Thrombo-inflammation in atherosclerosis has been described as a complex interplay between blood coagulation and inflammation that plays a critical role in cardiovascular diseases (CVD). Furthermore, thrombo-inflammation is associated with residual cardiovascular risk. Protease-activated receptor 1 (PAR1) is the thrombin receptor expressed on platelets, but it is also expressed in endothelial cells (ECs), vascular smooth muscle cells (VSMCs), and immune cells. Therefore, PAR1-signalling is central in mediating thrombo-inflammation. Nevertheless, biomarker of PAR1-mediated thrombo-inflammation have not been described.

Methods:

Target identification of PAR1-mediated thrombo-inflammation was made in 190 patients with chronic CVD who were recruited from our outpatient department. We measured plasma biomarkers (using ELISA) indicative of endothelial activation—vascular cell adhesion protein 1 (VCAM1), intercellular adhesion molecule 1 (ICAM1), endothelial-leukocyte adhesion molecule 1 (E-selectin)—and vascular inflammation (TNF-α, IL-6, CRP). We correlated these with the percentage of FIIa-activated PAR1–positive (in which the antibody detects fragments of activated PAR1–cleaved-Ser⁴² protein) circulating peripheral blood mononuclear cells (PBMCs) using flow cytometry.

Results:

Evaluation of plasma biomarkers revealed that PAR1 expression correlated with endothelial activation and vascular inflammation.

We identified targets of PAR1-mediated thrombo-inflammation. Therefore, FIIa-activated PAR1 (the antibody detects PAR1 cleaved by FIIa)-positive PBMCs were used as an indicator for augmented signaling through the TF/FXa/FIIa-PAR1-axis. Thrombin-cleaved PAR1 was associated with increased plasma biomarkers such as VCAM1 (r=0.7579, p<0.0001), ICAM-1 (r=0.7514, p<0.0001), and E-selectin (r=0.5218, p<0.0001) in 190 high-risk patients (CRP of mg/l 7.4 ± 6.7 mean ± SD) with chronic CVD due to atherosclerosis. These markers are prognostically relevant and indicative of endothelial activation.

In addition, FIIa-activated PAR1-positive PBMC correlated with pro-inflammatory markers such as TNF-α (r=0.7006, p<0.0001), IL-6 (r=0.7757, p<0.0001), and CRP (r=0.7615, p<0.0001), showing vascular inflammation.

Conclusions:

Taken together, our findings contribute to the understanding of the atheroprotective effects of FXa/FIIa/PAR1-inhibitors observed in large clinical trials. Beyond thrombus formation, TF/FXa/FIIa-initiated PAR1/TLR2/4 signalling contributes to endothelial dysfunction and vascular inflammation. PAR1-mediated thrombo-inflammation is a potential target to treat atherosclerosis and prevent adverse atherothrombotic events in patients with a high residual ischemic risk.