Abstract-Activated platelets can express CD40 ligand (CD40L) and trigger inflammatory response and tissue factor (TF) expression in endothelial cells through interaction with CD40. This pathway is also important for T cell-induced monocyte and endothelial cell procoagulant activity. We have studied the potential role of the CD40-CD40L pathway in platelet-induced TF expression in a monocytic cell line and in whole-blood monocytes. In vitamin D 3 -differentiated U-937 cells, thrombin-stimulated platelets increased TF expression as measured by mRNA quantification, flow cytometry, and procoagulant activity. Maximum antigen expression occurred after 2 hours. Neutralizing anti-P-selectin antibody yielded a 50% suppression of procoagulant activity, whereas antibody to CD40L had no effect. In thrombin receptor activator-stimulated citrated blood, monocytes were up to 77% TF-positive, with peak expression after only 15 minutes. However, no TF mRNA was detectable at that time. Anti-P-selectin antibody reduced TF by 50%, whereas antibody to CD40L gave a 17% reduction. Thus, we conclude that P-selectin exposed on activated platelets induces the expression of TF in both U-937 cells and whole-blood monocytes but by different mechanisms. Platelet CD40L does not display any significant effect on U-937 cells but may be of some importance on whole-blood monocytes. This suggests a possible functional difference between U-937 and monocyte CD40. Another important finding in this study is the rapid appearance of surface TF on monocytes without detectable mRNA formation. This indicates that TF may be stored intracellularly in these cells and can be exposed on the surface independent of de novo protein synthesis.
Objective-Tissue factor (TF) has, among other factors, a prominent role in acute coronary syndrome (ACS). Our goal was to investigate whether single nucleotide polymorphisms (SNP) in the TF gene (F3) are associated with plasma TF, risk, and outcome in patients with ACS. Moreover, we wanted to investigate the impact of associated TF SNPs on mRNA production in human monocytes. Methods and Results-In 725 patients with ACS [Fragmin and Fast Revascularization during Instability in CoronaryArtery Disease II (FRISC-II) study] and 376 controls, 13 SNPs were genotyped and plasma TF measured. Thereafter, the 5466 AϾG and the Ϫ1812 CϾT were genotyped among all of the FRISC-II participants (nϭ3143) and assessed concerning clinical outcome. Associated SNPs were genotyped in 92 healthy blood donors for comparison of TF activity and TF mRNA expression. None of the SNPs were associated with patient/control status. The 5466 AϾG SNP was associated with cardiovascular death (odds ratio, 1.8; Pϭ0.025). The CG haplotype by Ϫ1812 CϾT and 5466 AϾG was associated with a 3-fold increased risk of death (PϽ0.001). TF mRNA and basal TF activity was significantly lower among 5466 AG carriers, whereas the increase in monocyte TF activity on lipopolysaccharide stimulation was significantly stronger (Pϭ0.04). Key Words: acute coronary syndrome Ⅲ tissue factor Ⅲ single nucleotide polymorphism Ⅲ outcome Ⅲ mRNA A cute coronary syndrome (ACS) is a multifactorial disease in which a multitude of lifestyle and genetic factors contribute to the development and outcome of the disease. 1 The genetic component of ACS seems to be relatively high, because family history is a strong risk factor, and heritability studies have shown a large genetic contribution to cardiac disease. 2,3 Single nucleotide polymorphisms (SNPs) are the most common genetic variations in the genome and may influence factors involved in atherosclerosis, plaque destabilization, and thrombosis, all of which are fundamental aspects of ACS etiology. 3 Variations in genes that encode for hemostatic factors are considered to be likely candidates for modifiers of the procoagulant phenotype, usually observed in patients with myocardial infarction (MI). 4 A procoagulant phenotype may have a substantial effect on ACS outcome, because a larger sized superimposed thrombus is directly related to the severity of myocardial ischemia. 5 A major player in this respect is tissue factor (TF), which rapidly initiates blood coagulation and is generally recognized as an important thrombogenic factor in atherosclerotic lesions. 6 TF is present as a membrane-bound molecule in different cell types in the vessel wall and within plaques, including endothelial cells, smooth muscle cells monocytes/macrophages, and foam cells. 7 Proteolytic cleavage of TF results in a soluble form of TF (plasma TF), which only contains the extracellular part of the protein and does not show significant procoagulant activity. 8 Moreover, the presence of an alternatively spliced form of TF has been reported recently. 9 Intravascula...
Interleukin (IL)-4, IL-10, IL-13 and transforming growth factor beta (TGF-beta) are known to regulate several monocyte functions, including inhibition of the synthesis of different cytokines. Using quantitative RT-PCR and flow cytometry analysis we investigated the effects of these cytokines on bacterial lipopolysaccharide (LPS)-induced tissue factor (TF) expression in human monocytes. The effects of IL-4 and IL-10 on monocyte chemoattractant protein-1 (MCP-1)-and C-reactive protein (CRP)-induced TF expression were also studied. A direct comparison revealed that IL-4, IL-10 and IL-13 all down-regulated LPS-induced TF expression in a concentration-dependent manner without the need for priming. In contrast, TGF-beta required 4 h of priming to inhibit TF expression induced by LPS. IL-10 was the most powerful inhibitor, causing almost complete inhibition at 5 ng/ml. IL-4 and IL-13 exhibited a significantly lower inhibitory capacity even at concentrations of 100 ng/ml. IL-4 and IL-10 showed similar concentration-dependent inhibition of MCP-1- and CRP-induced TF expression. We also showed that the regulatory effect of the interleukins occurred at the mRNA level. In vivo, these inhibitory cytokines may play an important regulatory role in preventing thrombosis. IL-10, in particular, may be a possible candidate as a TF-preventing drug.
Summary.Interleukin 4 (IL-4), IL-10 and IL-13 are all known to modulate several proinflammatory functions in human monocytes. They have also previously been shown to down-regulate lipopolysaccharide (LPS)-induced tissue factor (TF) expression in isolated cultured monocytes. In this study we investigated the effect of these three cytokines on the induction of monocytic TF in a whole blood environment at three levels: mRNA quantitation, surface antigen expression and procoagulant activity. We showed that IL-10 attenuated LPS-induced monocyte TF expression and activity in whole blood in a concentration-dependent manner, both when added to the blood prior to LPS and, although to a lesser extent, when added up to 1 h subsequent to LPS challenge. Maximum inhibition occurred at 5 ng/ml of IL-10 when the cytokine was added before LPS. IL-4 and IL-13, however, did not exhibit any inhibitory effect in the whole blood environment, contrary to the reported findings in cell culture experiments. Our results confirm the potential of IL-10 as an anti-inflammatory, TF-preventing drug, whereas the effects of IL-4 and IL-13 on monocytes in whole blood seem more complex, and require further investigation.
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