Basic Evaluation of the Newly Developed "Lias Auto P-FDP" Assay and the Influence of Plasmin-α2 Plasmin Inhibitor Complex Values on Discrepancy in the Comparison with "Lias Auto D-Dimer Neo" Assay.

BACKGROUND: Laboratory determination of fibrin/fibrinogen degradation products (FDP) levels, along with that of the D-dimer, is important for assessing the fibrinolytic situation. Recently, we developed a new FDP reagent "Lias Auto P-FDP", which can detect various FDP fragments. The purpose of this study was to evaluate the basic performance of the newly developed Lias Auto P-FDP and compare it with Lias Auto D-Dimer Neo assay. METHODS: The within-run precision of Lias Auto P-FDP and Lias Auto D-Dimer was determined 20 times in low and high value controls. The between-day precision was evaluated five times a day for five days. The linearity study was performed by diluting high value samples for 2 - 10-fold and 2 - 8-fold. The comparative study was performed using 172 patient samples with elevated FDP values. For the discrepancy analysis, the samples were divided into three groups by the discrepancy percentage between the FDP and D-dimer values. The groups were defined as follows: lower discrepancy group, less than -20%; no discrepancy group, -20% to 20%; upper discrepancy group, more than 20%. RESULTS: The coefficient of variation % (CV%) in within-run and between-day precision were within 3.8% for both FDP and the D-dimer. The correlation coefficients were more than 0.999 and the linearity was high. In the comparative study, the values of FDP were higher than that of the D-dimer in all samples. The median FDP and D-dimer values of lower discrepancy, no discrepancy, and upper discrepancy groups were 11.8, 20.3, and 51.4, and 8.0, 11.3, and 13.1, respectively. FDP showed an increasing tendency but D-Dimer showed constant values. Thus, the possible cause of discrepancy between FDP and D-dimer values were the elevated FDP values. In addition, the values of plasmin-α2 plasmin inhibitor complex (PIC) in the upper discrepancy group were higher than that of the lower and no discrepancy groups, indicating progression of fibrinolysis. CONCLUSIONS: In this study, we evaluated the newly developed Lias Auto P-FDP reagent and confirmed that the basic performance was acceptable. FDP was elevated in samples with high PIC values, which indicated progression of fibrinolysis. Determination of fibrinolysis conditions by FDP measurement is important.

[A Case of Lupus Anticoagulant-Hypoprothrombinemia Syndrome with Phosphatidylserine-Dependent Anti-Prothrombin Antibody].

Lupus anticoagulant-hypothrombinemia syndrome (LAHS) is a rare disease involving hemorrhagic diathe- sis due to hypothrombinemia with lupus anticoagulant. We report a 28-week-pregnant woman at twenty years of age, who had been hospitalized with jaundice. In laboratory data, AST, ALT, and bilirubin were elevated and the prothrombin time (PT) and activated partial thromboplastin time (APTT) were prolonged. Although the liver failure was improved after she delivered a baby by Caesarean section, postoperative intraperitoneal bleeding persisted. The diagnosis by liver biopsy was autoimmune hepatitis. Although the bleeding was stopped on the seventh postoperative day, the prolongation of PT and APTT remained. LA was positive in the diluted Russell's viper venom time. Anti-cardiolipin and anti-beta-2-glycoprotein anti- bodies were also positive. The prothrombin activity was reduced. A high titer of phosphatidylserine- dependent antiprothrombin antibody (aPS/PT), which causes bleeding, was observed. Based on these data, she was diagnosed with LAHS. The liver dysfunction and prolongation of PT and APTT were normalized following the administration of corticosteroids. In this case, aPS/PT may have contributed to the pathological physiology of LAHS. [Case Report].

[Development of acquired hemophilia A during maintenance therapy for immune thrombocytopenia].

Acquired hemophilia A (AHA) is a rare coagulation disorder caused by autoantibodies against coagulation factor VIII (FVIII). We report herein a very rare case of AHA complicated by immune thrombocytopenia (ITP). A 30-year-old woman was hospitalized with severe thrombocytopenia. Her platelet count was 5,000/µl on admission, at which time APTT was normal. ITP was diagnosed and she was treated with γ-globulin, platelet transfusion, and prednisolone at 1 mg/kg/day. She was discharged after platelet count normalization and prednisolone was tapered to 5 mg/day. During the prednisolone tapering, purpura appeared on both thighs and in the left inguinal region, and APTT was found to be prolonged. She was referred to our hospital for examination of APTT prolongation. FVIII activity was markedly decreased to 7.7% and the FVIII inhibitor was positive (1.5 BU/ml), based on which AHA was diagnosed. We carefully followed this patient without intensification of immunosuppressive therapy for 7 weeks, but her platelet count decreased from 150,000/µl to 70,000/µl and the FVIII inhibitor increased to 4 BU/ml. We therefore increased prednisolone to 30 mg/day, after which her platelet count increased and complete remission of AHA was achieved by day 42. In addition, we examined the relationship of the FVIII inhibitor and FVIII binding antibody in this case.

[Pathogenesis and Laboratory Findings in Antiphospholipid Syndrome, Especially Associated with Lupus Anticoagulant].

Antiphospholipid syndrome (APS), an acquired thrombotic condition, is a complex clinical state characterized by the presence of circulating antiphospholipid antibodies in patients with thrombosis or pregnancy morbidity. Revised APS classification criteria are used for diagnosis, which include at least one clinical criterion (thrombosis or pregnancy loss) and at least one of the laboratory criteria [anticardiolipin antibodies, anti-ß2GPI antibodies, lupus anticoagulant (LA)]. LA is also an independent risk factor for developing thrombosis, though some LA-positive cases have been reported to have a bleeding symptom. Lupus anticoagulant-hypoprothrombinemia syndrome (LAHPS) is a rare disorder characterized by a bleeding tendency due to low prothrombin activity in patients with LA, and has recently been reported not only in children but also in adults We have encountered LA cases with bleeding and low coagulation factor activities except for prothrombin. Based on our findings, we propose that LA-positive cases with a bleeding symptom and characterized by low coagulation factor activity including prothrombin be termed lupus anticoagulant-associated coagulopathy (LAAC). Furthermore, coagulation factor autoantibodies are often detected in LAAC patients; thus, correct measurement of LA is important to distinguish LAAC patients from those possessing an inhibitor to coagulation factors such as acquired hemophilia A as well as to select the optimal therapeutic strategy.

Endoplasmic reticulum stress suppresses lipin-1 expression in 3T3-L1 adipocytes.

Lipin-1 plays crucial roles in the regulation of lipid metabolism and cell differentiation in adipocytes. In obesity, adipose lipin-1 mRNA expression is decreased and positively correlated with systemic insulin sensitivity. Amelioration of the lipin-1 depletion might be improved dysmetabolism. Although some cytokines such as TNF-α and interleukin-1ß reduces adipose lipin-1 expression, the mechanism of decreased adipose lipin-1 expression in obesity remains unclear. Recently, endoplasmic reticulum (ER) stress is implicated in the pathogenesis of obesity. Here we investigated the role of ER stress on the lipin-1 expression in 3T3-L1 adipocytes. We demonstrated that lipin-1 expression was suppressed by the treatment with ER stress inducers (tunicamycin and thapsigargin) at transcriptional level. We also showed that constitutive lipin-1 expression could be maintained by peroxisome proliferator-activated receptor-γ in 3T3-L1 adipocytes. Activation of peroxisome proliferator-activated receptor-γ recovered the ER stress-induced lipin-1 suppression. These results suggested that ER stress might be involved in the pathogenesis of obesity through lipin-1 depletion.

Measurement of coagulation factor antibody levels is useful for diagnosis and determining therapeutic efficacy in hemorrhagic patients with autoantibodies to coagulation factor VIII and factor V: results from a single center in Japan.

Coagulation factor inhibitors (CFIs) sometimes cause fatal bleeding conditions. Determination of an inhibitor titer (INH-titer) using the Bethesda method is essential for diagnosing diseases associated with CFIs and examining the effects of immunosuppressive therapy. We reviewed 17 cases with CFIs (acquired hemophilia A, n = 11; FV inhibitor, n = 6) to examine the usefulness of determining quantities of an autoantibody to a coagulation factor (CF-IgG) by ELISA for diagnosis and therapeutic efficacy, as compared with INH-titer. One patient with an INH-titer and no evidence of CF-IgG was lupus anticoagulant (LA)-positive, and thus the positive INH-titer may have been a false positive caused by LA. Although INH-titer alone was insufficient to correctly identify patients with CFI, determination of CF-IgG appeared to be useful. In addition, even after INH-titer disappearance, hemorrhagic conditions recurred when CF-IgG was detected. These findings suggest that the presence of a clearance antibody against the coagulation factor might reduce the activity of that coagulation factor even after disappearance of the corresponding neutralizing antibody. Although the diagnosis and therapeutic efficacy can also be determined by INH-titer disappearance and improvement of corresponding coagulation factor activity, determination of CF-IgG by ELISA can improve the accuracy of these assessments.

Suppression of lipin-1 expression increases monocyte chemoattractant protein-1 expression in 3T3-L1 adipocytes.

Lipin-1 plays a crucial role in the regulation of lipid metabolism and cell differentiation in adipocytes. Expression of adipose lipin-1 is reduced in obesity, and metabolic syndrome. However, the significance of this reduction remains unclear. This study investigated if and how reduced lipin-1 expression affected metabolism. We assessed mRNA expression levels of various genes related to adipocyte metabolism in lipin-1-depleted 3T3-L1 adipocytes by introducing its specific small interfering RNA. In lipin-1-depleted adipocytes, mRNA and protein expression levels of monocyte chemoattractant protein-1 (MCP-1) were significantly increased, although the other genes tested were not altered. The conditioned media from the cells promoted monocyte chemotaxis. The increase in MCP-1 expression was prevented by treatment with quinazoline or salicylate, inhibitors of nuclear factor-κB activation. Because MCP-1 is related to adipose inflammation and systemic insulin resistance, these results suggest that a reduction in adipose lipin-1 in obesity may exacerbate adipose inflammation and metabolism.

Characterization of fibrin/fibrinogen degradation products reagents and their utility in critical care patients with enhanced fibrinolysis.

INTRODUCTION: Fibrin/fibrinogen degradation products (FDP) values reflect coagulation and fibrinolysis status, and FDP levels are helpful for diagnosis and classification of disseminated intravascular coagulation (DIC). FDP measurement has always played a key role in diagnosing DIC, a phenomenon that has recently gained renewed attention because of its occurrence in coronavirus disease 2019 (COVID-19) patients. Although the evaluation of FDP is crucial for the management of critical care, the variability among FDP reagents is unclear. In this study, we aimed to compare LIASAUTO P-FDP with three FDP reagents and investigate their characteristics. METHODS: In total, 172 plasmas samples were used in the correlation. The sample data were divided into three groups including negative, no and positive discrepancy based on the discrepancy percentages calculated from each correlation between LIASAUTO P-FDP and other three reagents. D-dimer, plasmin-α2 plasmin inhibitor complex (PIC), fibrin monomer complex (FMC), fibrinogen (Fbg) and Plasmin-α2 Plasmin Inhibitor (α2 PI) were measured and included in data analysis. RESULTS: The positive discrepancy groups showed higher D-dimer, PIC and FMC values than the negative discrepancy groups. The data indicated that LIASAUTO P-FDP had higher reactivity to D-dimer than other reagents and the values were elevated in the fibrinolysis-enhanced samples with various FDP fragments. CONCLUSION: LIASAUTO P-FDP displayed the reactivity towards various fibrin/fibrinogen degradation products, and it might be useful for DIC diagnosis because the fibrinolytic status differed in the DIC types and stages.

Comparative evaluation of reagents for measuring protein S activity: possibility of harmonization.

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

[Laboratory examinations in antiphospholipid syndrome].

Antiphospholipid syndrome (APS), formerly detected by the association between clinical events (venous/arterial thrombosis or pregnancy morbidity) and positivity in at least one of the laboratory tests used to detect antiphospholipid antibodies (aPL), namely lupus anticoagulant (LA), IgG- or IgM-class anticardiolipin antibodies (aCL), and IgG or IgM-class anti-beta2GPI antibodies (abeta2GPI), is now diagnosed using revised APS classification criteria. However, there are a number of problems with the methods used to detect aPL. IgM-class aCL and abeta2GPI measurements are not covered by the health insurance system of Japan, while ELISA to measure abeta2GPI has not been standardized LA which is strongly associated with thrombosis in APS, can be determined using methods recommended by the Scientific and Standardization Committee of the International Society of Thrombosis and Haemostasis (ISTH-SSC). According to the guidelines for detection of LA recently published by ISTH-SSC, APTT reagents with silica as an activator and double centrifugation-treated samples (including control plasma) are recommended to detect LA, while 50% patient plasma mixed with control plasma is recommended for a cross-mixing test. However, some APTT reagents with ellagic acid are sensitive for LA activity. In the present study, we obtained favorable results for LA detection with cross-mixing tests by measuring APTT in mixtures of control plasma with 0%, 10%, 20%, 50%, and 100% concentrations of patient plasma treated with a 0.2 microm filter. In the future, laboratory examinations for APS will change with diagnostic criteria, as APS has not yet been established as a distinct disease concept.

[Accuracy and reliability of international normalized ratio (INR) in monitoring of warfarin--comparison in the sensitivity to extrinsic coagulation factors between PT (INR) and TT (INR)].

Prothrombin time (PT) is widely used as the monitor of oral anticoagulant therapy using ISI/NR system which WHO recommended. However, a main clinical usefulness of an Owren-type combined reagent (TT) also remains in the monitoring of warfarin in Europe and Japan. Recently, a TT reagent utilizing a recombinant bovine tissue factor (TF) expressed by silkworm-baculovirus system has been developed. The purpose of this study is to investigate the sensitivity to coagulation factors (FII, FV, FVII, FX) of PT (rabbit brain and recombinant human) and TT (bovine brain and recombinant bovine) in INR, and the correlation among those reagents using 53 plasma samples from patients treated with warfarin. The INR results were calculated using a certified INR calibrator, "AK-CALIBRANT", according to the method of "Direct" INR determination. The sensitivity to FII, FVII and FX of those reagents results similar behavior in INR, but the sensitivity to FV of PT reagents were generally higher than that of TT reagents. The correlation coefficient between recombinant PT and recombinant TT was 0.979. There were a good agreement between two TT reagents (bovine brain and recombinant bovine) in INR (r = 0.998). It was apparent that a variance between PT and TT was dependent on the sensitivity to FV level during the course of warfarin treatment in a clinical case. In conclusion, PT and TT reagents gave generally acceptable correlation in INR, and our results indicate that the Owren-type TT reagent is also well suited for monitoring warfarin using local INR calibration according to WHO recommendation.

Magnesium-dependent activated partial thromboplastin time assay-Simple method for lupus anticoagulant detection.

INTRODUCTION: Detection of lupus anticoagulant (LA), an antiphospholipid (aPL) antibody, in a clotting time test is an important finding for diagnosis of antiphospholipid syndrome (APS). However, confirmation of LA requires several different testing procedures, some of which can be difficult and require time. We report here a simple and highly specific method for detecting LA. MATERIALS AND METHODS: We examined 66 plasma samples obtained from LA-positive (LA) and 75 from LA-negative (non-LA) subjects, which included patients with acquired hemophilia and coagulation disorders, as well as from 43 healthy volunteer samples as normal controls. Activated partial thromboplastin time (APTT) was determined by adding 20 mmol of CaCl2 (Ca-APTT) or 25 mmol of a mixture of Mg and Ca (Mg-APTT). The ratio of Mg-APTT/Ca-APTT was then calculated and used as the Mg/Ca Index. RESULTS: The Mg/Ca Index value for the LA group was significantly lower than that for the non-LA and normal control groups (P < .0001). When the cutoff value of the Mg/Ca Index was less than 1.00, the sensitivity of LA determination using the Mg-APTT assay was 80.3%, while specificity was 100%. CONCLUSION: Our findings indicate that the present Mg-APTT assay is a simple yet highly specific method for LA detection.

Knockdown of long noncoding RNA dreh facilitates cell surface GLUT4 expression and glucose uptake through the involvement of vimentin in 3T3-L1 adipocytes.

Glucose uptake in adipocytes is crucial for regulating systemic metabolism. Long noncoding RNAs (lncRNAs), defined as being transcripts with lengths exceeding 200 nucleotides that are not translated, are recently identified regulators of cellular functions. Previously, we have shown that an lncRNA, "down-regulated expression by hepatitis B virus X" (dreh), is involved in glucose transport in skeletal muscle cells. Here, we aimed to examine the involvement of dreh in glucose transport in 3T3-L1 adipocytes. Expression analysis showed that dreh was expressed in 3T3-L1 fibroblasts and adipocytes. Knockdown of dreh expression using its specific siRNAs lowered the glucose concentration of the medium and facilitated [3H]-2-deoxyglucose transport in adipocytes. Additionally, dreh silencing enhanced the protein expression of glucose transporter (GLUT4) in the plasma membrane of adipocytes. Treatment with siRNA against vimentin attenuated the glucose-lowering effect of dreh depletion. These results suggest that the repression of dreh facilitates glucose transport via increased GLUT4 expression in the plasma membrane through the involvement of vimentin in 3T3-L1 adipocytes. In conclusion, dreh is the first observed lncRNA that regulates glucose transport in adipocytes and could serve as a novel therapeutic target for diabetes by modulating adipocyte function. Considering the new function of dreh, we propose that dreh be renamed "down-regulated expression-related hexose/glucose transport enhancer."

Novel assay based on diluted prothrombin time reflects anticoagulant effects of direct oral factor Xa inhibitors: Results of multicenter study in Japan.

BACKGROUND: Direct oral anticoagulants targeting factor Xa (DXaIs) are administered as prophylaxis for various venothrombotic diseases without routine monitoring required. However, assessment of their anticoagulant effects is necessary to prevent severe events, including major bleeding and/or refractory thrombosis. OBJECTIVES: We examined the correlation of ratio of inhibited thrombin generation (RITG), determined using a novel assay based on dilute prothrombin time (dPT), with coagulant markers and laboratory test results to show drug effects. In addition, RITG usefulness as a confirmation test for DXaI therapy was investigated. METHODS: Citrated plasma samples were obtained from patients treated with rivaroxaban (n = 882), apixaban (n = 1214), or edoxaban (n = 820) at 4 different institutions in Japan. Laboratory tests, including prothrombin time (PT), activated partial thromboplastin time (APTT), D-dimer, and plasma concentrations of DXaIs, were conducted, with drug concentrations divided into peak and trough groups, within and after 5 h of administration. RESULTS: In each DXaI group, RITG was positively correlated with PT, APTT, and drug concentration, and negatively with D-dimer. RITG fluctuation during the peak and trough periods reflected the anticoagulant activity characteristic of each DXaI, which was different from blood concentration fluctuations. RITG showed a significant decrease in cases with thrombosis, while that was increased in those with hemorrhage. CONCLUSION: We developed RITG, a novel measurement method based on dPT. RITG represents residual coagulation ability in plasma samples, and is useful for assessment of bleeding and thrombotic tendencies in DXaI patients. RITG can be utilized to confirm the effectiveness of oral anticoagulation therapy with DXaI agents.

Expert consensus regarding standardization of sample preparation for clotting time assays.

Accurate clotting time assay results are vital, as the test is employed to indicate the amount of oral anticoagulant to be prescribed, while it is also used for screening the hemorrhagic and thrombotic diseases. The procedure chosen for preparation of a patient blood sample including centrifugation can contribute to significant differences in the results obtained. Thus, for the purpose of proposing a standardized method to appropriately prepare blood samples prior to assay, the Japanese Society of Laboratory Hematology organized the Working Group for Standardization of Sample Preparation for Clotting Time Assays (WG). Following reviews of previously announced guidelines and original experimental results, consensus was obtained by the WG, with the main findings as follows. (1) The recommended anticoagulant in the blood collection tube is sodium citrate solution at 0.105-0.109 M (3.13-3.2%). (2) Whole blood samples should be stored at room temperature (18-25 ˚C) within 1 h of collection from the patient. (3) For plasma preparation, centrifugation at 1500 × g should be performed for at least 15 min or at 2000 × g for at least 10 min at room temperature. (4) After the plasma sample is prepared, it should be stored at room temperature and assayed within 4 h.

Plasma soluble fibrin monomer complex as a marker of coronary thrombotic events in patients with acute myocardial infarction.

Soluble fibrin monomer appears in the bloodstream during the extremely early stage of blood coagulation and generally forms a complex with fibrinogen, termed soluble fibrin monomer complex (FMC). Determination of FMC can provide information regarding the state of thrombotic diseases; thus it is important to investigate whether FMC serves as an early indicator of myocardial infarction (MI). We investigated hemostatic and fibrinolytic parameters including FMC to determine their capabilities for indicating thrombotic conditions in the coronary artery. Blood samples from 47 patients with acute MI were obtained within 48 hours (acute phase) and during 120 - 600 hours (recovery phase), respectively, after MI onset. Plasma FMC was significantly elevated in the acute phase, compared with that during the recovery phase and in healthy controls (p = 0.001), suggesting that its elevation indicates thrombotic events in the coronary artery of MI patients. D-dimer, a marker of thrombus formation accompanied with fibrinolysis, was increased in both phases in the patients. In addition, FMC and D-dimer were significantly increased within 24 hours after onset as compared to 24 - 48 hours (p = 0.003 and p = 0.011). Furthermore, cardiac troponin T, a marker of myocardial damage, was significantly higher after 24 hours than within the first 24 hours (p = 0.001). Receiver operating characteristic (ROC) analysis of FMC for early MI diagnosis indicates that FMC, rather than D-dimer, is a better marker within 24 hours of onset. Measuring plasma FMC may be useful for early diagnosis of MI recurrence and deciding primary treatment.

[Cross-mixing test to detect lupus anticoagulant for diagnosis of antiphospholipid syndrome].

The activated partial thromboplastin time (APTT) is generally used to screen for intrinsic coagulation factor deficiency. APTT prolongation suggests a bleeding tendency, although it can also reveal the presence of lupus anticoagulant (LA), which is one of the antiphospholipid antibodies associated with a high risk of thrombosis. Therefore, differential diagnosis in patients with a prolonged APTT is important. Recently, a cross mixing test has been proposed as a useful laboratory examination for differential diagnosis in these patients. When a cross-mixing test utilizes a mixture of patient and normal plasma at various ratios, the APTT value of the control plasma is prolonged by the addition of a small amount (10-20%) of the plasma from a patient with LA, while a prolonged APTT in a patient with an intrinsic coagulation factor deficiency is shortened by the addition of control plasma(50%). We obtained more favorable results for LA detection with a cross mixing test by measuring APTT in 5 different mixtures of control plasma and 0, 10, 20, 50, and 100% concentrations of patient plasma with an LA-sensitive APTT reagent such as PTT-LA. Also, we utilized a 0.2 microm filter unit to remove residual platelets from both patient and control plasma samples, which we recommend as useful. We found that a cross-mixing examination with appropriate samples and reagents contributed to the detection of LA. However, some problems remain regarding the final determination and quantitative measurements of LA.

Lupus anticoagulant-hypoprothrombinemia syndrome and similar diseases: experiences at a single center in Japan.

Patients with lupus anticoagulant (LA), a thrombotic risk factor, along with decreased prothrombin (FII) activity are classified as lupus anticoagulant-hypoprothrombinemia syndrome (LAHPS) and occasionally show bleeding symptoms, although this is not essential for diagnosis. We treated 20 cases of LAHPS over a 3-year period. Median FII activity was 20.9% and the anti-prothrombin antibody (anti-II Ab), shown by ELISA findings, was detected in 55%. Bleeding symptoms were observed in 20%, although that finding was not correlated with FII activity or anti-FII Ab quantity. We also observed 21 LA cases with decreased activity of coagulation factors other than FII, which we have designated LAHPS-like syndrome (LLS). Among LLS patients, anti-FII Ab and bleeding symptoms were seen in 47.6% and 14.3%, respectively. Our findings suggest that bleeding in LAHPS and LLS cannot be explained only by FII activity decreased by anti-FII Ab. Low FVIII activity and the anti-FVIII antibody (anti-FVIII Ab) were detected in some LAHPS and LLS patients, making it difficult to distinguish those from acquired hemophilia A cases. Detection of anti-FVIII Ab quantity by ELISA may be useful for accurate determination, as that was not performed in our LAHPS or LLS patients.

Dreh, a long noncoding RNA repressed by metformin, regulates glucose transport in C2C12 skeletal muscle cells.

AIMS: The anti-hyperglycemic action of metformin on skeletal muscles is presently unclear. Long noncoding RNAs (lncRNAs) are implicated in multiple cellular functions. This study aims to explore the role of lncRNAs in the glucometabolic action of metformin on skeletal muscle cells. MAIN METHODS: Metformin accumulation was assessed using [14C]-metformin. A lncRNA array was used to investigate metformin-regulated lncRNAs in C2C12 skeletal muscle cells. Knockdown studies were applied to evaluate the function of lncRNA Dreh. A colorimetric assay was used for the measurement of medium glucose concentration; glucose transport was assessed using [3H]-2-deoxyglucose; real-time PCR was used for RNA expression analysis, and western blotting was used to assess protein expression in myotubes. A Dreh overexpression plasmid was transfected into the cells. KEY FINDINGS: Metformin accumulated in C2C12 myotubes. Metformin reduced medium glucose concentration and repressed lncRNA Dreh expression in the myotubes. Knockdown of Dreh in the myotubes resulted in reduced glucose concentration in the culture medium, increased glucose transport, and increased levels of GLUT4 protein in the plasma membrane. Overexpression of Dreh attenuated the glucose-lowering effect of metformin in myotubes. SIGNIFICANCE: The glucoregulatory actions of metformin are mediated in part by a lncRNA, Dreh, in the skeletal muscle cells. Dreh is a novel regulator for glucose transport and could be a therapeutic target for diabetes.