BACKGROUND:Neutrophil extracellular traps (NETs) and NOD-like receptor protein 3 (NLRP3) inflammation are key contributors to cholestatic liver disease (CLD). However, the relationship between NETs release and inflammasome activation, as well as its contribution to intrahepatic coagulation in CLD, remains unexplored. This study explores NETs-induced liver sinusoidal endothelial cells (LSECs) pyroptosis on intrahepatic coagulation in CLD. METHODS:Wild-type (WT) and PAD4-/- mice underwent bile duct ligation (BDL) or sham surgery for 7 or 14 days. The liver analysis assessed intrahepatic coagulation, inflammation, fibrosis, NETs release, and NLRP3 activation. Primary LSECs were exposed to NETs with or without MCC950. Pyroptosis and LSECs procoagulant activity were quantified. RESULTS:BDL mice exhibited significantly increased inflammation, tissue factor (TF), and fibrin deposition compared with controls. NETs release in the liver was increased significantly in WT BDL mice and was responsible for intrahepatic coagulation. PAD4 deficiency reduced TF and fibrin expression, improving hepatic sinusoid function. RNA-seq revealed BDL-induced enrichment of coagulation, neutrophil activation, and pyroptosis pathways. In vivo, NETs increased intrahepatic NLRP3 and IL-1β expression in BDL mice. However, NLRP3 inhibition (MCC950) or activation (BMS-986299) did not alter NETs release. Furthermore, NETs-induced NLRP3 activation increased intrahepatic coagulation, inflammation, and fibrosis. Finally, we demonstrated that NETs triggered LSECs dysfunction and pyroptosis, upregulating TF and phosphatidylserine production and enhancing procoagulant activity. CONCLUSIONS:NETs-induced LSECs pyroptosis exacerbates intrahepatic coagulation in cholestasis. Targeting NETs and LSECs pyroptosis holds promise for treating chronic liver injury in CLD.

Background: Cholestatic liver diseases induce local and systemic hypercoagulation, with neutrophil extracellular traps (NETs) serving as major drivers. These NETs have been linked to decreased liver function in patients with obstructive jaundice. However, the impact of NETs on liver hypercoagulation in cholestatic liver disease remains unknown. Methods: We utilized bile duct ligation to create experimental mice and analyzed NETs formation in the liver. Fibrin deposition, tissue factor expression, and inflammation in the liver were visualized through western blot and immunohistochemical techniques. LSECs were incubated with isolated NETs, and we detected endothelial procoagulant activity using coagulation protein production assays and measuring endothelial permeability. In both in vivo and in vitro settings, DNase I was applied to clarify the effect of NETs on intrahepatic hypercoagulability, hepatotoxicity, LSEC, and macrophage activation or injury. Results: Bile duct ligation mice exhibited significantly increased levels of NETs in liver tissue, accompanied by neutrophil infiltration, tissue necrosis, fibrin deposition, and thrombophilia compared to sham mice. Notably, NETs resulted in phosphatidylserine and tissue factor exposure on LSEC, enhancing coagulation Factor Xa and thrombin production. The enhanced procoagulant activity could be reversed by degrading NETs with DNase I. Additionally, NETs-induced permeability changes in LSECs, characterized by increased VE-cadherin expression and F-actin retraction, which could be rescued by DNase I. Meanwhile, NET formation is associated with KC activation and the formation of inflammatory factors. Conclusions: NETs promote intrahepatic activation of coagulation and inflammation, leading to liver tissue injury. Strategies targeting NET formation may offer a potential therapeutic approach for treating cholestatic liver disease.

BACKGROUND: Backbend-induced pediatric thoracic spinal cord injury without radiologic abnormality (BBPT-SCIWORA) in children is rare in clinical practice and leads to lower limb motor dysfunction. There are few clinical studies on BBPT-SCIWORA and even fewer on treatments for BBPT-SCIWORA-induced lower limb motor dysfunction. OBJECTIVE: To explore the therapeutic effect of acupuncture at bilateral spine acupoints combined with lower limb acupoints in BBPT-SCIWORA. CASE PRESENTATION: This study reported four cases of BBPT-SCIWORA after dancing, two of which received a unique medium-frequency electroacupuncture treatment. They were all females aged between 5 and 12 years old. They were diagnosed with BBPT-SCIWORA by magnetic resonance imaging (MRI), transferred to the rehabilitation department for lower limb dysfunction, and received rehabilitation treatments and acupuncture. Cases 1 and 2 received acupuncture treatment for lower limb acupoints, while Cases 3 and 4 received acupuncture treatment at the bilateral spine acupoints beside the lesion and lower limb acupoints. Cases 3 and 4 achieved better American spinal injury association (AIS) grades and lower extremity motor scores (LEMS) than Cases 1 and 2 after treatment. CONCLUSION: Acupuncture treatment of beside bilateral spine acupoints plus lower limb acupoints therapy might facilitate early lower limb motor function recovery in children with BBPT-SCIWORA.

BACKGROUND:Nonalcoholic steatohepatitis (NASH) patients are at a high risk of developing venous thromboembolism, with a high rate of morbidity and mortality. The role of neutrophil extracellular traps (NETs) in procoagulant activity (PCA) in patients with NASH remains unclear. Our study aimed to investigate the formation of NETs in NASH patients stimulated by specific pro-inflammatory factors. Moreover, we evaluated the pivotal role of NETs in the induction of hypercoagulability in NASH and the interaction between NETs and endothelial injury.METHOD:The levels of the NETs biomarkers were evaluated in the plasma samples of 27 NASH patients and 18 healthy subjects. The formation of NETs was visualized using immunofluorescence microscopy. The PCA of the NETs was assessed using coagulation time, purified coagulation complex, and fibrin formation assays. Confocal microscopy was further used to evaluate the interactions between the NETs and HUVECs.RESULTS:The levels of NETs markers in the plasma of NASH patients were significantly higher than healthy controls. NETs derived from NASH enhanced thrombin and fibrin formation and significantly reduced CT (p<0.05). The mixture of IL-6 and TNF-α triggered the NETs release in the plasma rather than them alone. Additionally, the NETs exerted cytotoxic effects on the endothelial cells, converting them to a procoagulant and pro-inflammatory phenotype, and DNase I could reverse these effects.CONCLUSION:Our results revealed the primary role of NETs in promoting the hypercoagulable state in NASH patients. Methods that prevent the formation of NETs may be a novel approach for the prevention and treatment of NASH.

During the coronavirus disease 2019 (COVID-19) pandemic, some patients with severe COVID-19 exhibited complications such as acute ischemic stroke (AIS), which was closely associated with a poor prognosis. These patients often had an abnormal coagulation, namely, elevated levels of D-dimer and fibrinogen, and a low platelet count. Certain studies have suggested that COVID-19 induces AIS by promoting hypercoagulability. Nevertheless, the exact mechanisms through which COVID-19 leads to a hypercoagulable state in infected patients remain unclear. Understanding the underlying mechanisms of hypercoagulability is of utmost importance for the effective treatment of these patients. The present review aims to summarize the current status of research on COVID-19, hypercoagulability and ischemic stroke. The present review also aimed to shed light into the underlying mechanisms through which COVID-19 induces hypercoagulability, and to provide therapies for different mechanisms for the more effective treatment of patients with COVID-19 with ischemic stroke and prevent AIS during the COVID-19 pandemic.

Background:The prevalence of deep vein thrombosis in hospitalized patients with COVID-19 is higher and is associated with adverse outcomes. However, the treatment options received by patients with different classifications are different, and previous studies have not discussed the differences in specific coagulation parameters between patients with mild, severe, and critically ill COVID-19. Aim:To investigate the change in coagulation function and the incidence of low limb venous thromboembolic events in mild/severe/critically ill patients with COVID-19. Methods:A retrospective analysis of coagulation parameters and lower extremity venous ultrasound examination results in 77 patients with laboratory-confirmed COVID-19 admitted to the first affiliated hospital of Harbin Medical University. We discussed the occurrence of vascular complications in patients with normal, severe and critically ill patients Rate and explore the nature of such vascular events. The anticoagulation dose was left to the discretion of the treating physician based on the individual risk of thrombosis and patients were classified as treated with prophylactic anticoagulation or therapeutic anticoagulation. Approval was obtained from the local institutional review board and all procedures were performed in accordance with the Declaration of Helsinki. Results:The incidence of low limb venous thromboembolic events in COVID-19 patients included in the study was 28.6% (22/77). A total of 22 cases with deep vein thrombosis, 13 of whom with multiple thrombosis events, and 9 cases with independent distal deep vein thrombosis. There were 0 cases, 8 cases (17.4%) and 14 cases (87.5%) of patients with deep vein thrombosis occurred in mild, severe and critically ill patients, respectively. There were 4 cases (50%) and 9 cases (64.29%) of severe and critically ill patients with multiple deep vein thrombosis events, respectively. There was no difference in age and gender between patients with lower extremity venous thrombosis and those without. The mortality rate of patients with thrombotic events has an upward trend; the mortality rate of patients with thrombosis is 18.18%, and the mortality rate of patients without thrombosis is 3.64%. Compared with mild patients, white blood cell counts, neutrophil percentage, fibrinogen, IL-6, IL-10 serum levels are higher in severe and critically ill patients. The patients whose ultrasonography reported thrombosis mostly showed a dynamic increase and/or a significant increase in D-dimer. The patients whose ultrasonography reported no thrombosis showed mildly elevated D-dimer or within the normal range. Conclusions:The development of massive venous thrombotic events, as observed in our study cohort, suggests the possibility of COVID-19 associated hypercoagulability and endothelial activation and/or dysfunction in affected individuals. Figure Disclosures No relevant conflicts of interest to declare.

Coronavirus disease 2019 (COVID-19) infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global pandemic disease with high morbidity and mortality. Inflammatory and thrombosis are its main manifestations. As an important organ of hemofiltration metabolism, the kidney is prone to blockage and destruction when filter high inflammatory and high viscous blood of COVID-19, resulting in the loss of a large amount of protein, aggravating blood concentration, and then worsening COVID-19 hypercoagulability, which may explain the phenomenon of erythrocytes aggregation blocking the capillary lumen and the main reason why the kidney has become the second largest involvement organs. Therefore, this review discusses the effects of pathophysiological mechanisms such as inflammatory storm, endothelial injury, phosphatidylserine expression, extracellular traps release on renal capillary thrombosis caused by COVID-19 infection. Meanwhile, in view of the above mechanisms, we put forward the potential targets of antithrombotic therapy, and graded management of patients, reasonable use of drugs according to the severity of the disease and the choice of time. And we support the view of prevention of thrombus before admission, continuous anticoagulation and drug choice after discharge. It is suggested that the symptomatic and supportive treatment of renal disease in critically ill patients should be combined with the concept of antithrombotic therapy. The ultimate goal is to reduce the occurrence and development of kidney disease, provide direction for the current management of COVID-19 with kidney disease, and reduce the mortality of COVID-19.

BACKGROUND:The role of vascular endothelium in acute promyelocytic leukaemia (APL) remains unknown. We aimed to investigate the mechanisms by which APL cells interact with endothelial cells (ECs) and to further explore how the endothelium affects bleeding as well as therapeutic interventions.METHOD:APL cells and an original APL cell line, NB4 cells, were used for experiments. The effects of leukaemic cells on ECs were analyzed in vitro and in vivo. Moreover, the endothelial barrier function and procoagulant activity were detected. An APL mouse model was established for in vivo studies.FINDINGS:APL cells interacted with ECs via ICAM-1 and VCAM-1 receptors to disrupt endothelial integrity. This binding activated MLCK signaling, resulting in the trans-endothelial passage of protein and red blood cells (RBCs). Combined treatment with asiatic acid or anti-adhesion receptor antibody inhibited the response of ECs to APL cells, thereby preventing APL-associated haemorrhage in vitro and in vivo. Activated ECs exhibited a procoagulant phenotype after phosphatidylserine exposure. Plasma from APL patients formed a thin fibrin network between procoagulant ECs, and this intercellular fibrin decreased the passage of albumin and RBCs. Ex vivo addition of fibrinogen further enhanced this barrier function in a dose-dependent manner.INTERPRETATION:Endothelial damage induced by leukaemic cell adherence promotes haemorrhaging in APL. Stabilization of ECs, decreasing adhesion receptor expression, and increasing fibrinogen transfusion levels may be a new therapeutic avenue to alleviate this fatal bleeding complication.FUNDING:National Science Foundation of China (81670128, 81873433).

Patients with pancreatic cancer (PC) are at increased risk of venous thrombosis, but the precise mechanisms of hypercoagulable state in PC remain unclear. We aimed to identify how phosphatidylserine positive (PS+) blood cells (BCs), PS+ microparticles (MPs) and neutrophil extracellular traps (NETs) regulate procoagulant activity (PCA) in PC, and to assess the relationship between PCA and PC staging. A total of 83 PC patients with different stages of disease were compared to 30 healthy controls, with confocal microscopy and flow cytometry used to assess MP and cellular PS exposure. MP and cell PCA was determined using both fibrin production assays and procoagulant enzyme complex analyses, and coagulation time was further measured. Patients with stage I PC and healthy controls exhibited significantly lower frequencies of PS+ MPs and BCs relative to those with more advanced disease, which may partly due to the increased levels of inflammation cytokines in advanced disease. Functional coagulation assays indicated that PS+ MPs and BCs derived from patients with stage II/III/IV PC directly contribute to elevated FXa, thrombin, and fibrin formation, and to more rapid coagulation relative to healthy control samples. In inhibition assays, lactadherin, which antagonizes PS, led to a roughly 80% inhibition of PCA. We further used isolated NETs to stimulate endothelial cells, revealing that this led to morphological changes including retraction from cell-cell junctions and a more pro-coagulative phenotype, with DNase I and activated protein C treatment reversing these changes. In patients with stage III PC, curative resection surgery significantly reduced PCA, whereas non-curative surgery did not have a marked impact based on studies of pre- and post-operative samples. These results highlight the pathogenic activity of PS+ cells, MPs, and NETs in promoting a prothrombotic environment within individuals suffering from advanced PC. Targeting PS and NETs in these patients may thus be a viable means of preventing pathological thrombosis.

BACKGROUND & AIMS:Patients with obstructive jaundice (OJ) are considered to be prothrombotic with increased risk of thromboembolism complications. The role of neutrophil extracellular traps (NETs) in procoagulant activity (PCA) and thrombosis risk in patients with OJ is unclear. In this study, we investigated NETs formation in OJ patients and the role of elevated unconjugated bilirubin (UCB) in inducing NETs, resulting in enhanced PCA and endothelial injury.METHODS:NETs of OJ patients and healthy controls were measured. NETs PCA was assessed via coagulation time (CT), fibrin formation and purified coagulation complex production assays. Visualization of NETs and mitochondrial reactive oxygen species (MitoROS) were performed with a fluorescence microscope. We further used confocal microscopy to quantify the exposure of phosphatidylserine (PS), fibrin strands and FVa/Xa on Human umbilical vein endothelial cells (HUVECs).RESULTS:Assessment of NETs components levels revealed greater NETs production in OJ patients than in healthy controls. Importantly, OJ-NETs were responsible for enhanced PCA. UCB induced NETs formation via MitoROS accumulation and mitochondrial mobilization. HUVECs cocultured with OJ NETs lost their cell-cell junctions and consequently converted to a procoagulant phenotype. The PCA was attenuated by using DNase I alone or in combination with lactadherin.CONCLUSIONS:Our results suggest that UCB-induced NETs play a prominent role in promoting the hypercoagulable and prothrombotic state in OJ patients. The increased MitoROS accumulation in neutrophils initiated NETosis. NETs are promising targets for indicating or improving coagulation disorders in OJ patients.

BACKGROUND AND AIMS:Despite the presence of neutrophil extracellular traps [NETs] in inflamed colon having been confirmed, the role of NETs, especially the circulating NETs, in the progression and thrombotic tendency of inflammatory bowel disease [IBD] remains elusive. We extended our previous study to prove that NETs constitute a central component in the progression and prothrombotic state of IBD.METHODS:In all 48 consecutive patients with IBD were studied. Acute colitis was induced by the treatment of C57BL/6 mice with 3.5% dextran sulphate sodium [DSS] in drinking water for 6 days. Peripheral blood neutrophils and sera were collected from IBD patients and murine colitis models. Exposed phosphatidylserine [PS] was analysed with flow cytometry and confocal microscopy. Procoagulant activity was evaluated using clotting time, purified coagulation complex, and fibrin formation assays.RESULTS:We observed higher plasma NET levels and presence of NETs in colon tissue in patients with active IBD. More importantly, NETs were induced in mice with DSS colitis, and inhibition of NET release attenuated colitis as well as colitis-associated tumorigenesis. NET degradation through DNase administration decreased cytokine levels during DSS-induced colitis. In addition, DNase treatment also significantly attenuated the accelerated thrombus formation and platelet activation observed in DSS-induced colitis. NETs triggered PS-positive microparticle release and PS exposure on platelets and endothelial cells partially through TLR2 and TLR4, converting them to a procoagulant phenotype.CONCLUSIONS:NETs exacerbate colon tissue damage and drive thrombotic tendency during active IBD. Strategies directed against NET formation may offer a potential therapeutic approach for the treatment of IBD.

2019 IERI International Conference on Medical Physics, Medical Engineering and Informatics (ICMMI 2019), Tokyo, Japan, 22–24 March 2019

Background:Relatively little is known about the role of phosphatidylserine (PS) in procoagulant activity (PCA) in patients with diabetic kidney disease (DKD). This study was designed to evaluate whether exposed PS on microparticles (MPs) and MP-origin cells were involved in the hypercoagulability in DKD patients. Methods:DKD patients (n = 90) were divided into three groups based on urinary albumin excretion rate, defined as normoalbuminuria (No-A) (<30 mg/24 h), microalbuminuria (Mi-A) (30-299 mg/24 h) or macroalbuminuria (Ma-A) (>300 mg/24 h), and compared with healthy controls (n = 30). Lactadherin was used to quantify PS exposure on MPs and their original cells. Healthy blood cells (BCs) and human umbilical vein endothelial cells (HUVECs) were treated with 25, 5 or 2.5 mmol/L glucose as well as 3-12 mg/dL uric acid and cells were evaluated by clotting time and purified coagulation complex assays. Fibrin production was determined by turbidity. PS exposure and fibrin strands were observed using confocal microscopy. Results:Using flow cytometry, we found that PS+ MPs (derived from platelets, erythrocytes, HUVECs, neutrophils, monocytes and lymphocytes) and BCs were significantly higher in patients than in controls. Furthermore, the number of PS+ MPs and BCs in patients with Ma-A was significantly higher than in patients with No-A. Similarly, we observed markedly elevated PS exposure on HUVECs cultured with serum from patients with Ma-A versus serum from patients with Mi-A or normoalbuminuria. In addition, circulating PS+ MPs cooperated with PS+ cells, contributing to markedly shortened coagulation time and dramatically increased FXa/thrombin generation and fibrin formation in each DKD group. Confocal microscopy images demonstrated colocalization of fibrin with PS on HUVECs. Moreover, blockade of exposed PS on MPs and cells with lactadherin inhibited PCA by ∼80%. In vitro, BCs and endothelial cells exposed more PS in hypoglycemia or hyperglycemia. Interestingly, reconstitution experiments showed that hypoglycemia-treated cells could be further activated or injured when recovery is obtained reaching hyperglycemia. Moreover, uric acid induced PS exposure on cells (excluding platelets) at concentrations >6 mg/dL. Linear regression analysis showed that levels of PS+ BCs and microparticles were positively correlated with uric acid and proteinuria, but negatively correlated with glomerular filtration rate. Conclusions:Our results suggest that PS+ MPs and MP-origin cells play procoagulant roles in patients with DKD. Blockade of PS could become a novel therapeutic modality for the prevention of thrombosis in these patients.

Objective:This study was designed to evaluate whether exposed tissue factor (TF) on platelets, leukocyte and microparticles were involved in the hypercoagulability of gastric cancer.Methods:Gastric cancer patients were divided into four groups based on tumor-node-metastasis (TNM) stage, defined as stage I (n=10), stage II (n=10), stage III (n=15) or stage IV (n=10), and compared with healthy controls (n=30).Flow cytometry was used to quantify the counts of tissue factor positive cells and microparticels.The procoagulant activity of cells and microparticles were tested by thrombin production assays.Results:Using flow cytometry, we found TF+platelets, neutrophils, monocytes and microparticles were significantly higher in stage III/IV patiens than in stage I/II patients and healthy controls.Moreover, the TF+blood cells cooperated with microparticels, contributing to the increased rate and levels of thrombin formation.In addition, blockade of exposed TF on cells and micrparticles with anti-TF antibody significantly inhibited the procoagulant activity.However, blockade of exposed PS with annexin V has little effect on the procoagulant activity.Meanwhile, we found that treatment with radical resection could reduce the number of TF+platelets, neutrophils, monocytes and microparticles.Conclusion:Our results suggest that TF+platelets, leukocyte and microparticles play procoagulant roles in patients with gastric cancer.Blockade of TF and inhibite the formation of thrombin maybe the therapeutic target to prevent coagulation in patients with stage III/IV gastric cancer.

Suramin inhibits immune responses and protects cells against inflammatory cell injury. However, little is known about its mechanisms. Using an in vitro model of glomerular mesangial cell (MC) lysis induced by antibodies plus complement, we investigated the potential protective effects and mechanisms of suramin on immunologic cell injury. Exposure of rat MCs to anti-Thy-1 antibody plus complement or anti-MC rabbit serum caused complement-dependent cell lysis, which was blocked by suramin and its structural analogue NF023 and NF049, but not by PPADS, an antagonist of purinergic receptors. Addition of exogenous ATP also failed to affect MC lysis. Further analysis revealed that suramin interfered with antibody binding to cell membrane antigens and suppressed antibody-induced phosphorylation of several proteins, including p38. Inhibition of p38 with chemical inhibitor significantly attenuated cell injury. Collectively, our results indicate that suramin protects cells against antibody-initiated and complement-dependent cell injury through inhibition of antibody binding to cell surface antigens and suppression of p38 activation. Our study thus provides novel mechanistic insights into the actions of suramin and suggests that suramin might be used to treat certain immune diseases.

Objective To identify and analyze the DNA methylation induced pathogenic genes in breast cancer.Methods Using genomic data provided by the international cancer genome,we carried out systematic a-nalysis of the abnormal gene expression in breast cancer and epigenetic regulation mechanism by bioinformatics methods.Results We identified 428 genes with abnormal expressions in breast cancer by t test.Functional enrich-ment analysis revealed genes up-regulation in breast cancer were closely related to the cell cycle,and those down-regulated were significantly enriched in the hormone response function.Study on DNA methylation revealed that breast cancer showed an unique DNA methylation pattern.Further analysis reveals 23 breast cancer genes induced by abnormal DNA methylation.Conclusion DNA methylation can mediate the abnormal expression of breast canc-er genes,and is an important biological marker for early diagnosis and prognosis of breast cancer.

Objective To investigate the effects of celecoxib on rats with 2,4,6-trinitrobenzenesulphonic acid(TNBS)-induced colitis in order to understand its underlying mechanisms in treatment of IBD.Methods 30 male Wistar rats were given 2,4,6-trinitrobenzenesulphonic acid-induced colitis and were randomly divided into three groups,SASP(200 mg/kg) treated group(group B) and celecoxib(10 mg/kg) treated group(group C),0.9% sodium chloride group(group D) with 10 each,another 10 rats were severed as normal control(group A).Except the rats in normal control group,all rats were intragastric administrated and were killed 7-day after colitis treatment and assessed with following variables including pathological change with HE staining of colon;the serum level of PGE2 was detected by enzymelinkde immunosorbent assay(ELISA),respectively.And the COX-2 was measured by immunohistochemistry.Results The DAI in group B,C and D increased since TNBS was administered rectally,but in group C,which was significantly higher than that in group B(P0.01),and was similar to that in the group D(P=0.418);the serum level of PGE2 in group A(75.28±7.55) was significantly lower than that in group B,C and D(P0.05).But in group C,which was significantly lower than that in group B and D(P0.01).Conclusion Celecoxib has no obvious therapeutic effect on TNBS induced ulcers colitis.The celecoxib can reduce the inflammation of colon tissues of experimental colitis induced by TNBS,and decrease expression of the serum PGE2,but even have the tendency of severity of the disease symptoms.Therefore,whether celecoxib can be used in the treatment of ulcerative colitis or not remains to be further researched.