Inflammatory thrombosis is a denominator of permanent platelet activation in chronic thromboembolic pulmonary hypertension

Abstract Introduction Chronic thromboembolic pulmonary hypertension (CTEPH) is a late sequela of acute pulmonary embolism (PE), but also carries features of arterial vascular disease. Increased levels of C-reactive protein, D-dimer, and myeloperoxidase are common risk factors for both conditions, and indicate a possible role of platelets in CTEPH pathogenesis. Therefore, we investigated platelet function at rest and under agonist stimulation. Methods Thrombi from CTEPH patients were collected after pulmonary endarterectomy and proteomic analysis was performed using liquid chromatography–mass spectrometry (n=41). In addition to healthy controls, we looked at patients with PE as comparator. Plasma and platelet lysate concentrations of platelet activation markers like soluble P-selectin, von Willebrand factor (vWF), platelet factor 4 and neutrophil protein S100A8/A9 were determined by immunoassays. Platelet surface P-selectin as well as circulating monocyte-, leukocyte-, and granulocyte-platelet aggregates were measured in whole blood by flow cytometry both at baseline and following stimulation with platelet agonists like adenosine diphosphate (ADP), convulxin (CVX), and thrombin receptor associated protein (TRAP). Immunoblotting was used to assess NF-kB signaling in isolated platelets. Statistical analysis was conducted in GraphPad Prism. Differential expression analysis was done in R using the limma package and multiple testing correction was performed using the Benjamini-Hochberg method. Results Mass spectrometry analysis demonstrated that fresh or partially organized CTEPH thrombus was enriched in proteins associated with platelet activation, coagulation and leukocyte activation pathways. Secreted P-selectin and vWF plasma concentrations were elevated in CTEPH, while platelet lysates demonstrated low P-selectin concentrations, and upregulation of NF-kB signaling. CTEPH patients with inflammatory bowel disease and anti-phospholipid syndrome had higher plasma levels of neutrophil-derived S100A8/A9 protein. Although surface P-selectin expression was elevated on CTEPH platelets, aggregate formation remained unchanged. However, these platelets exhibited heightened responsiveness to stimulation by the collagen-receptor agonist CVX. Conclusion Circulating CTEPH platelets are in a state of activation at baseline, and responsive to agonist-induced activation, particularly to stimulation via the collagen receptor. Increased activation of pro-inflammatory NF-kB signaling in CTEPH platelets speak to chronic vWF-driven thrombosis and inflammatory platelet activation in CTEPH.