Roflumilast N-oxide, a selective PDE4 inhibitor, curbs platelet-leukocyte interactions

Objective: COPD is associated with cardiovascular comorbidities. Platelet (Plt)–leukocyte interactions play a role in vascular disease. We tested the effect of the PDE4 inhibitor roflumilast N-oxide (RNO), the active metabolite of roflumilast approved for severe COPD in EU, on Plt-mediated neutrophil (Neu) recruitment and tissue factor (TF) expression in monocytes (Mn). Methods: Neu adhesion to spread Plt or endothelium (EC) and Plt-induced TF in Mn were analysed with RNO with or without formoterol (F) (100 nM).*p Results: In a flow adhesion assay RNO, alone or with F, concentration-dependently reduced the number of Neu firmly adhered on spread Plt at 10 dynes/cm 2 shear stress (C 106±18 and RNO [100nM] 71±7*; F 110±11 and RNO&F 58±7* Neu per field). RNO also inhibited P-selectin (C 59±11, RNO 35±7*, F 65±13, RNO&F 16±4* Neu per field) and Plt-induced (C 49±2, RNO 12±2*, F 50±11, RNO&F 2±0* Neu per field) Neu recruitment on EC. Src kinases and downstream effectors Pyk2 and PI(3)K mediate P-selectin-triggered PSGL-1-Mac-1 cross-talk. RNO curbed Src-mediated Pyk2 phosphorylation and PI3K-mediated Akt phosphorylation, while inducing phosphorylation of Csk, the major negative regulator of Src, by protein kinase A. In Mn exposed to activated Plt (24h), RNO dose-dependently reduced TF activity (C 67±7, F 56±6, RNO [100nM] 9.7±3.9*, RNO&F 2±0.8*, arbitrary units). In the presence of F, RNO at 1nM reduced TF activity to 24.5±7.8*. RNO (100nM) also reduced TF mRNA (C 72±10, RNO 9±3 fold increase). Conclusion: The PDE4 inhibitor RNO curbs Plt–leukocyte interactions.