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Patients with colorectal cancer (CRC) are at increased risk of venous thrombosis, but the precise mechanisms of thrombogenesis in CRC remain largely unknown. We aimed to identify the novel role of neutrophil extracellular traps (NETs) in the induction of procoagulant activity (PCA) in CRC, and to evaluate its interactions with platelets and endothelial cells (ECs). In this study, we first showed that the levels of NETs in the peripheral blood of CRC patients were increased in parallel with cancer progression and reached significance in stage II patients compared to healthy subjects. In addition, neutrophils from CRC patients were more prone to produce NETs, resulting in shortened coagulation time, significantly increased thrombin-antithrombin (TAT) complexes and fibrin fibrils compared to healthy controls. Furthermore, platelets from CRC patients stimulated healthy neutrophils to extrude NETs, which could be inhibited by the depletion of HMGB1. Conversely, NETs from CRC patients could also induce the exposure of PS on platelets, leading to markedly enhanced PCA. Importantly, ECs were also converted to a procoagulant phenotype when exposed to NETs from CRC patients. The PCA of NETs-activated platelets or ECs could be inhibited either by the cleavage of NETs with DNase1 or the blockage of histone with activated protein C (APC). Our results reveal the complex interactions between neutrophils, platelets and ECs and their potential role in the hypercoagulable state in CRC. We propose that NETs may provide new therapeutic targets to combat the thrombotic consequences of CRC.

Relatively little information is known about the definitive role of phosphatidylserine (PS) in the hypercoagulability of heart failure (HF). Our objectives were to assess the levels of PS exposure on microparticles (MPs) and blood cells (BCs) in each group of HF patients and to evaluate their procoagulant activity (PCA). HF patients in each NYHA functional class II–IV (II n = 30, III n = 30, IV n = 30) and healthy controls (n = 25) were enrolled in the present study. PS exposure on MPs, BCs was analyzed with flow cytometry. MPs were classified based on their cellular origin: platelets (CD41a+), neutrophils (CD66b+), endothelial cells (CD31+CD41a−), erythrocytes (CD235a+), monocytes (CD14+), T lymphocytes (CD3+), and B lymphocytes (CD19+). PCA was evaluated by clotting time, extrinsic/intrinsic FXa and prothrombinase production assays, as well as fibrin formation assays. Inhibition assays of PCA of PS+ BCs and MPs were performed by lactadherin. There was no significant difference in MP cellular origin between healthy and HF subjects. However, the total number of PS+ MPs was significantly increased in HF patients compared with healthy controls. In addition, circulating PS+ BCs cooperated with PS+ MPs to markedly shorten coagulation time and dramatically increase FXa/thrombin generation and fibrin formation in each HF group. Moreover, blockade of exposed PS on BCs and MPs with lactadherin inhibited PCA by approximately 80%. Our results lead us to believe that exposing PS on the injured BCs and MPs played a pivotal role in the hypercoagulability state in HF patients.

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.

The present study aimed to evaluate the procoagulant effects of phosphatidylserine (PS) exposure on blood cells and microparticles (MPs), and examine its role in predicting early recurrence atrial fibrillation (ERAF) in patients with atrial fibrillation (AF) treated with pulmonary vein isolation (PVI). Blood samples were obtained from 40 healthy controls and 56 patients with AF at baseline (prior to PVI), and 0, 1 h, 1 day, 3 days and 7 days following PVI. The exposure of PS (PS+) to blood cells (platelets, erythrocytes and leukocytes) and MPs was detected using flow cytometry. The procoagulant activity was evaluated by coagulation time, and the formation of factor Xa (FXa) and thrombin. In addition, independent factors associated with PS+ blood cells and MPs, and significant predictors of ERAF following PVI were investigated by statistical analyses. The numbers of PS+ blood cells and MPs were significantly increased by PVI (P355/µl were identified as independent predictors of ERAF (P

The mechanisms that eliminate activated platelets in inflammation-induced disseminated intravascular coagulation (DIC) in micro-capillary circulation are poorly understood. 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. Platelets in septic patients demonstrated increased levels of surface activation markers and apoptotic vesicle formation, and also formed aggregates with leukocytes. Activated platelets adhered were and ultimately digested by ECs in vivo and in vitro. Blocking PS on platelets or αvβ3 integrin on ECs attenuated platelet clearance resulting in increased platelet count in a mouse model of sepsis. Furthermore, platelet removal by ECs resulted in a corresponding decrease in platelet-leukocyte complex formation and markedly reduced generation of factor Xa and thrombin on platelets. Pretreatment with lactadherin significantly increased phagocytosis of platelets by approximately 2-fold, diminished PCA by 70%, prolonged coagulation time, and attenuated fibrin formation by 50%. Our results suggest that PS-mediated clearance of activated platelets by the endothelium results in an anti-inflammatory, anticoagulant, and antithrombotic effect that contribute to maintaining platelet homeostasis during acute inflammation. These results suggest a new therapeutic target for impeding the development of DIC.

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.

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

We evaluated cellular origin, numbers, and procoagulant activity of phosphatidylserine-positive microparticles (MPs) among subgroups in acute coronary syndromes (ACS).Parameters were measured on admission, days 1 (within 24 h of admission), 2, 3, and 7. All ST-elevated myocardial infarction (STEMI) patients presented more than 3 h from symptom onset and received fibrinolysis treatment; controls included unstable angina and non-STEMI patients as well as healthy controls. Phosphatidylserine-positive MPs were detected by flow cytometry, whereas procoagulant activity was assessed by coagulation time, purified coagulation complex assays, and fibrin formation. MP-induced fibrins were visualized by confocal microscopy.On admission, the total MP count was ∼2.5-fold higher in the ACS groups compared with the healthy controls (P0.05), primarily originating from platelets and endothelial cells, and there were no significant differences among ACS subgroups. Specifically, leukocyte-derived and erythrocyte-derived MPs were higher in the STEMI group compared with unstable angina and non-STEMI groups (both P0.05). Further, MPs from the ACS groups reduced coagulation time by 27.5% and induced intrinsic and extrinsic FXase, prothrombinase, and fibrin formation by 2.8-, 2.3-, 2.5-, and 1.7-fold, respectively (P0.05 for all), whereas blocking phosphatidylserine with lactadherin inhibited ∼70% of procoagulant activity. MP number and concomitant coagulation decreased significantly by day 2 and continued to decrease gradually during the recovery period.This study shows that MP characteristics from circulating blood may be used as prognostic indicators to reflect the origin cell of activation and thrombophilic states found in ACS subgroups.

SummaryInflammatory bowel disease (IBD)-associated thromboembolic event often lacks precise aetiology. The aim of this study was to investigate the contribution of phosphatidylserine (PS) exposure and neutrophil extracellular traps (NETs) towards the hypercoagulable state in IBD. We demonstrated that the levels of PS exposed MPs and the sources of MP-origin, platelets, erythrocytes, leukocytes and cultured endothelial cells (ECs) were higher in IBD groups than in healthy controls using flow cytometry and confocal microscopy. Wright-Giemsa and immunofluorescence staining demonstrated that the elevated NETs were released by activated IBD neutrophils or by control neutrophils treated with IBD sera obtained from patients with the active disease. MPs and MP-origin cells in IBD groups, especially in active stage, markedly shortened coagulation time and had increased levels of fibrin, thrombin and FXa production as assessed by coagulation function assays. Importantly, we found that on stimulated ECs, PS rich membranes provided binding sites for FXa and FVa, promoting fibrin formation while TNF blockage or IgG depletion attenuated this effect. Treatment of control neutrophils with TNF and isolated IgG from PR3-ANCA-positive active IBD patients also resulted in the release of NETs. Blockade of PS with lactadherin prolonged coagulation time, decreased fibrin formation to control levels, and inhibited the procoagulant enzymes production in the MPs and MP-origin cells. NET cleavage by DNase I partly decreased PCA in IBD or stimulated neutrophils. Our study reveals a previously unrecognised link between hypercoagulable state and PS exposure or NETs, and may further explain the epidemiological association of thrombosis within IBD patients.

The mechanisms that eliminate activated platelets in inflammation-induced disseminated intravascular coagulation (DIC) in micro-capillary circulation are poorly understood. 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. Platelets in septic patients demonstrated increased levels of surface activation markers and apoptotic vesicle formation, and also formed aggregates with leukocytes. Activated platelets adhered were and ultimately digested by ECs in vivo and in vitro. Blocking PS on platelets or αvβ3 integrin on ECs attenuated platelet clearance resulting in increased platelet count in a mouse model of sepsis. Furthermore, platelet removal by ECs resulted in a corresponding decrease in platelet-leukocyte complex formation and markedly reduced generation of factor Xa and thrombin on platelets. Pretreatment with lactadherin significantly increased phagocytosis of platelets by approximately 2-fold, diminished PCA by 70%, prolonged coagulation time, and attenuated fibrin formation by 50%. Our results suggest that PS-mediated clearance of activated platelets by the endothelium results in an anti-inflammatory, anticoagulant, and antithrombotic effect that contribute to maintaining platelet homeostasis during acute inflammation. These results suggest a new therapeutic target for impeding the development of DIC.

BACKGROUND Relatively little information is available about phosphatidylserine positive (PS(+)) microparticles (MPs) and their originating cells in IgA nephropathy (IgAN) despite well-established intraglomerular coagulation. Our objectives were to detect PS exposure on MP membranes and MP-origin cells and to evaluate its role in procoagulant activity (PCA) and fibrin formation and their association with pathological lesions in the disease. METHODS Patients with IgAN and healthy controls were studied. Lactadherin was used to quantify PS exposure on MPs and MP-origin cells. PCA of MPs and MP-origin cells was evaluated by clotting time and purified coagulation complex assays. Fibrin production was determined by turbidity. PS exposure, fibrin strands and FVa/Xa binding were observed on MPs/cells using confocal microscopy. RESULTS Using flow cytometry, we found that IgAN patients had high levels of PS(+) MPs derived from lymphocytes, monocytes, neutrophils, platelets, erythrocytes and endothelial cells (ECs). The PS exposure on MP-origin cells also increased in these patients. MPs and MP-origin cells (leukocytes, platelets and erythrocytes) isolated from IgAN patients and ECs cultured with IgAN serum had a significantly shorter median coagulation time (P < 0.001), higher median intrinsic FXa (P < 0.001) and higher thrombin (P < 0.001) generation than controls. These coagulation functional assays were associated with the glomerular lesions. The lesions were also correlated with glomerular fibrin deposition (all P < 0.05). In the presence of patient MPs or their related cells, fibrin formation peaked faster with a higher maximum turbidity when compared with healthy controls. Blocking PS with lactadherin in the IgAN group prolonged coagulation time to control levels, inhibited the PCA up to 80% and markedly reduced fibrin formation. More importantly, we observed that fibrin strands formed on MPs and ECs in the same regions that bound lactadherin, similar to the FVa/Xa costaining. CONCLUSIONS We find that high levels of PS(+) MPs and the MP-origin cells are associated with the coagulation process in IgAN, and this may provide a previously unrecognized contribution to intraglomerular coagulation.

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.

Multiple myeloma (MM) is characterized by an increased incidence of thromboembolic events, particularly when treated with immunomodulatory drugs (IMiDs) in combination with dexamethasone. The optimal prophylactic strategy to prevent the hypercoagulable state of patients with MM is still debated. The aim of the current study was to investigate the definitive role of phosphatidylserine (PS) in supporting procoagulant activity (PCA) in patients with MM. Patients with MM (n=20) and healthy subjects (n=15) were recruited for the present study. PS analyses were performed by flow cytometry and confocal microscopy. The PCA was evaluated by clotting time, purified coagulation complex assays and fibrin production assays. The percentage of PS+ blood cells was significantly higher in patients with MM than in healthy subjects. Additionally, the patient serum induced more PS exposure on endothelial cells (ECs) in vitro than serum from healthy subjects. Isolated blood cells from patients with MM and ECs cultured with patient serum in vitro demonstrated significantly shortened coagulation time, greatly intrinsic/extrinsic factor Xa generation and increased thrombin formation. In addition, the levels of PS+ erythrocytes, platelets, leukocytes, and ECs incubated with IMiDs and dexamethasone were higher than with IMiDs alone. The findings support the hypothesis that increased PS exposure on blood cells and ECs participates in the hypercoagulable state in patients with MM. Thus, blocking PS may be a novel therapeutic target for the prevention of thrombosis in these patients.

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.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.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 concentrations6 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.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.

The mechanisms of thrombogenicity in essential thrombocythemia (ET) are complex and not well defined. Our objective was to explore whether phosphatidylserine (PS) exposure on blood cells and endothelial cells (ECs) can account for the increased thrombosis and distinct thrombotic risks among mutational subtypes in ET. Using flow cytometry and confocal microscopy, we found that the levels of PS-exposing erythrocytes, platelets, leukocytes, and serum-cultured ECs were significantly higher in each ET group [JAK2, CALR, and triple-negative (TN) (all P

The procoagulant status of patients with non-small cell lung cancer (NSCLC) after chemotherapy is poorly characterized and the role of platelets in hypercoagulative state of NSCLC is unknown. The aim of this study was to evaluate the procoagulant activity (PCA) of platelets in NSCLC before and after chemotherapy. The subjects were 52 patients newly diagnosed with NSCLC. The patients had decreased clotting time compared with healthy subjects, and the thrombin-antithrombin complex increased 2.5-fold after chemotherapy. Platelets in the patients after chemotherapy had enhanced phosphatidylserine (PS) exposure, and shortened coagulation time as well as increased thrombin and fibrin formation of platelets compared with those before chemotherapy. Platelet-derived microparticles increased 2-fold at day 1 and peaked at day 2 post-chemotherapy. Treatment of cisplatin in vitro also resulted in upregulated intrinsic FXa and thrombin formation on platelets with a dose-dependent manner. Platelets treated with aspirin significantly decreased PCA. However, lactadherin blocked PS and inhibited the PCA approximately by 70%. Seven days after chemotherapy, PCA of platelets restored to the baseline as that before chemotherapy, indicating that within a week of chemotherapy patient platelets are highly procoagulant and effective intervention should be taken in case of thrombosis. Our results suggested that platelets after chemotherapy had elevated PCA and may contribute to the hypercoagulative state of NSCLC. Prophylactic anti-coagulant combined with anti-platelet therapy may play an inhibitory role in thrombotic complications in NSCLC.

OBJECTIVEPhosphatidylserine (PS) is a major component of the inner leaflet of membrane bilayers. During cell activation or apoptosis, PS is externalized to the outer membrane, providing an important physiological signal necessary for the release of the microparticles (MPs) that are generated through the budding of cellular membranes. MPs express PS and membrane antigens that reflect their cellular origin. PS exposure on the cell surface and the release of MPs provide binding sites for factor Xa and prothrombinase complexes that promote thrombin formation. Relatively little is known about the role of PS exposure on blood cells and MPs in patients with internal carotid artery (ICA) stenosis who have undergone carotid artery stenting (CAS). The authors aimed to investigate the extent of PS exposure on blood cells and MPs and to define its role in procoagulant activity (PCA) in the 7 days following CAS.METHODSThe study included patients with ICA stenosis who had undergone CAS (n = 70), matched patients who had undergone catheter angiography only (n = 30), and healthy controls (n = 30). Blood samples were collected from all patients just before the procedure after an overnight fast and at 2, 6, 24, 48, and 72 hours and 7 days after the CAS procedure. Blood was collected from healthy controls after an overnight fast. Phosphatidylserine-positive (PS+) MPs and blood cells were analyzed by flow cytometry, while PCA was assessed with clotting time analysis, purified coagulation complex assays, and fibrin formation assays.RESULTSThe authors found that levels of PS+ blood cells and PS+ blood cell–derived MPs (platelets and platelet-derived MPs [PMPs], neutrophils and neutrophil-derived MPs [NMPs], monocytes and monocyte-derived MPs [MMPs], erythrocytes and erythrocyte-derived MPs [RMPs], and endothelial cells and endothelial cell–derived MPs [EMPs]) were increased in the 7 days following the CAS procedure. Specifically, elevation of PS exposure on platelets/PMPs, neutrophils/NMPs, and monocytes/MMPs was detected within 2 hours of CAS, whereas PS exposure was delayed on erythrocytes/RMPs and EMPs, with an increase detected 24 hours after CAS. In addition, PS+ platelets/PMPs peaked at 2 hours, while PS+ neutrophils/NMPs, monocytes/MMPs, and erythrocytes/RMPs peaked at 48 hours. After their peak, all persisted at levels above baseline for 7 days post-CAS. Moreover, the level of PS+ blood cells/MPs was correlated with shortened coagulation time and significantly increased intrinsic and extrinsic Xase, thrombin generation, and fibrin formation. Pretreatment of blood cells with lactadherin at their peak time point after CAS blocked PS, resulting in prolonged coagulation times, decreased procoagulant enzyme activation, and fibrin production.CONCLUSIONSThe results of this study suggest that increased exposure of PS on blood cells and MPs may contribute to enhanced PCA in patients with ICA stenosis who have undergone CAS, explaining the risk of perioperative thromboembolic complications in these patients. PS on blood cells and MPs may serve as an important biomarker for predicting, and as a pivotal target for monitoring and treating, acute postoperative complications after CAS.■ CLASSIFICATION OF EVIDENCE Type of question: association; study design: prospective cohort trial; evidence: Class I.

The mechanism of thrombogenicity in liver cirrhosis is largely unknown. Our objective was to study the relationship between phosphatidylserine on blood cells and endothelial cells and the hypercoagulable state in cirrhotic patients. Patients with liver cirrhosis and healthy controls were studied. Lactadherin was used to quantify phosphatidylserine exposure on blood cells and endothelial cells. Procoagulant activity of cells was evaluated using clotting time and purified coagulation complex assays. Fibrin production was determined by turbidity. Phosphatidylserine exposure, fibrin strands, and FVa/Xa binding on cells were observed using confocal microscopy. Our study showed that phosphatidylserine exposure on erythrocytes, platelets, and leukocytes in cirrhotic patients increased progressively with Child-Pugh categories. Additionally, we found that endothelial cells treated with cirrhotic serum in vitro exposed more phosphatidylserine than those exposed to healthy serum. The exposed phosphatidylserine supported a shorter coagulation time and increased FXa, thrombin, and fibrin formation. Notably, phosphatidylserine(+) erythrocytes also promoted shorter coagulation times and more fibrin generation in cirrhotic microparticle-depleted plasma, regardless of Child-Pugh categories. Confocal microscopy data showed that the FVa/FXa complex and fibrin fibrils colocalized with phosphatidylserine on endothelial cells. Lactadherin significantly inhibited FXa and thrombin generation and consequently decreased fibrin production in normal or cirrhotic plasma. These results lead us to believe that exposed phosphatidylserine on activated or injured erythrocytes, platelets, leukocytes, and endothelial cells plays an important role in the hypercoagulable state in cirrhotic patients. Thus, blocking phosphatidylserine binding sites might be a new therapeutic target for preventing thrombosis. This article is protected by copyright. All rights reserved.

Introduction:Fibrinolysis plays an important role in the treatment of ST-elevated myocardial infarction (STEMI) when percutaneous coronary intervention is not readily available. Early and successful myocardial reperfusion with thrombolytic therapy effectively reduces the infarct size and improves the clinical outcome. However, the process of restoring blood flow to the ischemic myocardium can induce injury and reduce the beneficial effects of myocardial reperfusion. Previous studies had shown that platelets, leukocytes and TF play important role in thrombotic complications after fibrinolysis in AMI. However, there are still 10-15% patients who have risk for re-occlusion after antiplatelet and anticoagulant therapies. Thus, we speculate that there may be other mechanisms involved in the hypercoagulability after STEMI fibrinolysis. Neutrophil extracellular traps (NETs) are double-edge swords that could ensnare and kill microbial pathogens but also contribute to thrombosis. However, the role of NETs during STEMI fibrinolysis-induced re-occlusion is largely unknown. Our aims were to determine the procoagulant role of NETs after successful thrombolysis, and to elucidate its interaction with endothelial cells (ECs). Methods:31 STEMI patients with successfully fibrinolysis and 12 healthy controls were enrolled. Patient blood samples were collected at 0 h, 2 h, 6 h, 12 h and 24 h after fibrinolysis. Cell-free DNA (cf-DNA) was quantified using the Quant-iT PicoGreen dsDNA Assay Kit. ELISA was used to detect MPO-DNA complexes and TAT (thrombin-antithrombin) complexes. Wright-Giemsa and immunofluorescence confocal microscope were used to analyze and quantify NETs formation in neutrophil cells. ECs were incubated in growth media containing 20% pooled serum obtained from healthy donors in the presence or absence of 20-fold concentrated neutrophil extracellular chromatin. The procoagulant activity (PCA) of neutrophils and ECs was measured by clotting time and purified coagulation complex assays. DNase I or anti-TF were included in the inhibition assays. Results: We found that cf-DNA, MPO-DNA and TAT are significantly reduced at 2 hours in STEMI patients with successful fibrinolysis. Their levels then increased and peaked at 6 hours (Figure 1A, B, E). Interestingly, the level of cf-DNA at 6 hours in STEMI thrombotic patients was positively correlated with TAT (r=0.959; p Conclusions: Our study reveals that the PCA of STEMI following fibrinolytic administration decrease after 2 hours, then increase and peak at 6 hours, which is at least partly due to the release of NETs induced by activated PMNs. Additionally, NETs partly contribute to ECs injury after myocardial reperfusion. DNase I can disconnect NETs and may therefore serve as a promising therapeutic target in STEMI reinfarction and recurrent ischemia. Disclosures No relevant conflicts of interest to declare.

Introduction:Venous thromboembolism(VTE), encompassing pulmonary embolism (PE) and deep vein thrombosis (DVT), is a major contributor to global morbidity and mortality, especially among cancer patients. However, the factors that contribute to PE, a prevalent and fatal complication of DVT, remain poorly understood.Despite a common pathology relating PE to DVT, this leaves a grey area in the differences among DVT, confirmed PE, or DVT with PE, and the definitive relationship between VTE and this hypercoagulable state.However, relatively little is known about the definitive role of phosphatidylserine (PS), one of the blood constituents in the coagulant pathway, in the hypercoagulability of VTE and their altered plasma fibrin clot properties. Our objectives were to assess the levels of PS exposure on microparticles (MPs), blood cells, endothelial cells(ECs) and cell-derived MPs in each group of VTE and to evaluate their procoagulant activity (PCA), as well as the correlation between initial PS exposure and their altered plasma fibrin clot properties. Methods:DVT alone (n=27), definitely PE alone (n=22), DVT with PE (n=19) and healthy controls (n=30) were enrolled in the present study. PS exposure on MPs, blood cells and cultured ECs treated with VTE serum in vitro was analyzed with flow cytometry and confocal microscopy. MPs were classified based on their cellular origin: platelets (CD41a+, PMPs), neutrophils (CD66b+, NMPs), endothelial cells(CD31+CD41a-, EMPs), erythrocytes (CD235a+, RMPs), monocytes (CD14+, MMPs), T lymphocytes (CD3+, T LyMPs), and B lymphocytes (CD19+, B LyMPs). PCA was evaluated by clotting time, extrinsic/intrinsic FXa and prothrombinase production assays, as well as fibrin formation assays. Inhibition assays of PCA of PS+MPs, blood cells and ECs were performed by lactadherin. Abnormal fibrin clot properties measured ex vivo in plasma was examined by fibrin clot analysis and scanning electron microscopy (SEM). Results: There was no significant difference in MP cellular origin between healthy and VTE subjects. However, the total number of PS+MPs was significantly increased (P < 0.001 for total MP and all MP subtypes) in VTE patients compared with healthy controls. In addition, circulating PS+ MPs cooperated with PS+blood cells and cultured ECs to markedly shorten coagulation time (Figure 1A) and dramatically increase FXa/thrombin generation and fibrin formation in each VTE group (all P < 0.05). Confocal microscopy images showed that the FVa/FXa complex and fibrin strands co-stained with PS on ECs. Moreover, blockade of exposed PS on MPs and intravascular cells with lactadherin inhibited PCA by approximately 80% (Figure 1B). Fibrin clot structure was evaluated in 15 VTE patients (DVT=5, PE=5 and DVT with PE=5) and 5 healthy subjects.Compared with those from DVT and PE, clots from plasma of DVT with PE subjects showed lower fiber density and faster clot lysis time, whereas there were no differences in lag time, rate of clot formation and maximum absorbance of turbidity. Conclusions: Our results suggest that compared with healthy subjects, each group of VTE patients had markedly higher levels of circulating PS+MPs, PS+blood cells. Additional, ECs treated with VTE patient serum had elevated percentages of PS+cells versus those treated with serum from healthy controls. Blockade of PS with lactadherin significantly inhibits PCA of blood cells, MPs and VTE serum-treated ECs. In addition, differences in fibrin clot properties and clot structure among DVT, PE and DVT with PE subjects may provide important insights into the pivotal mechanisms that regulate embolization. Disclosures No relevant conflicts of interest to declare.

Sepsis is invariably accompanied by altered coagulation cascade; however, the precise role of phosphatidylserine (PS) in inflammation-associated coagulopathy in sepsis has not been well elucidated. We explored the possibility of exposed PS on microparticles (MPs), blood cells, as well as on endothelium, and defined its role in procoagulant activity (PCA) in sepsis. PS-positive MPs and cells were detected by flow cytometry, while PCA was assessed with clotting time, purified coagulation complex, and fibrin formation assays. Plasma levels of PS MPs derived from platelets, leukocytes (including neutrophils, monocytes, and lymphocytes), erythrocytes, and endothelial cells were elevated by 1.49-, 1.60-, 2.93-, and 1.53-fold, respectively, in septic patients. Meanwhile, PS exposure on blood cells was markedly higher in septic patients than in controls. Additionally, we found that the endothelial cells (ECs) treated with septic serum in vitro exposed more PS than with healthy serum. Isolated MPs/blood cells from septic patients and cultured ECs treated with septic serum in vitro demonstrated significantly shortened coagulation time, greatly enhanced intrinsic/extrinsic FXa generation, and increased thrombin formation. Importantly, confocal imaging of MPs or septic serum-treated ECs identified binding sites for FVa and FXa to form prothrombinase, and further supported fibrin formation in the area where PS exposure was abundant. Pretreatment with lactadherin blocked PS on MPs/blood cells/ECs, prolonged coagulation time by at least 25%, reduced FXa/thrombin generation, and inhibited fibrin formation by approximately 85%. Our findings suggest a key role for PS exposed on MPs, blood cells, and endothelium in augmenting coagulation in sepsis. Therefore, therapies targeting PS may be of particular importance.

Hypercoagulability in gastric cancer is a common complication and a major contributor to poor prognosis. This study aimed to determine procoagulant activity of blood cells and microparticles (MPs) in gastric cancer patients. Phosphatidylserine-positive blood cells and MPs, and their procoagulant properties in particular, were assessed in 48 gastric cancer patients and 35 healthy controls. Phosphatidylserine-positive platelets, leukocytes, and MPs in patients with tumor-node-metastasis stage III/IV gastric cancer were significantly higher than those in stage I/II patients or healthy controls. Moreover, procoagulant activity of platelets, leukocytes, and MPs in stage III/IV patients was significantly increased compared to the controls, as indicated by shorter clotting time, higher intrinsic and extrinsic factor tenase, and prothrombinase complex activity. Interestingly, lactadherin, which competes with factors V and VIII to bind phosphatidylserine, dramatically prolonged clotting time of the cells and MPs by inhibiting factor tenase and prothrombinase complex activity. Although anti-tissue factor antibody significantly attenuated extrinsic tenase complex activity of leukocytes and MPs, it only slightly prolonged clotting times. Meanwhile, treatment with radical resection reduced phosphatidylserine-positive platelets, leukocytes, and MPs, and prolonged the clotting times of the remaining cells and MPs. Our results suggest that phosphatidylserine-positive platelets, leukocytes, and MPs contribute to hypercoagulability and represent a potential therapeutic target to prevent coagulation in patients with stage III/IV gastric cancer.

Introduction: Inflammatory bowel disease (IBD) arises from a combination of genetic susceptibility and environmental factors, which trigger inappropriate mucosal inflammatory responses, the pathogenesis of which still remains exclusive. Therapeutic strategies for IBD mainly focus on controlling the active inflammation, intestinal epithelial barrier destruction and thrombotic tendency. Our group has recently demonstrated, for the first time, that patients with active IBD exhibited enhanced neutrophil extracellular traps (NETs) release, leading to a hypercoagulable state (He Z et al, Thromb Haemost 2016). However, the mechanisms by which NETs drive the pathogenesis of IBD remain unclear. The aim of this study was to further identify the novel role of NETs in the initiation and progression of IBD. Methods: 51 consecutive patients with IBD were studied. Disease activity was assessed by using the Mayo Score (MS) for patients with (ulcerative colitis) UC and (Crohn's disease) CD. Acute disease was induced by the treatment of C57BL/6 mice with 3.5% DSS in drinking water for 6 days. For an inhibition assay, DNase I perfusion or neutrophil depletion with anti-Ly6G antibody (1A8 clone) was performed. Cell-free DNA (cf-DNA) was quantified using the Quant-iT PicoGreen dsDNA Assay Kit. ELISA was used to detect MPO-DNA complexes, TAT (thrombin-antithrombin) complexes, nucleosomes, chemokines, and cytokines. Results: Compared to subjects with inactive UC or CD, patients with active UC or CD had significantly increased levels of cf-DNA, nucleosomes and NETs formation (MPO-DNA complexes). Neutrophils from active CD and UC demonstrated more spontaneous NET release as compared to inactive patients and controls. In DSS-induced colitis, significantly higher levels of serum cf-DNA and NETs formation were found in mice on day 4 and day 6 after DSS initiation. Western blot analysis and immunofluorescent staining of colonic tissues from mice with DSS-induced colitis showed increased NETs release and deposition. Mice treated with DNase I were protected from DSS-induced colitis, showing slighter weight loss, lower disease activity index, improved survival rate, diminished colon length shortening and decreased histologic signs of inflammation compared with controls. Furthermore, DNase I perfusion also decreased MPO levels by 62% on day 4 and by 58% on day 6, indicating DNase down-regulated neutrophil infiltration during DSS-induced colitis. The expression of Interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, CXCL2, CXCL10 and monocyte chemotactic protein-1(MCP-1) messenger RNA in colonic extracts were lower in DNase I perfused DSS-induced mice compared with saline perfused DSS-induced mice. Incubation of normal platelets with NETs from active IBD patients, but not inactive IBD patients, significantly enhanced their procoagulant activity by 32% and the ability to support fibrin formation by 42%. This effect was blocked by DNase I treatment. Conclusions: We have extended our previous study and demonstrated that NETs constitute a central component in the initiation and progression of colitis through mediating inflammation cell infiltration, driving cytokines release and thrombotic tendency. NET degradation through DNase I perfusion protected mice from severe DSS-induced colitis. Thus, strategies focusing on the application of DNase I to accelerate the degradation of excessive NET release and deposition may offer potential therapeutic benefits for patients with inflammatory bowel disease. Disclosure of Interest: None declared. Disclosures No relevant conflicts of interest to declare.

Cardiac dysfunction caused by excessive alcohol consumption is a specific disease, alcoholic cardiomyopathy (ACM). High-dose alcohol has been found to induce oxidation stress and apoptosis in cardiomyocytes, but the signaling link between alcohol-induced oxidation stress and apoptosis in cardiomyocytes remains to be elucidated. To address the issue, we exposed primary cardiomyocytes from neonatal mouse hearts to high doses of alcohol (50mM, 100mM, and 200 mM). We found that alcohol induced dose-dependent phosphorylation of p66shc, and reactive oxygen species (ROS) production increased in parallel with phosphorylation levels of p66shc. Exposure to alcohol also led to loss of mitochondrial membrane potential and cytochrome c release. Depletion of p66Shc and inhibition of protein kinase C-β (PKC-β) successfully reversed all the effects and suppressed alcohol-induced apoptosis in cardiomyocytes. Collectively, our study provides a molecular basis for signaling transduction of alcohol-induced oxidation stress and apoptosis of cardiomyocytes, which may facilitate the prevention and treatment of ACM.

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.

Acute promyelocytic leukemia (APL) cells exhibit disrupted regulation of cell death and differentiation, and therefore the fate of these leukemic cells is unclear. Here, we provide the first evidence that a small percentage of APL cells undergo a novel cell death pathway by releasing extracellular DNA traps (ETs) in untreated patients. Both APL and NB4 cells stimulated with APL serum had nuclear budding of vesicles filled with chromatin that leaked to the extracellular space when nuclear and cell membranes ruptured. Using immunofluorescence, we found that NB4 cells undergoing ETosis extruded lattice-like structures with a DNA–histone backbone. During all-trans retinoic acid (ATRA)-induced cell differentiation, a subset of NB4 cells underwent ETosis at days 1 and 3 of treatment. The levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were significantly elevated at 3 days, and combined treatment with TNF-α and IL-6 stimulated NB4 cells to release ETs. Furthermore, inhibition of autophagy by pharmacological inhibitors or by small interfering RNA against Atg7 attenuated LC3 autophagy formation and significantly decreased ET generation. Our results identify a previously unrecognized mechanism for death in promyelocytes and suggest that ATRA may accelerate ET release through increased cytokines and autophagosome formation. Targeting this cellular death pathway in addition to conventional chemotherapy may provide new therapeutic modalities for APL.

SummaryAbundant senescent neutrophils traverse the vascular compartment and may contribute to pathologic conditions. For example, they become procoagulant when undergoing apoptosis and may contribute to thrombosis or inflammation. Our previous studies demonstrated a dominant clearance pathway in which the neutrophils can be phagocytosed by liver macrophages. The aim of this study was to explore an alternate pathway of neutrophil clearance by endothelial cells. Phagocytosis of the neutrophils by endothelial cells was performed using various experimental approaches including flow cytometry, confocal microscopy and electron microscopy assays in vitro and in vivo. Procoagulant activity of cultured neutrophils was evaluated by coagulation time, factor Xase and prothrombinase assays. Lactadherin functioned as a novel probe for the detection of phosphatidylserine on apoptotic cells, an opsonin (bridge) between apoptotic cell and phagocyte for promoting phagocytosis, and an efficient anticoagulant for inhibition of factor Xase and thrombin formation. When cultured, purified human neutrophils spontaneously entered apoptosis and developed procoagulant activity that was directly related to the degree of phosphatidylserine exposure. Co-culture of aged neutrophils and endothelial cells resulted in phagocytosis of the neutrophils and prolonged coagulation time. Lactadherin diminished the procoagulant activity and increased the rate of neutrophil clearance. In vivo, neutrophils were sequestered by endothelial cells after blockade of Kupffer cells, a process that was dependent upon both phosphatidylserine exposure and P-selectin expression. Thus, the ability of endothelial cells to clear senescent neutrophils may limit the procoagulant and/or inflammatory impact of these cells.

The mechanisms contributing to an increased risk of thrombosis in uremia are complex and require clarification. There is scant morphological evidence of membrane-dependent binding of factor Xa (FXa) and factor Va (FVa) on endothelial cells (EC) in vitro. Our objectives were to confirm that exposed phosphatidylserine (PS) on microparticle (MP), EC, and peripheral blood cell (PBC) has a prothrombotic role in uremic patients and to provide visible and morphological evidence of PS-dependent prothrombinase assembly in vitro. We found that uremic patients had more circulating MP (derived from PBC and EC) than controls. Additionally, patients had more exposed PS on their MPs and PBCs, especially in the hemodialysis group. In vitro, EC exposed more PS in uremic toxins or serum. Moreover, reconstitution experiments showed that at the early stages, PS exposure was partially reversible. Using confocal microscopy, we observed that PS-rich membranes of EC and MP provided binding sites for FVa and FXa. Further, exposure of PS in uremia resulted in increased generation of FXa, thrombin, and fibrin and significantly shortened coagulation time. Lactadherin, a protein that blocks PS, reduced 80% of procoagulant activity on PBC, EC, and MP. Our results suggest that PBC and EC in uremic milieu are easily injured or activated, which exposes PS and causes a release of MP, providing abundant procoagulant membrane surfaces and thus facilitating thrombus formation. Blocking PS binding sites could become a new therapeutic target for preventing thrombosis.