title: PKC and AKT Modulate cGMP/PKG Signaling Pathway on Platelet Aggregation in Experimental Sepsis.
abstract: Sepsis severity has been positively correlated with platelet dysfunction, which may be due to elevations in nitric oxide (NO) and cGMP levels. Protein kinase C, Src kinases, PI3K and AKT modulate platelet activity in physiological conditions, but no studies evaluated the role of these enzymes in platelet aggregation in sepsis. In the present study we tested the hypothesis that in sepsis these enzymes positively modulate upstream the NO-cGMP pathway resulting in platelet inhibition. Rats were injected with lipopolysaccharide (LPS, 1 mg/kg, i.p.) and blood was collected after 6 h. Platelet aggregation was induced by ADP (10 μM). Western blotting assays were carried out to analyze c-Src and AKT activation in platelets. Intraplatelet cGMP levels were determined by enzyme immunoassay kit. Phosphorylation of c-SRC at Tyr416 was the same magnitude in platelets of control and LPS group. Incubation of the non-selective Src inhibitor PP2 (10 μM) had no effect on platelet aggregation of LPS-treated rats. LPS increased intraplatelet cGMP levels by 5-fold compared with control group, which was accompanied by 76% of reduction in ADP-induced platelet aggregation. The guanylyl cyclase inhibitor ODQ (25 μM) and the PKG inhibitor Rp-8-Br-PET-cGMPS (25 μM) fully reversed the inhibitory effect of LPS on platelet aggregation. Likewise, the PKC inhibitor GF109203X (10 μM) reversed the inhibition by LPS of platelet aggregation and decreased cGMP levels in platelets. AKT phosphorylation at Thr308 was significantly higher in platelets of LPS compared with control group, which was not reduced by PI3K inhibition. The AKT inhibitor API-1 (20 μM) significantly increased aggregation and reduced cGMP levels in platelets of LPS group. However, the PI3K inhibitor wortmannin and LY29004 had no effect on platelet aggregation of LPS-treated rats. Therefore, inhibition of ADP-induced platelet aggregation after LPS injection is mediated by cGMP/PKG-dependent mechanisms, and PKC and AKT act upstream upregulating this pathway.
authors.affiliation: Department of Pharmacology, Faculty of Medical Sciences, University of Campinas (UNICAMP), Campinas (SP), Brazil.
authors.forename: M Elisa
authors.initials: ME
authors.lastname: Lopes-Pires
authors.affiliation: Department of Pharmacology, Faculty of Medical Sciences, University of Campinas (UNICAMP), Campinas (SP), Brazil.
authors.forename: Ana C Antunes
authors.initials: AC
authors.lastname: Naime
authors.affiliation: Department of Pharmacology, Faculty of Medical Sciences, University of Campinas (UNICAMP), Campinas (SP), Brazil.
authors.forename: Nádia J
authors.initials: NJ
authors.lastname: Almeida Cardelli
authors.affiliation: Department of Pharmacology, Faculty of Medical Sciences, University of Campinas (UNICAMP), Campinas (SP), Brazil.
authors.forename: Débora J
authors.initials: DJ
authors.lastname: Anjos
authors.affiliation: Department of Pharmacology, Faculty of Medical Sciences, University of Campinas (UNICAMP), Campinas (SP), Brazil.
authors.forename: Edson
authors.initials: E
authors.lastname: Antunes
authors.affiliation: Department of Pharmacology, Faculty of Medical Sciences, University of Campinas (UNICAMP), Campinas (SP), Brazil.
authors.forename: Sisi
authors.initials: S
authors.lastname: Marcondes
mesh_terms: D000818:Animals; D001780:Blood Coagulation Tests; D015153:Blotting, Western; D006152:Cyclic GMP / Q000378:metabolism*; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D010766:Phosphorylation / Q000187:drug effects; D010974:Platelet Aggregation / Q000187:drug effects; D010979:Platelet Function Tests; D011493:Protein Kinase C / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D018805:Sepsis / Q000139:chemically induced / Q000378:metabolism / Q000473:pathology*; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D008070:Lipopolysaccharides; D051057:Proto-Oncogene Proteins c-akt; D017869:Cyclic GMP-Dependent Protein Kinases; D011493:Protein Kinase C; D006152:Cyclic GMP