovery rate. Immunological anti-FV antibody detection is recommended to avoid missing AiFVD cases for the presence of nonneutralizing anti-FV autoantibodies. Further investigation is necessary to clarify the long-term prognosis and optimal management of AiFVD., Competing Interests: None declared., (Thieme. All rights reserved.)

Coagulation factor X (F10) amplifies the clotting reaction in the middle of the coagulation cascade, and thus F10 deficiency leads to a bleeding tendency. Isolated acquired F10 deficiency is widely recognized in patients with immunoglobulin light-chain amyloidosis or plasma cell dyscrasias. However, its occurrence as an autoimmune disorder is extremely rare. The Japanese Collaborative Research Group has been conducting a nationwide survey on autoimmune coagulation factor deficiencies (AiCFDs) starting in the last decade; we recently identified three patients with autoimmune F10 deficiency (AiF10D). Furthermore, an extensive literature search was performed, confirming 26 AiF10D and 28 possible cases. Our study revealed that AiF10D patients were younger than patients with other AiCFDs; AiF10D patients included children and were predominantly male. AiF10D was confirmed as a severe type of bleeding diathesis, although its mortality rate was not high. As AiF10D patients showed only low F10 inhibitor titers, they were considered to have nonneutralizing anti-F10 autoantibodies rather than their neutralizing counterparts. Accordingly, immunological anti-F10 antibody detection is highly recommended. Hemostatic and immunosuppressive therapies may help arrest bleeding and eliminate anti-F10 antibodies, leading to a high recovery rate. However, further investigation is necessary to understand the basic characteristics and proper management of AiF10D owing to the limited number of patients., Competing Interests: None declared., (Thieme. All rights reserved.)

Autoimmune haemorrhaphilia XIII/13 (AH13) is an acquired life-threatening bleeding disorder due to anti-factor XIII (FXIII) autoantibodies (auto-Abs). AH13 patients may die of haemorrhage without correct diagnosis and proper treatment because of lack of awareness and the absence of rapid easy-to-use tests specific for this disease. Currently, the definitive diagnosis is established by cumbersome and time-consuming laboratory tests such as dot-blot assays and enzyme-linked immunosorbent assays (ELISA), and therefore these tests are generally not carried out. To save AH13 patients' lives, there is an urgent necessity for developing a rapid test for FXIII auto-Abs. We first generated and characterised mouse monoclonal antibodies (mAb) against human FXIII A subunit (FXIII-A), and then developed a rapid immunochromatographic test (ICT) for detection of anti-FXIII-A auto-Abs using one mAb with a dissociation constant of 9.3 × 10⁻¹¹ M. The auto-Ab-FXIII-A complex was captured by the mAb on a nitrocellulose membrane and visualised by Au-conjugated anti-human IgG Ab. Mixing with healthy control plasma improved the detection of auto-Abs in patients having extremely low levels of FXIII-A. The specificity and sensitivity of the ICT were 87 % and 94 %, respectively. We also detected auto-Abs against activated FXIII (FXIIIa) in three patients by pre-converting FXIII to FXIIIa by thrombin treatment. ICT values were significantly inversely correlated with FXIII activity levels, indicating an association between the quantity of anti-FXIII autoantibodies and AH13. This reliable rapid ICT assay can be applied to a point-of-care test to detect anti-FXIII-A auto-Abs, and will contribute to early diagnosis and treatment of AH13.

Antihistamines play an important role in medicine when it comes to relieving seasonal or non-seasonal rhinitis, the common cold, and itching. They have also shown many various combinations of pharmacological properties such as anti-inflammatory and analgesic activities. Phenothiazines and ethylenediamines are 2 important classes of antihistamines with analgesic activities in addition to other pharmacological effects. In this study, some new derivatives of these compounds (V-IX) were synthesized and their antinociceptive behaviors were examined by pharmacological tests. The results indicated that new analogue with methyl groups produced a better analgesic activity than chlorine atoms but less than III (without any substitutions) in ethylenediamine class. Also in phenothiazine class, adding pyrimidine and pyridine substituted showed the better analgesic activity compared to other groups. Moreover, the analgesic activities proved that dimethylamine is the best group in amino alkyl side chain of these molecules relative to the substituted piperazines in new analogues., (© Georg Thieme Verlag KG Stuttgart · New York.)

Factor XIII (FXIII) is a fibrin-stabilising factor consisting of catalytic A subunits (FXIII-A) and carrier B subunits (FXIII-B). FXIII-B prevents the fast clearance of FXIII-A from the circulation. Congenital FXIII-A deficiency is a rare bleeding disorder, and congenital FXIII-B deficiency is even rarer. Through our recent nationwide survey on "acquired haemophilia-like disease due to anti-FXIII autoantibodies," we newly diagnosed severe congenital FXIII-B deficiency in a Japanese man. He developed thrombocytopenia and gingival bleedings at the age of 73, and his FXIII activity was as low as 10% of the normal. When he suddenly developed spontaneous intramuscular haematoma, the bleeding was arrested by infusing FXIII concentrates. However, his FXIII activity remained around 10% of the normal. At the age of 74, ELISA and western blotting assay unexpectedly revealed complete absence of FXIII-B in the patient's plasma. A dot blot assay detected anti-FXIII-B alloantibodies for the first time in this disease, which could be attributed to the infusion of exogenous FXIII. He was found to be homozygous for a Japanese founder-effect mutation of F13B. Repeated infusions of exogenous FXIII for hemostasis increased anti-FXIII-B alloantibodies that resisted FXIII substitution. To the best knowledge of the authors, none of the remaining 10 reported cases of congenital FXIII-B deficiency developed alloantibodies to exogenous FXIII-B of plasma FXIII. An originally mild bleeding phenotype of severe congenital FXIII-B deficiency can be exaggerated by additional acquired conditions. Physicians should consider congenital FXIII-B deficiency when they encounter cases of unexplained bleeding disorders.

Protein Z (PZ)-dependent protease inhibitor (ZPI) is a serine protease inhibitor which efficiently inactivates activated factor X, when ZPI is complexed with PZ in plasma. Reduced plasma levels of ZPI and PZ have been reported in association with thrombosis. It has also been reported that PZ increases during pregnancy and that its partial deficiency is related to early pregnancy loss or recurrent miscarriage (RM). However, until now there has been no report on ZPI in pregnancy. To explore the possible role(s) of ZPI in the maintenance of pregnancy, we studied 42 non-pregnant normal women, 32 women with normal pregnancies, and 134 cases of unexplained RM in Japan, as well as 64 non-pregnant normal German females. Plasma ZPI was measured by in-house ELISA. There were significantly higher concentrations of plasma ZPI in normal pregnancies compared to non-pregnant women. The present study also confirmed that both factor X, the major target of ZPI, and protein Z increased during normal pregnancies. This increased ZPI and PZ may counteract the increased activated factor X, which may in turn contribute to the maintenance of normal placental circulation. Plasma ZPI levels were unchanged in non-pregnant RM women, while the plasma PZ level was slightly reduced, a finding consistent with existing reports. The exact relationship between RM and this unaltered ZPI with mild PZ reduction relative to normal pregnancies warrants further investigation.

Factor XIII (FXIII) is a proenzyme of plasma transglutaminase consisting of enzymatic A subunits (FXIII-A) and non-catalytic B subunits (FXIII-B), and acts in haemostasis and wound healing. We generated mice lacking either FXIII-A or FXIII-B to investigate the physiological functions of FXIII in vivo. A longitudinal study was carried out using the gene-targeted mice to explore the possible effects of FXIII deficiency on aging. Survival rates of FXIII-A(-/-) males decreased to approximately 50% at 10 months after birth, although most FXIII-A(-/-) females and both genders of wild-type mice survived. Four FXIII-A(-/-) males died of severe intra-thoracic haemorrhage, and a large haematoma was found in their hearts. Haemorrhage, haemosiderin deposition and/or fibrosis were observed in the hearts of other dead FXIII-A(-/-) males. Fibrosis together with haemosiderin deposition was also found in the hearts of FXIII-A(-/-) males sacrificed. The in-vivo cardiac function was normal in FXIII-A(-/-) mice when compared with wild-type mice despite the presence of significant cardiac fibrosis. Although survival rates for both genders of the FXIII-B(-/-) and wild-type mice did not differ, mild fibrosis together with haemosiderin deposits were only found in the hearts of the sacrificed FXIII-B(-/-) males. Carditis and fibrosis in FXIII-deficient mice might be caused by a faulty or delayed reparative process that was initiated by abnormal haemorrhagic events within heart tissue. It is important therefore to examine possible cardiac involvement in human patients with congenital FXIII deficiency.

Factor XIII is a proenzyme for a plasma transglutaminase. Factor XIII in plasma is a tetramer (A2B2) held together by noncovalent bonds, and the A subunit contains the active site. Recently, the three-dimensional structure of the A subunit has been determined by x-ray crystallography. To understand the structure-function relationships of the factor XIII molecule and its clinical implications in factor XIII deficiency, we characterized its genetic defects and closely examined its gene products, including mRNA and protein levels. A variety of missense and nonsense mutations (Arg260-Cys, Tyr283-Cys, Gly562-Arg) and deletions/insertions with or without out-of-frame shift/premature termination and splicing abnormalities (4-bp deletion with 464Stop, T insertion at the exon IV/intron D boundary with exon IV-skipping, 20-bp deletion at the exon I/intron A boundary) has been identified in cases demonstrating A subunit deficiency. In some cases, the A subunit mRNA levels were severely reduced. Their molecular and cellular bases have also been explored by expression experiments in mammalian cells and by molecular modeling. In most cases, impaired folding and/or conformational changes of the mutant A subunits lead to both intra- and extracellular instability, which is responsible for the A subunit deficiency in the patients.

We previously concluded that genetic defects in the B subunit of factor XIII were the basis for former Type I deficiency (i.e. factor XIII B subunit deficiency). When we examined an Italian patient with the disease at the DNA level, restriction digestion and sequencing analyses of amplified DNAs revealed that the proband and her family members possessed an AAC insertion within the codon for Tyr-80 in exon III in the gene for the B subunit. a nucleotide polymorphism (A-G) in its 3'-noncoding region in exon XII, and a short tandem repeat polymorphism of (TTTA9, in the 3'-flanking region. These mutations and 3'-polymorphisms were also identified in another Italian family reported in a previous study (10). suggesting that a founder effect is responsible for factor XIII B subunit deficiency in Italians.