Search

In this paper, we consider a priori and a posteriori error estimates of the H(curl2)-conforming finite element when solving the quad-curl eigenvalue problem. An a priori estimate of eigenvalues with convergence order 2(s − 1) is obtained if the corresponding eigenvector u ∈ Hs − 1(Ω) and ∇ × u ∈ Hs(Ω). For the a posteriori estimate, by analyzing the associated source problem, we obtain lower and upper bounds for the errors of eigenvectors in the energy norm and upper bounds for the errors of eigenvalues. Numerical examples are presented for validation.

Current lung cancer diagnosis methods encounter delayed visual confirmation of tumor foci and low-resolution metrics in imaging findings, which delays the early treatment of tumors. Here, we developed a potent lung cancer imaging and treatment strategy centered around a nanotransformational concept of tumor iron mineralization in situ, which employs Prussian blue/calcium peroxide nanocomposites as a precursor. The resultant iron mineralization in tumor cells greatly facilitates the early and differential diagnosis of lung carcinoma from benign nodules via medical imaging, meanwhile introducing oxidative stress to activate the cellular apoptosis and ferroptosis pathways, resulting in inhibition of the malignant behavior of tumor cells. Tumor-microenvironment-triggered iron mineralization enables integration of the detection and prevention of tumor metastasis at its early stages with no assistance of toxic drugs, which offers a potential solution for the precise management of lung cancer with ideal outcomes.

In this paper, we propose an H ⁢ ( curl 2 ) \boldsymbol{H}(\mathbf{curl}^{2}) -conforming spectral elements to solve the quad-curl problem on cubic meshes in three dimensions. Starting with generalized vectorial Jacobi polynomials, we first construct the basis functions of the H ⁢ ( curl 2 ) \boldsymbol{H}(\mathbf{curl}^{2}) -conforming spectral elements using the contravariant transform together with the affine mapping from the reference cube onto each physical element. Falling into four categories, interior modes, face modes, edge modes, and vertex modes, these H ⁢ ( curl 2 ) \boldsymbol{H}(\mathbf{curl}^{2}) -conforming basis functions are constructed in an arbitrarily high degree with a hierarchical structure. Next, H ⁢ ( curl 2 ) \boldsymbol{H}(\mathbf{curl}^{2}) -conforming spectral element approximation schemes are established to solve the boundary value problem as well as the eigenvalue problem of quad-curl equations. Numerical experiments demonstrate the effectiveness and efficiency of the ℎ-version and the 𝑝-version of our H ⁢ ( curl 2 ) \boldsymbol{H}(\mathbf{curl}^{2}) -conforming spectral element method.

We investigate numerical solutions of high-order $\operatorname {curl}$ problems with various formulations and finite elements. We show that several classical conforming finite elements lead to spurious solutions, while mixed formulations with finite elements in complexes solve the problems correctly. To explain the numerical results, we clarify the cohomological structures in high-order $\operatorname {curl}$ problems by relating the partial differential equations to the Hodge–Laplacian boundary problems of the $\operatorname {grad}\operatorname {curl}$ complexes.

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 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.

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).

In this article, we propose a practical scheme for constructing global superconvergent approximations for Maxwell’s equations in both two and three dimensions. Superconvergence of order $$O(h^{p+1})$$ is established in a discrete norm. This superconvergence result, combined with the polynomial-preserving recovery postprocessing technique, leads to global superconvergence of order $$O(h^{p+1})$$ for recovered quantities in energy norms. Numerical experiments are provided to confirm our theoretical findings.

Background The definitive role of phosphatidylserine (PS) in the prothrombotic state of non-valvular atrial fibrillation (NVAF) remains unclear. Our objectives were to study the PS exposure on blood cells and microparticles (MPs) in NVAF, and evaluate their procoagulant activity (PCA). Methods NVAF patients without ( n = 60) and with left atrial thrombi ( n = 18) and controls ( n = 36) were included in our study. Exposed PS was analyzed with flow cytometry and confocal microscopy. PCA was evaluated using clotting time, factor Xa (FXa), thrombin and fibrin formation. Results PS + blood cells and MPs were significantly higher in NVAF patients without and with left atrial thrombi (both P + platelets, neutrophils, erythrocytes and MPs compared with patients without thrombi (all P + blood cells and MPs contributed to markedly shortened coagulation time and dramatically increased FXa/thrombin/fibrin (all P + platelets and MPs was positively correlated with thrombus diameter (all p Conclusions Our results suggest that exposed PS on blood cells and MPs play a procoagulant role in NVAF patients. Blockade of PS prior to thrombus formation might be a novel therapeutic approach in these patients.

Despite the high efficacy and safety of arsenic trioxide (ATO) in treating acute promyelocytic leukemia (APL) and eradicating APL leukemia-initiating cells (LICs), the mechanism underlying its selective cytotoxicity remains elusive. We have recently demonstrated that APL cells undergo a novel cell death program, termed ETosis, through autophagy. However, the role of ETosis in ATO-induced APL LIC eradication remains unclear. For this study, we evaluated the effects of ATO on ETosis and the contributions of drug-induced ETosis to APL LIC eradication. In NB4 cells, ATO primarily increased ETosis at moderate concentrations (0.5–0.75 μM) and stimulated apoptosis at higher doses (1.0–2.0 μM). Furthermore, ATO induced ETosis through mammalian target of rapamycin (mTOR)-dependent autophagy, which was partially regulated by reactive oxygen species. Additionally, rapamycin-enhanced ATO-induced ETosis in NB4 cells and APL cells from newly diagnosed and relapsed patients. In contrast, rapamycin had no effect on apoptosis in these cells. We also noted that PML/RARA oncoprotein was effectively cleared with this combination. Intriguingly, activation of autophagy with rapamycin-enhanced APL LIC eradication clearance by ATO in vitro and in a xenograft APL model, while inhibition of autophagy spared clonogenic cells. Our current results show that ATO exerts antileukemic effects at least partially through ETosis and targets LICs primarily through ETosis. Addition of drugs that target the ETotic pathway could be a promising therapeutic strategy to further eradicate LICs and reduce relapse.

Despite routine treatment of unselected acute promyelocytic leukemia (APL) with all-trans-retinoic acid (ATRA), early death because of hemorrhage remains unacceptably common, and the mechanism underlying this complication remains elusive. We have recently demonstrated that APL cells undergo a novel cell death program, termed ETosis, which involves release of extracellular chromatin. However, the role of promyelocytic extracellular chromatin in APL-associated coagulation remains unclear. Our objectives were to identify the novel role of ATRA-promoted extracellular chromatin in inducing a hypercoagulable and hyperfibrinolytic state in APL and to evaluate its interaction with fibrin and endothelial cells (ECs). Results from a series of coagulation assays have shown that promyelocytic extracellular chromatin increases thrombin and plasmin generation, causes a shortening of plasma clotting time of APL cells, and increases fibrin formation. DNase I but not anti-tissue factor antibody could inhibit these effects. Immunofluorescence staining showed that promyelocytic extracellular chromatin and phosphatidylserine on APL cells provide platforms for fibrin deposition and render clots more resistant to fibrinolysis. Additionally, coincubation assays revealed that promyelocytic extracellular chromatin is cytotoxic to ECs, converting them to a procoagulant phenotype. This cytotoxity was blocked by DNase I by 20% or activated protein C by 31%. Our current results thus delineate the pathogenic role of promyelocytic extracellular chromatin in APL coagulopathy. Furthermore, the remaining coagulation disturbance in high-risk APL patients after ATRA administration may be treatable by intrinsic pathway inhibition via accelerating extracellular chromatin degradation.

In this paper, nonconforming mixed finite element method is proposed to simulate the wave propagation in metamaterials. The error estimate of the semi-discrete scheme is given by convergence order O(h2), which is less than 40 percent of the computational costs comparing with the same effect by using Nédélec-Raviart element. A Crank-Nicolson full discrete scheme is also presented with O(τ2 + h2) by traditional discrete formula without using penalty method. Numerical examples of 2D TE, TM cases and a famous re-focusing phenomena are shown to verify our theories.

Background Relatively little is known about the role of phosphatidylserine (PS) in procoagulant activity (PCA) in patients with non-ST-elevated myocardial infarction (NSTEMI) after stent implantation. This study was designed to evaluate whether exposed PS on microparticles (MPs) and blood cells were involved in the hypercoagulable state in NSTEMI patients with stent implantation. Methods NSTEMI patients (n=90) and healthy controls (n=20) were included in our study. PS exposure on MPs and blood cells was analyzed with flow cytometer and confocal microscope. PCA was evaluated by clotting time, purified coagulation complex assays and fibrin production assays. Results Baseline levels of MPs and PS + blood cells were significantly higher (P + blood cells. Specifically, PS + MPs, PS + platelets and erythrocytes peaked at 18h following stent implantation, while PS + leukocytes peaked on day 2. In addition, circulating MPs (mostly derived from platelets, leukocytes, erythrocytes and endothelial cells) cooperating with PS + blood cells, contributed to markedly shortened coagulation time and markedly increased FXa/thrombin/fibrin (all P Conclusions Our results suggest that PS + MPs and blood cells play a procoagulant role in NSTEMI patients following stent implantation. Blockade of PS could become a novel therapeutic modality for the prevention of thrombosis in these patients.

The role of phosphatidylserine (PS)-mediated procoagulant activity (PCA) in stroke remains unclear. To ascertain this role, early dynamic evolution of PS exposure on blood cells and released microparticles (MPs) and the corresponding PCA were evaluated in patients with acute ischemic stroke (AIS). Flow cytometry analyses revealed that initial levels of PS exposure on erythrocyte, platelet, and leukocyte were 2.40-, 1.36-, and 1.38-fold higher, respectively, in AIS than the risk factor-matched (RF) controls. Concomitantly, total PS+ MPs were increased in AIS (1949 ± 483/μl) compared with the RF group (1674 ± 387/μl; P = 0.019) and healthy controls (1052 ± 179/μl; P

The mechanism of hypercoagulable state following transcatheter closure of atrial septal defects (ASDs) remains unclear. We evaluated the exposure of phosphatidylserine (PS) on released microparticles (MPs) and also the cells of their origin from peripheral blood, and the associated increase in procoagulant activity (PCA) following transcatheter ASD closure. We demonstrate that PS(+) MP levels were elevated immediately after device implantation (P0.002), peaked at 24hour (P0.002), and persisted at high levels for 1-week post procedure (P0.002). Flow cytometry analysis indicated that PS(+) MPs were mainly derived from platelets, endothelial cells, and the red blood cells (RBCs). Concomittantly, PS(+) platelet and RBC count also increased after transcatheter closure of ASDs, while PS(+) leukocytes levels remained the same. Compared to the baseline, coagulation time of PS(+) MPs, platelets, and RBCs at 24hours post procedure decreased by about 18.7% (P0.004), 21.5% (P0.001), and 26.8% (P0.001), respectively. Intrinsic factor Xa and prothrombinase were produced abundantly by platelets, RBCs, and MPs leading to materialization of fibrin by 24hours. Additionally, Xase complex formation and thrombin generation was inhibited by about 74% by the addition of lactadherin to the assays. Our results thus demonstrate that PS exposure on MPs, platelets, and RBCs play an important role in hypercoagulability following transcatheter ASD closure.

Introduction: Disorders of coagulation are common in sepsis, with disseminated intravascular coagulation (DIC) occurring in approximately 35 % of severe cases, contributing to microvascular dysfunction and death. Intensive platelet activation in sepsis facilitates platelet aggregation, leading to the formation of microthrombi and platelet depletion. This results in the development of DIC and sepsis-associated thrombocytopenia. Therefore, platelets must be cleared locally and quickly in the early phase of activation. Previous studies mainly focused on the clearance of activated cold-stored and aging platelets as well as platelets in immune-mediated thrombocytopenia. However, platelet activation and their clearance in sepsis are poorly understood. Platelets can form aggregates with leukocytes resulting in leukocyte death, the release of extracellular traps (ETs), increased endothelial permeability, and aggravated thrombosis. This study explored an alternate pathway for platelet disposal mediated by endothelial cells (ECs) through phosphatidylserine (PS) and examined the effect of platelet clearance on procoagulant activity (PCA) in sepsis. Methods: The subjects were septic patients (n=48) and healthy controls (n=48). Platelet engulfment by ECs was observed by electron microscopy, immunofluorescence, or immunochemistry both in vitro and in animal models. The PCA of platelets was measured by clotting time, purified coagulation complex assays, and fibrin formation. Results: Platelets in septic patients demonstrated increased levels of surface activation markers and apoptotic vesicle formation, and also formed aggregates with leukocytes. Activated platelets adhered to and were ultimately digested by ECs in vivo and in vitro. Blocking PS on platelets or integrin on ECs attenuated platelet clearance, resulting in increased platelet count in a mouse model of sepsis (p Conclusions: Our results suggest that PS-mediated clearance of activated platelets by the endothelium results in an anti-inflammatory, anticoagulant, and antithrombotic effect that contributes to maintaining platelet homeostasis during acute inflammation. Antiplatelet treatment has been suggested as a novel strategy in sepsis, and we speculate that promoting efficient removal of activated and apoptotic platelets could further improve patient outcomes. Therefore, clearance of activated platelets earlier in the disease process could hasten recovery of homeostasis in circulation by eliminating catalytic platforms for the coagulation pathway, protecting blood cells from excessive activation, and restoring their normal function. Endothelium, at least in part, contributes to platelet disposal and may further improve the hypercoagulable status in inflammation. It is noteworthy that PS-mediated and lactadherin-strengthened platelet engulfment may modify coagulopathy, and thus provide a new modality for treatment of septic clotting disorders. Figure 1 Phagocytosis of platelets by endothelial cells in vitro. Figure 1. Phagocytosis of platelets by endothelial cells in vitro. Figure 1 Effect of lactadherin-mediated phagocytosis on procoagulant activity and fibrin formation. Figure 1. Effect of lactadherin-mediated phagocytosis on procoagulant activity and fibrin formation. Disclosures No relevant conflicts of interest to declare.

Introduction: Acute ischemic stroke (AIS) is a hypoxic ischemic disorder associated with a sterile inflammatory reaction. Neuronal injury from ischemic stroke is aggravated by invading peripheral polymorphonuclear cells (PMNs). Neutrophils not only have a remarkable neurotoxic effect from the release of proteolytic enzymes, but also foster coagulation cascade by exposing membrane phosphatidylserine (PS), aggregating to platelets and releasing neutrophil extracellular traps (NETs). However, the role of circulating neutrophils in hypercoagulation state after AIS remains unclear. Our aims were to determine the procoagulant role of circulating neutrophils after AIS, and to elucidate the mechanism of neutrophils-induced thrombophilia. Methods: 73 newly diagnosed AIS patients and 35 risk factors matched controls were enrolled. Patient blood samples were collected at 6 h, 12 h, 24 h, 3 d, and 7 d after the onset of clinical symptoms. PS exposure on neutrophils and neutrophil-platelet aggregation was measured by flow cytometry and confocal microscopy. The percentage of NETs-releasing PMNs was quantified by confocal microscopy. Double-stranded DNA and myeloperoxidase-DNA complexes were also measured as in vivo markers of NETosis. Specifically, dsDNA was quantified using the Quant-iT PicoGreen dsDNA Assay Kit. Myeloperoxidase-DNA complex was measured using a capture ELISA.The procoagulant activity (PCA) of neutrophils was measured by clotting time and purified coagulation complex assays. Plasma levels of coagulation activation were evaluated by thrombin-antithrombin experiment. Results: The initial levels of PS+ neutrophils and neutrophil-platelet aggregation were 2.1- and 1.8-fold higher, respectively, in AIS than controls. Specifically, PS+ neutrophils peaked at 24 hours and returned to their initial levels at 3 days while neutrophil-platelet aggregation elevated at initial and remained high for the later time points. Patient neutrophils supported significantly shortened clotting times and increases in Xase activity and thrombin formation compared to controls (P < 0.001 for all). Interestingly, treatment with lactadherin, a PS antagonist, effectively restored patient PCA to control levels. The amounts of NETs+ cells, dsDNA and myeloperoxidase-DNA complexes were elevated at 3 days after stroke and positively correlated with thrombin generation. Furthermore, cultured endothelial cells were intensely activated by neutrophils of stroke patients and subsequently supported massive fibrin formation. Moreover, blockade of NET formation with DNAse I inhibited fibrin formation by approximately 60%. In a subanalysis, 27 patients with early infections were matched with 27 patients without infections according to S100B peak levels. These two patient subgroups showed significant differences in the temporal pattern of PS+ neutrophils, neutrophil-platelet aggregation, NETs%, markers of NET formation, and coagulation activation. The levels of coagulation markers were significantly higher in patients with early infections than in patients without (P < 0.05 for all). Conclusions: The thrombophilic susceptibility could be partly due to the activation of neutrophils after ischemic stroke. AIS patients with early infections are more prone to thrombosis. Our studies identify PS exposure on neutrophils and formation of NET as potentially novel therapeutic targets in the treatment of AIS. Figure 1. The changes in the number of neutrophils, PS+ neutrophils, and neutrophil-platelet aggregation (NPA) measured within 7 days of stroke. *P < 0.05 vs. control. Figure 1. The changes in the number of neutrophils, PS+ neutrophils, and neutrophil-platelet aggregation (NPA) measured within 7 days of stroke. *P < 0.05 vs. control. Disclosures No relevant conflicts of interest to declare.

Introduction:Despite treatment with all-trans-retinoic acid, the early death rate in unselected acute promyelocytic leukemia (APL) due to hemorrhage still remains unacceptably high. It is attractive to speculate whether other uncovered procoagulants exist which are not attenuated by ATRA. We have recently demonstrated that APL cells undergo a novel cell death program, termed ETosis, which involves release of extracellular chromatin (Ma R et al, Cell Death Dis 2016). However, the role of promyelocytic extracellular chromatin in APL-associated coagulation disorder remains unclear. The aims of this study were to identify the novel role of extracellular chromatin in induction of the hypercoagulable state in APL, and to evaluate its interaction with fibrin and endothelial cells (ECs). Methods:Twenty-two newly diagnosed APL patients were included. Fresh APL blasts from bone marrow specimens were treated with 1 μM ATRA or phosphate buffered saline (PBS). ETosis was distinguished by rounded cells whose nuclei stained with PI and whose nuclear contents diffused throughout the cell. Cell-free DNA (cf-DNA) was quantified using the Quant-iT PicoGreen dsDNA Assay Kit. Elastase-DNA complexes and TAT (thrombin-antithrombin) complexes were detected by ELISA. ECs were incubated in growth media containing 20% pooled serum obtained from healthy donors in the presence or absence of 20-fold concentrated extracellular chromatin. Procoagulant activity (PCA) of ECs and APL cells was evaluated by one-stage recalcification time assay, pro-thrombinase assay and fibrin formation assay. DNase I or anti-TF were included in the inhibition assays. Results: ATRA treatment induced markedly increased cf-DNA release in a time-dependent manner compared with no ATRA group. Furthermore, ETosis was the major cell death pattern in the ATRA-treated group while apoptosis was predominant in the no-treatment group until the third day, indicating that the increased cell-free DNA triggered by ATRA was mainly from ETosis. NE-DNA, defined as marker of ETosis, peaked on day 3 and showed no significant elevation to day 5, indicating that increased part of cf-DNA from day 3 to day 5 was mainly from apoptosis. Additionally, thrombin generation was found to parallel the change in the releasing of promyelocytic extracellular chromatin induced by ATRA. Pretreatment with DNase I inhibited thrombin generation by 47%, diminished PCA by 35%, prolonged coagulation time, and attenuated fibrin formation by 50%, while neutralizing anti-TF antibody produced no effect. Confocal microscopy showed that fibrin was preferentially deposited on promyelocytic chromatin from ETosis or apoptosis and exposed PS. Lastly, we found that extracellular chromatin from the ATRA group significantly triggered PS exposure on ECs, converting them to a pro-coagulant phenotype. This cytotoxity was blocked by DNase I by 20% or activated protein C (APC) by 31% indicating that DNA scaffold and histones were both necessary for the cytotoxic effect of extracellular chromatin. Conclusions:ATRA promotes procoagulant promyelocytic extracellular chromatin mainly through ETosis. Extracellular chromatin fosters excess thrombin generation, increases fibrin deposition, and causes endothelium damage. To improve the remaining coagulation disturbance in APL patients of high risks during ATRA administration, therapeutic strategies focusing on combined application of DNase I and APC to accelerate the degradation of overwhelmed promyelocytic extracellular chromatin would be of great interest in the future. Disclosures No relevant conflicts of interest to declare.

Background: We recently reported that phosphatidylserine on blood and endothelial cells played an important role in the hypercoagulable state of liver cirrhosis (Wu et al, Liver Int 2016). Recent studies showed that enoxaparin could reduce the incidence of portal vein thrombosis and alleviate liver decompensation. Thus, we speculate that there may be other mechanisms involved in the hypercoagulability of cirrhosis. Neutrophil extracelluar traps (NETs) play an important role in thrombosis and organ dysfunction. Whether the benefit of enoxaparin is due to inhibiting NETs in cirrhotic patients remains unknown. Our objectives were to study the formation of NETs and their role in the hypercoagulable state and liver damage in cirrhotic patients. Methods:Cirrhotic patients (n = 36), healthy controls (n = 10), and mice treated with CCl4 (n = 50) were studied. Immunofluorescence confocal microscope was used to analyze and quantify NETs formation in neutrophil cells from human and mice. TAT and fibrin formation assays in normal and cirrhotic plasma were performed to analyze the procoagulant activity of NETs. Calibrated automated thrombography (CAT) was performed with and without 6 nM thrombomodulin I to analyze TM resistance changes induced by NETs. Liver injury was assessed by plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity, determined spectrophotometrically, and by morphometric evaluation of left lateral liver lobes. DNase I and activated protein C (APC) were used to abolish NETs. Results: We observed that cirrhotic platelets induced 1.9 fold higher NETs formation in neutrophil cells (p = 0.021). Increased NETs promoted higher TAT (Fig 1A, B) and fibrin formation in normal and cirrhotic plasma (p < 0.05). DNase I abolished the procoagulant activity of NETs in corn trypsin inhibitor (CTI ) or anti-TF pre-treated plasma (p < 0.001) (Fig 1C). TM pre-induced with NETs for 24 h showed a higher endogenous thrombin potential (ETP) than no treatment in normal plasma using CAT (p = 0.032). APC and de-N-sulfated heparin (de-N-Hep), but not DNase I, can decrease the ETP ratio (with/without TM) in cirrhotic plasma (p < 0.05) (Fig 1D). Mice treated with CCl4 showed a higher ALT, AST, necrotic area (Fig 2), and NETs formation than control (p < 0.05). DNase I and enoxaparin decreased ALT, AST, necrotic area (Fig 2) and NETs formation (p < 0.05). Conclusion s:Cirrhotic patients have enhanced NETs formation compared to healthy controls. NETs initiated intrinsic and extrinsic coagulation pathway and impaired anticoagulant function of TM. Additionally, NETs partly contributed to tissue injury in the liver of cirrhotic mice. DNase I and enoxaparin can disconnect NETs and decreased the toxin to liver. Figure 1 Neutrophil extracelluar traps (NETs) were isolated and incubated with plasma from healthy controls (A) or cirrhotic patents (B). TAT complexes were measured by ELISA. (C) TAT complexes formation of NETs were analyzed in normal plasma pre-treated with corn trypsin inhibitor (CTI ) or anti-TF in absence or presence of DNase I. (D) Calibrated automated thrombography was performed with and without 6 nM thrombomodulin (TM) in cirrhotic plasma. APC, activated protein C; de-N-Hep, de-N-sulfated heparin. *p < 0.05 vs. Control in figure A, B; *p < 0.001 vs. NETs in figure C; *p = 0.008 and #p = 0.039 vs. PBS in figure D. Figure 1. Neutrophil extracelluar traps (NETs) were isolated and incubated with plasma from healthy controls (A) or cirrhotic patents (B). TAT complexes were measured by ELISA. (C) TAT complexes formation of NETs were analyzed in normal plasma pre-treated with corn trypsin inhibitor (CTI ) or anti-TF in absence or presence of DNase I. (D) Calibrated automated thrombography was performed with and without 6 nM thrombomodulin (TM) in cirrhotic plasma. APC, activated protein C; de-N-Hep, de-N-sulfated heparin. *p < 0.05 vs. Control in figure A, B; *p < 0.001 vs. NETs in figure C; *p = 0.008 and #p = 0.039 vs. PBS in figure D. Figure 2 The necrotic area of liver tissue in control mice, cirrhotic mice treated with PBS, DNase I, enoxaparin or a combination of DNase I with enoxaparin. Enox, Enoxaparin. *p < 0.05 vs. PBS Figure 2. The necrotic area of liver tissue in control mice, cirrhotic mice treated with PBS, DNase I, enoxaparin or a combination of DNase I with enoxaparin. Enox, Enoxaparin. *p < 0.05 vs. PBS Disclosures No relevant conflicts of interest to declare.

Introduction: Atrial fibrillation (AF) is the most common sustained arrhythmia in clinical practice, contributing directly to morbidity and mortality largely through thromboembolism events such as stroke. The risk of stroke in patients with AF is five times higher than in patients without AF. The pathophysiological of thromboembolism in AF is multifactorial and is not only related to stasis in a poorly contractile left atrium. Indeed, there is an increasing body of evidence to support that Virchow triad, including abnormal blood stasis, endothelial damage, and changes in blood constituents, is the main cause of thromboembolism or prothrombotic state in AF patients. However, relatively little is known about the precise role of phosphatidylserine (PS), one of the blood constituents, in the pro-thrombotic state or hypercoagulability of non-valvular AF (NVAF). Our objectives were to study the increased PS exposure on microparticles (MPs) and the outer membrane of MP-origin blood cells in NVAF patients, and to evaluate their procoagulant activity (PCA). Methods: Our study included NVAF patients without (n = 60) and with left atrial thrombosis (n = 30) and healthy controls (n = 30). PS exposure on MPs and blood cells was analyzed with flow cytometry and confocal microscopy. PCA was evaluated using clotting time, extrinsic/intrinsic FXa and prothrombinase production, and fibrin formation assays. Inhibition assays of PCA of PS+ MPs and blood cells were performed using lactadherin. Results: The number of PS+ MPs was significantly higher in NVAF patients (1.8-fold, P < 0.001) and NVAF patients with left atrial thrombosis (2.7-fold, P < 0.001) compared with healthy subjects, respectively. Furthermore, the number of PS+ MPs in NVAF patients with left atrial thrombosis was significantly higher than that in NVAF patients without (1.4-fold, P < 0.01, Figure 1A). Moreover, the PS+ MPs were originated from platelets (CD41a+, PMPs), neutrophils (CD66b+, NMPs), mononuclear cells (CD14+, MMPs), lymphocytes (CD3/19+, LMPs), erythrocytes (CD235a+, ErMPs), and endothelial cells (CD31+ CD41a-, EMPs) (Figure 1B). The levels of PS+ platelets, neutrophils, mononuclear cells, lymphocytes, and erythrocytes were significantly higher (all P < 0.001) in each NVAF group than those in healthy controls. And the levels of PS+ platelets, neutrophils, and erythrocytes in NVAF patients with left atrial thrombosis were significantly higher than those in NVAF patients (all P < 0.01, Figure 1C). In addition, circulating PS+ MPs cooperated with PS+ blood cells, contributing to markedly shortened coagulation time and dramatically increased FXa/thrombin generation and fibrin formation in each NVAF group (all P < 0.01). Moreover, blockade of exposed PS on MPs and blood cells with lactadherin inhibited PCA by approximately 80%. Conclusions: Our results suggest that increased PS exposure on MPs and blood cells play a procoagulant role in NVAF patients with or without left atrial thrombosis. Blockade of PS prior to left atrial thrombosis could become a novel therapeutic strategy to prevent thrombosis in these patients. Figure 1 Flow cytometry analyses of PS+ MPs and blood cells in each NVAF group and control subjects. (A and B) Events were selected for lactadherin-Alexa Fluor 488 binding. Lactadherin-positive MPs were further examined for expression of other antigens by co-labeling with Alexa Fluor 488- and Alexa Fluor 647-labeled antibodies as follows: PMPs (Alexa Fluro 647-CD41a+), NMPs (Alexa Fluro 647-CD66b+), MMPs (Alexa Fluro 488-CD14+), LMPs (Alexa Fluor 647-CD3+/Alexa Fluor 488-CD19+), ErMPs (Alexa Fluor 488-CD235a+), and EMPs (Alexa Fluro 488-CD31+/Alexa Fluor 647-CD41a-). (C) Lactadherin-binding counts of PLT/PMN/MNC/LYM/RBC from controls (n = 30), NVAF patients without (n = 60) and with left atrial thrombosis (n = 30) were measured. MPs, microparticles; NVAF, non-valvular atrial fibrillation; Thr, thrombosis; PMPs, platelet-derived MPs; NMPs, neutrophil-derived MPs; MMPs, mononuclear cell-derived MPs; LMPs, lymphocyte-derived MPs; ErMPs, erythrocyte-derived MPs; EMPs, endothelial-derived MPs; PLT, platelet; PMN, polymorphomuclear cells; MNC, mononuclear cell; LYM, lymphocyte; RBC, red blood cell. *P Disclosures No relevant conflicts of interest to declare.