Elevated platelet-leukocyte aggregates as markers of chronic inflammatory thrombosis in chronic thromboembolic pulmonary hypertension

Chronic thromboembolic pulmonary hypertension (CTEPH) is a late sequela of acute pulmonary embolism (APE), but also carries features of arterial vascular disease. Therefore, we investigated platelet function in CTEPH.CTEPH platelets were characterized in samples of our prospective CTEPH registry and biobank (n=85). Platelet function was measured by cone and platelet analyzer and multiplate analyzer. Plasma and platelet lysate concentrations of platelet activation markers (soluble P-selectin (sP-sel), platelet factor 4), granule proteins (von Willebrand factor, plasminogen activator inhibitor-1) and neutrophil protein S100A8/A9 were determined by ELISAs. Platelet surface P-selectin and activated glycoprotein (GP) IIb/IIIa, as well as circulating monocyte-, leukocyte-, and neutrophil-platelet aggregates were measured in whole blood by flow cytometry. Nuclear factor-κB (NF-κB) signaling was assessed by IκB kinase β (IKKβ) immunoblotting in lysates of isolated platelets. We found that a large proportion of CTEPH platelets were complexed with leukocytes. As compared to APE, monocyte- and neutrophil-platelet aggregates were significantly elevated in CTEPH patients after the addition of agonists. CTEPH platelet lysates showed low sP-sel concentrations, and high ratios of phosphorylated IKKα/β to IKKβ. CTEPH patients with ongoing systemic inflammation like inflammatory bowel disease and/or anti-phospholipid syndrome, demonstrated higher plasma levels of neutrophil-derived S100A8/A9. CTEPH platelets form high levels of aggregates with leukocytes which might be mediated by increased NF-κB signaling. These data speak to chronic inflammatory thrombosis in CTEPH.