A questionnaire on COVID-19-related thrombosis in patients hospitalized before Aug 31, 2020, was sent to 399 hospitals throughout Japan. Responses were received from 111 (27.8%) with information on ...6,202 COVID-19 patients. Of these, 333 and 56 required ventilation or extracorporeal membrane oxygenation (ECMO), respectively, and 212 died (3.4%). D-dimer levels were measured in 75.0% of the patients, revealing that 9.2% and 7.6% exhibited D-dimer increases of 3-8-fold and ≥8-fold the reference value, respectively. Thrombotic events occurred in 108 patients (1.86% of the 5,807 patients with available data) including symptomatic cerebral infarction in 24, myocardial infarction in 7, deep vein thrombosis in 41, pulmonary thromboembolism in 30, and other thrombotic events in 22. Some patients developed multiple thrombotic events. Thrombosis occurred in 32 patients with mild or moderate COVID-19 severity (0.59% of those with data available) and in 52 patients on ventilation or ECMO (13.5% of severe patients for whom data were available). Thrombosis occurred in 67 patients during worsening clinical condition and in 26 during recovery. Anticoagulant therapy was provided to 893 patients (14.6% of the 6,119 patients with available data), the main reasons being provided as elevated D-dimer levels and worsening clinical condition.
The blood glycoprotein von Willebrand factor (VWF) plays an important role in hemostasis and thrombosis.VWF is produced and secreted as large multimers by endothelial cells and megakaryocytes. It is ...then cleaved in a sheer-stress dependent manner by a specific protease, ADAMTS13, into multimers consisting of 2–80 subunits. Among VWF multimers, high molecular weight (HMW) multimers play important roles in platelet aggregation. Therefore, their loss induces a hemostatic disorder known as von Willebrand disease (VWD) type 2A. Various cardiovascular diseases, such as aortic stenosis, hypertrophic obstructive cardiomyopathy (HOCM), and several congenital structural diseases, as well as mechanical circulatory support systems, generate excessive high shear stress in the bloodstream. These cause excessive cleavage of VWF multimers resulting in a loss of HMW multimers, known as acquired von Willebrand syndrome (AVWS), a hemostatic disorder similar to VWD type 2A. Bleeding often occurs in the gastrointestinal tract since a fragile angiodysplasia develops associated with these diseases. Radical treatment for AVWS is to remove the pathological high shear causing AVWS.
Metformin is the first-line drug in the treatment of type 2 diabetes. In addition to its hypoglycemic effect, metformin has an anti-inflammatory function, but the precise mechanism promoting this ...activity remains unclear. High mobility group box 1 (HMGB1) is an alarmin that is released from necrotic cells and induces inflammatory responses by its cytokine-like activity and is, therefore, a target of anti-inflammatory therapies. Here we identified HMGB1 as a novel metformin-binding protein by affinity purification using a biotinylated metformin analogue. Metformin directly bound to the C-terminal acidic tail of HMGB1. Both in vitro and in vivo, metformin inhibited inflammatory responses induced by full-length HMGB1 but not by HMGB1 lacking the acidic tail. In an acetaminophen-induced acute liver injury model in which HMGB1 released from injured cells exacerbates the initial injury, metformin effectively reduced liver injury and had no additional inhibitory effects when the extracellular HMGB1 was blocked by anti-HMGB1-neutralizing antibody. In summary, we report for the first time that metformin suppresses inflammation by inhibiting the extracellular activity of HMGB1. Because HMGB1 plays a major role in inflammation, our results suggest possible new ways to manage HMGB1-induced inflammation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Ykt6 is an evolutionarily conserved SNARE protein regulating Golgi membrane fusion and other diverse membrane trafficking pathways. Unlike most SNARE proteins, Ykt6 lacks a transmembrane domain but ...instead has a tandem cysteine motif at the C-terminus. Recently, we have demonstrated that Ykt6 undergoes double prenylation at the C-terminal two cysteines first by farnesyltransferase and then by a newly identified protein prenyltransferase named geranylgeranyltransferase type-III (GGTase-III). GGTase-III consists of a novel α subunit prenyltransferase alpha subunit repeat containing 1 (PTAR1) and the β subunit of Rab geranylgeranyltransferase. PTAR1 knockout (KO) cells, where Ykt6 is singly prenylated with a farnesyl moiety, exhibit structural and functional abnormalities in the Golgi apparatus with delayed intra-Golgi trafficking and impaired protein glycosylation. It remains unclear whether the second prenylation of Ykt6 is required for proper trafficking of lysosomal hydrolases from Golgi to lysosomes. Here, we show that lysosomal hydrolases, cathepsin D and β-hexosaminidase, were missorted at the trans-Golgi network and secreted into the extracellular space in PTAR1 KO cells. Moreover, maturation of these hydrolases was disturbed. LC3B, an autophagy marker, was accumulated in PTAR1 KO cells, suggesting defects in cellular degradation pathways. Thus, doubly prenylated Ykt6, but not singly prenylated Ykt6, is critical for the efficient sorting and trafficking of acid hydrolases to lysosomes.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Background and Purpose
Upon stimulation, neutrophils release their nuclear contents called neutrophil extracellular traps (NETs), which contain unfolded chromatin and lysosomal enzymes. NETs have ...been demonstrated to play a critical role in host defence, although the role of PGE2, a bioactive substance generated in inflammatory tissues, in the formation of NETs remains unclear.
Experimental Approach
The effects of PGE2, agonists and antagonists of its receptors, and modulators of the cAMP–PKA pathway on the formation of NETs were examined in vitro in isolated neutrophils and in vivo in a newly established mouse model.
Key Results
PGE2 inhibited PMA‐induced NET formation in vitro through EP2 and EP4 Gαs‐coupled receptors. Incubation with a cell‐permeable cAMP analogue, dibutyryl cAMP, or various inhibitors of a cAMP‐degrading enzyme, PDE, also suppressed NET formation. In the assay established here, where an agarose gel was s.c. implanted in mice and NET formation was detected on the surface of the gel, the extent of the NET formed was inhibited in agarose gels containing rolipram, a PDE4 inhibitor, and butaprost, an EP2 receptor agonist.
Conclusions and Implications
PGE2 inhibits NET formation through the production of cAMP. These findings will contribute to the development of novel treatments for NETosis‐related diseases.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
The immune system exhibits circadian rhythms, and its response to viral infection is influenced by the circadian clock system. Previous studies have reported associations between the time of day of ...vaccination against COVID-19 and production of anti-SARS-CoV-2 antibody titer. We examined the effect of vaccination time of day on anti-SARS-CoV-2 antibody titer after the first dose of vaccination with the mRNA-1273 (Moderna) COVID-19 vaccine in an adult population. A total of 332 Japanese adults participated in the present study. All participants were not infected with SARS-CoV-2 and had already received the first dose of mRNA-1273 2 to 4 weeks prior to participating in the study. The participants were asked to provide basic demographic characteristics (age, sex, medical history, allergy, medication, and mean sleep duration), the number of days after the first dose of vaccination, and the time of day of vaccination. Blood was collected from the participants, and SARS-CoV-2 antibody titers were measured. Ordinary least square regression was used for assessing the relationship between basic demographic characteristics, number of days after vaccination, time of day of vaccination, and the log10-transformed normalized antibody titer. The least square mean of antibody titers was not associated with the vaccination time and sleep durations. The least square means of antibody titers was associated with age; the antibody titers decreased in people aged 50 to 59 years and 60 to 64 years. The present findings demonstrate that the vaccination time with mRNA-1273 was not associated with the SARS-CoV-2 antibody titer in an adult population, suggesting that these results do not support restricting vaccination to a particular time of day. The present findings may be useful in optimizing SARS-CoV-2 vaccination strategies.
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NUK, OILJ, SAZU, UKNU, UL, UM, UPUK
Von Willebrand factor (vWF) plays a crucial role in hemostasis, acting as a key factor for platelet adhesion/aggregation and as a transport protein for coagulation factor VIII. vWF is secreted as a ...giant multimer, and it undergoes shear stress-dependent cleavage by a specific metalloproteinase in plasma. Among vWF multimers, high-molecular-weight (large) multimers are essential for hemostasis. Acquired von Willebrand syndrome, linked to various conditions, is a hemostatic disorder due to reduced vWF activity. Extracorporeal membrane oxygenation (ECMO), utilized recently for out-of-hospital cardiac arrest patients, generates high shear stress inside the pump. This stress may induce a conformational change in vWF, enhancing cleavage by a specific metalloproteinase and thereby reducing vWF activity. However, no study has investigated the effects of ECMO on vWF-related factors in patients receiving or not receiving ECMO. This study aimed to elucidate the relationship between ECMO treatment and acquired von Willebrand syndrome-related factors in patients with out-of-hospital cardiac arrest.
This study included patients with cardiogenic out-of-hospital cardiac arrest admitted to our hospital. The patients were categorized into two groups (ECMO and non-ECMO) based on the presence or absence of ECMO treatment. Plasma samples were collected from patients admitted to the emergency department (days 0-4). The vWF antigen (vWF: Ag), vWF ristocetin cofactor activity (vWF: RCo), and factor VIII activity were measured. Additionally, a large multimer of vWF was evaluated through vWF multimer analysis, utilizing western blotting to probe vWF under non-reducing conditions.
The ECMO and non-ECMO groups included 10 and 22 patients, respectively. The median ECMO treatment in the ECMO group was 64.6 h. No differences in vWF: Ag or factor VIII activity were observed between the two groups during the observation period. However, the ECMO group exhibited a decrease in large vWF multimers and vWF: RCo during ECMO. Strong correlations were observed between vWF: RCo and vWF: Ag in both groups, although the relationships were significantly different between the two groups.
ECMO treatment in patients with out-of-hospital cardiac arrest resulted in the loss of large vWF multimers and decreased vWF activity. Hence, decreased vWF activity should be considered as a cause of bleeding during ECMO management.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
The small GTPases RalA and RalB are members of the Ras family and activated downstream of Ras. Ral proteins are found in GTP‐bound active and GDP‐bound inactive forms. The activation process is ...executed by guanine nucleotide exchange factors, while inactivation is mediated by GTPase‐activating proteins (GAPs). RalGAPs are complexes that consist of a catalytic α1 or α2 subunit together with a common β subunit. Several reports implicate the importance of Ral in pancreatic ductal adenocarcinoma (PDAC). However, there are few reports on the relationship between levels of RalGAP expression and malignancy in PDAC. We generated RalGAPβ‐deficient PDAC cells by CRISPR‐Cas9 genome editing to investigate how increased Ral activity affects malignant phenotypes of PDAC cells. RalGAPβ‐deficient PDAC cells exhibited several‐fold higher Ral activity relative to control cells. They had a high migratory and invasive capacity. The RalGAPβ‐deficient cells grew more rapidly than control cells when injected subcutaneously into nude mice. When injected into the spleen, the RalGAPβ‐deficient cells formed larger splenic tumors with more liver metastases, and unlike controls, they disseminated into the abdominal cavity. These results indicate that RalGAPβ deficiency in PDAC cells contributes to high activities of RalA and RalB, leading to enhanced cell migration and invasion in vitro, and tumor growth and metastasis in vivo.
Ral is a Ras family small GTPase that have recently been noted as a novel signaling factor that regulates progression of pancreatic ductal adenocarcinoma (PDAC). In this study, by characterizing newly generated RalGAP‐deficient PDAC cells, we first demonstrated that Ral GTPase–activating proteins played a critical role in not only cell migration and invasion in vitro but also tumor growth and metastasis in vivo in PDAC.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Chronic blood trauma caused by the shear stresses generated by mechanical circulatory support (MCS) systems is one of the major concerns to be considered during the development of ventricular assist ...devices. Large multimers with high-molecular-weight von Willebrand factor (VWF) are extended by the fluid forces in a shear flow and are cleaved by ADAMTS13. Since the mechanical revolving motions in artificial MCSs induce cleavage in large VWF multimers, nonsurgical bleeding associated with the MCS is likely to occur after mechanical hemodynamic support. In this study, the shear stress (~ 600 Pa) and exposure time related to hemolysis and VWF degradation were investigated using a newly designed mechanical shuttle shear flow tester. The device consisted of a pair of cylinders facing the test section of a small-sized pipe; both the cylinders were connected to composite mechanical heads with a sliding-sleeve structure for axial separation during the withdrawing motion. The influence of exposure time, in terms of the number of stress cycles, on hemolysis and VWF degradation was confirmed using fresh goat blood, and the differences in the rates of dissipation of the multimers were established. The plasma-free hemoglobin levels showed a logarithmic increase corresponding to the number of cycles, and the dissipation of large VWF multimers occurred within a few seconds under high shear stress flow conditions.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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