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

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.

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.

Background Coagulopathy is a major cause of early death when arsenic trioxide (As2O3) therapy fails. In addition to the procoagulant properties of blast cells, the cytotoxic therapy may contribute to the coagulation disorders. The aim of the present study was to evaluate the possible impact of As2O3 on membrane alterations, including phosphatidylserine (PS) exposure and microparticle generation, and the consequent procoagulant properties of endothelial cells. Methods Procoagulant activity (PCA) of human umbilical vein endothelial cells (HUVECs) was assessed by measuring clotting time and through purified coagulation complex assays. PS exposure on HUVEC membrane was observed by confocal microscopy and quantified with flow cytometry. In addition, counts and PCA of endothelial microparticles were determined by flow cytometry and plasma coagulation assay. Results As2O3 increased the ability of HUVECs to accelerate coagulation process and promote formation of coagulation complexes. Procoagulant activity corresponded to PS exposed on HUVECs. In coincidence with the PS externalization, As2O3 increased the production of PS-bearing microparticles, which then accelerated fibrin strand formation significantly. By blocking PS, lactadherin was able to inhibit over 90% of the intrinsic tenase/prothrombinase activity of As2O3-treated HUVECs, and restored coagulation times of As2O3–treated cells and microparticles to control levels. Conclusions As2O3 increases PCA of HUVECs through PS exposure and PS-bearing microparticle generation, which might cause thrombosis and act as a contributing factor in As2O3 therapy-related coagulopathy.

Introduction Thromboembolism is a serious complication in patients with acute lymphoblastic leukemia (ALL). Coagulation disorders can be induced and worsened by cytotoxic drugs; however, the mechanisms are largely unknown. Our study aims to investigate the effects of daunorubicin (DNR) and L-asparaginase (L-ASP) on phosphatidylserine (PS) exposure and the procoagulant activity (PCA) of Jurkat/ALL cells. The anticoagulant properties of lactadherin were also explored. Materials and Methods Jurkat cells and cells from 10 newly diagnosed patients with ALL were treated with DNR or L-ASP. Flow cytometry and confocal microscopy were used to quantify and locate PS exposure, respectively. PCA was evaluated using coagulation assays and purified coagulation complex assays. Lactadherin, a glycoprotein of the milk fat globule membrane with stereospecific binding to phosphatidyl-L-serine, was used as a probe for the detection of exposed PS. Results Untreated Jurkat/ALL cells exhibited higher PS exposure and greater PCA than mononuclear cells (MNCs). The PCA of cells treated with DNR or L-ASP was markedly increased. Flow cytometry and confocal microscopy indicated that the increased PCA occurred in parallel with PS exposure. The blocking of PS with lactadherin prolonged the coagulation time and inhibited approximately 85-90% of the activities of procoagulant enzyme complexes in Jurkat/ALL cells. Conclusions Our results indicate that DNR and L-ASP increased the PCA of Jurkat/ALL cells through PS exposure and played a critical role in inducing thrombosis in ALL patients. Lactadherin is an ideal probe for PS detection at an early stage and a potential anticoagulant to improve the hypercoagulability of ALL patients.

Introduction Cytotoxic chemotherapy induces or worsens hemostatic disorders in patients with acute myeloid leukemia. Procoagulant release and tissue factor expression on tumor cells are considered to contribute to chemotherapeutic agents-associated coagulopathy. The role of red blood cells during this process has not been determined; although they lack tissue factor, they may contribute suitable membranes for the amplification phase of blood coagulation. The present study aims to evaluate the possible impact of daunorubicin on phosphatidylserine exposure and consequent procoagulant property of red blood cells. Materials and methods Red blood cells from acute myeloid leukemia patients and healthy donors were treated with daunorubicin as well as all-trans-retinoic acid, arsenic trioxide or etoposide for 0-48 h. Procoagulant activity was assessed by measurement of a clotting time and by purified coagulation complex assays. Lactadherin was used as a probe for phosphatidylserine. Results Daunorubicin treatment increased procoagulant activity of red blood cells regardless of the cell origin. Moreover, coagulation complexes assays supported daunorubicin-evoked procoagulant response. The procoagulant property of red blood cells was not affected by the other three agents. The modulating effect of procoagulant activity was concomitant with and dependent on level of phosphatidylserine on the outer surface. Blockade of phosphatidylserine with lactadherin inhibited over 90% of tenase generation and prothrombinase activity and prolonged the coagulation time. Conclusions We conclude that daunorubicin interacts with red blood cells in a manner that increases phosphatidylserine exposure and consequent procoagulant activity. Lactadherin is an efficient anticoagulant of this process.