Neutrophil subpopulation-specific ratio and neutrophil extracellular traps are dynamic and sensitive biomarkers of thrombo-inflammation in patients with acute coronary syndrome – a prospective study

Abstract Neutrophils have recently emerged as important effectors of thrombo-inflammation that contribute to the initiation and progression of atherothrombosis. Neutrophils are comprised of high-density neutrophil (HDN) and low-density neutrophil (LDN) sub-populations that differ in function and in thrombo-inflammatory potential, partially via differential formation capacity of neutrophil extracellular traps (NETs). In this prospective study, we investigated the dynamics of NET formation and of HDN and LDN dynamics over time in patients with acute coronary syndromes (ACS). Patients with ACS, admitted to the Cardiac Intensive Care Unit in a large tertiary center, were enrolled and assessed for NETosis biomarkers and for LDN to HDN ratio during the acute coronary event (up to 24h from admission), at 48h after admission and after 1 month. Neutrophils were isolated from peripheral blood samples and LDN and HDN sub-populations were separated via percoll gradient. NET formation was evaluated in equivalent LDN and HDN sub-population cell counts by assessing NET-specific neutrophil elastase activity, nucleosome release and NET associated markers such as citrullinated-histone H3 and MPO. In addition, LDN to HDN ratio was assessed by trypan blue staining and flow cytometry. Ten patients with ACS were enrolled to date. In LDNs, neutrophil elastase activity peaked at <24h after the cardiac index event with a value of 29.79 mU/ml and declined to 14.53 mU/ml and 4.14 mU/ml at 48h and 1 month, respectively (p<0.05). HDNs showed an opposite trend with elastase activity increasing from 22.71 mU/ml to 30.90 mU/ml and 37.70 mU/ml at <24h, 48h and 1 month form the index cardiac event (p<0.05).Nucleosome release mirrored this pattern, progressively decreasing in LDNs (0.78→0.52) while increasing in HDNs (0.59→0.83). LDN to HDN ratio significantly changed over time. The ratio was highest during the acute cardiac event and decreasing thereafter (p<0.05). This ongoing prospective study is the first, to our knowledge, to demonstrate neutrophil sub-population specific NET formation dynamics over time in patients with ACS. The LDN to HDN ratio represents a potential practical novel biomarker to follow the burden of thrombo-inflammation and NET formation in patients with ACS. Clinical trial enrollment is ongoing.