Introduction:Breast cancer is the most common cancer in Chinese women, with invasive breast cancer (IBC) accounting for more than 80% of these diagnoses. Recent studies have shown that increased arterial and venous thromboses occur in IBC and may predict poor prognosis. Uncontrolled activation of coagulation cascade is epidemic in cancer treated with neo-adjuvant chemotherapy (NAC), contributing to hypercoagulability resulting in venous thromboembolism and leading to significant morbidity and mortality. Microparticles (MPs) contribute to coagulation and thrombogenesis. However, the role of tumor-derived microparticles (TMPs) in coagulation during NAC is not fully understood, including clearance of TMPs and their effect on activated platelets and endothelial cells (ECs). Previous studies have shown that elevated circulating MPs has clinical significance in patient prognosis. However, the changes in TMP count, morphology and procoagulant activity (PCA) after NAC in IBC are unclear. This study aimed to explore the properties of MPs derived from breast cancer cells (BCMPs) following exposure to high- or low- dose chemotherapeutic agents and evaluate thrombogenic effects of these BCMPs on platelets and ECs.

Background: Patients with colorectal cancer (CRC) are at increased risk of venous thromboembolism (VTE), but the precise mechanisms of hypercoagulability in CRC remain largely unknown. Neutrophil extracellular traps (NETs) are web-like chromatin structures decorated with cytoplasmic, granular and nuclear components of neutrophils, which can participate in both antimicrobial responses and contribute to a number of autoimmune and thrombotic diseases. However, a definitive role of NETs in the hypercoagulable state in CRC patients is still unclear. The aims of this study were to identify the novel role of NET in the induction of procoagulant activity (PCA) in CRC, and to evaluate its interactions with platelets and endothelial cells (ECs). Methods: Ninety-two CRC patients and 30 healthy controls were included. The presence of NETs was assessed using immunofluorescence microscopy. Cell-free DNA (cf-DNA) was quantified using the Quant-iT PicoGreen dsDNA Assay Kit, myeloperoxidase (MPO)-DNA complex was measured using a capture enzyme linked immunosorbent assay (ELISA). Thrombin-antithrombin (TAT) complex of NETs was evaluated by ELISA. Coagulation time of NETs, platelets and ECs was assessed by coagulation time (CT) using one-stage recalcification time assays, purified coagulation complex and fibrin turbidity were measured using ELISA. PS exposure on platelets and ECs, and fibrin formation on ECs were detected with flow cytometry and confocal microscopy. Results: We showed that the levels of cf-DNA and MPO-DNA complexes in the peripheral blood of CRC patients were increased in parallel with cancer progression and reached significance in stage III and IV patients compared to healthy subjects (all P<0.01). In addition, NETs released by CRC patients shortened coagulation time (CT), significantly enhanced the generation of TAT complexes and the formation of fibrin fibrils compared to healthy controls (all P<0.05). Moreover, DNase1-mediated degradation of NETs resulted in decreased PCA in patients with CRC (P<0.001). Furthermore, platelets from CRC patients stimulated healthy neutrophils to extrude NETs, which could be inhibited by the depletion of HMGB1 (P<0.01). Conversely, NETs from CRC patients could also induce the exposure of PS on platelets and the release of platelet MPs (PMPs), leading to markedly enhanced intrinsic/extrinsic FXa and FIIa, as well as shortened CT (all P<0.05). Importantly, endothelial cells (ECs) were 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) (all P<0.05). Our study also showed that the levels of NETs in CRC patients was positively correlated with TAT complexes and D-dimer (all P<0.05). Conclusion: Our results suggest that activated platelets promote NETs formation through the release of HMGB1 and result in an elevated PCA in CRC patients. In turn, NETs induce platelet PS exposure and PMPs release, forming a vicious cycle. In addition, NETs could also induce a procoagulant phenotype of ECs, indicating the complex relationship among these cellular constituents and highlighting the procoagulant role and cytotoxic effects of NETs in CRC. We propose that the rapid developments in the field of NETs may provide new therapeutic targets to combat the thrombotic consequences of CRC. No relevant conflicts of interest to declare.

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

Introduction:Oral squamous cell carcinoma (OSCC) is the most common cancer of the head and neck area, and the incidence remains high.Despite advances in diagnosis and treatment, the poor prognosis of OSCC is characterized by a high rate of local recurrence and the overall five-year survival rate remains at approximately 50%. Therefore, the mechanisms underlying the development of OSCC still need to be clarified. Patients with cancer tend to develop a hypercoagulable state which predisposes them to thromboembolic events. Cancer increases the risk of venous thrombosis several fold with varying degree of relative risks (range 4-7). A recent study has reported that microparticles (MPs) increased procoagulant activity (PCA) in OSCC. MPs are small membrane vesicles of 0.1-1 µm containing negatively charged, procoagulant phosphatidylserine (PS), which plays an important role in thrombosis. The definitive role of PS in the hypercoagulable state in patients with OSCC remains unclear. Our objectives were to measure the PS exposure on MPs, blood cells, and endothelium, and to evaluate their PCA in different stages of OSCC.

Introduction: Lupus nephritis (LN) is associated with a hypercoagulable state and an increased risk for thrombosis. Glomerular microthrombosis is detected in approximately 33% of LN patients. The incidence of thrombotic disease in patients with LN is 1.35 to 6.2 times higher than in other systemic lupus erythematosus (SLE) patients. While the immune complex is thought to elicit most forms of injury in LN, thromboembolic complications may be another important cause of renal injury and kidney dysfunction. However, the mechanisms specific to LN that promote a hypercoagulable state have not yet been identified. Antiphospholipid antibodies (aPLs) have been found to severely affect the pathophysiology of LN. But several authors have reported that the presence of aPLs is not sufficient for thrombus formation in vivo. Despite the clinical efficacy of the anti-inflammatory and immunosuppressive properties of glucocorticoid (GC) in treating LN, increased risk of thrombosis has been observed in GC users. Therefore, further studies should aim at finding other appropriate targets and effective treatments to correct coagulation abnormalities in LN. Recent studies have shown that phosphatidylserine (PS), a membrane constituent, plays an important role in thrombosis. However, the procoagulant role of PS in LN is not fully understood. Our objective was to elucidate the effects of PS exposure on microparticles (MPs) and their originating cells in LN.

Introduction: Serving as the preferred treatment for patients with acute kidney injury (AKI), continuous renal replacement therapy (CRRT) is mostly interrupted by clotting caused by procoagulant state despite of persistent improvements in anticoagulant technology, reducing therapy time, enhancing cost and resulting in blood lost. Due to hemodynamics disorders and foreign material circuit, blood cells undergo definite activation. However, considered as markers of cell activation, the role of phosphatidylserine (PS) and microparticles (MPs) in the hypercoagulability remains largely unexplored. The aim of this study is to measure PS exposure on blood cells and MPs at baseline and after different therapy duration in AKI patients.

Background: Despite dramatic improvement in treatment for acute promyelocytic leukemia (APL), early death due to hemorrhage remains a major obstacle to achieving a complete cure. In contrast to classical disseminated intravascular coagulation, APL-associated coagulopathy is characterized by rare microvascular fibrin thrombi. Thus, it is attractive to speculate whether other unknown mechanisms depleting coagulation factors and unrecognized fibrin-deposition location exist. Procoagulant activity associated with APL cells plays a direct role in bleeding complicationsin. We have shown that exposed phosphatidylserine (PS) on APL cells supports purified prothrombinase (Zhou J et al, JTH 2010) and fibrin preferentially deposits on promyelocytic chromatin from ETosis or apoptosis (Cao M et al, Blood 2017). However, relatively little is known about the PS-driven prothrombinase complex assembly and in situ fibrin deposition on APL cells.

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).

Introduction: Patients with diabetes mellitus (DM) are considered to be prothrombotic with major disturbances in hemostasis that are associated with an increased risk of venous thromboembolism, especially when microvascular complications of diabetes mellitus occur, such as diabetic kidney disease (DKD). Additionally, microthrombosis in glomerular capillaries could decrease the kidney blood flow and induce renal insufficiency. However, patients with DKD do not receive routine antithrombotic therapy. Therefore, more study is needed on the mechanisms of coagulation in DKD with the aim of identifying therapeutic targets. Recent studies have shown that phosphatidylserine (PS), a membrane constituent, plays an important role in the thrombosis. However, relatively little is known about the precise role of PS in the prothrombotic state of DKD. Our objectives were to study the effect of increased PS exposure on microparticles (MPs) and the outer membrane of MP-origin cells in DKD patients, and to evaluate its effect on procoagulant activity (PCA).