Yayan Bi

Phosphotidylserine exposure and neutrophil extracellular traps enhance procoagulant activity in patients with inflammatory bowel disease

Inflammatory 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.

CIRCULATING MICROPARTICLES, BLOOD CELLS, AND ENDOTHELIUM INDUCE PROCOAGULANT ACTIVITY IN SEPSIS THROUGH PHOSPHATIDYLSERINE EXPOSURE.

ABSTRACT 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.

Endothelial cell phagocytosis of senescent neutrophils decreases procoagulant activity

Abundant 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 includingflow 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.

Phosphatidylserine exposing-platelets and microparticles promote procoagulant activity in colon cancer patients

BackgroundColon cancer is invariably accompanied by altered coagulation activity; however, the precise role of phosphatidylserine (PS) in the hypercoagulable state of colon cancer patients remains unclear. We explored the exposure of PS on platelets and microparticles (MPs), and evaluate its role in procoagulant activity in colon cancer patients.MethodsPS-positive platelets and MPs, mainly from platelets and endothelial cells, were detected by flow cytometry and confocal microscopy, and their procoagulant activity was assessed with purified coagulation complex assays, clotting time, and fibrin turbidity.ResultsPlasma levels of PS-positive platelets increased gradually from stage I to IV and were higher in all stages of the patients than in the healthy control, while PS-positive platelet-derived MPs only increased significantly in stage III/IV patients. Meanwhile, PS-positive MPs and endothelial-derived MPs in stage II/III/IV patients were markedly higher than ones in controls but no difference with stage I. Tissue factor positive MPs were higher in all 4 stages of colon cancer patients than in the healthy control. Platelets and MPs from the patients demonstrated significantly enhanced intrinsic/extrinsic FXa and thrombin generation, greatly shortened coagulation time, and increased fibrin formation. Combined treatment with PS antagonist lactadherin, strongly prolonged the coagulation time and reduced fibrin formation by inhibiting factor tenase and prothrombinase complex activity. In contrast, pretreatment with anti tissue factor antibody played a lesser role in suppression of procoagulant activity.ConclusionOur results suggest that PS-positive platelets and MPs contribute to hypercoagulability and represent a potential therapeutic target to prevent coagulation in patients with colon cancer.

Thrombotic Role of Blood and Endothelial Cells in Uremia through Phosphatidylserine Exposure and Microparticle Release

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.

Contributions of phosphatidylserine-positive platelets and leukocytes and microparticles to hypercoagulable state in gastric cancer patients.

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.

Promyelocytic extracellular chromatin exacerbates coagulation and fibrinolysis in acute promyelocytic leukemia.

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.

Extracellular DNA traps released by acute promyelocytic leukemia cells through autophagy.

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.

Microparticles and blood cells induce procoagulant activity via phosphatidylserine exposure in NSTEMI patients following stent implantation

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<0.001) in the patients than in controls. After stent implantation, a remarkable increase was observed in both MPs and PS+ 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<0.01) generation in patient group. Moreover, blockade of exposed PS on MPs and cells with lactadherin inhibited PCA by approximately 70%. 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.

Arsenic trioxide promoting ETosis in acute promyelocytic leukemia through mTOR-regulated autophagy

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.