Tissue Factor Levels Research Articles

Coagulation, Protease-Activated Receptors, and Diabetic Kidney Disease: Lessons from eNOS-Deficient Mice.

Endothelial nitric oxide synthase (eNOS) dysfunction is known to exacerbate the progression and prognosis of diabetic kidney disease (DKD). One of the mechanisms through which this is achieved is that low eNOS levels are associated with hypercoagulability, which promotes kidney injury. In the extrinsic coagulation cascade, the tissue factor (factor III) and downstream coagulation factors, such as active factor X (FXa), exacerbate inflammation through activation of the protease-activated receptors (PARs). Recently, it has been shown that the lack of or reduced eNOS expression in diabetic mice, as a model of advanced DKD, increases renal tissue factor levels and PAR1 and 2 expression in their kidneys. Furthermore, pharmaceutical inhibition or genetic deletion of coagulation factors or PARs ameliorated inflammation in DKD in mice lacking eNOS. In this review, we summarize the relationship between eNOS, coagulation, and PARs and propose a novel therapeutic option for the management of patients with DKD.

BAY11-7082 inhibits the expression of tissue factor and plasminogen activator inhibitor-1 in type-II alveolar epithelial cells following TNF-α stimulation via the NF-κB pathway.

Pulmonary inflammation strongly promotes alveolar hypercoagulation and fibrinolytic inhibition. NF-κB signaling regulates the expression of molecules associated with coagulation and fibrinolytic inhibition in type-II alveolar epithelial cells (AECII) stimulated by lipopolysaccharide. However, whether TNF-α-induced alveolar hypercoagulation and fibrinolysis inhibition is also associated with the NF-κB pathway remains to be determined. The aim of the present study was to determine whether BAY11-7082, an inhibitor of the NF-κB pathway, inhibits the expressions of tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1) in AECⅡ in response to TNF-α. Rat AECII were treated with BAY11-7082 for 24 h and stimulated with TNF-α for 1 h. The expression of TF and PAI-1 were determined using western blotting and reverse transcription-quantitative PCR. The concentrations of TF and PAI-1 in culture supernatant were also measured by ELISA. Moreover, levels of NF-κB p65 (p65), phosphorylated (p)-p65 (p-p65), inhibitor of NF-κB α (IκBα) and p-IκBα were also evaluated. Immunofluorescence was used to detect p65 levels in cell nuclei. TNF-α significantly promoted TF and PAI-1 expression either at the mRNA or protein level in AECII cells. Concentrations of TF and PAI-1 in supernatant also significantly increased upon TNF-α stimulation. Furthermore, TNF-α upregulated the levels of p-IκBα, p65, and p-p65 in the cytoplasm. Immunofluorescence analysis indicated that TNF-α increased p65 translocation from the cytoplasm to the nucleus. However, AECII pre-treated with BAY11-7082 expressed lower levels of TF and PAI-1 following TNF-α treatment. Levels of p-IκBα, p65 and p-p65 in the cytoplasm also decreased, and translocation of p65 from cytoplasm into the nucleus was inhibited by BAY11-7082 pretreatment. These findings suggest that BAY11-7082 improves the hypercoagulation and fibrinolytic inhibition induced by TNF-α in alveolar epithelial cells via the NF-κB signaling pathway. BAY11-7082 might represent a therapeutic option for alveolar hypercoagulation and fibrinolytic inhibition in acute respiratory distress syndrome.

Effects of Astragalus Polysaccharides Nanoparticles on Cerebral Thrombosis in SD Rats.

ObjectiveTo investigate the efficacy and improvement of Astragalus polysaccharides (APS) and APS-nano on cerebral thrombosis in rats.MethodsA total of 72 SD rats were randomly divided into NC group, Model group, APS-Nano group, and APS group. The cerebral thrombosis Model of SD rats was established by injecting compound thrombus inducer into the internal carotid artery. After 14 days of different intervention treatments, the TTC staining of brain tissue were performed, and A/left brain wet weight ratio, left brain/right brain wet weight ratio, blood rheology indexes, and coagulation function indexes of cerebral thrombosis were measured. ELISA was used to measure the contents of thromboxane 2 (TXB2), 6-keto-prostaglandin F1α (6-Keto-PGF1α), tissue factor (TF), neuron-specific enolase (NSE), S-100β, catenin (CAT), superoxide dismutase (SOD), as well as malondialdehyde (MDA). The binding specificity between miR-885-3p and TF was verified by the double-luciferin reporting experiment, and western blot was used to measure the expression level of TF protein.ResultsCompared with the Model group, after treatment with APS-nano or APS, the ratio of left brain/right brain wet weight decreased significantly. Whole blood low shear viscosity (WBLSV), whole blood high shear viscosity (WBHSV), plasma viscosity (PV), and erythrocyte aggregation index (Arbc) was all reduced. In addition, prothrombin time (PT) and activated partial thromboplastin time (APTT) were increased, and fibrinogen (FIB) content was decreased. The expression of TXB2, 6-Keto-PGF1α, and TF showed a downward trend. Similarly, the expression of TF protein was decreased. Furthermore, the contents of NSE and S-100β proteins were all decreased, whereas the contents of CAT and SOD were increased, and the contents of MDA was decreased. At the same dose, compared with APS treatment, APS-nano treatment had a significant inhibitory effect on cerebral thrombosis in rats. Finally, we found that TF is a target gene of miR-885-3p and specifically binds to miR-885-3p.ConclusionAPS has a significant inhibitory effect on the formation of cerebral thrombosis induced by compound thrombus inducers. Moreover, APS-nano has a more significant inhibitory effect on cerebral thrombosis. Meanwhile, the regulation of miR-885-3p regulating TF expression may be related to the occurrence of cerebral thrombosis.

The Level of Certain Interleukins and Tissue Factor in Conditions of Intestinal Ischemia and Reperfusion in Rats

Background: The aim of this study was to estimate the blood levels of interleukins (ILs) and tissue factor (TF) in rat blood when modeling acute mesenteric ischemia (AMI) in various stages and during reperfusion. Methods and Results: The study was performed on white non-linear male rats, weighing 200±25g. AMI was simulated by ligating the cranial mesenteric artery at the mesentery root. Then, at certain time intervals, a relaparotomy was performed, blood was collected, and the animals were subjected to reperfusion at different time intervals. During the experiment, the animals were divided into 3 groups depending on the time of ischemia (3 hours [n=10], 6 hours [n=10], and 8 hours [n=10]) followed by reperfusion after the indicated time of ischemia. The level of ILs (IL-6, IL-8, and IL-10) and TF in rat blood serum was determined by EIA. We found that AMI in rats is accompanied by pronounced changes in the immune system and the development of a systemic inflammatory reaction syndrome, which is aggravated by reperfusion. At the same time, the restoration of blood flow after 3-hour ischemia was characterized by an increase in the concentration of IL-6, IL-8, and IL-10; after 6-hour ischemia – by an increase in the IL-6, IL-8 content and TF, and stabilization of the IL-10 concentration. Reperfusion after 8-hour ischemia was accompanied by an increase in the IL-6 concentration, a decrease in the levels of IL-8, IL-10, and TF, which can lead to the progression of necrotic changes in the intestine. Conclusion: The detected changes can serve as laboratory markers that characterize the course and stage of acute mesenteric ischemia.

Progress curve analysis of microtitre plate plasma clotting assays. Assessment of tissue factor levels

MTP plasma clotting assays monitor the time course of fibrin formation in re-calcified plasma by absorbance measurements and are increasingly used as alternatives to traditional one-point clot time assays employed in clinical laboratories to detect thrombotic disorders. The parameters derived from these analyses are analogous to thromboelastography viz. time, rate and maximum extent of clot formation. The derived parameters, based on the whole course of the clotting reaction are more robust, informative and quantitative than single-point clot time assays. However, the parameters themselves are usually obtained arbitrarily by crude graphical analysis of subjectively selected points of progress curves. The current work aimed to investigate the sensitivity and reproducibility of an MTP clotting assay and examine its suitability for measuring tissue factor (TF) levels in cell culture medium and patient urine. The results demonstrate that progress curves can be analysed by fitting a logistic equation, derived from a simplified autocatalytic clot formation model. The parameters, maximum amplitude (Fm), rate constant (k), time to half-maximum amplitude (tm) and maximum rate of clot formation (vm), fit a power curve showing limiting effects with increasing TF concentration. Log/log plots of tm and k against TF concentration provide standard curves for assessment of unknowns.

Coagulation Changes during Central Hypovolemia across Seasons.

Lower body negative pressure (LBNP) application simulates hemorrhage. We investigated how seasons affect coagulation values at rest and during LBNP. Healthy participants were tested in cold (November–April) and warm (May–October) months. Following a 30-min supine period, LBNP was started at −10 mmHg and increased by −10 mmHg every five minutes until a maximum of −40 mmHg. Recovery was for 10 min. Blood was collected at baseline, end of LBNP, and end of recovery. Hemostatic profiling included standard coagulation tests, calibrated automated thrombogram, thrombelastometry, impedance aggregometry, and thrombin formation markers. Seven men (25.0 ± 3.6 years, 79.7 ± 7.8 kg weight, 182.4 ± 3.3 cm height, and 23.8 ± 2.3 kg/m2 BMI) and six women (25.0 ± 2.4 years, 61.0 ± 8.4 kg weight, 167 ± 4.7 cm height, and 21.8 ± 2.4 kg/m2 BMI) participated. Baseline levels of prothrombin (FII), tissue factor (TF) and markers for thrombin generation F1+2 and the thrombin/antithrombin complex (TAT) were higher during summer. Factor VIII, prothrombin fragment 1+2 (F1+2), TAT and the coagulation time showed significant increases during LBNP in both seasons. Some calibrated automated thrombography variables (Calibrated automated thrombography (CAT): lag, time to peak (ttPeak), peak) shifted in a procoagulant direction during LBNP in summer. Red blood cell counts (RBC), hemoglobin and white blood cell counts (WBC) decreased during LBNP. LBNP application reduced prothrombin time in winter and activated partial thromboplastin time in summer. Greater levels of FII, TF, F1+2, and TAT—a more pronounced LBNP-induced procoagulative effect, especially in CAT parameters (lag time (LT), Peak, ttPeak, Velindex)—were seen in summer. These results could have substantial medical implications.

Oxidative Stress Product, 4-Hydroxy-2-Nonenal, Induces the Release of Tissue Factor-Positive Microvesicles From Perivascular Cells Into Circulation.

TF (Tissue factor) plays a key role in hemostasis, but an aberrant expression of TF leads to thrombosis. The objective of the present study is to investigate the effect of 4-hydroxy-2-nonenal (HNE), the most stable and major oxidant produced in various disease conditions, on the release of TF+ microvesicles into the circulation, identify the source of TF+ microvesicles origin, and assess their effect on intravascular coagulation and inflammation. Approach and Results: C57BL/6J mice were administered with HNE intraperitoneally, and the release of TF+ microvesicles into circulation was evaluated using coagulation assays and nanoparticle tracking analysis. Various cell-specific markers were used to identify the cellular source of TF+ microvesicles. Vascular permeability was analyzed by the extravasation of Evans blue dye or fluorescein dextran. HNE administration to mice markedly increased the levels of TF+ microvesicles and thrombin generation in the circulation. HNE administration also increased the number of neutrophils in the lungs and elevated the levels of inflammatory cytokines in plasma. Administration of an anti-TF antibody blocked not only HNE-induced thrombin generation but also HNE-induced inflammation. Confocal microscopy and immunoblotting studies showed that HNE does not induce TF expression either in vascular endothelium or circulating monocytes. Microvesicles harvested from HNE-administered mice stained positively with CD248 and α-smooth muscle actin, the markers that are specific to perivascular cells. HNE was found to destabilize endothelial cell barrier integrity. HNE promotes the release of TF+ microvesicles from perivascular cells into the circulation. HNE-induced increased TF activity contributes to intravascular coagulation and inflammation.

Human monocyte-derived macrophages: Pathogenetic role in plaque rupture associated to systemic inflammation

BackgroundMacrophages play a key role in coronary plaque destabilization. In-vitro human monocyte-derived macrophages (MDMs) are used to study macrophages infiltrating tissue. Optical coherence tomography (OCT) provides an in-vivo insight of the coronary arteries. We compared the MDMs morpho-phenotype and culprit plaque features at OCT in acute coronary syndrome (ACS) patients according to the underlying plaque pathobiology. MethodsSixty-six patients undergoing coronary angiography and pre-angioplasty OCT of the culprit vessel were allocated to three groups according to mechanism of ACS at OCT and C-reactive protein levels (cut-off: 2 mg/Ll): 1) plaque rupture with systemic inflammation; 2) plaque rupture without systemic inflammation, 3) plaque with intact fibrous cap. A blood sample was collected to obtain MDMs, categorized as having “round” or “spindle” morphology. ResultsThirty-two patients (48.5%) were assigned to Group 1, 10 (15.2%) to Group 2 and 24 (36.4%) to Group 3. The “round” MDMs were significantly more frequent in Group 1 (39.25 ± 4.98%) than in Group 2 (23.89 ± 3.10%) and Group 3 (23.02 ± 7.89%), p = 0.008. MDMs in Group 1 as compared to Groups 2 and 3 showed lower efferocytosis (8.74 ± 1.38 vs 9.74 ± 2.15 vs 11.41 ± 2.41; p = 0.012), higher tissue factor levels (369.84 ± 101.13 vs 301.89 ± 59.78 vs 231.74 ± 111.47; p = 0.001) and higher heme oxygenase-1 expression (678.78 ± 145.43 vs 419.12 ± 74.44 vs 409.78 ± 64.33; p = 0.008). ConclusionsMDMs of ACS patients show morpho-phenotypic heterogeneity with prevalence of pro-thrombotic and pro-oxidative properties in case of plaque rupture and systemic inflammation. Such MDMs subpopulation may take part to the cellular pathways leading to fibrous cap rupture with the subsequent thrombus formation.

Elevated Levels of Procoagulant Microvesicles in Patients With Dengue Fever

Background: The levels of procoagulant microvesicles (MVs) and tissue factor (TF)-bearing MVs may be increased in many conditions, including dengue fever (DF). This study aimed to measure the levels of MVs and TF-bearing MVs in patients with DF and matched healthy controls. Materials & methods: Levels of MVs and TF-bearing MVs in the plasma of patients with DF and matched healthy controls were measured using functional assay. Results: The patient group had significantly elevated levels of MVs (p < 0.001) and slightly increased levels of TF-bearing MVs (p = 0.454) compared with the matched healthy controls. Conclusion: Elevated levels of MVs and TF-bearing MVs could be used as biomarkers to evaluate the hemostatic function of patients with DF.

Mathematical Models for the Prediction of Coagulation Activity in Patients with Paroxysmal Atrial Fibrillation

Our previous studies showed activation of coagulation in the early hours of the clinical manifestation of paroxysmal atrial fibrillation (PAF). Plasma coagulation activity of factor II, factor V, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII, vWF, tissue factor levels, FVIII, vWF, prothrombin fragment 1+2(F1 + 2) and fibrinopeptide A (FPA) were significantly increased as early as the first twenty-four hours of the disease. The results suggest that there is a correlation between the studied parameters and development of the disease. Aim: To search for a statistical model that predicts coagulation activity in PAF patients. Materials and methods: Coagulation parameters were examined in 51 PAF patients (26 males, 25 females; mean age 59.84 ±1.60 years, onset of PAF episode < 24h prior to hospitalization). Controls included 52 individuals (26 males, 26 females; mean age 59.50 ± 1.46 years) with no prior anamnestic or ECG AF data, corresponding to patients in sex, age, BMI and comorbidities. A linear regression model was used to predict coagulation activity in PAF. Regression models showed good correlation between the duration of arrhythmia and six of the fourteen coagulation parameters studied: F1+2 (r = 0.83, p <0.001), FPA (r = 0.84, p <0.001), FVIII levels (r = 0.85, p <0.001) as well as activity of FII (r = 0.83, p <0.001), FVIII (r = 0.83, p <0.001) and FXII (r = 0.78, p <0.001). Changes in F1+2 plasma levels were most sensitive to PAF duration, where the contribution of duration to the values of the indicator is the greatest (b = 15.31). Conclusion: Linear regression analysis allowed us to create models with a high correlation coefficient for predicting the values of F1+2, FPA, FVIII levels, as well as activity of FII, FVIII and FXII in PAF patients. These models could allow for quantification of the procoagulatory process and thrombotic potential of the disease.

Prognostic significance of tissue factor in patients with pancreatic cancer: a systematic review protocol.

IntroductionPancreatic cancer is a highly aggressive digestive system tumour with poor prognosis. Venous thromboembolism (VTE) is a well-known complication of pancreatic cancer, and tissue factor (TF) contributes to the generation of a hypercoagulable state and thrombotic disease in pancreatic cancer. Several studies showed that an elevated TF level was related to the development of VTE and influenced the survival of patients with pancreatic cancer. Thus, we wish to conduct a systematic review of literature to clarify the prognostic significance of TF in pancreatic cancer.Methods and analysisStudies comparing the circulating microparticle-associated TF (MP TF) level between patients who had pancreatic cancer with and without VTE will be included to evaluate the roles of TF in VTE development. Studies comparing the survival data between patients with high TF expression and low TF expression will also be included to explore the association of TF expression with patient survival. The outcomes are plasma MP TF level and survival endpoints (overall and progression-free survival), respectively. Primary studies of any type published in English will be included. Two reviewers will search Medline, EMBASE and Cochrane databases from inception to June 2020, retrieve relevant studies, and independently select the literatures and extract data from the included studies. The quality of each included study will be assessed by the Newcastle-Ottawa Scale score. The HR and 95% CI of each study will be pooled for survival outcome, and the standardised mean difference (SMD) with 95% CIs will be used for continuous outcomes. If meta-analysis is inappropriate, the result will only be reported qualitatively. Subgroup and sensitivity analyses will be considered to identify sources of heterogeneity. The Grades of Recommendation, Assessment, Development and Evaluation method will be applied to assess the level of evidence of this systematic review.Ethics and disseminationThere are no concerning ethical issues. The results will be published.PROSPERO registration numberCRD42019133665.

Bleeding Disorders in Bothrops atrox Envenomations in the Brazilian Amazon: Participation of Hemostatic Factors and the Impact of Tissue Factor.

Bleeding is a common hemostatic disorder that occurs in Bothrops envenomations. We evaluated the changes in coagulation, fibrinolysis components, and platelets in Bothrops atrox envenomations with bleeding. This is an observational study with B. atrox snakebite patients (n = 100) treated in Manaus, Brazilian Amazon. Bleeding was recorded on admission and during hospitalization. We found that the platelet count in our patients presented a weak correlation to tissue factor, factor II, and plasminogen. Tissue factor presented weak correlation to factor V, II, D-dimer, plasminogen, alpha 2-antiplasmin, and moderate correlation to fibrinogen and fibrin/fibrinogen degradation product (FDP). Patients with systemic bleeding (n = 20) presented low levels of factor V, II, fibrinogen, plasminogen, and alpha 2-antiplasmin, and high levels of tissue factor and FDP compared to those without bleeding. Patients with only local bleeding (n = 41) and without bleeding showed similar levels of hemostatic factors. Thrombocytopenia was observed mainly in patients with systemic bleeding and increased levels of serum venom. No association was found between venom levels and systemic bleeding, or between venom levels and clinical severity of envenomation. This is the first report that shows the participation of the extrinsic coagulation pathway in the consumption coagulopathy of B. atrox envenomations with systemic bleeding due to tissue factor release.

Extracellular Vesicles Reflect the Efficacy of Wheatgrass Juice Supplement in Colon Cancer Patients During Adjuvant Chemotherapy.

IntroductionColorectal cancer (CC) is the third most common type of cancer, accounting for 10% of all cancer cases. Adjuvant chemotherapy is recommended in stages II–III CC. Wheatgrass juice (WGJ) from wheat seeds has high nutritional values, may induce synergistic benefits to chemotherapy and may attenuate chemotherapy-related side effects. Extracellular vesicles (EVs) are subcellular membrane blebs. EVs include exosomes (generated in the endosome, in size <150 nm) and microvesicles (shed from the plasma cell membrane) provide information on their parental cells and play a role in intercellular communication. We aimed to elucidate the effects of chemotherapy administration with supportive treatment of WGJ on CC patients’ EVs characteristics.MethodsEVs were isolated from the blood samples of 15 healthy controls (HCs) and 50 CC patients post-surgery, treated by chemotherapy, with or without additional daily WGJ. Blood samples were taken before, during, and at the end of chemotherapy. EVs were characterized by size, concentration, membrane antigens and cytokine content using nanoparticle-tracking analysis, western blot, flow cytometry, and protein array methods.ResultsEVs were found to be similar by size and concentration with reduced levels of exosome markers (CD81) on samples at the end of combined treatment (chemotherapy and WGJ). Higher levels of endothelial EVs, which may indicate impairment of the vascular endothelial cells during treatment, were found in CC patients treated by chemotherapy only compared to those with chemotherapy and daily WGJ. Also, EVs thrombogenicity was lower in patients added WGJ compared to patients who had only chemotherapy (levels of tissue factor p = 0.029 and endothelial protein C receptor p = 0.005). Following treatments, levels of vascular endothelial growth factor receptors (VEGFR-1) and the majority of growth-factors/pro-inflammatory cytokines were higher in EVs of patients treated by chemotherapy only than in EVs obtained from patients with the combined treatment.ConclusionDaily consumption of WGJ during chemotherapy may reduce vascular damage and chemotherapy-related thrombogenicity, growth factors and cytokines, as reflected by the characteristics of patient’s EVs.

AMPK activation by ozone therapy inhibits tissue factor-triggered intestinal ischemia and ameliorates chemotherapeutic enteritis.

Chemotherapeutic enteritis is a major dose-limiting adverse reaction to chemotherapy, with few effective drugs in clinic. Intestinal ischemic injury plays prominent role in chemotherapeutic enteritis clinically. However, mechanism is not clear. In this article, irinotecan (CPT-11) was used to establish chemotherapeutic enteritis mice model. Western blotting, gelatin zymography, immunohistochemistry (IHC), Laser Doppler flowmetry (LDF) were used to detect the pathogenesis of ischemia-hypoxia injury. CPT-11 increased levels of tissue factor (TF) both in the blood and in intestines, and decreased the intestinal blood flow in mice. Interestingly, the elevation of TF in the blood displayed "double-peak," which was consistent with the intestinal mucosal "double-strike" injury trend. Intestinal microthrombus and mixed thrombus formation were detectable in chemotherapeutic enteritis. Furthermore, ozone therapy relieved chemotherapeutic enteritis in mice. Ozone inhibited TF expression induced by CPT-11 via activating AMPK/SOCS3, and effectively ameliorated the intestinal mucosal injury in mice. Moreover, ozone autotransfusion therapy effectively attenuated chemotherapeutic enteritis and the blood hypercoagulability in patients. For the first time, we proposed that TF-induced thrombotic intestinal ischemic injury is a core trigger pathological mechanism of chemotherapeutic enteritis, and provided a new treatment strategy, ozone therapy, to suppress TF expression and treat chemotherapeutic enteritis.

Increased tissue factor activity promotes thrombin generation at type 1 diabetes onset in children

ObjectiveIn type 1 diabetes (T1D), a prothrombotic status due to elevated coagulation factors coincides with metabolic derailment. In a previous study, we discovered altered thrombin generation profiles in children with T1D. These alterations are potentially most pronounced at T1D onset and ameliorated after insulin treatment. We tested this hypothesis in a longitudinal study, measuring thrombin generation together with coagulation parameters in children at T1D onset and during follow‐up.Materials and methodsTwenty‐three children (12 female, age: 9.4 [2.7‐17.3] years; median [range]) were tested at T1D onset and after long‐term insulin treatment. Thrombin generation was measured using calibrated automated thrombography. Tissue factor (TF) activity and tissue factor pathway inhibitor (TFPI) activity were measured using enzyme‐linked immunosorbent assay (ELISA).ResultsA procoagulant shift was observed in thrombin generation traces at T1D onset compared to follow‐up (time to peak: 5.67 [4.11‐7.67] min vs 6.39 [4.89‐10.44] min, P < .001). These alterations at T1D onset coincided with increased TF activity (5.18 [0.01‐12.97] pmol/L vs 2.67 [0.04‐10.41] pmol/L, P < .05) and increased TFPI activity (0.051 [0.038‐0.074] U/mL vs 0.035 [0.026‐0.056] U/mL, P < .05).ConclusionThe procoagulant shift in thrombin generation at T1D onset is a result of increased TF activity, but this effect is partially counterbalanced by increased TFPI levels. Elevated TF and TFPI levels hint to a fragile hemostatic balance at the endothelial lining of blood vessels. Additional prothrombotic stimuli may tip over this balance explaining the increased thrombotic risk of children with T1D.

Evaluation of different commercial antibodies for their ability to detect human and mouse tissue factor by western blotting

BackgroundWestern blotting is used to measure protein expression in cells and tissues. Appropriate interpretation of resulting data is contingent upon antibody validation. ObjectivesWe assessed several commercial anti‐human and anti‐mouse tissue factor (TF) antibodies for their ability to detect TF by western blotting. Material and MethodsWe used human pancreatic cancer cell lines expressing different levels of TF and a mouse pancreatic cancer cell line expressing TF with a matched knockout derivative. ResultsHuman and mouse TF protein detected by western blotting correlated with levels of TF mRNA in these cell lines. The apparent molecular weight of TF is increased by N‐linked glycosylation and, as expected, deglycosylation decreased the size of TF based on western blotting. We found that four commercial anti‐human TF antibodies detected TF in a TF‐positive cell line HPAF‐II whereas no signal was observed in a TF‐negative cell line MIA PaCa‐2. More variability was observed in detecting mouse TF. Two anti‐mouse TF antibodies detected mouse TF in a TF‐positive cell line and no signal was observed in a TF knockout cell line. However, a third anti‐mouse TF antibody detected a nonspecific protein in both the mouse TF‐positive and TF‐negative cell lines. Two anti‐human TF antibodies that are claimed to cross react with mouse TF either recognized a nonspecific band or did not detect mouse TF. DiscussionOur results indicate that there is a range in quality of commercial anti‐TF antibodies. ConclusionWe recommend that all commercial antibodies should be validated to ensure that they detect TF.

Changes of Tissue Factors and Tissue Factor Pathway Inhibitors in the Acute Phase of Cardiogenic Stroke in Patients With Atrial Fibrillation

Abstract Background To investigate changes of tissue factor (TF) and tissue factor pathway inhibitor (TFPI) in the acute phase of cardiogenic stroke in patients with atrial fibrillation (AF). Methods A total of 152 patients with nonvalvular persistent AF, 103 cardiogenic stroke patients with nonvalvular persistent AF, and 126 cases with noncardiogenic stroke were enrolled. Non-AF patients (n = 138) without stroke served as controls. The clinical data, biochemical indicators, and plasma levels of von Willebrand factor (vWF), TF, and TFPI were determined. Results Compared with controls, the plasma levels of vWF and TF were significantly higher, while TFPI and TFPI/TF ratio were lower in noncardiogenic stroke patients (all P &amp;lt; 0.05). Compared with cardiogenic stroke patients, noncardiogenic stroke subjects showed significantly higher plasma vWF and TF levels while TFPI levels and TFPI/TF ratio were significantly lower (all P &amp;lt; 0.05). Compared with AF patients, plasma levels of vWF, TF, and TFPI were higher while TFPI/TF ratios were decreased in the cardiogenic ischemic stroke subjects (all P &amp;lt; 0.05). Logistic regression analysis showed that a lower TFPI/TF ratio was an independent predictor of cardiogenic stroke in patients with nonvalvular AF (odds ratio = 3.78, 95% confidence interval: 1.86–7.31). Conclusions During the acute stage of stroke, TFPI/TF ratio is decreased in both cardiogenic and noncardiogenic stroke patients while plasma TFPI levels show divergent changes.

Differential biomarker profiles between unprovoked venous thromboembolism and cancer

Background The relationship between cancer and venous thromboembolic disease (VTD) are complex because the activated coagulation factors are not only involved in thrombosis but also in malignant processes, such as angiogenesis and metastasis. Objective To compare phenotypes of extracellular vesicles (EVs), and levels of D-dimer, soluble P-selectin (sP-selectin) and antigenic tissue factor (TF) between unprovoked VTD patients, who did not develop cancer during one-year follow-up, and those with advanced stage of cancer but not associated with VTD. Methods A prospective study in which we included 138 unprovoked VTD patients and 67 advanced cancer patients, who did not develop thrombosis. Levels of EVs of different cellular origin (platelet, endothelium and leukocyte), EVs positive for tissue factor (TF) and P-selectin glycoprotein ligand 1 were quantified by flow cytometry. D-dimer, soluble P-selectin (sP-selectin) and antigenic TF were determined by ELISA. Results TF-positive EVs, D-dimer, and sP-selectin were markedly elevated in unprovoked VTD patients compared to cancer patients without association with thrombosis. Conclusions Levels of TF-positive EVs, D-dimer and sP-selectin are able to discriminate between unprovoked VTD patients not related to cancer and cancer patients not associated with VTD. These results could lead to the application of EVs as biomarkers of both diseases. Key messages: Circulating EVs, specifically TF-positive EVs, in combination with plasmatic markers of hypercoagulable states, such as D-dimer, sP-selectin and antigen TF, are able to discriminate between cancer patients without thrombosis and patients with unprovoked VTD. Research fields could be opened. Future studies will assess if these biomarkers together serve as predicting thrombotic events in cancer populations

Expression of tissue factor mRNA in thrombosis associated with antiphospholipid syndrome.

Tissue factor (TF) is a procoagulant protein associated with increased risk of thrombotic events in antiphospholipid syndrome (t-APS). The mechanisms by which TF levels are increased in APS have not yet been established. The aim of this study was to investigate whether TF mRNA expression is associated with TF levels and thrombosis in APS. We compared levels of circulating TF and high sensitivity C-reactive protein (hs-CRP) between t-APS and controls (individuals without thrombosis). The association between TF mRNA expression, quantified by real time quantitative polymerase chain reaction, and t-APS was accessed using regression analysis. We included 41 controls and 42t-APS patients, mean age was 41years old (SD 14) in both groups. Hs-CRP and TF levels were higher in t-APS patients (mean hs-CRP levels 0.81mg/dL [SD 1.88] and median TF levels 249.0pg/mL [IQR 138.77-447.61]) as compared to controls (mean hs-CRP levels 0.24mg/dL [SD 0.26] and median TF levels 113.0pg/mL [IQR 81.17-161.53]; P = 0.02 and P < 0.0001, respectively). There was no correlation between TF mRNA expression and TF levels in t-APS (r -0.209, P = 0.19). TF mRNA expression was not associated with t-APS (adjusted OR 1.16; 95%CI 0.72-1.87). Despite circulating TF levels being higher in patients with t-APS than in controls, TF mRNA expression was similar between groups. The results demonstrate that TF mRNA expression is not associated with levels of circulating TF and hypercoagulability in t-APS.

Elevated Microparticles, Thrombin-antithrombin and VEGF Levels in Colorectal Cancer Patients Undergoing Chemotherapy

Hypercoagulable state and neoangiogenesis are common phenomena associated with malignancy. Cancer patients have increased levels of circulating endothelium-derived microparticles (EMPs), which have been hypothesized to be involved in numerous pathophysiological processes. Hemostasis and angiogenesis are also activated in colorectal cancer (CRC) patients. The study aimed to investigate potential influence of chemotherapy on EMPs, thrombin anti-thrombin complex (TAT) and vascular endothelial growth factor (VEGF) levels in CRC patients undergoing chemotherapy. The study group consisted of 18 CRC patients: 8 stage III colon cancer (CC) and 10 stage IV rectal cancer (RC) patients. EMPs, TAT and VEGF levels were assessed before chemotherapy and after the third course. Results were compared with 10 healthy subjects. EMP concentration was measured by flow cytometry, while TAT and VEGF concentrations were assayed employing ELISA. Compared to the control group, CC and RC patients had significantly higher levels of tissue factor (TF)-bearing and non-TF-bearing EMPs before and after three courses of chemotherapy. VEGF concentrations in CRC patients were higher than in the control groups and increased following chemotherapy. TAT levels were elevated in CRC patients before chemotherapy compared to healthy subjects and significantly increased after the third course of chemotherapy. No significant correlation was found either between EMP and TAT levels, or between EMP concentrations and VEGF levels in the study group. CRC patients have increased EMPs, and TAT as well as VEGF levels tend to increase during chemotherapy.