Roflumilast inhibits leukocyte-platelet interactions and prevents the prothrombotic functions of polymorphonuclear leukocytes and monocytes.
ESSENTIALS: Thrombosis is a major comorbidity in patients with chronic obstructive pulmonary disease (COPD). Roflumilast is a selective phosphodiesterase type-4 (PDE4) inhibitor approved for treatment of severe COPD. PDE4 blockade by roflumilast inhibits prothrombotic functions of neutrophils and monocytes. PDE4 inhibitors may reduce thrombotic risk in COPD as well as in other vascular diseases.


BACKGROUND
Roflumilast, an oral selective phosphodiesterase type 4 inhibitor, is approved for the treatment of severe chronic obstructive pulmonary disease (COPD). A recent meta-analysis of trials on COPD revealed that treatment with roflumilast was associated with a significant reduction in the rate of major cardiovascular events. The mechanisms of this effect remain unknown.


OBJECTIVE
We tested the hypothesis that roflumilast N-oxide (RNO), the active metabolite of roflumilast, curbs the molecular mechanisms required for leukocyte-platelet (PLT) interactions and prevents the prothrombotic functions of polymorphonuclear leukocytes (PMNs) and monocytes (MNs).


METHODS
Using well-characterized in vitro models, we analysed the effects of RNO on: (i) PMN adhesiveness; (ii) the release of neutrophil extracellular traps (NETs); and (iii) tissue factor expression in MNs. Key biochemical events underlying the inhibitory effects of RNO were defined.


CONCLUSIONS
In PMNs, RNO prevented phosphoinositide 3-kinase (PI3K)-dependent phosphorylation of Akt on Ser473, and Src family kinase (SFK)-mediated Pyk2 phosphorylation on Tyr579-580, while inducing protein kinase A-mediated phosphorylation of C-terminal Src kinase, the major negative regulator of SFKs. Modulation of these signaling pathways by RNO resulted in a significant impairment of PMN adhesion to activated PLTs or human umbilical vein endothelial cells, mainly mediated by inhibition of the adhesive function of Mac-1. Moreover RNO curbed SFK/PI3K-mediated NET release by PMNs adherent on fibrinogen-coated surfaces. In MNs interacting with activated PLTs, RNO curbed PI3K-mediated expression of tissue factor. The efficacy of RNO was significantly potentiated by formoterol, a long acting β-adrenergic receptor agonist. This study reveals novel antithrombotic activities by which roflumilast may exert protective effects against cardiovascular comorbodities in COPD.
Laboratory of Vascular Biology and Pharmacology, Fondazione Mario Negri Sud, Santa Maria Imbaro, Italy.
L
L
Totani
Laboratory of Vascular Biology and Pharmacology, Fondazione Mario Negri Sud, Santa Maria Imbaro, Italy.
C
C
Amore
Laboratory of Vascular Biology and Pharmacology, Fondazione Mario Negri Sud, Santa Maria Imbaro, Italy.
A
A
Di Santo
Laboratory of Vascular Biology and Pharmacology, Fondazione Mario Negri Sud, Santa Maria Imbaro, Italy.
G
G
Dell'Elba
Laboratory of Vascular Biology and Pharmacology, Fondazione Mario Negri Sud, Santa Maria Imbaro, Italy.
A
A
Piccoli
Laboratory of Vascular Biology and Pharmacology, Fondazione Mario Negri Sud, Santa Maria Imbaro, Italy.
N
N
Martelli
Takeda Pharmaceuticals International GmbH, Glattpark-Opfikon, Switzerland.
H
H
Tenor
Takeda Pharmaceuticals International GmbH, Glattpark-Opfikon, Switzerland.
R
R
Beume
Laboratory of Vascular Biology and Pharmacology, Fondazione Mario Negri Sud, Santa Maria Imbaro, Italy.
V
V
Evangelista
D000631:Aminopyridines / Q000494:pharmacology*; D000818:Animals; D001549:Benzamides / Q000494:pharmacology*; D001792:Blood Platelets / Q000187:drug effects*; D002318:Cardiovascular Diseases / Q000517:prevention & control; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4 / Q000378:metabolism; D003521:Cyclopropanes / Q000494:pharmacology; D065206:Extracellular Traps; D005340:Fibrinogen / Q000737:chemistry; D006801:Humans; D007962:Leukocytes / Q000187:drug effects*; D016177:Macrophage-1 Antigen / Q000235:genetics; D051379:Mice; D018613:Microscopy, Confocal; D009000:Monocytes / Q000166:cytology* / Q000187:drug effects; D009504:Neutrophils / Q000166:cytology* / Q000187:drug effects; D019007:P-Selectin / Q000235:genetics / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D058988:Phosphodiesterase 4 Inhibitors / Q000494:pharmacology; D010766:Phosphorylation; D010973:Platelet Adhesiveness / Q000187:drug effects; D029424:Pulmonary Disease, Chronic Obstructive / Q000188:drug therapy; D012306:Risk; D013925:Thromboplastin / Q000378:metabolism; D013927:Thrombosis / Q000097:blood*
D000631:Aminopyridines; D001549:Benzamides; D003521:Cyclopropanes; D016177:Macrophage-1 Antigen; D019007:P-Selectin; D058988:Phosphodiesterase 4 Inhibitors; C424423:Roflumilast; D005340:Fibrinogen; D013925:Thromboplastin; D019869:Phosphatidylinositol 3-Kinases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; C517734:roflumilast N-oxide
blood platelets; leukocytes; pharmacology; signal transduction; thrombosis