Monocyte Tissue Factor Antigen Research Articles

LPS-induced expression and release of monocyte tissue factor in patients with haemophilia

In haemophilia, thrombin generation and fibrin deposition upon vascular injury critically depend on the tissue factor (TF)-driven coagulation pathway. TF expression by monocytes/macrophages and circulating microvesicles contributes to haemostasis, thrombosis and inflammation. Inflammation is a hallmark of blood-induced joint disease. The aim of this study is to correlate TF production by whole-blood monocytes with inflammatory markers and clinical parameters in patients with moderate-to-severe haemophilia A or B (n = 43) in comparison to healthy males (n = 23). Monocyte TF antigen and microvesicle-associated TF procoagulant activity (MV TF PCA) were measured immediately after blood draw (baseline) and following incubation of whole blood with buffer or lipopolysaccharide (LPS) using two-colour flow cytometry and chromogenic FXa generation assay, respectively. Patients with HIV or uncontrolled HBV/HCV infections were excluded. TF was hardly detectable and not different in baseline and buffer-treaded samples from both groups. Stimulation with LPS, however, induced monocyte TF production, with increased TF-specific mean fluorescence intensity (P = 0.08) and MV TF PCA (P < 0.05) in patients compared to controls. Patients also had elevated hs-CRP and IL-6 serum levels (P < 0.001), which correlated with LPS-induced TF parameters. Further exploratory analyses revealed that the presence of systemic (low-grade) inflammation and boosted LPS-induced monocyte TF production were mainly restricted to patients with clinically controlled HBV and/or HCV infection (n = 16), who were older and also had a significantly worse orthopaedic joint score than patients with no history of viral hepatitis (P < 0.01). Our study delineates a previously unrecognised link between systemic inflammation and inducible monocyte TF production in patients with haemophilia A or B.

Functional and Antigenic Detection of Intravascular Tissue Factor (TF).

Recently, intravascular TF has been the focus of tremendous research activity in the fields of hematology, oncology, and cardiovascular medicine. The biological relevance of this so-called blood-borne TF, however, remains controversial. This may, at least in part, be due to a lack of controlled studies comparing functional and antigenic detection systems. The following study was conducted to provide insight into this area of uncertainty. Incubation of citrated blood with LPS (4 h) increased monocyte TF antigen from 0–2 to 7–35% and reduced plasma recalcification times (RTs) from 648±87 to 388±23 s (n=10), an effect that was completely reversed by inhibitory TF mAb. In all donors, monocyte-platelet-conjugates, as assessed by CD41 labeling of CD14+ events, decreased following LPS stimulation (47±15 vs. 58±16%, P<0.01), suggesting that microparticles (MPs) shed from monocytes into plasma were associated with platelet membrane components. Plasma TF antigen, as measured by a commercial ELISA using non-lipidated, full-length recombinant human TF (rhTF1–263) as standard and 0.1% Triton X-100 for sample dilution, was increased from 21±33 to 160±110 pg/ml (P<0.01). TF antigen levels were unaffected by ultracentrifugation or 0.2-μm filtration, both of which reduced cellular MPs by >90% and significantly prolonged clotting times. After 18 h, LPS further shortened plasma RTs to 170±31 s, whereas a difference in plasma TF antigen was no longer detectable (80±66 vs. 127±103 pg/ml, P=0.48). We used a highly sensitive clotting assay ± TF mAb to determine the TF-specific procoagulant activity (PCA) of washed and concentrated plasma MPs. Clotting times were calibrated against serial dilutions (1/10–1/105) of relipidated rhTF1–263, showing a linear correlation in a log-log plot with R2>0.99. MP-associated TF PCA was increased >10fold after LPS stimulation. However, by flow cytometry using PE-conjugated TF mAb (HTF-1) and microspheres for size calibration (1 μm) and sample flow standardization, we could not reliably detect an increase in TF+ MPs. Furthermore, a chromogenic assay, which was 10fold less sensitive than the clotting assay, failed to detect significant TF-VIIa-dependent Xa generation by LPS-induced MPs. Similarly, incubation of strongly TF-expressing adenocarcinomatous HT29 cells in citrated blood under stirring conditions (1 h) shortened plasma RTs from 694±26 to 152±2 s and increased MP-associated TF PCA >100fold, whereas plasma TF antigen was elevated by only 18%. In an 84-year-old patient with metastatic adenocarcinoma and Trousseau's syndrome, who presented with DIC, bilateral deep vein thrombosis, pulmonary embolism, intracardiac thrombi, and extensive cutaneous necrosis due to thrombotic microangiopathy, we found significant expression of TF PCA on tumor cells derived from pleural effusion. Whereas TF-specific PCA of MPs isolated from the patient's plasma on three different occasions was 8–97fold higher than in five healthy controls, plasma TF antigen was only slightly increased (110 vs. 45±26 pg/ml). In summary, functional appear superior to antigenic assays in detecting intravascular TF, with clotting assays being most sensitive and reproducible. Particularly, commercial ELISAs may not adequately reflect MP-associated TF in plasma. Instead, increased TF antigen after short-term exposure of whole blood monocytes to LPS may indicate preferential detection of soluble variants such as alternatively spliced TF.

Effect of glycoprotein IIb/IIIa antagonist abciximab on monocyte-platelet aggregates and tissue factor expression.

Activated platelets rapidly adhere to monocytes and upregulate the expression of tissue factor (TF), the major trigger of the coagulation cascade. In this study, we examined the effect of abciximab, a nonselective glycoprotein IIb/IIIa-receptor antagonist, on monocyte TF expression in thrombin receptor activator-stimulated whole blood in vitro. Abciximab (50 microg/mL) reduced the mass of platelets attached to monocytes, measured by the mean fluorescence intensity (MFI) of CD42b on CD14+ cells, 1 (CD42b, 471+/-197 versus 1073+/-217 MFI, mean+/-SD, P<0.05), 5, and 10 minutes after thrombin receptor activator stimulation of whole blood to the same extent as anti-P-selectin (50 microg/mL; 288+/-177 MFI, P<0.05) when determined by flow cytometry. In parallel, the expression of the platelet activation marker P-selectin colocalized with CD14+ monocytes was reduced up to 25% by abciximab at the same time points. Expression of monocyte TF antigen (CD14+/TF+, 39.9+/-8.7% versus 66.3+/-19.9%, P<0.05), chromogenic TF-activity (TF, 8.4+/-1.9 versus 13.2+/-2.8 U, arbitrary units, P<0.05), and TF mRNA was suppressed in the presence of abciximab as a consequence of reduced platelet mass attached to monocytes. Our data suggest that heterotypic monocyte-platelet aggregates are a target for abciximab, which suppresses monocyte TF because of a reduction of monocyte-platelet cross talk.

Enhanced monocyte expression of tissue factor by oxidative stress in patients with antiphospholipid antibodies: effect of antioxidant treatment

In a first study, we performed a cross-sectional analysis of urinary excretion of isoprostanes, IPF(2alpha-III) and (VI), and monocyte tissue factor (TF) antigen and activity between 11 antiphospholipid (APL) antibody-positive patients and 13 APL negative subjects. In a second study, 11 APL positive patients were randomly supplemented either with (n = 6) or without (n = 5) antioxidants (vitamin E at 900 IU day(-1), vitamin C at 2000 mg day(-1)) for 6 weeks. In a third study, TF and superoxide anion were measured in human monocytes incubated with anti-beta(2) glycoprotein 1 (beta(2)GP(1)) or control IgG, either with or without vitamin E. APL-positive patients had higher values of isoprostanes (P < 0.05) and monocyte TF antigen (P = 0.001) and activity (P = 0.0001) than APL-negative subjects. Only in APL positive patients did monocyte TF antigen correlate significantly with IPF(2alpha-III) (rho 0.79; P < 0.003) and IPF(2alpha-VI) (rho = 0.87; P < 0.0001). In patients who received antioxidant supplementation, we found a significant decrease of isoprostanes (P < 0.05) and monocyte TF antigen (P < 0.01) and activity (P < 0.007). In vitro experiments demonstrated that anti-beta(2)GP(1) antibodies dose-dependently enhanced the monocyte production of the superoxide anion and TF, which were significantly inhibited by vitamin E. This study demonstrates that in APL-positive patients, oxidative stress contributes to activate the clotting system via over-expression of monocyte TF. We suggest that anti-beta(2)GP(1) antibodies could play a pivotal role by enhancing the monocyte production of oxygen free radicals.

Vitamin E Reduces Monocyte Tissue Factor Expression in Cirrhotic Patients

Clotting activation may occur in liver cirrhosis, but the pathophysiological mechanism has not been fully elucidated. Because a previous study demonstrated that lipid peroxidation is increased in cirrhosis, we analyzed whether there is a relationship between lipid peroxidation and clotting activation. Thirty cirrhotic patients (19 men and 11 women; age, 34 to 79 years) and 30 controls matched for sex and age were investigated. In all subjects, monocyte expression of tissue factor (TF) antigen and activity; plasma levels of prothrombin fragment 1+2 (F1+2), a marker of thrombin generation; and urinary excretion of Isoprostane-F2-III, a marker of lipid peroxidation, were measured. Furthermore, the above-reported variables were re-evaluated after 30 days of treatment with standard therapy (n = 5) or standard therapy plus 300 mg vitamin E twice daily (n = 9). In addition, we analyzed in vitro if vitamin E (50 μmol/L) influenced monocyte TF expression and F1+2 generation. Cirrhotic patients had higher values of Isoprostane-F2-III (P &lt; .0001), F1+2 (P &lt; .0001), and monocyte TF antigen (P &lt; .0001) and activity (P &lt; .03) than controls. Isoprostane-F2-III was significantly correlated with F1+2 (Rho = 0.85; P &lt; .0001) and TF antigen (Rho = 0.95; P &lt; .0001) and activity (Rho = 0.94;P &lt; .0001). After vitamin E treatment, Isoprostane-F2-III (P = .008), F1+2 (P &lt; .008), and monocyte TF antigen (P = .012) and activity (P = .008) significantly decreased; no changes of these variables were detected in patients not receiving vitamin E. In vitro, vitamin E significantly reduced the expression of monocyte TF antigen (−52%; P = .001) and activity (−55%; P= .003), as well as F1+2 generation (−51%; P = .025). This study shows that vitamin E reduces both lipid peroxidation and clotting activation and suggests that lipid peroxidation may be an important mediator of clotting activation in liver cirrhosis.

Vitamin E Reduces Monocyte Tissue Factor Expression in Cirrhotic Patients

AbstractClotting activation may occur in liver cirrhosis, but the pathophysiological mechanism has not been fully elucidated. Because a previous study demonstrated that lipid peroxidation is increased in cirrhosis, we analyzed whether there is a relationship between lipid peroxidation and clotting activation. Thirty cirrhotic patients (19 men and 11 women; age, 34 to 79 years) and 30 controls matched for sex and age were investigated. In all subjects, monocyte expression of tissue factor (TF) antigen and activity; plasma levels of prothrombin fragment 1+2 (F1+2), a marker of thrombin generation; and urinary excretion of Isoprostane-F2α-III, a marker of lipid peroxidation, were measured. Furthermore, the above-reported variables were re-evaluated after 30 days of treatment with standard therapy (n = 5) or standard therapy plus 300 mg vitamin E twice daily (n = 9). In addition, we analyzed in vitro if vitamin E (50 μmol/L) influenced monocyte TF expression and F1+2 generation. Cirrhotic patients had higher values of Isoprostane-F2α-III (P < .0001), F1+2 (P < .0001), and monocyte TF antigen (P < .0001) and activity (P < .03) than controls. Isoprostane-F2α-III was significantly correlated with F1+2 (Rho = 0.85; P < .0001) and TF antigen (Rho = 0.95; P < .0001) and activity (Rho = 0.94;P < .0001). After vitamin E treatment, Isoprostane-F2α-III (P = .008), F1+2 (P < .008), and monocyte TF antigen (P = .012) and activity (P = .008) significantly decreased; no changes of these variables were detected in patients not receiving vitamin E. In vitro, vitamin E significantly reduced the expression of monocyte TF antigen (−52%; P = .001) and activity (−55%; P= .003), as well as F1+2 generation (−51%; P = .025). This study shows that vitamin E reduces both lipid peroxidation and clotting activation and suggests that lipid peroxidation may be an important mediator of clotting activation in liver cirrhosis.