Plasma Levels Of Tissue Factor Research Articles

Hypoxic Exercise Training Affects Thrombin Generation by Modulating Platelet‐derived Procoagulant Microparticles under Shear Flow

This study investigated how exercise training with/without hypoxia affects thrombin generation (TG) mediated by platelet‐derived micropraticles (PDMPs) under physio‐pathological shear flows. Seventy five sedentary males were randomly divided into five groups. The subjects in each group (n=15) were exposed to 21% (NC) or 15% (HC) O2 at rest or were trained at 50% of Wmax under 21% (NT) or 15%O2 (HAT), or 50% of heart rate reserve under 15%O2 (HRT) for 30 minutes daily, 5 days weekly for 4 weeks. Before the intervention, strenuous exercise increased the levels of coagulant factor V/Va (FV/FVa)‐/FVIII‐/tissue factor (TF)‐rich and phosphatidylserine (PS)‐exposed PDMPs and the rate of TG in PDMP‐rich plasma (MRP) under 100 dyne/cm2 of shear stress. The NT and HRT, but not NC and HC, for 4 weeks attenuated the enhancements of shear‐induced FV/FVa‐/FVIII‐/TF‐rich and PS‐exposed PDMP formation and TG rate in MRP by severe exercise. Conversely, the HAT regimen promoted shear‐induced formation and procoagulant activation of PDMPs, concurrence with increasing plasma FV and TF levels at rest or following exercise. We conclude that a hypoxic exercise regimen, that performs a relative‐intensive workload according to exercise heart rate, may depress shear‐mediated procoagulant activity of PDMPs during exercise.

Preeclampsia: the role of tissue factor and tissue factor pathway inhibitor

Preeclampsia (PE) is a multi-system disorder of human pregnancy, whose etiology remains poorly understood. Preeclamptic women are known to have an increased hypercoagulable state that result in excess fibrin deposition in several organs, which compromises their function. Tissue factor (TF) is the main physiological initiator of blood coagulation and its activity is regulated by a specific inhibitor known as Tissue factor pathway inhibitor (TFPI). Based on the important role of TF and TFPI in hemostasis, we hypothesize that their levels may change in the severe PE contributing to exacerbate hypercoagulable state. Some studies have assessed the balance between TF and TFPI in preeclamptic women, but results are inconsistent. Therefore, the aim of this study was to examine these inconsistencies and to assess TF and TFPI plasma levels in three groups of age matched women; pregnant with severe PE (n = 60), normotensive pregnant (n = 50) and normotensive non-pregnant women (n = 50). There was not significantly different among the three groups for TF plasma levels; severe PE women: 338.4 pg/mL (248.1-457.6), normotensive pregnant women: 301.5 pg/mL (216.4-442.9) and normotensive non-pregnant women 393 pg/mL (310.3-522.9). TFPI plasma levels were higher in severe PE comparing to normotensive pregnant women and normotensive non-pregnant women, 115.8 ng/mL (75-149.8); 80.3 ng/mL (59.6-99.7) and 74.5 ng/mL (47.1-98.0), respectively No difference was found between normotensive pregnant women and normotensive non-pregnant women. As for gestational age, a significant difference in TFPI levels was found between severe PE and normotensive pregnant women up to the 33rd week of pregnancy (p = 0.001), and severe PE and non-pregnant women up to the 34th (p = 0.01). In summary, our results indicated that TF plasma levels did not vary in the studied groups, while TFPI plasma levels were significantly increased in severe PE compared to normotensive pregnant and normotensive non-pregnant women. So, our data do not explain the exacerbated hypercoagulability state observed in severe PE. Further studies evaluating genes expression, TF activity and antigen, total and free TFPI and TFPI-2, both in plasma and obstetric tissues, throughout the pregnancy in PE (mild and severe forms) are required.

Hydroxyurea Reduces the Hypercoagulable State In Sickle Cell Anemia

Abstract 1068Sickle cell anemia (SCA) is associated with a hypercoagulable state, through mechanisms not yet clearly defined. SCA patients present an elevated rate of thrombotic complications and increased biological markers of coagulation activation. Currently one of the major pillars of SCA management is hydroxyurea (HU), a drug used primarily as an inductor of fetal hemoglobin (HbF), but with many other pleiotropic effects. Tissue factor (TF) is the major initiator of blood coagulation and has been shown to be up regulated in several inflammatory conditions. In the present study, we evaluated the effect of HU on coagulation activation by studying the expression of TF and final markers of coagulation activation: thrombin-antithrombin complex (TAT) and prothrombin fragment F 1+2 (F 1+2). We also correlated these measurements with HbF levels, lactate dehydrogenase (LDH), markers of endothelial activation (soluble thrombomodulin [sTM]) and inflammation (tumor necrosis factor-alpha [TNF-α] and white blood cell counts, including leukocyte, monocyte and neutrophil counts). We studied a cohort of 48 adult SCA patients (all with genotype SS), median age of 37 years (minimum: 20 – maximum: 50) and 25 healthy age and race matched controls. The patients included were all in steady state and 23 of them were receiving HU (SSHU). We analyzed leukocyte TF mRNA expression by real time quantitative RT-PCR and TF protein plasma levels by ELISA. TAT, F 1+2, sTM and TNF-α were all measured by ELISA. Statistical analyses were performed using Mann-Whitney’s U test and Spearman’s correlation test. Fisher’s exact test was used to compare plasma TF levels, on the basis of detectable levels. Leukocyte TF mRNA expression was up regulated in SCA patients, in comparison to healthy controls (5.29 vs. 1.16; P = 0.0005). HU was effective in inhibiting this expression significantly (5.29 vs. 2.34; P = 0.0083). These results were confirmed by the measurements of protein plasma levels of TF. Only 27.8% (5/18) of SSHU patients had detectable plasma levels of TF, in comparison to 78.5% (11/14) in the group without the drug (P = 0.01). SCA patients also showed higher levels of TAT (11.34 vs. 2.44; P <0.0001), F 1+2 (301.5 vs. 145.2; P = 0.0003), sTM (3.11 vs. 2.58; P = 0.0008) and TNF-α (2.49 vs. 0; P < 0.0001) when compared to controls. HU therapy was able to effectively reduce all of these markers (TAT: 11.34 vs. 6.53, P = 0.019; F1+2: 301.5 vs. 216.7, P = 0.05; sTM: 3.11 vs. 2.52, P = 0.0075; TNF-α: 2.49 vs. 0.27, P = 0.0003). Levels of TF mRNA showed a strong negative correlation with HbF levels (r=−0.47) and hemoglobin (r=−0.54), and a positive correlation with sTM (r=0.6), TNF-α (r= 0.52), leukocyte (r=0.41), neutrophil (r=0.46) and monocyte (r=0.54) counts (P values ≤0.01). TAT and F 1+2 presented a positive association with LDH and TAT also presented a significant negative association with HbF levels (P values <0.05). sTM showed a significant negative correlation with HbF and a positive correlation with LDH and inflammation markers (P values <0.05). Additionally, TNF-α presented a negative association with HbF and a positive association not only with TF and sTM, but also with LDH and white blood cell counts (P values < 0.001). Our results clearly demonstrated that HU therapy reduces the hypercoagulability encountered in SCA. We showed that HU was capable of inhibiting TF expression and decreasing both TAT and F1+2 levels, final markers of thrombin generation. In addition, HU reduced the endothelial marker sTM, as well as the pro-inflammatory marker TNF-α. Correlation analyses indicated that TF inhibition was proportional to the increase in HbF levels and a reduction in LDH, a relevant marker of hemolysis and disease severity in SCA. In parallel, TF down-regulation was associated with a reduction in the endothelial marker sTM, the inflammatory markers TNF-α and white blood cell counts. Hemolysis, endothelial activation and inflammation are pathways closely connected to each other and to the activation of coagulation. Thus, as HU is a drug capable of modulating all of these pathways, most likely all of these mechanisms are involved in the inhibitory effect of HU on activation of coagulation. Disclosures:No relevant conflicts of interest to declare.

A 24-h Work Shift in Intensive Care Personnel: Biological Pathways between Work Stress and Ill Health

This study evaluated inflammatory, coagulation and microvascular responses to a continuous 24-h work day in 13 healthy intensive care physicians. Inflammatory markers (interleukin [IL]-2, IL-6, IL-10, tumour necrosis factor-α, matrix metalloproteinase [MMP]-9 and adiponectin), adhesion molecules (vascular cellular adhesion molecule-1 and intercellular adhesion molecule-1 [ICAM-1]), coagulation parameters (thrombin-anti thrombin, von Willebrand factor and tissue factor) and sublingual micro circulation were assessed before and after a 24-h work shift. The 24-h work shift had no effect on inflammatory markers and ICAM-1. Direct visualization of micro-circulation did not reveal stress-related perfusion abnormalities. A 24-h work shift in the intensive care unit was associated with significantly increased plasma levels of tissue factor - a potentially important mechanism linking acute job strain, haemostasis and atherosclerosis. The long-term consequences warrant further evaluation.

Increased plasma level of asymmetric dimethylarginine in hypertensive rats facilitates platelet aggregation: role of plasma tissue factor

This study was designed to explore the role of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthesis, in platelet aggregation in hypertension and its possible mechanisms. Spontaneously hypertensive rats (SHR) and L-NAME-induced hypertensive rats were orally administered with L-arginine (1 g/(kg·day) for 14 days. Systolic blood pressure, platelet aggregation, and plasma tissue factor (TF) level and activity were measured. The plasma concentration of ADMA in SHR was determined. In vitro, platelet-rich plasma isolated from Wistar rats was prepared in order to observe the effect of exogenous ADMA on platelet aggregation and TF level and (or) activity in platelet-rich plasma. In both types of hypertensive rats, systolic blood pressure, platelet aggregation, and the level and activity of plasma TF were elevated compared with corresponding control animals. Plasma ADMA level was also increased in SHR. Treatment with L-arginine, a competitor of ADMA, lowered blood pressure and inhibited platelet aggregation concomitantly with a decrease in plasma TF level and activity in both types of hypertensive rats. We also found that exogenous ADMA promoted platelet aggregation and increased TF level and (or) activity in platelet-rich plasma, an effect that was inhibited by pretreatment with L-arginine. Importantly, the enhanced platelet aggregation induced by exogenous ADMA was reduced by pretreatment with anti-TF antibody. The results suggest that endogenous ADMA may be involved in platelet hyperaggregation status in hypertension, and the facilitation of platelet aggregation by ADMA is related to upregulation of the level and activity of plasma TF.

Silencing of tissue factor by antisense deoxyoligonucleotide prevents monocrotaline/LPS renal injury in mice

Tissue factor (TF) is involved in monocrotaline (MCT)/lipopolysaccharide (LPS) hepatotoxicity. It is not known whether MCT/LPS can cause renal toxicity and whether TF is involved in this toxicity. Thus, the present study was undertaken to investigate the potential renal toxicity after MCT/LPS co-treatment and the involvement of TF in this toxicity. MCT was delivered to ND4 male mice (200 mg/kg) per os followed 4 h later by treatment with LPS ip (6 mg/kg) to investigate its effect on kidney. We injected TF antisense oligonucleotide (TF-AS) intravenously (i.v) in mice prior to LPS treatment, to block TF, and measured their blood urea nitrogen (BUN), creatinine (CRE), alkaline phosphatase (ALP), and potassium. In MCT/LPS co-treated group, fibrin was detected on the glomerular capillary lumina, distal tubules of renal cortex, and the necrotic tubules of renal medulla. An elevation of BUN, creatinine, and the BUN/creatinine ratio was seen in mice with MCT/LPS co-treatment, compared to animals receiving LPS or MCT alone. Simultaneously, an aggressive tubular necrosis was seen in the medullary tubules in the same group which may account for the oliguria observed in these animals. Fourfold inductions in the plasma TF level was detected at 10 h after MCT/LPS co-treatment which increased to 18-fold at 24 h. Increased blood level of leptin, interleukin-6 (IL-6) and downregulation of tubular chemokine (C-X-C motif) ligand 16 (CXCL16) are characteristic features in MCT/LPS co-treated animal. On the other hand, mice injected with TF-AS in the presence of MCT/LPS co-treatment showed no elevation of the blood BUN, creatinine, potassium, and normal levels of the proinflammatory molecules. TF-AS injection significantly prevented glomerular and tubular fibrin deposition, tubular necrosis, and improvement of the animal survivability. Renal toxicity involving TF can be prevented successfully by the use of TF-AS.

Plasma Tissue Factor Levels and Salivary Tissue Factor Activities of Periodontitis Patients with and without Cardiovascular Disease

The association between periodontal and cardiovascular disease has received considerable attention. Studies have demonstrated a higher incidence of atherosclerotic complications in patients with periodontal disease. Tissue factor (TF) has been known as a key initiator of the coagulation cascade, and the TF pathway is the primary physiological mechanism of initiation of blood coagulation. Recently, it has been shown that the circulating pool of TF in blood is associated with increased blood thrombogenicity in patients with coronary artery disease (CAD). Various tissues and saliva have been known to have TF activity. Consequently, the aim of this study was to investigate plasma TF levels and TF activity of saliva in periodontitis patients with and without diagnosed CAD. Twenty-six patients with a diagnosis of CAD and 26 systemically healthy patients were examined in the dental clinic, and the Community Periodontal Index Treatment Needs (CPITN) scores were recorded. Plasma TF levels were determined using commercially available ELISA kit. Salivary TF activities were determined according to Quick’s one-stage method. Plasma TF levels were significantly increased in patients with CAD when compared with the control group. There was no difference in salivary TF activities between the 2 groups, but there was a strong and negative correlation between salivary TF activities and CPITN indexes in both groups. In order to determine the possible role of TF activity as a salivary marker in CAD and periodontitis and to fully understand the negative correlation between salivary TF activities and CPITN, TF activity of gingival crevicular fluid that may also affect saliva can be evaluated.

Plasma factor VII: A potential marker of pre-eclampsia

Introduction Normal pregnancy is associated with a local hypercoagulable state that becomes more profound in certain obstetric complications such pre-eclampsia (P-EC). Current literature on the levels of individual haemostatic factors in women with P-EC is limited and results are inconsistent. In this study we provide detailed investigation on the tissue factor (TF)-dependent pathway in women with P-EC. Materials and Methods Enzyme-linked immunosorbent assays (ELISA) were used to measure plasma factor (F) FVII, FVIIa, TF and tissue factor pathway inhibitor (TFPI) in healthy non-pregnant women (n = 22), normal pregnant women (n = 15), and women with P-EC (n = 20). All subjects were age matched. In addition, pregnant women were matched for gestational age, parity and were all at the third trimester. Results Plasma FVII levels were significantly higher in women with P-EC compared to the healthy non-pregnant (P < 0.001) or the normal pregnant groups (P < 0.001). No such significant trends were observed for plasma FVIIa, TF or TFPI levels. Plasma FVII levels can distinguish women with P-EC from healthy non-pregnant women or normal pregnant women at the third trimester, with high sensitivity (90%), specificity (80%), positive and negative predictive values (86%). Conclusions Plasma FVII levels are significantly elevated in women with P-EC, in the absence of comparable changes in other TF-dependent pathway factors (FVIIa, TF and TFPI). We propose the use of plasma FVII as a marker for P-EC.

Tissue Factor, Tissue Factor Pathway Inhibitor and Factor VII Activity in Cardiovascular Complicated Type 2 Diabetes Mellitus

Tissue factor (TF) is the main initiator of the extrinsic coagulation pathway through factor VII (FVII) activation, which is physiologically inhibited by tissue factor pathway inhibitor (TFPI). Alteration of this pathway has been described in Type 2 diabetes mellitus (T2DM). The aim of this study is to assess TF and TFPI plasma levels and FVII coagulant activity (FVIIa) in T2DM in relation to cardiothrombotic disease and their correlation to metabolic and clinical behavior of the patients. The study was conducted on 80 T2DM patients divided to accordingly; groupI: 40 patients without a history or clinically detected heart disease, and groupII: 40 patients with a history of myocardial infarction compared to 30 controls. The patients were recruited from Ain Shams University diabetes clinic from September 2007 to February 2009 after informed consent was obtained. Peripheral blood samples were taken for measurement of plasma TF and TFPI levels using ELISA technique and quantitative FVIIa using FVII deficient plasma. Plasma levels of TF, TFPI and FVIIa were significantly higher in T2DM patients compared to the controls (p<0.001). TF (236.50±79.23)and TFPI (242.33±85.84)were significantly higher in group II, compared to group I (150.33±81.16), (152.8± 82.46), (p<0.001). TF and TFPI were significantly correlated to body mass index and glycemic control. Also, TF and TFPI were significantly higher in hypertensives (p=0.001) and dyslipidemics (p=0.006) but not in smokers (p=0.64), (p=0.11) respectively. There was a correlation between high TF, TFPI plasma levels, FVIIa activity and cardiothrombotic complications in T2DM especially in the presence of high risk factors such as poor glycemic control, dyslipidemia and obesity. Future target therapy against TF may be beneficial for T2DM patients.

The Relation between Plasma Tissue Factor and Oxidized LDL Levels in Acute Coronary Syndromes

Aim: Tissue factor (TF) is a low-molecular-weight glycoprotein responsible for the initiation of the coagulation cascade. The relation between oxidized low-density lipoprotein (Ox-LDL), that has been shown to be involved in atherogenesis, and TF has not been evaluated before in circulating plasma. The aim of this study was to determine plasma levels of TF and Ox-LDL in acute coronary syndrome (ACS) and stable coronary artery disease (SCAD). Methods: The study group consisted of 41 patients with ACS and 26 patients with SCAD. Among the ACS patients, 12 were diagnosed with unstable angina pectoris (UAP) and 29 were diagnosed with acute myocardial infarction (AMI). The control group consisted of 30 healthy volunteers. TF and Ox-LDL levels were evaluated by ELISA kits. Results: Ox-LDL levels were significantly higher in UAP and AMI patients compared with the control (p < 0.001) and SCAD (p < 0.01 and p < 0.001, respectively) groups. TF levels were significantly higher in the UAP, AMI and SCAD groups compared with the control group (p < 0.001, p < 0.001 and p < 0.01, respectively). In the AMI group a significant increase was observed in TF levels when compared with the SCAD group (p < 0.01). Plasma Ox-LDL levels were significantly and positively correlated with TF levels in the UAP and AMI groups (p < 0.05, r = 702, and p < 0.0001, r = 0.679, respectively). Conclusion: Thepotential link between Ox-LDL and TF in circulating blood in ACS may strengthen the evidence supporting a relationship between oxidant stress, lipids and thrombosis and consequently may contribute to understanding the mechanism through which Ox-LDL and TF may mediate the pathogenesis of CAD.

The relationship between plasma and placental tissue factor, and tissue factor pathway inhibitors in severe pre-eclampsia patients

Objective To investigate the mechanism underlying the hypercoagulable state in severe pre-eclampsia. Methods Plasma tissue factor (TF) and tissue factor pathway inhibitor (TFPI) expression from pre-eclampsia patients and healthy pregnant controls were determined by ELISA. Placental TF and TFPI gene and protein expression were detected by quantitative RT-PCR, immunohistochemistry, and Western analysis. Results The plasma TF level in the pre-eclampsia group was significantly higher than the control group (p < 0.01), and surprisingly, the plasma TFPI-1 and TFPI-2 in the pre-eclampsia group were significantly lower (p < 0.01). Placental TF gene and protein expression levels in the pre-eclampsia group was significantly higher than the control group, while TFPI-1 and TFPI-2 levels were significantly lower (p < 0.05). Lastly, a significant correlation was found between plasma and placental TF protein levels in the pre-eclampsia group (p < 0.01). Conclusion Higher expression and/or release of TF from the placenta may contribute towards a pathological hypercoagulable state in pre-eclampsia patients.

Venous Thrombosis and Procoagulant Factors in High-risk Neuroblastoma

The mechanism of increased thrombin production has been investigated in children with high-risk neuroblastoma (NB), to detect any possible association between catheter- related venous thrombosis (VT) and prothrombotic factors. Consecutive children with high-risk NB were studied by color-doppler ultrasonography of the upper vein system and thrombophilia factors assessment. Plasma levels of Tissue Factor (TF), Vascular Endothelial Growth Factor (VEGF), Prothrombin Activation Fragment 1+2, and Thrombin-Antithrombin Complex were evaluated. Moreover, inherited thrombophilia factors (homocystein, antithrombin, protein C, protein S, factor V Leiden, activated protein C resistence, mutation H1299R and G1691A of factor V, mutation G20210A of prothrombin, mutation T677 and A1298C of methylenetetrahydrofolate reductase, and allele 4G of plasminogen activator inhibitor-1) were tested to exclude congenital disorders. Six patients with mean age: 48.8 months---were studied. Five patients were affected by stage 4 NB and another one by stage 3 NB with Myc-N amplification. All children had a central venous line (mean duration: 8.5 mos). Four patients (67%) had asymptomatic catheter-related VT visualized by color-doppler ultrasonography. No patient had major inherited thrombophilia factors. The levels of plasma TF and plasma VEGF were found elevated in all patients. Mean value of TF (nv 20.3+/-6.6) was 82 pg/mL with a range of 39 to 131 pg/mL. Mean value of VEGF (nv 24.3 pg/mL) was 78.5 pg/mL with a range of 31 to 142 pg/mL. The increased risk of catheter-related VT detected in our small series of high-risk NB patients, was associated with elevated levels of circulating TF and VEGF. Further studies are needed to evaluate if elevated levels of TF/VEGF are involved both in the hypercoagulable state and in advanced childhood cancer.

Increased levels of tissue factor activity and procoagulant phospholipids during treatment of children with acute lymphoblastic leukaemia

The use of L-asparaginase (L-ASP) in paediatric patients with acute lymphoblastic leukaemia (ALL) is associated with thrombotic complications. We evaluated the activities of tissue factor (TFa), thrombomodulin (TMa) and procoagulant phospholipids (PPL) in 26 consecutive children with ALL (25 B-ALL and one T-ALL) treated by the French Acute Lymphoblastic Leukemia group (FRALLE)-2000 protocol. Samples were obtained at diagnosis, after glucocorticoid (GC) therapy, during the induction phase with L-ASP, vincristine (VCR) and adriamycin (ADR), during the re-induction and within the week after treatment. Plasma levels of TFa, TMa and PPL increased gradually and significantly during the different phases of the treatment, with higher levels observed during the induction period, and decreased after treatment discontinuation. In vitro studies showed that the different drugs used for ALL treatment could induce a weak expression of TF and procoagulant activity (PCA) on normal and leukaemia blood cells, while a marked effect was observed on endothelial cells. In conclusion, these data indicate that, in addition to the well-identified increased in coagulation factors and inhibitor deficiencies, the injury of the endothelium could lead to the release of TF and PPL and could contribute to the hypercoagulability of children treated for ALL.

Detection of endogenous tissue factor levels in plasma using the calibrated automated thrombogram assay

Detection of endogenous tissue factor levels in plasma using the calibrated automated thrombogram assay

Plasma thrombomodulin activity, tissue factor activity and high levels of circulating procoagulant phospholipid as prognostic factors for acute myocardial infarction

Several studies have indicated an association between haemostatic markers and acute myocardial infarction, but few or no studies refer to their activity. We studied plasma levels of 10 coagulation factors (fibrinogen, protein C, protein S, von Willebrand factor, D-dimers, factor VIIa, free tissue factor pathway inhibitor, tissue-type plasminogen activator, plasminogen activator inhibitor-1, thrombomodulin) and using new specific assays analysed the activity of plasma tissue factor (TFa), thrombomodulin (TMa), and procoagulant phospholipid in 46 consecutive patients with acute myocardial infarction at the time of hospital admission, and compared them with 34 healthy normal volunteers. Plasma levels of TFa, TMa, and procoagulant phospholipid were significantly higher in cases than in control patients (P < 0.001). In addition the ratio of TFa/free tissue factor pathway inhibitor was higher in patients than in controls, whereas the tissue-type plasminogen activator (t-PA)/plasminogen activator inhibitor-1 ratio was lower in patients. Interestingly, patients with an unfavourable outcome during a 2-month follow-up had higher levels of TFa, TMa, procoagulant phospholipid, a higher ratio of TFa/free tissue factor pathway inhibitor and a lower ratio of t-PA/plasminogen activator inhibitor-1 than patients who recovered. The combination of these different parameters reveals an increase in procoagulant activity as well as impaired fibrinolytic activity during the acute phase of an acute myocardial infarction. The association of the level of the activity of these three factors may provide a new tool to assess the prognosis of acute myocardial infarction. Further studies are needed to support our findings and to elucidate the clinical interest of measuring these factors.

Abstract 4939: Hypercholesterolemia Increases Microparticle Tissue Factor and Activates Coagulation in LDL Receptor Deficient Mice

Objective: Tissue factor (TF) is the initiator of the extrinsic pathway of coagulation and contributes to thrombosis. Previous studies demonstrated that the risk of arterial thrombosis is elevated in patients with increased plasma cholesterol concentrations. In addition, increased levels of circulating TF, bound to microparticles (MPs), may induce thrombosis. Therefore, we tested the hypothesis that hypercholesterolemia increases plasma levels of MP TF and activation of coagulation in low density lipoprotein receptor deficient (LDLr −/− ) mice. Methods and Results: Male LDLr −/− mice were fed a high fat diet (HFD) consisting of 21% saturated fat, and 0.15% cholesterol for 0, 1, 2, 4, and 12 weeks (n=3–7 each time point). Mice were then euthanized, blood was collected from the inferior vena cava (into 3.8% sodium citrate), and plasma prepared. Mice had a sustained elevation in plasma cholesterol concentrations after 1 week of feeding HFD, which were unchanged in all subsequent feeding intervals (945±35 mg/dL). MP TF was analyzed by detection of Factor Xa generation in the presence or absence of an inhibitory rat anti mouse TF antibody. MP TF activity increased in a time-dependent manner at 1 (75%), 2 (112%), 4 (650%), and 12 (1140%) weeks (all times versus 0 week control; P&lt;0.001). Importantly, a similar time-dependent increase in plasma thrombin-antithrombin (TAT) activity was observed at 2 (362%), 4 (1203%), and 12 (927%) weeks (all times versus 0 week control; P&lt;0.001). Moreover, the levels of MP TF activity and TAT were linearly correlated (R=0.70, P&lt;0.001). The inflammatory cytokine IL-6 was also increased in a time-dependent manner from 0 weeks (22.7±10.1 pg/ml) to 12 weeks (112.3±20.2 pg/ml; P&lt;0.023). Conclusion: These data indicate that hypercholesterolemia results in increased MP TF activity and subsequently activates coagulation. Further studies are required to determine if TF positive MPs are required for hypercholesterolemia -induced coagulation.

Tissue factor gene promoter haplotype associates with carotid intima-media thickness in subjects in cardiovascular risk prevention

Objective Tissue factor (TF), key initiator of coagulation, has been ascribed roles not only in thrombosis but also in atherosclerosis. TF gene promoter haplotypes modulate TF expression, thereby potentially affecting atherosclerosis. The objective of this study was to evaluate functionally relevant TF promoter haplotypes as determinants of carotid intima-media thickness (C-IMT), a marker of atherosclerosis. Methods The haplotype-tagging TF A-603G polymorphism and C-IMT were determined in 611 subjects referred to cardiovascular risk prevention. Subjects were aged 59.7 (58.1–61.1) years (mean (95% C.I.)), 79% were male, and 74% in secondary prevention with a history of coronary, cerebrovascular, or peripheral atherosclerotic disease. TF plasma levels were measured in 120 subjects. Results Significant dose-dependent associations were found between A-603G genotype and both IMT max and IMT mean-max after adjusting for potential confounders. IMT values were highest in A/A ( n = 173), intermediate in A/G ( n = 312) and lowest in G/G ( n = 126). IMT max values (average and 95% C.I., in mm) for A/A, A/G and G/G, were 2.22 (2.11–2.34), 2.09 (2.01–2.18) and 2.02 (1.90–2.15), respectively ( p trend = 0.019), and IMT mean-max values were 1.28 (1.23–1.32), 1.25 (1.22–1.28) and 1.21 (1.16–1.26), respectively ( p trend = 0.041). A significant interaction between A-603G genotype and prevention status was found ( p = 0.046 and p = 0.042 for IMT max and IMT mean-max, respectively). TF plasma levels did not differ between genotypes and were not determinants of C-IMT. Conclusion The haplotype-tagging TF A-603G polymorphism is associated with C-IMT, independently of TF levels in plasma. These findings provide clinical evidence of an involvement of TF in atherosclerosis, in addition to its well-known roles in hemostasis and thrombosis.

Detection of endogenous tissue factor levels in plasma using the calibrated automated thrombogram assay

Background The calibrated automated thrombogram (CAT) assay measures thrombin generation in plasma. Objective Use the CAT assay to detect endogenous tissue factor (TF) in recalcified platelet-rich plasma (PRP) and platelet-free plasma (PFP). Methods Blood from healthy volunteers was collected into citrate and incubated at 37 °C with or without lipopolysaccharide (LPS) for 5 hours. PRP and PFP were prepared and clotting was initiated by recalcification. Thrombin generation was measured using the CAT assay. Results The lag time (LT) was significantly shortened in PRP prepared from LPS-treated blood compared with untreated blood (10 ± 3 min versus 20 ± 6 min), and this change was reversed by the addition of inactivated human factor VIIa. LPS stimulation did not change the peak thrombin. Similar results were observed in PFP (21 ± 4 min versus 35 ± 5 min). LPS stimulation also significantly reduced the LT of PRP and PFP derived from blood containing citrate and a factor XIIa inhibitor. Finally, a low concentration of exogenous TF shortened the LT of PFP prepared from unstimulated, citrated blood without affecting the peak thrombin. Conclusion Changes in LT in the CAT assay can be used to monitor levels of endogenous TF in citrated plasma.

Time-course of tissue factor plasma level in patients with acute coronary syndrome

Enhanced expression of tissue factor (TF) may result in thrombosis contributing to acute clinical consequences of coronary artery disease. Several studies demonstrated elevated plasma levels of TF in patients with acute coronary syndrome (ACS). The aim of our study was to compare the concentrations of TF in coronary sinus (CS), proximal part of the left coronary artery (LCA) and peripheral vein (PV) of patients with ACS and stable coronary artery disease (SCAD). Time course of the TF plasma levels in PV was followed on day 1 and day 7 after index event of ACS presentation and was compared to day 0 values. No heparin was given prior to the blood sampling. Twenty-nine patients in the ACS group (age 63.6+/-10.8 years, 20 males, 9 females) and 24 patients with SCAD (age 62.3+/-8.1 years, 21 males, 3 females) were examined. TF plasma level was significantly higher in patients with ACS than in those with SCAD (239.0+/-99.3 ng/ml vs. 164.3+/-114.2 ng/ml; p=0.016). There was no difference in TF plasma levels in PV, CS and LCA (239.0+/-99.3 ng/ml vs. 253.7+/-131.5 ng/ml vs. 250.6+/-116.4 ng/ml, respectively). TF plasma levels tended to decrease only non-significantly on the day 7 (224.4+/-109.8 ng/ml). Significant linear correlation between TF and high sensitivity CRP (hs-CRP) levels on day 0 was found. In conclusion, TF plasma levels are elevated in patients with ACS not only locally in CS but also in systematic circulation. Our data support the relationship between TF production and proinflammatory mediators.

Platelet turnover, coagulation factors, and soluble markers of platelet and endothelial activation in essential thrombocythemia: Relationship with thrombosis occurrence and JAK2 V617F allele burden

Patients with essential thrombocythemia (ET) have an increased frequency of thrombosis, but the relationship of both thrombosis and JAK2 V617F allele burden with platelet turnover, acquired activated protein C resistance (aAPCR), and levels of coagulation factors and soluble markers of platelet, and endothelial activation is not well known. In 53 ET patients (26 with a history of thrombosis), reticulated platelets (RP) percentage, aAPCR, platelet tissue factor (TF) expression, and plasma levels of TF, coagulation factors, soluble P-selectin (sP-selectin), soluble CD40 ligand (sCD40L), von Willebrand factor antigen (VWF:Ag), soluble thrombomodulin (sTM), D-dimer and prothrombin fragment 1 + 2 were compared with those in matched healthy individuals and correlated with thrombosis occurrence and JAK2 mutational load. ET patients with thrombosis had significantly higher values for RP percentage, aAPCR, and levels of factors V and VIII, VWF:Ag, sP-selectin, and sCD40L than patients without thrombosis and controls. At multivariate study, RP percentage, factor V levels, and aAPCR were independently associated with an increased risk of thrombosis. Patients with JAK2 mutation had significantly lower levels of free protein S (PS) and higher levels of TF, sP-selectin, sCD40L, VWF:Ag, and sTM than those with wild-type allele. A mutant allele dosage effect (>or= 12%) was observed for TF, sP-selectin, sCD40L, VWF:Ag, and PS levels. These results support a role for platelet turnover, factor V, and aAPCR in the thrombosis of ET as well as the association between JAK2 V617F allele burden and either decreased free PS or increased TF and soluble markers of platelet and endothelial activation.