BIO15: Inhibition of Leukocytes Decreases Thrombin Generation in a In Vitro ECMO model

Abstract

Background: Despite systemic anti-coagulation, blood exposure to extracorporeal membrane oxygenation circuits (ECMO) generates excess thrombin, often resulting in thrombus formation, device failure, and life-threatening thromboembolism. Activated leukocytes and platelets promote ECMO thrombosis under controlled static and flow conditions. Previous studies document that phosphodiesterase type 4 inhibitor (PDE4) can selectively inhibit the pro-thrombotic function of leukocytes. Our hypothesis is that roflumilast (PDE4 inhibitor) decreases thrombin generation in an in vitro ECMO model using human blood. Methods: Simulated neonatal roller pump with a KIDS D100 oxygenator circulated human whole blood at a nominal flow rate (0.5 L/min) for six hours or until clot formation caused device failure. Samples were collected hourly from the ECMO model that received heparin alone or heparin plus roflumilast, a PDE4 inhibitor. Expression of pro-thrombotic proteins on leukocytes and extracellular vesicles (EV) was measured with high-resolution flow cytometry. A calibrated automated thrombogram without a trigger measured differences in time to thrombin and peak thrombin generation. Results: The addition of roflumilast to heparin sustained patency in 75% of tested circuits (3/4) after six hours, compared to 28% of circuits treated with heparin alone (2/7). Roflumilast decreased extracellular leukocytes expressing tissue factor. Roflumilast plus heparin prolonged time to thrombin generation at 13.9 minutes, compared to heparin alone at 6.8 minutes at the 3-hour time point (p<0.04). The addition of roflumilast significantly reduced peak thrombin generation compared to heparin alone (69 vs. 207 nm, p<0.02). Conclusions: Inhibition of leukocytes may be an effective adjunct to systematic anti-coagulation in extracorporeal life support.