Background: Protease-activated receptor 1 (PAR1) and toll-like receptors (TLRs) are inflammatory mediators contributing to atherogenesis and atherothrombosis. Vorapaxar, which selectively antagonizes PAR1-signaling, is an approved, add-on antiplatelet therapy for secondary prevention. The non-hemostatic, platelet-independent, pleiotropic effects of vorapaxar have not yet been studied. Methods and Results: Cellular targets of PAR1 signaling in the vasculature were identified in three patient cohorts with atherosclerotic disease. Evaluation of plasma biomarkers (n = 190) and gene expression in endomyocardial biopsies (EMBs) (n = 12) revealed that PAR1 expression correlated with endothelial activation and vascular inflammation. PAR1 colocalized with TLR2/4 in human carotid plaques and was associated with TLR2/4 gene transcription in EMBs. In addition, vorapaxar reduced atherosclerotic lesion size in apolipoprotein E–knock out (ApoEko) mice. This reduction was associated with reduced expression of vascular adhesion molecules and TLR2/4 presence, both in isolated murine endothelial cells and the aorta. Thrombin-induced uptake of oxLDL was augmented by additional TLR2/4 stimulation and abrogated by vorapaxar. Plaque-infiltrating pro-inflammatory cells were reduced in vorapaxar-treated ApoEko mice. A shift toward M2 macrophages paralleled a decreased transcription of pro-inflammatory cytokines and chemokines. Conclusions: PAR1 inhibition with vorapaxar may be effective in reducing residual thrombo-inflammatory event risk in patients with atherosclerosis independent of its effect on platelets.

Diabetes mellitus is characterized by chronic vascular disorder and presents a main risk factor for cardiovascular mortality. In particular, hyperglycaemia and inflammatory cytokines induce vascular circulating tissue factor (TF) that promotes pro-thrombotic conditions in diabetes. It has recently become evident that alterations of the post-transcriptional regulation of TF via specific microRNA(miR)s, such as miR-126, contribute to the pathogenesis of diabetes and its complications. The endothelial miR-19a is involved in vascular homeostasis and atheroprotection. However, its role in diabetes-related thrombogenicity is unknown. Understanding miR-networks regulating procoagulability in diabetes may help to develop new treatment options preventing vascular complications. Plasma of 44 patients with known diabetes was assessed for the expression of miR-19a, TF protein, TF activity, and markers for vascular inflammation. High miR-19a expression was associated with reduced TF protein, TF-mediated procoagulability, and vascular inflammation based on expression of vascular adhesion molecule-1 and leukocyte count. We found plasma expression of miR-19a to strongly correlate with miR-126. miR-19a reduced the TF expression on mRNA and protein level in human microvascular endothelial cells (HMEC) as well as TF activity in human monocytes (THP-1), while anti-miR-19a increased the TF expression. Interestingly, miR-19a induced VCAM expression in HMEC. However, miR-19a and miR-126 co-transfection reduced total endothelial VCAM expression and exhibited additive inhibition of a luciferase reporter construct containing the F3 3′UTR. While both miRs have differential functions on endothelial VCAM expression, miR-19a and miR-126 cooperate to exhibit anti-thrombotic properties via regulating vascular TF expression. Modulating the post-transcriptional control of TF in diabetes may provide a future anti-thrombotic and anti-inflammatory therapy.

Background: Atrial myopathy and atrial fibrillation (AF) accompany thrombo-inflammation. This facilitates disease progression and promotes major adverse cardiovascular events (MACEs). Thrombin receptor (protease-activated receptor 1, PAR1) signalling is central in mediating thrombo-inflammation. We hypothesised that PAR1 signalling links coagulation and inflammation through cytotoxic CD8⁺ T lymphocytes in patients presenting with first-diagnosed AF (FDAF). Methods: A total of 210 patients were studied. We included data and blood samples from patients presenting with FDAF (n = 160), cardiac tissue from patients with paroxysmal AF (n = 32) and 20 controls. Results: During early AF, a pro-inflammatory and cytotoxic subset of T lymphocytes (CD8⁺) circulated more frequently when compared to patients with chronic cardiovascular disease but without AF, accompanied by elevated plasma levels of CD8⁺ effector molecules, which corresponded to biomarkers of adverse cardiac remodelling and atrial dysfunction. Activation of tissue factor (TF) and PAR1 was associated with pro-inflammatory and cytotoxic effector functions. PAR1-related CD8⁺ cell activation was more frequent in FDAF patients that experienced a MACE. Conclusions: In patients with FDAF, the TF-factor Xa-factor IIa-axis contributes to thrombo-inflammation via PAR1 in CD8⁺ T cells. Intervening in this cascade might be a promising synergistic approach to reducing disease progression and the vascular complications of AF.

Background: According to the leaky gut concept, microbial products (e.g., lipopolysaccharide, LPS) enter the circulation and mediate pro-inflammatory immunological responses. Higher plasma LPS levels have been reported in patients with various cardiovascular diseases, but not specifically during early atrial fibrillation (AF). Methods: We studied data and blood samples from patients presenting with first-diagnosed AF (FDAF) (n = 80) and 20 controls. Results: Circulating biomarkers that are suggestive of mucosal inflammation (zonulin, mucosal adhesion molecule MAdCAM-1) and intestinal epithelium damage (intestinal fatty acid binding protein, IFABP) were increased in the plasma of patients with FDAF when compared to patients with chronic cardiovascular diseases but without AF. Surrogate plasma markers of increased intestinal permeability (LPS, CD14, LPS-binding protein, gut-derived LPS-neutralising IgA antibodies, EndoCAbs) were detected during early AF. A reduced ratio of IgG/IgM EndoCAbs titres indicated chronic endotoxaemia. Collagen turnover biomarkers, which corresponded to the LPS values, suggested an association of gut-derived low-grade endotoxaemia with adverse structural remodelling. The LPS concentrations were higher in FDAF patients who experienced a major adverse cardiovascular event. Conclusions: Intestinal barrier dysfunction and microbial translocation accompany FDAF. Improving gut permeability and low-grade endotoxaemia might be a potential therapeutic approach to reducing the disease progression and cardiovascular complications in FDAF.

Inflammation-induced coagulopathy is a common complication associated with coronavirus disease 2019 (COVID-19). We aim to evaluate the association of NETosis and complement markers with each other as well as their association with thrombogenicity and disease severity in COVID-19. The study included hospitalized patients with an acute respiratory infection: patients with SARS-CoV2 infection (COVpos, n = 47) or either pneumonia or infection-triggered acute exacerbated COPD (COVneg, n = 36). Our results show that NETosis, coagulation, and platelets, as well as complement markers, were significantly increased in COVpos patients, especially in severely ill COVpos patients. NETosis marker MPO/DNA complexes correlated with coagulation, platelet, and complement markers only in COVpos. Severely ill COVpos patients showed an association between complement C3 and SOFA (R = 0.48; p ≤ 0.028), C5 and SOFA (R = 0.46; p ≤ 0.038), and C5b-9 and SOFA (R = 0.44; p ≤ 0.046). This study provides further evidence that NETosis and the complement system are key players in COVID-19 inflammation and clinical severity. Unlike previous studies that found NETosis and complement markers to be elevated in COVID-19 patients compared to healthy controls, our findings show that this characteristic distinguishes COVID-19 from other pulmonary infectious diseases. Based on our results, we propose that COVID-19 patients at high risk for immunothrombosis could be identified via elevated complement markers such as C5.

Galectin-3 is a beta-galactoside-binding lectin involved in inflammation and lung fibrosis and postulated to enhance thrombosis. In COVID-19, it is considered to be a prognostic marker of severity. The aim of this study was to evaluate whether galectin-3 is associated with thrombogenicity in COVID-19. Patients with moderate-to-severe COVID-19 (COVpos; n = 55) and patients with acute respiratory diseases, but without COVID-19 (COVneg; n = 35), were included in the study. We measured the amount of galectin-3, as well as other platelet and coagulation markers, and correlated galectin-3 levels with these markers of thrombogenicity and with the SOFA Score values. We found that galectin-3 levels, as well as von Willebrand Factor (vWF), antithrombin and tissue plasminogen activator levels, were higher in the COVpos than they were in the COVneg cohort. Galectin-3 correlated positively with vWF, antithrombin and D-dimer in the COVpos cohort, but not in the COVneg cohort. Moreover, galactin-3 correlated also with clinical disease severity, as measured by the SOFA Score. In patients with acute respiratory diseases, galectin-3 can be considered as a marker not only for disease severity, but also for increased hypercoagulability. Whether galectin-3 might be a useful therapeutic target in COVID-19 needs to be assessed in future studies.