The effects of gaseous nitric oxide on platelets and leukocytes in membrane oxygenators

Abstract

Nitric oxide (NO), known as a potent endogenous platelet anti-adhesive, anti-aggregating, and disaggregating radical, was shown to reduce platelet trapping in microporous membrane oxygenators [1]. In an additional study we investigated the effects of gaseous NO on platelet and leukocyte activation markers during extracorporeal circulation. Two parallel separated extracorporeal circuits (n = 6) were filled with heparinized (1 IE/ml) fresh drawn blood from one healthy volunteer. The gas inlets of both oxygenators (M8 Jostra/Germany) received dry gas (21% oxygen, 5% carbon dioxide, 84% nitrogen); gaseous NO (20 ppm) was added to the gas inlet of one of the oxygenators, whereas the other one was used for control. Blood samples obtained from a venous reservoir and from the blood donor were investigated by flow cytometry for the following markers: leukocytes-CD11a, CD11b, HLA-DR, CD62L (L-selectin), and CD14. Platelets-P-selectin (CD62P), CD42b (GPlb), CD41a (GPIIbIIIa), and activated conformationally changed GP-IIbIIIa. Additional, platelets were stimulated with ADP (10 µM), epinephrine (10 µM), or both (each 5 µM) to investigate platelet reactivity. Further analysis included: coagulation parameters (fibrinogen, ATIII, heparin-time, prothrombin-time, fibrin monomers); platelet counts (in quadruplet); blood gas analysis; and leukocyte differential count. The main results are: (i) NO significantly attenuated platelet trapping within the membrane oxygenator (Fig ​(Fig1);1); (ii) In both oxygenators only small amounts (1-2%) of circulating activated (P-selectin or activated GPIIbIIIa) platelets were detectable over time; (iii) platelet reactivity to stimulating agents decreased during circulation, indicating platelet damage; (iv) NO seemed to preserve platelet reactivity to some degree which was pronounced with duration of circulation (Fig ​(Fig2);2); (v) there was no significant difference in loss of leukocytes, ie trapping of PMN and monocytes (lymphocytes remained stable) between the two oxygenators; (vi) leukocyte adhesion molecule expression was significantly altered during circulation, however, no differences were found between NO and control: HLA-DR and CD14 increased on monocytes, CDlla increased on lymphocytes, CDllb increased on monocytes and PMN, and L-selectin was reduced on monocytes and PMN, whereas L-selectin expression on lymphocytes increased over time; (vii) no significant differences were found lor coagulation parameters or blood gas analysis. Figure 1 Platelet (squares) and hemoglobin (circles) values in percent from baseline (start). *P < 0.05, **P < 0.01, paired t-test. Figure 2 Activated GPIIb-IIIa on platelets stimulated with 10 µM ADP (A), 10 µM epinephrine (EPI) (B), or 5 µM ADP + 5 µM EPI (C) at different timepoints. Note the time-dependent loss of reactivity to stimulating agents in both ...