Procoagulant soluble tissue factor is released from endothelial cells in response to inflammatory cytokines

Inflammatory cytokines alter the hemostatic balance of endothelial cells (ECs). Alternatively spliced human tissue factor (asHTF), a soluble isoform of tissue factor (TF), has recently been detected in ECs, possibly contributing to procoagulability. Agonists regulating asHTF expression and release are yet unknown. This study examines the effect of TNF-alpha and IL-6 on the endothelial expression of both TF variants and delineates the impact of asHTF on the procoagulability of extracellular fluids. asHTF and TF mRNA were assessed by real-time PCR, and asHTF, TF, and tissue factor pathway inhibitor (TFPI) proteins by Western blot and fluorescence microscopy before and after stimulation with TNF-alpha (10 ng/mL) or IL-6 (10 ng/L). The procoagulability of cell supernatant was analyzed by a chromogenic assay with or without phospholipid vesicles. We found asHTF mRNA to be maximally increased 10 minutes after TNF-alpha and 40 minutes after IL-6 treatment (asHTF/GAPDH ratio 0.0223+/-0.0069 versus 0.0012+/-0.0006 for control, P<0.001 and 0.0022+/-0.0004 versus 0.0012+/-0.0007, P<0.05, respectively). Not only was asHTF increased, but also TFPI decreased after cytokine treatment. asHTF was found in the supernatant as early as 5 hours after TNF-alpha stimulation, supporting factor Xa generation after relipidation (6.55+/-1.13 U versus 2.99+/-0.59 U in control supernatant, P<0.00001). Removal of asHTF from supernatants by immunoprecipitation diminished its procoagulability to baseline. The soluble TF isoform expressed and released from ECs in response to inflammatory cytokines becomes procoagulant in the presence of phospholipids. Thus, asHTF released from ECs is a marker for and a contributor to imbalanced hemostasis.     

Viral myocarditis and coagulopathy: increased tissue factor expression and plasma thrombogenicity

We investigated the effects of viral infection on Tissue Factor (TF) expression and activity in mice within the myocardium to understand increased thrombosis during myocarditis. Mice were infected with coxsackie virus B3 (CVB3) and the hearts were collected at day 4, 8 and 28 post infection (p.i.). Myocardial TF expression and cellular activity as well as plasma activity were analyzed from CVB3 infected mice by Western blot, chromogenic Factor Xa generation assay, in situ staining for active TF and immunohistochemistry. In addition to TF expression, hemodynamic parameters were measured during the time course of infection. Furthermore, we analyzed myocardial tissues from patients with suspected inflammatory cardiomyopathy. TF protein expression was maximally 5-fold elevated 8 days p.i. in mice and remained increased on day 28 p.i. (P < 0.001 vs. non-infected controls). Alterations in TF expression were associated with fibrin deposits within the myocardium. The TF pathway inhibitor protein expression in the myocardium was not altered during myocarditis. Active cellular TF co-localized with CD3 positive cells and VCAM-1 positive endothelial cells in the myocardium. The TF expression was positively correlated with the amount of infiltrating CD3 and Mac3 positive cells (Spearman-Rho ρ = 0.749 P < 0.0001 for CD3⁺ and ρ = 0.775 P < 0.0001 for Mac3⁺; N = 35). Increased myocardial TF expression was associated with a 2-fold elevated plasma activity (P < 0.05 vs. non-infected controls). In the human hearts, the TF expression correlated postively with an endothelial cell activation marker (ρ = 0.523 P < 0.0001 for CD62E; N = 54). Viral myocarditis is a hypercoagulative state which is associated with increased myocardial TF expression and activity. Upregulation of TF contributes to a systemic activation of the coagulation cascade.     

Endothelial dysfunction of peripheral arteries in patients with immunohistologically confirmed myocardial inflammation correlates with endothelial expression of human leukocyte antigens and adhesion molecules in myocardial biopsies

OBJECTIVES: The aim of this study was to investigate whether myocardial inflammation (MC) and endothelial activation are associated with clinically detectable endothelial dysfunction. BACKGROUND: In patients with MC, immunohistologic evaluation of myocardial biopsies demonstrates a cellular infiltrate of lymphocytes in the myocardium and endothelial activation, as indicated by enhanced expression of human leukocyte antigen (HLA)-1, HLA-DR and intercellular adhesion molecule (ICAM)-1. This chronic inflammatory process may be associated with endothelial dysfunction. METHODS: In 65 patients with suspected MC, endothelial function of the radial artery was noninvasively assessed. By means of high-resolution ultrasound, diameter changes in response to reactive hyperemia (endothelium-dependent), as compared with glyceroltrinitrate (endothelium-independent), were analyzed. In the myocardial biopsies, MC was confirmed by immunohistology in 53 patients; 12 patients with normal myocardial biopsies served as controls. Endothelial expression of HLA-1, HLA-DR and ICAM-1 was semiquantitatively evaluated by immunohistology. To minimize other factors influencing endothelial function, patients with coronary artery disease, diabetes, severely impaired left ventricular function or more than one arteriosclerotic risk factor were excluded from this study. RESULTS: Endothelial function, as determined by flow-mediated vasodilation (FMD), in patients with MC was impaired (FMD(MC) 4.28%), as compared with controls (FMD(Co) 10.10%). The severity of endothelial dysfunction in patients with MC correlated significantly with the extent of endothelial expression of HLA-1, HLA-DR and ICAM-1 in myocardial biopsies. Endothelium-independent vasodilation was not affected by MC or endothelial activation. CONCLUSIONS: Myocardial inflammation is associated with endothelial dysfunction of peripheral arteries. The severity of endothelial dysfunction correlates with the extent of endothelial activation.     

Doubling the clopidogrel dose in patients with reduced responsiveness to the standard dose is associated with a limited effectiveness as evaluated by impedance aggregometry

BackgroundDifferent methods are available for quantifying platelet function inhibition. Measuring vasodilator-stimulated phosphoprotein (VASP) phosphorylation is currently the most specific method for assessing the clopidogrel effect. The aim of our study was to compare different tests in view of a clinically applicable bedside test. Further, we examined whether doubling the clopidogrel dose to 150 mg/d in clopidogrel low-responder would lead to a reduction in platelet reactivity.Methods and resultsADP-, ADP Hs-, and TRAP-induced platelet aggregation were measured by impedance aggregometry in 100 patients with CAD and 18 healthy controls. Moreover, platelet aggregation was assessed by flow cytometrical detection of VASP-phosphorylation and surface P-selectin in a subgroup of 34 patients and in healthy controls. Another 10 patients with CAD, identified as low-responder, were treated with a clopidogrel dose of 150 mg/d. Thereafter, ADP-induced platelet aggregation was assessed by impedance aggregometry. Significant correlations were observed between ADP-induced platelet aggregation assessed by VASP-phosphorylation and by impedance aggregometry. Doubling the dose of clopidogrel to 150 mg/d was associated with a reduction of ADP-induced platelet aggregation in only 60% of the patients.ConclusionsImpedance aggregometry is a valuable bedside test to assess platelet function inhibition. Doubling the clopidogrel dose is not effective to reduce high on-treatment platelet reactivity in almost half of these patients, pointing to the need of a more powerful platelet inhibitor.     

Annual distribution of ventricular tachycardias and ventricular fibrillation

Background

Ischemic events and coronary deaths show seasonal variability with a peak during December and January. It remains unclear whether ventricular tachycardias (VT) and ventricular fibrillation (VF) follow a similar pattern. The purpose of this study was to investigate the annual distribution of malignant ventricular arrhythmias.

Methods

Over a period of 11 years, all appropriate shock episodes (SE) after VT and VF in patients with an implantable cardioverter defibrillator (lCD) were analyzed with respect to the month of occurrence. An appropriate SE was defined as out-of-hospital VT/VF terminated by lCD shocks. Multiple shocks within 1 week were defined as 1 SE.

Results

Two hundred and thirty-three of 308 patients with an lCD had appropriate SE during follow-up. In these patients the seasonal variation of 753 SE was calculated. Most SE occurred during January (93 SE), and the fewest SE occurred during June (39 SE). The seasonal pattern was statistically significant with a peak during winter (P = .001). The seasonal pattern did not differ between patients with an ischemic and those with a nonischemic underlying cardiac disease.

Conclusion

Appropriate shock episodes due to out-of-hospital VT/VF in patients with an lCD show seasonal variation with a significant peak during winter. The pattern is similar in patients with ischemic and nonischemic cardiac disease.     

Functional iron deficiency and diastolic function in heart failure with preserved ejection fraction

Background

Functional iron deficiency (FID) is an independent risk factor for poor outcome in advanced heart failure with reduced EF, but its role in heart failure with preserved EF (HFPEF) remains unclear. We aimed to investigate the impact of FID on cardiac performance determined by pressure–volume loop analysis in HFPEF.

Methods

26 HFPEF patients who showed an increase in LV stiffness by pressure–volume (PV) loop analysis obtained by conductance-catheterization, performed exercise testing, echocardiographic examination including tissue Doppler and determination of iron metabolism: serum iron, ferritin and transferrin saturation. HFPEF patients who provided ferritin < 100 μg/l or ferritin of 100–299 μg/l in combination with transferrin saturation < 20% were defined as having FID. In 14 patients the expression of transferrin receptor was determined from available endomyocardial biopsies.

Results

Fifteen out of 26 HFPEF patients showed FID without anemia. Compared to control subjects and HFPEF patients without FID, HFPEF patients with FID showed an up-regulation of the myocardial transferrin receptor expression (p < 0.05). No differences between HFPEF patients with and without iron deficiency were found in heart dimensions, systolic and diastolic function obtained by PV-loop and echocardiography analysis. According to the linear regression analysis, LV stiffness was correlated with peak oxygen uptake (r = − 0.636, p < 0.001) but not with the ferritin level or transferrin saturation. No relation was found between FID and exercise capacity. The association of LV stiffness with exercise performance was independent from the level of iron deficiency.

Conclusion

In non-anemic HFPEF patients, cardiac dysfunction and impaired exercise capacity occur independently of FID.