Microparticle Tissue Factor Activity Research Articles

Elevated Microparticle Tissue Factor Activity Differentiates Patients With Venous Thromboembolism in Anti-neutrophil Cytoplasmic Autoantibody Vasculitis

IntroductionVenous thromboembolism (VTE) is a life-threatening complication of anti-neutrophil cytoplasmic autoantibody (ANCA) vasculitis whose mechanism remains incompletely elucidated. We tested the hypothesis that elevated microparticle tissue factor activity (MPTFa) or anti-plasminogen antibodies (anti-Plg) may identify patients at risk for VTE.MethodsIn this prospective study, patients were enrolled during active disease and followed longitudinally. Twelve patients who experienced a VTE (VTEpos) were compared with patients without VTE (VTEneg, n = 29) and healthy controls (HC, n = 70). MPTFa, anti-Plg, interleukin-6, high-sensitivity C-reactive protein (hs-CRP), D-dimer, serum creatinine, and serum albumin were assessed. Fisher’s exact tests and Wilcoxon tests compared categorical and continuous variables, respectively. Cox regression for time to VTE or last follow-up was performed.ResultsVTEpos patients had higher MPTFa (peak median = 14.0, interquartile range = 4.3–36.6) than HC (0, 0–3.5) and VTEneg patients (0, 0–1.4). In time-to-event analysis, MPTFa was associated with VTE when measured during both active disease (hazard ratio [HR]; 95% confidence interval [CI]: 1.04; 1.01–1.08) and remission (1.4; 1.11–1.77). Anti-Plg during remission was also associated with VTE (1.17; 1.03–1.33). Each g/dl decrease of serum albumin was associated with a 4-fold increase in VTE risk (4.4; 1.5–12.9). Adjusting for estimated glomerular filtration rate (eGFR), anti-Plg during remission remained significantly associated with VTE.ConclusionElevated MPTFa and increased anti-Plg in remission are strong indicators of VTE independent of renal function. Association of anti-Plg during remission with VTE implies hypercoagulability even during disease quiescence. Hypoalbuminemia strongly portends VTE risk, which is a novel finding in ANCA vasculitis. A thrombotic signature would allow improved management of patients to minimize VTE risk and complications of anticoagulation.

Elevated Microparticle Tissue Factor Activity Is Associated With Carotid Artery Plaque in HIV-Infected Women.

Expression of tissue factor (TF) on the surface of activated monocytes may trigger thrombosis, leading to clotting risk, inflammation, and atherosclerosis. TF-positive microparticles (MP-TF) represent a functionally active form of TF that may be promulgated by long-term HIV infection. We hypothesized that greater MP-TF activity is associated with carotid artery plaque in HIV+ women. In a case-control study nested within the Women's Interagency HIV Study (WIHS), eligible HIV+ participants underwent B-mode carotid artery ultrasound at 2 study visits occurring 7 years apart. Cases were defined by the presence of at least 1 carotid artery plaque assessed at either visit. Cases were matched 1:2 to controls who were found not to have carotid artery plaques. Conditional logistic regression estimated the association of MP-TF activity with the presence of carotid artery plaque, adjusting for demographic and behavioral characteristics, HIV-related factors, cardiometabolic risk factors, and serum inflammation biomarkers (high-sensitivity C-reactive protein, IL-6, sCD14, sCD163, Gal-3, and Gal-3BP). Elevated MP-TF activity (>0.537 pg/mL) was found to be significantly associated with greater odds of plaque (adjusted odds ratio 3.86, 95% confidence interval: 1.06 to 14.07, P = 0.04). The association was attenuated after further adjustment for IL-6 but was unaffected by adjustment for other biomarkers including those denoting monocyte activation. Our findings suggest a link among HIV infection, innate immune system perturbation, coagulation, and atherosclerosis.

The Release of Monocyte-Derived Tissue Factor-Positive Microparticles Contributes to a Hypercoagulable State in Idiopathic Membranous Nephropathy.

Aim: Idiopathic membranous nephropathy (IMN) is an immune-mediated inflammatory disease characterized by a high risk of thromboembolic complications. Microparticles (MPs), a type of extracellular vesicles, have procoagulant properties, especially when they display tissue factor (TF). This study aimed to investigate whether circulating TF-positive MPs contributed to the hypercoagulable state in patients with IMN.Methods: Twenty adult IMN patients and fourteen healthy subjects were included in the study. The basic indexes of a routine biochemical examination and coagulative function were determined. The plasma levels of MPs were detected by flow cytometry, and TF activity of MPs was examined using an assay kit. The plasma levels of lipopolysaccharide (LPS) were measured by an enzyme-linked immunosorbent assay.Results: Total circulating MPs were not increased in patients with IMN compared with healthy controls. Circulating CD14+/TF+MPs were significantly increased in IMN patients, but this achieved significance was not observed in CD41+/TF+MPs between the two groups. Interestingly, the circulating TF-positive MPs were increased significantly. Plasma MPs TF assays revealed high procoagulant activity, which was positively associated with the D-dimer level in IMN. In addition, circulating LPS in IMN patients were significantly higher than those in the controls. Furthermore, after two hours' incubation with healthy whole blood, LPS enhanced the release of circulating TF-positive MPs and the TF activity of MPs.Conclusion: Increased circulating LPS may mediate the release of monocyte-derived TF-positive MPs, which further contributes to the hypercoagulable state in IMN patients. These findings provide an additional mechanism by which patients with IMN have a higher risk of thromboembolic complication.

Plasma Levels of IL-8 and Microparticle Tissue Factor Activity Are Associated with Mortality in Patients with Primary Influenza A/H1N1 Infection

Background and Objective: Treatment and triage decisions during influenza remain difficult due to lack of reliable severity of illness predictive score. Influenza A/H1N1 induces the expression and release of tissue-factor bearing microparticles (MP-TF), contributing to a prothrombic milieu. However, there are no studies reporting levels of circulating TF-expressing MPs during the course of human influenza. We sought to determine if MP-TF are an early predictor of mortality in critically ill patients with influenza A/H1N1.Methods: This was a prospective, multicenter, case-cohort pilot study of three academic intensive care units. We prospectively studied 15 patients with primary influenza A/H1N1 that included 7 survivors and 8 non-survivors. For comparison, 27 healthy, medication-free, age- and gender-matched control subjects were also prospectively studied. Plasma was prepared from blood drawn upon ICU admission in influenza patients. MP-TF activity, thrombin-antithrombin complexes (TATc), and D-dimers were measured as markers of activation of coagulation. Plasma cytokine levels were measured on the same blood samples. Patients were followed for the primary outcome of 28-day mortality.Results: The average admission APACHE II score of the influenza patients was 25.5±9.3, 60% of patients had shock, and the 28-day mortality rate was 53.3% (n=8/15). Compared to healthy controls, influenza patients had significantly higher plasma fibrinogen, C-reactive protein (CRP) MP-TF activity, TATc, D-dimer and a prolonged prothrombin time. However, of these procoagulant markers, only MP-TF activity predicted influenza related mortality (5.6±1.2 pg/ml in non-survivors vs. 1.8±0.8 pg/mL in survivors, p < 0.05; Table 1 and Fig. 1A). MP-TF activity, TATc levels, and D-dimer did not correlate with APACHE II score, platelet count, fibrinogen levels, CRP, or age or between patients with severe sepsis versus septic shock. Influenza non-survivors also had significantly higher plasma IL-8 levels compared with survivors (71.8±29.1 pg/ml vs. 17.3±3.7 pg/mL, p < 0.05; Figure 1B). MP-TF activity and IL-8 levels were significantly and positively correlated (r2 = 0.60, P =0.003; Figure 1C). Other cytokines, TATc, and D-dimer were not different between non-survivors and survivors.Conclusions: This study demonstrates that plasma IL-8 and MP-TF activity measured upon admission in patients with severe, primary influenza A/H1N1 infection is associated with subsequent mortality. Thus, these biomarkers may serve as very early prognostic markers for patients with influenza A/H1N1.Table 1Characteristics of the influenza patients. Laboratory values, coagulation markers, and plasma cytokines were measured within 24 hours of ICU admission.All Influenza A/H1N1 Patients(n=15)Influenza Non-Survivors(n=8)Influenza Survivors(n=7)P valueAdmission CharacteristicAge, years43.3±11.045.8±4.640.4±15.50.37Male Gender, n (%)7 (47%)3 (38%)4 (57%)0.45Weight, kg95.5±25.286.6±20.9105.7±27.40.15BMI, kg/m233.3±7.231.5±5.035.4±9.10.31Obesity (BMI³30 kg/m2)10 (67%)5 (63%)5 (71%)0.71Tobacco Use, n (%)4 (27%)3 (38%)1 (14%)0.31APACHE II Score25.5±9.327.9±9.122.9±9.40.31Mechanically Ventilated, n (%)15 (100%)8 (100%)7 (100%)--Shock, n (%)9 (60%)6 (75%)3 (43%)0.21P/F Ratio83±2884±3482±220.89Clinical OutcomesICU Length of Stay, days21.9±7.723.0±7.920.7±7.80.58Duration of Ventilation, days8.2±1.08.5±0.97.6±0.90.11Secondary Bacterial Infection, n (%)5 (33%)4 (50%)1 (14%)0.28Overt DIC, n (%)15 (100%)8 (100%)7 (100%)--Laboratory ValuesPlatelets, 103/µL213±138154±104280±1500.08White Blood Cells, K/µL7.3±4.48.5±4.95.9±3.50.28Hemoglobin, g/dL11.7±1.911.5±2.012.0±1.80.66Serum Creatinine, mg/dL1.1±0.71.14±0.741.07±0.590.84Coagulation MarkersFibrinogen, mg/dL571±240461±253700±1570.07C-reactive Protein, mg/L11.0±7.611.8±7.410.2±8.20.70PT, sec20±7.019.2±4.720.9±9.30.67aPTT, sec53.0±22.955.1±29.050.7±15.00.72MP-TF, pg/mL3.8±0.95.6±1.21.8±0.8< 0.05TATc, ng/mL11.8±2.714.3±3.59.1±4.10.35D-dimer, ng/mL2439±862568±982292±1340.11 [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Hepatocyte tissue factor contributes to the hypercoagulable state in a mouse model of chronic liver injury

Patients with chronic liver disease and cirrhosis have a dysregulated coagulation system and are prone to thrombosis. The basis for this hypercoagulable state is not completely understood. Tissue factor (TF) is the primary initiator of coagulation in vivo. Patients with cirrhosis have increased TF activity in white blood cells and circulating microparticles. The aim of our study was to determine the contribution of TF to the hypercoagulable state in a mouse model of chronic liver injury. We measured levels of TF activity in the liver, white blood cells and circulating microparticles, and a marker of activation of coagulation (thrombin-antithrombin complexes (TATc)) in the plasma of mice subjected to bile duct ligation for 12days. We used wild-type mice, mice with a global TF deficiency (low TF mice), and mice deficient for TF in either myeloid cells (TF(flox/flox),LysMCre mice) or in hepatocytes (TF(flox/flox),AlbCre). Wild-type mice with liver injury had increased levels of white blood cell, microparticle TF activity and TATc compared to sham mice. Low TF mice and mice lacking TF in hepatocytes had reduced levels of TF in the liver and in microparticles and exhibited reduced activation of coagulation without a change in liver fibrosis. In contrast, mice lacking TF in myeloid cells had reduced white blood cell TF but no change in microparticle TF activity or TATc. Hepatocyte TF activates coagulation in a mouse model of chronic liver injury. TF may contribute to the hypercoagulable state associated with chronic liver diseases in patients.

A matched cross-sectional study of the association between circulating tissue factor activity, immune activation and advanced liver fibrosis in hepatitis C infection.

BackgroundTissue factor (TF) is a protein that mediates the initiation of the coagulation cascade. TF expression is increased in patients with poorly-controlled HIV, and may be associated with increased immune activation that leads to cardiovascular morbidity. The role of TF in immune activation in liver disease in hepatitis C virus (HCV)-monoinfection and HIV/HCV-coinfection has not been explored.MethodsFifty-nine patients were stratified: A) HIV-monoinfection (N = 15), B) HCV-monoinfection with chronic hepatitis C (CHC) (N = 15), C) HIV/HCV-coinfection with CHC (N = 14), and D) HIV/HCV-seropositive with cleared-HCV (N = 15). All HIV+ patients had undetectable HIV viremia. Whole blood was collected for CD4/CD8 immune activation markers by flow cytometry and plasma was assayed for microparticle TF (MPTF) activity. Subjects underwent transient elastography (TE) to stage liver fibrosis. Undetectable versus detectable MPTF was compared across strata using Fisher's Exact test.ResultsMPTF activity was more frequently detected among patients with HCV-monoinfection (40%), compared to HIV-monoinfection and HIV/HCV-seropositive with cleared HCV (7%) and HIV/HCV-coinfection with CHC (14%) (p = 0.02). Mean TE-derived liver stiffness score in kPa was higher in patients with detectable MPTF (12.4 ± 8.5) than those with undetectable MPTF (6.4 ± 3.0) (p = 0.01). Mean CD4 + HLADR+ and CD4 + CD38-HLADR+ expression were higher in those with detectable MPTF (44 ± 9.8% and 38 ± 8.7%, respectively) than those with undetectable MPTF (36 ± 11% and 31 ± 10.4% respectively) (p = 0.05 and 0.04 respectively).ConclusionsHCV-monoinfection and HIV/HCV-coinfection with CHC were associated with MPTF activity. MPTF activity is also associated with advanced liver fibrosis and with CD4 + HLADR+ immune activation.

血漿中マイクロパーティクルtissue factor 活性測定法の比較検討

血漿中マイクロパーティクルtissue factor 活性測定法の比較検討

Circulating microparticle tissue factor activity Is increased in patients with cirrhosis

Cirrhosis is associated with a procoagulant imbalance.1 Reduced expression of the anticoagulants protein C and antithrombin and increased expression of the procoagulants factor VIII and von Willebrand factor may increase coagulation in these patients.1 Levels of procoagulant microparticles (MPs) and particularly MPs carrying the highly procoagulant protein tissue factor (TF) have not been investigated in cirrhosis patients. To determine if MP TF activity is increased in patients with cirrhosis, we analyzed 33 patients with alcoholic and/or hepatitis C virus related cirrhosis (10 Child-Pugh A, 9 Child-Pugh B and 14 Child-Pugh C) using a previously reported assay.2 None used drugs known to interfere with coagulation, had hepatocellular carcinoma, infection or transfusion within two weeks before blood draw. We also used 9 healthy volunteers as controls. All patients and controls gave their informed consent to participate to the study, which was approved by the Institutional Review Board. Levels of markers of activation of coagulation and fibrinolysis increased with cirrhosis severity (Fig. 1A, B). As expected, plasma from healthy individuals contained very low levels of MP TF activity.2 Importantly, MP TF activity increased with cirrhosis severity and was 17 fold higher in Child-Pugh C patients compared with controls (Fig. 1C). MP TF activity correlated with MELD score (Spearman’s correlation: r = 0.523; P = 0.002). Similarly, when restricting the analyses to the 22 patients with a relatively stable condition (outpatients or planned hospitalization), we observed higher MP TF activity in patients with cirrhosis than in controls (p = 0.007). A previous study reported that MP TF activity was increased 38-fold in acute liver injury/acute liver failure patients.3 This study used platelet poor plasma while we used platelet free plasma.4 Figure 1 MP Procoagulant Activity In Patients With Cirrhosis We also assessed levels of total MPs, but did not observe any difference between patients and controls either using flow cytometry (annexin V binding; Fig. 1D) or an activity assay (Zymuphen MP Activity, Hyphen BioMed, Fig. 1E). This lack of difference in levels of phosphatidylserine positive MPs may be because the majority of these MPs are derived from platelets and we have found that platelet-derived MPs are not increased in cirrhosis.4 In conclusion, patients with cirrhosis have increased levels of circulating MP TF activity that may contribute to the activation of coagulation and thrombosis in these patients.

Plasma Microparticle Tissue Factor Activity in Patients With Antiphospholipid Antibodies With and Without Clinical Complications

Antiphospholipid syndrome (APS) is defined by the association of autoantibodies to certain phospholipid-binding proteins with arterial or venous thrombosis ('AT' or 'VT', respectively), and/or pregnancy-related morbidity (PM). Antiphospholipid antibodies (aPLA) promote activation of several cell types including monocytes, resulting in procoagulant tissue factor (TF) expression that may contribute to the vascular complications. Since TF synthesis by monocytes is frequently accompanied by release of TF-bearing microparticles, we hypothesized that plasma microparticle TF activity (MP-TF) may be elevated in APS patients and contribute to thrombosis and/or PM. Platelet-poor plasma specimens were obtained from 30 patients with definite APS and 72 patients with asymptomatic aPLA from the Antiphospholipid Syndrome Collaborative Registry (APSCORE). MP-TF was measured by an in-house factor Xa generation assay. The two groups were well matched for gender, age, ethnicity, proportions with underlying SLE, and aPLA profiles. MP-TF (median and (IQR)) in asymptomatic aPLA subjects was 0.09 pg/mL (0.05-0.14) compared to 0.13 pg/mL (0.10-0.17) in APS (p < 0.001). No differences in MP-TF levels were observed between APS subjects with PM, thrombosis, or PM+thrombosis. Similarly, among subjects with either APS or asymptomatic aPLA, MP-TF did not differ in the presence or absence of underlying SLE. Prospective studies will be required to determine if plasma MP-TF activity is causally related to thrombotic or gestational complications in APS.

Plasma Microparticle Tissue Factor Activity in Patients With Antiphospholipid Antibodies With and Without Clinical Complications

Plasma Microparticle Tissue Factor Activity in Patients With Antiphospholipid Antibodies With and Without Clinical Complications

Tumor-derived tissue factor activates coagulation and enhances thrombosis in a mouse xenograft model of human pancreatic cancer

AbstractCancer patients often have an activated clotting system and are at increased risk for venous thrombosis. In the present study, we analyzed tissue factor (TF) expression in 4 different human pancreatic tumor cell lines for the purpose of producing derivative tumors in vivo. We found that 2 of the lines expressed TF and released TF-positive microparticles (MPs) into the culture medium. The majority of TF protein in the culture medium was associated with MPs. Only TF-positive cell lines activated coagulation in nude mice, and this activation was abolished by an anti–human TF Ab. Of the 2 TF-positive lines, only one produced detectable levels of human MP TF activity in the plasma when grown orthotopically in nude mice. Surprisingly, < 5% of human TF protein in plasma from tumor-bearing mice was associated with MPs. Mice with TF-positive tumors and elevated levels of circulating TF-positive MPs had increased thrombosis in a saphenous vein model. In contrast, we observed no difference in thrombus weight between tumor-bearing and control mice in an inferior vena cava stenosis model. The results of the present study using a xenograft mouse model suggest that tumor TF activates coagulation, whereas TF on circulating MPs may trigger venous thrombosis.

Abstract 42: Monocyte Tissue Factor-Dependent Activation of Coagulation in Hypercholesterolemic Mice and Monkeys Is Inhibited by Simvastatin

Hyperlipidemia is associated with a prothrombotic state that includes activation of platelets. However, the molecular pathway that leads to the activation of the coagulation cascade has not been defined. Hyperlipidemia leads to the presence of low levels of oxidized lipoproteins, such as oxidized LDL (oxLDL), in the circulation and the accumulation of oxLDL in atherosclerotic lesions.. Monocytes and macrophages are activated by oxLDL binding to a TLR4/TLR6/CD36 receptor complex. We hypothesized that oxLDL induction of monocyte tissue factor (TF) expression and the release of TF-positive microparticles (MPs) produces a prothrombotic state. In support of this hypothesis, we found that oxLDL induced TF expression in human monocytic cells and monocytes. In addition, patients with familial hypercholesterolemia had elevated levels of plasma MP TF activity. Furthermore, a western diet induced a time-dependent increase in plasma MP TF activity and activation of coagulation in both LDL receptor-deficient mice and African green monkeys. Inhibition of TF or a genetic deficiency of TF in bone marrow cells reduced coagulation in hypercholesterolemic mice, consistent with a major role for monocyte TF in the activation of coagulation. Similarly, a deficiency of either TLR4 or TLR6 reduced levels of MP TF activity consistent with a role of the TLR4/TLR6/CD36 complex in oxLDL induction of monocyte TF expression. Importantly, simvastatin treatment of hyperlipidemic mice and monkeys reduced oxLDL, monocyte TF expression, MP TF activity, activation of coagulation, and inflammation without affecting total cholesterol levels. Our results suggest that the prothrombotic state associated with hyperlipidemia is caused by oxLDL-mediated induction of TF expression in monocytes via engagement of a TLR4/TLR6/CD36 receptor complex. Furthermore, simvastatin appears to act at multiple levels to reduce the prothrombotic state.

Monocyte tissue factor–dependent activation of coagulation in hypercholesterolemic mice and monkeys is inhibited by simvastatin

Hypercholesterolemia is a major risk factor for atherosclerosis. It also is associated with platelet hyperactivity, which increases morbidity and mortality from cardiovascular disease. However, the mechanisms by which hypercholesterolemia produces a procoagulant state remain undefined. Atherosclerosis is associated with accumulation of oxidized lipoproteins within atherosclerotic lesions. Small quantities of oxidized lipoproteins are also present in the circulation of patients with coronary artery disease. We therefore hypothesized that hypercholesterolemia leads to elevated levels of oxidized LDL (oxLDL) in plasma and that this induces expression of the procoagulant protein tissue factor (TF) in monocytes. In support of this hypothesis, we report here that oxLDL induced TF expression in human monocytic cells and monocytes. In addition, patients with familial hypercholesterolemia had elevated levels of plasma microparticle (MP) TF activity. Furthermore, a high-fat diet induced a time-dependent increase in plasma MP TF activity and activation of coagulation in both LDL receptor-deficient mice and African green monkeys. Genetic deficiency of TF in bone marrow cells reduced coagulation in hypercholesterolemic mice, consistent with a major role for monocyte-derived TF in the activation of coagulation. Similarly, a deficiency of either TLR4 or TLR6 reduced levels of MP TF activity. Simvastatin treatment of hypercholesterolemic mice and monkeys reduced oxLDL, monocyte TF expression, MP TF activity, activation of coagulation, and inflammation, without affecting total cholesterol levels. Our results suggest that the prothrombotic state associated with hypercholesterolemia is caused by oxLDL-mediated induction of TF expression in monocytes via engagement of a TLR4/TLR6 complex.

Pre-analytical and analytical variables affecting the measurement of plasma-derived microparticle tissue factor activity

Pre-analytical and analytical variables affecting the measurement of plasma-derived microparticle tissue factor activity

Pre-analytical and analytical variables affecting the measurement of plasma-derived microparticle tissue factor activity

Introduction Elevated levels of tissue factor positive (TF +) microparticles (MPs) are observed in plasma from a variety of patients with an increased risk of thrombosis. We and others have described the measurement of TF activity in MPs isolated from plasma. The aim of this study was to investigate the effects of pre-analytical and analytical variables on TF activity of MPs isolated from blood of healthy volunteers either untreated or treated ex vivo with bacterial lipopolysaccharide. Materials and methods We evaluated the following parameters: use of different centrifugation speeds to isolate the MPs; comparison of TF activity of MPs isolated from platelet poor plasma versus platelet free plasma; effect of freeze/thaw on MP TF activity; and comparison of the MP TF activity assay with the measurement of TF protein by ELISA or flow cytometry. Results MPs prepared from platelet poor plasma by centrifugation at 20,000 × g or 100,000 × g for 15 minutes had similar levels of TF activity. However, significantly less TF activity was found in MPs isolated from platelet free plasma compared with platelet poor plasma. Interestingly, freeze/thawing of the plasma showed donor to donor variation in MP TF activity, with a moderate increase in some individuals. Conclusion TF + MPs can be quantitatively isolated from platelet poor or platelet free plasma by centrifugation at 20,000 × g for 15 minutes. Measurement of MP TF activity in plasma may be used to detect a prothrombotic state in patients with various diseases.

Male mice have increased thrombotic potential: Sex differences in a mouse model of venous thrombosis

Introduction Our objectives were to characterize sex differences during venous thrombosis, using the electrolytic inferior vena cava model of the disease. Materials and methods Male and female C57BL/6 mice (6-8 weeks) underwent inferior vena cava thrombosis. Time points included 6 hours, day 2, day 6, and day 14 post surgery, along with surgically naïve true controls and surgical shams. Analyses included thrombus weight, vein wall morphometrics, vein wall protein and gene expression for P-selectin, interleukin-1β, and tumor necrosis factor-α; hematology, soluble P-selectin, and plasma microparticle tissue factor activity assays. Results Male venous thrombi were significantly larger than females at days 2 (13.1 ± 1.0 vs. 6.8 ± 0.5 × 10 -3 grams, p < 0.01), 6 (10.4 ± 0.8 vs. 5.4 ± 0.5 × 10 -3 grams, p < 0.01) and 14 (6.3 ± 0.5 vs. 4.1 ± 0.3 × 10 -3 grams, p < 0.01). Both male and female mice exhibited significantly increased vein wall P-selectin at 6 hours, vs. true controls (p < 0.05). Males had increased vein wall interleukin-1β, versus females, at 6 hours (180.926 ± 24.596 vs. 60.417 ± 10.478 pg/mL, p < 0.05) and day 6 (76.966 ± 13.081 vs. 33.834 ± 4.198 pg/mL, p < 0.01). Males showed decreased tumor necrosis factor-α expression (-66 %) at 6 hours. Females had increased tumor necrosis factor-α expression at 6 hours (+ 541%) and day 6 (+ 539%). Both sexes demonstrated decreased peripheral platelets at 6 hours (p < 0.05), coinciding with thrombogenesis. Plasma P-selectin increased in both sexes, versus controls, through day 6 (p < 0.05). Conclusions Males had significantly larger venous thrombi than females. Sex differences in vascular anatomy and response to inflammation may influence thrombus formation in our mouse thrombosis model.

Anthracycline Treatment of the Monocytic Leukemia Cell Line THP-1 Increases Phosphatidylserine Exposure and Tissue Factor Activity.

AbstractAbstract 1136 Introduction:Cancer associated thrombosis is a well-recognized phenomenon that results in considerable patient morbidity and mortality. While malignancy alone conveys an increased risk for thrombosis, treatment with cytotoxic chemotherapeutics further elevates this risk. However, the pathogenic mechanisms underlying this process remain poorly defined. We hypothesized that treatment of tumor cells with cytotoxic chemotherapeutic drugs leads to phosphatidylserine (PS) exposure on the cell surface, as well as increased cellular tissue factor (TF) activity and release of TF positive microparticles (MPs). Aim:The aim of this study was to determine the effect of anthracyclines on TF activity and MP release from the human monocytic leukemia cell line THP-1. Materials and Methods:THP-1 cells were treated with different concentrations of doxorubicin (0-10μg/ml) or daunorubicin (0-1μg/ml) for 0–24 hours. These concentrations are similar to those found in plasma of cancer patients. The intact cells were analyzed for TF activity using a two stage chromogenic assay. TF antigen was evaluated by ELISA and TF mRNA by realtime PCR. In addition, TF activity of MPs released into the culture supernatant was measured using a two stage chromogenic assay. PS exposure on cells and MPs were analyzed by flow cytometry and PS exposure on MP was also evaluated in an annexin V capture assay. Cell viability was assayed by a dye exclusion assay as well as by flow cytometry. Results:We found that doxorubicin and daunorubicin increased PS exposure on the cell surface. Treatment with anthracyclines increased both cellular TF and MP TF activity in a concentration-dependent manner that also resulted in concentration dependent increase in apoptosis. Furthermore, this increased TF dependent activity was abolished with the addition of annexin V or lactadherin supporting a role of PS exposure in this elevated activity. Conclusion:These results support our hypothesis that treatment of THP-1 cells with chemotherapeutic agents induces apoptosis and increases cellular TF activity. This increased activity appears primarily mediated by an increase in cell surface expression of PS. Additionally, the anthracyclines increase the release of TF positive MPs from the THP-1 cells. We propose that this increase in cellular TF activity in tumor cells and increased numbers of TF positive MPs may contribute to thrombosis in cancer patients treated with chemotherapeutic drugs. Disclosures:No relevant conflicts of interest to declare.

PO-56 Potential contribution of microparticles to the thrombotic risk in high grade glioma

poorly defined. We hypothesized that treatment of tumor cells with cytotoxic chemotherapeutic drugs increases phosphatidylserine (PS) on the cell surface, cellular tissue factor (TF) activity and release of TF positive microparticles (MPs). Aim: The aim of the study was to determine the effect of anthracyclines on TF activity and MP release from the human monocytic leukemia cell line THP-1. Materials and Methods: THP-1 cells were treated with different concentrations of doxorubicin (0–10mg/ml) and daunorubicin (0–10mg/ml) for 24 hours. These concentrations are similar to those found in plasma of cancer patients. The intact cells were analyzed for TF activity using a two stage clotting assay and PS exposure was analyzed by flow cytometry. In addition, TF activity of MPs released into the culture supernatant was measured using a two stage clotting assay. Results: We found that doxorubicin and daunorubicin both increased cellular TF and MP TF activity in a concentration-dependent manner. This effect was partially abrogated by the caspase inhibitor ZVAD-FMK. Increased PS exposure on the cell surface was also noted in cells treated with anthracyclines. Conclusions: These results support our hypothesis that treatment of THP-1 cells with chemotherapeutic agents induces apoptosis and increases cellular TF activity. This increased activity appears primarily mediated by an increase in cell surface expression of PS. Additionally, the chemotherapeutics increase the release of TF positive MPs from the THP-1 cells. We propose that this increase in cellular TF activity in tumor cells and increased numbers of TF positive MPs leads to an increase in thrombosis in cancer patients treated with chemotherapeutic drugs.

Increased microparticle procoagulant activity in mice lacking alpha(1,3)‐fucosyltransferase‐IV and –VII

Fucosylation of selectin ligand glycans by alpha(1,3)‐fucosyltransferases (FucT)‐IV or ‐VII is required for sialyl Lewis x formation and selectin‐binding activity. FucT‐IV(−/−)/FucT‐VII(−/−) mice (DKO) have a 43% decrease in the time to arterial thrombosis due in part to enhanced whole blood platelet aggregation. We evaluated the contribution of the coagulation system to enhanced thrombosis. Fibrinometric determinations of intrinsic and extrinsic pathway activity were not different between WT (n=8) and DKO (n=8) mice [prothrombin time: WT = 12.4±0.3 vs DKO = 12.1±0.4, p=0.11; activated partial thromboplastin time: WT = 29.5±1.9 vs DKO = 28.2±2.6, p=0.28]. Similarly, parameters of the calibrated automated thrombography assay were not different between WT (n=10) and DKO (n=10) mice [lag time: WT = 4.1±1.4 vs DKO = 4.2±0.8, p=0.82; time to peak: WT = 10.4±2.1 vs DKO = 9.4±1.8, p=0.28; peak height: WT = 21.2±6.0 vs DKO = 23.7±4.3, p=0.29; endogenous thrombin potential: WT = 359±123 vs DKO = 336±77, p=0.6]. In contrast, total microparticle procoagulant activity was elevated 3 fold in DKO (n=8) mice compared to WT (n=8) mice [WT = 0.21±0.2 vs DKO = 0.6±0.2, p=0.02], due primarily to microparticle tissue factor‐dependent activity [WT = 0.08±0.1 vs. DKO = 0.23±0.22, p=0.1]. Microparticle tissue factor activity contributes in part to the enhanced thrombosis in DKO mice. Support by AHA FTF Award 0275023N and RO1‐HL090823.

Increased microparticle tissue factor activity in cancer patients with Venous Thromboembolism

Increased microparticle tissue factor activity in cancer patients with Venous Thromboembolism