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Acquired von Willebrand syndrome (aVWS) develops with various underlying diseases. We herein report an individual with aVWS associated with mucosa-associated lymphoid tissue lymphoma in the lungs complicated by hyperviscosity syndrome, Sjögren's syndrome, and hypothyroidism. This patient developed life-threatening hemorrhaging during a lung biopsy despite transfusion of concentrate of plasma-derived VWF/factor VIII. The use of rituximab caused remission of the lymphoma and hyperviscosity syndrome in parallel with the resolution of aVWS. Thus, lymphoma and hyperviscosity might result in aVWS. Invasive procedures with a risk of bleeding should be avoided in individuals with aVWS.

Autoimmune factor X (FX or F10) deficiency (AiF10D) is an extremely rare acquired haemorrhagic disorder characterized by a severe reduction in FX activity due to autoantibodies against FX.Anti-FX autoantibodies were investigated in four patients with suspected AiF10D, and their properties were analysed.Anti-FX auto antibodies in plasma were detected by ELISA with three of four cases. One case of anti-FX autoantibody negativity was later diagnosed as AL-amyloidosis. IgG1 and IgG3 coexisted in all anti-FX autoantibodies of the three patients with AiF10D (cases X1, X2, and X3). Western blot analysis showed that the antibodies were bound to the FX light chain for cases X2 and X3, but the binding was weak for case X1. When the fusion proteins of a secretory luciferase with full-length FX or its γ-carboxylated glutamic acid (Gla) domain were added to the plasma of the three patients, both fusion proteins were immunoprecipitated as antigen-antibody complexes. Contrarily, the latter fusion protein produced in the presence of warfarin demonstrated a decrease in the collection rate, suggesting that their autoantibodies recognized the light chain and regions containing Gla residues. Since all three patients were essentially negative for FX inhibitors, it was concluded that the anti-FX autoantibodies for these cases were predominantly non-neutralizing. The concentration of the FX antigen also significantly reduced in these patients, suggesting that anti-FX autoantibodies promote the clearance of FX.Immunological anti-FX autoantibody detection is highly recommended to ensure that AiF10D cases are not overlooked, and to start necessary immunosuppressive therapies.

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.

Autoimmune coagulation factor deficiency (AiCFD) is characterized by sudden excessive bleeding due to autoantibodies against coagulation factors. This occurs primarily in elderly patients with no family history or previous clotting issues. However, the detailed mechanisms underlying autoantibody development are not well understood. Here, we evaluated the plasma proteome in patients with AiCFD and compared it with that of 22 healthy controls and 17 patients with non-autoimmune acquired factor XIII deficiency (acF13D). We identified eighteen proteins whose plasma levels were higher in AiCFD than in either healthy controls or patients with acF13D. Most of these proteins were found to be acute-phase reactants or immunoglobulins. Patients with acF13D had lower levels of nine of these proteins and higher levels of the remaining nine compared to healthy controls. However, in all cases, these protein levels were much higher in patients with AiCFD than in those with acF13D. These results suggest that an inflammatory response independent of the acute inflammation caused by bleeding can occur, which may lead to the development of AiCFD. Therefore, we believe that severe and/or chronic inflammation, probably due to an underlying disease or aging, is the most important factor in autoantibody development.

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.

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 (A2B2 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 A2B2 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 A2B2 heterotetramer blocked the access of thrombin to the FXIII-A cleavage site, as indicated by the reaction of the alloantibodies to the A2B2 heterotetramer and FXIII-B, but not to FXIII-A. Conclusion Anti-FXIII-B antibodies binding to the A2B2 heterotetramer and FXIII-B inhibited FXIII activation and its crosslinking function despite being directed against its noncatalytic subunit (FXIII-B).

not applicable.

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.

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.

The early diagnosis and prompt treatment of autoimmune coagulation factor deficiencies (AiCFDs) are challenging for physicians when patients present with pseudo-deficiencies or pseudo-inhibitors of multiple coagulation factors. A reason for this is the diagnostic confusion caused by the apparent reduction in coagulation factor activity when using common one-stage coagulation factor measurement assays.After confirming the presence of autoantibodies against each coagulation factor, we retrospectively examined the activity of factors X, VIII, and IX (FX, FVIII, and FIX, respectively) and each coagulation factor inhibitor using their chromogenic substrates among 33 patients with AiCFD.Because the apparent coagulation factor deficiency was completely or partially restored in the chromogenic assay, 4, 9, and 22 patients with AiCFD were suspected of having pseudo-FX, pseudo-FVIII, and pseudo-FIX deficiencies, respectively. Moreover, in the chromogenic assay, the specific activities of FX, FVIII, and FIX (determined by their antigen levels) were higher than those in the one-stage assay. The titers for FV inhibitors showed negative correlations with the ratios of FX, FVIII, and FIX activities measured via the one-stage assay and the chromogenic assay. An especially high titer of one coagulation factor inhibitor tends to either cause pseudo-deficiencies or get mistaken for being a pseudo-inhibitor of other coagulation factors.Chromogenic assays appear to be superior to conventional one-stage assays when measuring coagulation factor activity in AiCFD cases. Detection of anti-coagulation factor autoantibodies is recommended to avoid overlooking the presence of non-neutralizing autoantibodies.

Background Dairy products are known as health-promoting foods. This study prospectively examined the association between milk and yogurt intake and mortality in a community-based population. Methods The study population comprised of 14,264 subjects aged 40–74 years who participated in an annual health checkup. The frequency of yogurt and milk intake was categorized as none (> 1/day) intake. The association between yogurt and milk intake and total, cardiovascular, and cancer-related mortalities was determined using the Cox proportional hazards model. Results During the follow-up period, there were 265 total deaths, 40 cardiovascular deaths and 90 cancer-related deaths. Kaplan–Meier analysis showed that the total mortality in high/moderate/low yogurt intake and moderate/low milk intake groups was lower than that in none group (log-rank, P Conclusions Our study showed that yogurt and milk intake was independently associated with a decrease in total and cancer-related mortalities in the Japanese population.

von Willebrand factor (VWF) is a glycoprotein that plays a central role in the initiation of blood coagulation. VWF performs two important functions: it acts as a molecular bridge between platelets and as a carrier for coagulation factor VIII (FVIII). von Willebrand disease (VWD) and acquired von Willebrand syndrome (AVWS) are caused by the absence of and/or abnormality in VWF. The pathophysiology of VWD and AVWS is complex and, therefore, it is difficult to diagnose them by conducting the same laboratory tests in all patients. To develop useful monoclonal antibodies (mAbs) for the diagnosis of VWD and AVWS, rat mAbs against human VWF were generated. Immunoblotting analysis revealed that mAbs recognized the reduced and nonreduced VWF protein and endogenous VWF in normal human plasma. Furthermore, we developed a highly sensitive monoclonal antibody-based sandwich enzyme-linked immunosorbent assay technique. In conclusion, the development of VWF-specific mAbs would be useful in the diagnosis of VWD and AVWS.

Introduction Autoimmune factor XIII (FXIII) deficiency (AiF13D) due to anti-FXIII autoantibodies is an extremely rare, life-threatening bleeding disorder that mostly occurs in the elderly. The number of patients diagnosed with AiF13D has been increasing in Japan, probably because of the nationwide survey on AiF13D supported by the Japanese Ministry of Health, Labour and Welfare. Aim To explore the pathologic characteristics of coagulation parameters in AiF13D. Methods AiF13D-suspected cases were consulted, and underwent unified/integrated coagulation screening and were definitively diagnosed as AiF13D separately. Results AiF13D patients had lower FXIII antigen levels than non-AiF13D patients, but their values overlapped. Among a series of 22-item screening tests and their resulting parameters, the 'FXIII inhibitory potential' yielded by a 1:1 mixing test of the patient's and healthy control's plasma and its 'residual FXIII activity' in 54 AiF13D cases were most distinguishable from 139 non-AiF13D cases, followed by FXIII activity per se and FXIII-specific activity. While the cross-linked α2 -plasmin inhibitor level reduced, the levels of D-dimer, fibrin/fibrinogen degradation products and plasmin-plasmin inhibitor complex increased, probably because the patients' haematoma nonspecifically induced secondary fibrinolysis in both AiF13D and non-AiF13D patients. Conclusion AiF13D appears to induce a hypocoagulopathy combined with a hyper-fibrinolytic state secondary to severe FXIII deficiency caused by anti-FXIII autoantibodies, and the consequent bleeding further modifies its pathological conditions. In addition, the 1:1 mixing test of FXIII activity was confirmed to be a reliable screening method for AiF13D, especially when its derivative parameter, such as the 'FXIII inhibitory potential' or 'FXIII inhibitory potential ratio', is employed.

Background Social support, defined as the exchange of support in social relationships, plays a vital role in maintaining healthy behavior and mitigating the effects of stressors. This study investigated whether functional aspect of social support is related to 5-year mortality in health checkup participants. Methods This study recruited 16,651 subjects (6797 males, 9854 females). Social support was evaluated using five-component questions: Do you have someone 1) whom you can consult when you are in trouble? 2) whom you can consult when your physical condition is not good? 3) who can help you with daily homework? 4) who can take you to hospital when you don’t feel well? and 5) who can take care of you when you are ill in bed? The association between the component of social support and all-cause and cardiovascular mortality was examined using Cox proportional hazard analysis. Results The percentage of subjects without social support components was 7.7–15.0%. They were more likely to be male, non-elderly, and living alone. During the follow-up period, there were 166 all-cause and 38 cardiovascular deaths. Cox proportional analysis adjusted for confounders showed that only the lack of support for transportation to hospital was significantly associated with all-cause (hazard ratio [HR] 2.01, 95% confidence interval [CI] 1.26–3.05) and cardiovascular mortality (HR 3.30, 95% CI 1.41–6.87). These associations were stronger in males than females. Conclusion This study showed that the lack of social support for transportation to the hospital was independently associated with all-cause and cardiovascular mortality in a community-based population.

Autoimmune factor V deficiency (AiF5D) is caused by autoantibodies to coagulation factor V (FV); its clinical manifestations range from asymptomatic to fatal hemorrhage. Herein, we report the case of a 68-year-old man who was diagnosed with end-stage renal disease at the time of a femoral fracture and developed AiF5D after initiating hemodialysis. A wound infection that occurred after joint replacement was treated with antibiotics; however, it was poorly controlled. One month after the procedure, his coagulation time prolonged. The infection was improved by debridement and antibiotics; however, the coagulation time was not decreased and poor hemostasis at the shunt was still persistent. Because ELISA detected anti-FV-binding IgG with FV activity of2.8% and FV inhibitor levels were 11.8 BU/ml, AiF5D was diagnosed. Oral prednisolone (PSL) was started. Dialysis was initially performed without anticoagulants, but blood clots were not found in the circuit. Anticoagulants were resumed when the coagulation time decreased. After achieving complete remission, PSL dose was tapered and finally discontinued. Few reports have described the management of AiF5D via dialysis. We consider that our report would be useful for the management of patients with similar manifestations.

Acquired factor V inhibitor (AFV-I) is a rare bleeding disorder wherein autoantibodies are developed against coagulation factor V (FV). The clinical symptoms are variable, from laboratory abnormalities without bleeding to life-threatening hemorrhage. We report herein the case of a patient with AFV-I with two relapses 4 years after the first remission. A 66-year-old male was diagnosed with AFV-I in March 20XX-4. He was treated with prednisolone (PSL) at 50 mg/day and achieved remission within 1 month. PSL dose was tapered to oral administration of 2.5 mg every other day, and long-term remission was maintained. He had been treated with dual antiplatelet therapy (DAPT) for old myocardial infarction. FV activity was markedly reduced to 3.4%, and FV inhibitor was detected (1.0 BU/ml) in May 20XX. We followed the patient without increasing the treatment dose for 2 months, but no spontaneous improvement was seen. Because DAPT was ongoing, we judged that the bleeding risk was high, although only minor bleeding symptoms appeared. PSL was therefore increased to 40 mg/day in June. FV inhibitor rapidly disappeared. When PSL dose was gradually decreased, FV activity decreased, and subcutaneous bleeding occurred in February 20XX＋1. PSL dose was increased again for the second relapse, and the patient achieved remission. Few reports have described recurrent AFV-I, and no cases of two relapses have been reported. We believe that this case report is useful for examining the long-term management of AFV-I.

A 64-year-old man first developed ligneous conjunctivitis at the age of 58 years after right pulmonary resection because of suspected cancer; otherwise, he had been healthy. Since then, he began to suffer from various forms of chronic pseudomembranous mucositis. Laboratory tests demonstrated that he had 7.8 % of plasminogen activity and 5.9 % of the normal antigen level. Thus, he was diagnosed as having severe type I plasminogen deficiency, making him the third case in Japan. DNA sequencing and PCR-restriction fragment length polymorphism analyses revealed that this patient was a compound heterozygote of a G-to-A missense mutation (G266E) in exon VIII and a g-to-a mutation at the obligatory splicing acceptor site in intron 12 (IVS12-1g>a). These two mutations were confirmed to be novel. Molecular modeling and splice site strength calculation predicted conformational disorder(s) for the Glu266 mutant and a drastic decrease in splicing efficiency for intron 12, respectively. Western blot analysis demonstrated that the patient contained a small amount of the normal-sized plasminogen protein. Mass spectrometric analysis of the patient's plasminogen revealed a peptide containing the wild-type Gly266 residue and no peptides with mutations at Glu266. However, he had never suffered from thrombosis. Low levels of fibrinogen/fibrin degradation products (FDP), D-dimer, and plasmin-α2-plasmin inhibitor complex clearly indicated a hypo-fibrinolytic condition. However, his plasma concentration of elastase-digested crosslinked FDPs was 4.8 U/mL, suggesting the presence of an on-going plasmin(ogen)-independent "alternative" fibrinolytic system, which may protect the patient from thrombosis. The patient has been free from recurrence of ligneous conjunctivitis for approximately 2.5 years.

Introduction Coagulation factor XIII (FXIII) is a plasma fibrin-stabilizing factor comprising A and B subunits (FXIII-A and FXIII-B, respectively) in the form of a heterotetramer (FXIII-A 2 B 2 ). A humanized monoclonal antibody to the interleukin-6 receptor (tocilizumab, TCZ) has emerged as an effective treatment for rheumatoid arthritis (RA), because it drastically reduces the inflammation of RA. We previously reported that two TCZ-treated RA patients with acquired FXIII deficiency developed pelvic hemorrhage. Methods Because TCZ treatment had been shown to be related to low FXIII ammonia release activity and FXIII antigen in the two RA cases, we further examined FXIII-related parameters in 36 TCZ-treated RA patients and compared to 29 healthy controls by employing functional and immunologic assays for FXIII. Results FXIII-A antigen and FXIII amine incorporation and ammonia release activities were significantly lower in the TCZ-treated group than the control group. The TCZ-treated group also showed mildly low FXIII-A 2 B 2 and FXIII-B levels, and their fibrinogen levels were the lower limit of normal. A significant correlation between FXIII-B and fibrinogen was observed in the control and the TCZ groups, suggesting a common metabolic mechanism(s) for these two hepatic proteins. Because the specific activities of FXIII were normal and neither anti-FXIII-A nor anti-FXIII-B antibody was detected, the overall low FXIII level may have resulted from its impaired synthesis under an unbalanced cytokine milieu caused by TCZ treatment. Conclusion Concomitant deficiencies in multiple hemostatic factors, including FXIII, may lead to an increased risk for hemorrhage in TCZ-treated RA patients.

A 68-year-old man presented with intracranial hemorrhage in the right frontal lobe, which rapidly increased the day after admission. We performed hematoma removal with a biopsy of the cortex around the hematoma. The day after the operation, a subcutaneous hematoma over the craniotomy appeared, and the computed tomography showed a recurrent hemorrhage with an acute subdural hematoma. We were aware of a bleeding tendency, and a detailed hematologic examination by hematologists revealed autoimmune acquired factor XIII deficiency due to an antifactor XIII antibody. Specimens taken around the hematomas were pathologically diagnosed as cerebral amyloid angiopathy (CAA) on immunohistochemical examination. We considered that acquired factor XIII deficiency had induced lobar hemorrhage in the frontal lobe affected with CAA, and the coagulation disorder induced postoperative rebleeding. The patient died from repeated lobar hemorrhage 3 years after the surgery. There is no routine screening coagulation test including the active partial thromboplastin time and the prothrombin time for factor XIII deficiency. It is important for neurologists and neurosurgeons to be aware of this rare disease in patients with a bleeding tendency.

An 87-year-old man with diabetes mellitus was admitted to control recurrent bleeding from hemodialysis puncture sites. He was a smoker and had been diagnosed with arteriosclerosis obliterans. His PT and APTT were markedly prolonged, and all coagulation factors were markedly decreased (factor V [FV] activity

Autoimmune hemorrhaphilia due to anti-factor XIII (FXIII) antibodies (AH13) is a life-threatening disease associated with high risk of surgical bleeding. Since AH13 occurs mainly in the elderly, patients of AH13 tend to be complicated with other life-threatening diseases that may require surgical procedures. During our nation-wide survey on AH13, supported by the Japanese Ministry of Health, Labor, and Welfare, patients with unexplained bleeding were examined for FXIII-related parameters and anti-FXIII autoantibodies. A 64-year-old man had previously been tentatively diagnosed with AH13 and received immunosuppressive therapies, as FXIII inhibitor was detected by functional cross-mixing studies. About 2 years later, he was definitively diagnosed with AH13, because our immuno-chromatographic test and enzyme-linked immuno-sorbent assay detected FXIII-bound anti-FXIII-A subunit autoantibodies. Since routine endoscopic examination revealed suspected esophageal carcinoma, a preparatory FXIII pharmacokinetic (PK) analysis was performed by infusing FXIII concentrates prior to biopsy. Consequently, biopsy of this lesion was done without bleeding complications. One month later, a second PK study was carried out before surgery, and esophageal bypass surgery was completed successfully under FXIII replacement therapy. Our experience with this case suggests that operations can be performed safely and with confidence even in patients with such life-threatening hemorrhagic diseases.

Introduction Protein Z (PZ)-dependent protease inhibitor (ZPI) is a serine protease inhibitor that efficiently inhibits activated factor X when ZPI is in complex with PZ. We previously reported significantly higher concentrations of plasma ZPI (and PZ) in women during normal pregnancy than in non-pregnant women. Methods We explored the possible contribution of estrogen to the ZPI levels in patients with or without bilateral oophorectomy (OVX), which induces artificial menopause where blood estrogen levels drastically decrease. One hundred ninety-one pre-menopausal Japanese women who underwent open hysterectomy owing to neoplasms participated in this study and were divided into two groups: 98 OVX and 93 Non-OVX cases. Plasma ZPI was measured by ELISA. Results and Conclusion Contrary to our working hypothesis, plasma ZPI levels increased significantly in the OVX group after surgery when compared with the pre-operation levels. When these patients were individually analyzed, their ZPI value also rose significantly from pre-operation to post-operation levels. In contrast, plasma PZ levels remained unchanged. The significantly increased ZPI and unchanged PZ levels were also observed in the Non-OVX group. The increased ZPI levels were not significantly related to 17β-estradiol, luteinizing hormone or follicular stimulating hormone levels, clearly indicating that estrogen did not contribute to the plasma ZPI concentrations. Typical acute phase reactants fibrinogen and C-reactive protein (CRP) were also significantly elevated after surgery in both OVX and Non-OVX groups. However, only weakly significant linear relationships were observed between ZPI and fibrinogen or CRP, indicating the presence of alternative regulatory mechanisms underlying their plasma concentrations.

Summary Background Autoimmune hemophilia-like disease (hemorrha-philia or hemorrhagic disorder) caused by anti-factor XIII antibodies (termed AH13) or ‘autoimmune FXIII deficiency’ is a life-threatening bleeding disorder. AH13 was thought to be rare worldwide. Objectives Because the number of diagnosed AH13 cases has recently been increasing, at least in Japan, we conducted a nationwide survey supported by the Japanese Ministry of Health, Labor, and Welfare, and explored the pathologic mechanism(s) of AH13. Methods We diagnosed AH13 cases during the last 11 years according to the presence of anti-FXIII autoantibodies confirmed by a dot blot assay and ELISA, and characterized 33 of these both immunologically and biochemically. Results The AH13 cases were immunologically classified into three types, Aa, Ab, and B. Type Aa autoantibodies, observed in 27 cases, were directed against the native FXIII A subunit (FXIII-A), and blocked FXIII activation. The autoantibodies not only prevented assembly of new FXIII-A2B2 heterotetramers, but also removed FXIII-A from native FXIII-A2B2 heterotetramers by forming an FXIII-A–IgG complex. Type Ab autoantibodies, detected in three cases, preferentially bound to activated FXIII-A and inhibited its activity. Type Aa and Ab autoantibodies were ‘neutralizing’ FXIII antibodies (or FXIII inhibitors), and thus could be screened with functional assays. Type B antibodies, detected in two cases, were non-neutralizing anti-FXIII B subunit (FXIII-B) autoantibodies that possibly accelerated the clearance of FXIII, and thus could be diagnosed exclusively with immunologic methods. Conclusion There are three major types of anti-FXIII autoantibody, with distinct targets and mechanisms that cause AH13.

Although the incidence of autoimmune hemorrhaphilia due to anti-Factor XIII (FXIII, not FVIII or FXII to avoid confusion) antibodies (AH13) or hemorrhagic "acquired FXIII deficiency due to anti-FXIII autoantibodies" was previously considered rare, it has been on the increase in the twenty-first century, at least in Japan. An 83-year-old woman with an unexplained hemorrhage was admitted to our hospital for intramuscular hematoma and severe anemia. Her FXIII activity was reduced to 10 % of normal; since FXIII inhibitors and anti-FXIII-A subunit autoantibodies were detected, she was definitively diagnosed with AH13. Despite developing cardiac tamponade due to pericardial hemorrhage, she clinically recovered from AH13 after hemostatic therapy with FXIII-concentrates and immunosuppressive treatment with rituximab and cyclophosphamide. However, her FXIII activity remained low and she died of hemorrhage 3.5 years after admission. AH13 patients should be monitored for a prolonged period, as this disease is very likely a chronic intractable hemorrhagic disorder.

Complete remission achieved by steroid pulse therapy following rituximab treatment in a case with autoimmune haemorrhaphilia due to anti-factor XIII antibodies

Factor XIII (FXIII) is a fibrin-stabilizing factor consisting of catalytic A subunits (FXIII-A) and carrier B subunits (FXIII-B). Congenital FXIII deficiency is a rare bleeding disorder. Acquired FXIII deficiency resulting from FXIII hypo-synthesis and/or hyperconsumption is a relatively common disorder in which patients seldom bleed. On the contrary, 'autoimmune/acquired hemorrhaphilia XIII/13 due to anti-FXIII antibodies (AH13)' is a rare but life-threatening bleeding disorder. Through a nationwide survey of AH13, we diagnosed aggressive AH13 in a 66-year-old woman. She consulted our department because of a spontaneous hematoma in her hand. After 1.5 months, she also developed an intramuscular hematoma but retained approximately half (52%) of the normal FXIII activities. The patient's bleeding symptoms were aggravated to catastrophic massive bleedings in the large abdominal muscles and intrapelvic and intraperitoneal spaces. Two months after the bleeding onset, she died despite undergoing plasma exchange, which was performed because we were deeply suspicious of the presence of an anti-FXIII inhibitor. Seven days after her death, extremely low FXIII activity (6%) and positive data on anti-FXIII inhibitor were reported by a commercial laboratory. Our dot blot assay detected anti-FXIII-A autoantibodies, afterwards. Thus, the diagnosis of aggressive AH13 as early as possible is necessary to save patients' lives.

Autoimmune hemophilia-like disease (hemorrhaphilia) due to anti-factor XIII (FXIII) antibodies (AH13) is a very rare, life-threatening bleeding disorder. A 77-year-old woman developed macrohematuria and a right renal pelvic hematoma. The coagulation times were not prolonged, but FXIII activity and antigen levels were severely and moderately reduced to 9 and 29% of normal values, respectively. Accordingly, the FXIII-specific activity turned out to be low. FXIII inhibitor and anti-FXIII-A subunit autoantibodies were detected by a 1:1 crossmixing test and immunoblot and immunochromatographic assays. She was therefore diagnosed with “definite AH13” and treated with plasma-derived FXIII concentrates to arrest the hemorrhage. In addition to a highly compressed inferior vena cava by a huge renal pelvic hematoma, deep vein thrombosis (DVT) and pulmonary thromboembolism (PE) were identified by systemic computed tomography. The patient was immediately started on anticoagulation therapy with low-dose heparin. Emboli disappeared quickly, probably because under-crosslinked thrombi caused by severe FXIII deficiency are vulnerable to fibrinolysis. After about 1.5 years, anti-FXIII-A subunit autoantibodies still remained despite the use of rituximab, steroid pulse therapy, oral prednisolone, and oral cyclophosphamide treatments. In conclusion, an extremely rare AH13 case complicated by DVT and PE was successfully managed by balancing anticoagulation therapy with hemostatic therapy.

Congenital factor XIII (FXIII) deficiency is a severe bleeding disorder. We previously identified an Arg260Cys missense mutation and an exon-IV deletion in patients' A subunit genes, F13A. To characterize the molecular/cellular basis of this disease, we expressed a wild type and these mutant A subunits in baby hamster kidney (BHK) cells. The mutant proteins were expressed less efficiently than the wild type. These mutants gradually decreased inside BHK cells, whereas the wild type remained largely unchanged. The decline/decrease in these mutants was completely blocked/restored by a potent proteasome inhibitor, MG-132. This was consistent with the prediction by molecular modelling that the mutant molecules would lose the native structure of wild-type molecule, leading to their instability and degeneration and ultimately to degradation. These mutants might have significantly altered conformations, resulting in the rapid degradation by the proteasome inside the synthesizing cells, and ultimately leading to FXIII deficiency.

Factor XIII (FXIII) is the final enzyme in the coagulation cascade. Acquired FXIII deficiency is caused by inhibitors of FXIII or decreased synthesis and/or increased consumption of FXIII, which leads to severe bleeding. Recently, we experienced a case of hemorrhagic-acquired factor XIII deficiency that occurred during treatment with the IL-6 inhibitor tocilizumab for rheumatoid arthritis. A 48-year-old man was referred because of right hip pain due to a hematoma. Laboratory findings showed that routine coagulation tests were normal, while FXIII activity was slightly low (52.4 %). The patient was successfully treated with plasma-derived factor XIII concentrates. The time course of recovery suggests that tocilizumab might have inhibited FXIII production. To our knowledge, this is the first report of acquired factor XIII deficiency associated with administering of tocilizumab. When recurrent bleeding is seen during administering of tocilizumab, acquired factor XIII deficiency may have been induced, thus attending physicians should consider this disease in a differential diagnosis.

Blood coagulation factor XIII (FXIII) is a pro-enzyme of plasma transglutaminase (TGase) consisting of two catalytic A (FXIII-A) and two carrier B subunits (FXIII-B) (1–3). In the final stage of the blood coagulation cascade, FXIII is activated by thrombin and promotes clot stability by forming covalent bonds between fibrin molecules; it also cross-links fibrin with other proteins including fibronectin and α2-plasmin inhibitor. Accordingly, FXIII deficiency results in defective crosslinking reactions, leading to a life-long bleeding tendency and abnormal wound healing in affected patients, and recurrent miscarriage in affected females (1, 4). Because FXIII is composed of FXIII-A and FXIII-B, and FXIII-B protects FXIII-A in vitro (5, 6) and in vivo (7, 8), a genetic defect of either subunit leads to FXIII deficiency. Accordingly, FXIII deficiency is classified into two categories at the DNA level: FXIII-A deficiency and FXIII-B deficiency (9). This classification was approved by the scientific and standardization committee of the international society of thrombosis and haemostasis in 1999 (http://www.med.unc.edu/isth/99FXIII.html). The gene for FXIII-A spans ∼180 kb and consists of 15 exons interrupted by 14 introns (10). The FXIII-A cDNA encodes a mature protein of 731 amino acids, including an activation peptide of 37 residues at the amino-terminus and a catalytic Cys314 residue (11). To date, ∼100 mutations in the gene for FXIII-A have been identified in patients with FXIII-A deficiency (http://www.f13-database.de) (12, 13). These mutations are highly heterogeneous and include a variety of nonsense and missense mutations, deletions and insertions with or without frame-shift/premature termination, splicing abnormalities, etc. (9). Many missense mutations of F13A have been confirmed to be causative, as they resulted in the generation of reduced amounts of proteins in mammalian cells (14–18). Most of these missense mutations have been predicted to change the conformation of their translated products, resulting in protein instability (14–17, 19); however, direct experimental evidence for this has not been demonstrated to date (no hit on Pubmed until the submission date of this manuscript), to the best of the authors’ knowledge. We previously identified two mutations in the F13A genes of Japanese patients with severe FXIII-A deficiency: an R260C missense mutation (20) and an exon IV-deletion (Del-IV) caused by a splicing defect (21). The former mutation was also found in four other ethnic groups, i.e. Swiss, Iranian, Polish and Italian (18, 22–24), indicating this being a naturally recurring mutation. In this study, to obtain direct evidence for the molecular abnormalities of these two mutations, we expressed large quantities of the mutant proteins in yeast cells to perform detailed biochemical experiments. We demonstrate here, for the first time, that instability of the mutant recombinant FXIII-As (rFXIII-As), likely due to impaired dimer assembly, lead to their extensive aggregation as well as degeneration. We therefore propose a new molecular basis for FXIII deficiency.

SummaryProtein 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.This work was presented at the 23rd International Society on Thrombosis and Haemostasis meetings in Kyoto, July 2011.

Coagulation factor XIII/13 (FXIII/13) stabilizes fibrin molecules by creating crosslinks with other fibrin molecules as well as with α2-plasmin inhibitor (α2-PI). “Hemorrhagic acquired FXIII/13 deficiency” was formerly considered rare, but has been increasing recently in Japan. During the 10 months of our nationwide campaign, we diagnosed five new patients with “acquired hemorrhaphilia due to anti-FXIII/13 autoantibodies,” after examining 20 newly suspected cases of “hemorrhagic acquired FXIII/13 deficiency.” When FXIII/13 activity was reduced to less than 50% of normal, it was proportional to the difference in α2-PI levels between plasma and serum (plasma–serum α2-PI), likely due to its cross-linking to fibrin by activated FXIII/13. Accordingly, decreased amounts of the plasma–serum α2-PI ex vivo may reflect reduced FXIII/13 activity in vivo. The plasma–serum α2-PI may thus also be a useful diagnostic marker for severe FXIII/13 deficiency.

Protein Z is a vitamin K–dependent plasma glycoprotein that is involved in the regulation of blood coagulation. Plasma concentrations of protein Z vary widely between subjects and are greatly reduced during warfarin therapy. We developed a sensitive and quantitative assay for protein secretion using a secretory luciferase to explore the mode of secretion of protein Z compared with that of factor X. Protein Z secretion was much less efficient than factor X and was totally dependent upon added vitamin K, while factor X secretion was not. Protein Z secretion was highly sensitive to warfarin treatment of the synthesizing cells. In contrast, although factor X secretion was not precluded by warfarin, its γ-carboxylation was completely blocked. An exchange of the propeptide and/or γ-carboxyglutamic acid domain between protein Z and factor X reproduced the inefficient and warfarin-sensitive secretion pattern of protein Z, and vice versa. Joining of the propeptide and γ-carboxyglutamic acid domain to luciferase also demonstrated that the γ-carboxyglutamic acid domain of protein Z was responsible for its warfarin-sensitive secretion. Thus, it was concluded that the difference observed in secretion patterns of protein Z and factor X was mainly based on the structure of their γ-carboxyglutamic acid domains.