PKC and AKT Modulate cGMP/PKG Signaling Pathway on Platelet Aggregation in Experimental Sepsis

Dermot Cox,M Elisa Lopes-Pires,Ana C Antunes Naime,Sisi Marcondes,Edson Antunes,Débora J Anjos,Nádia J Almeida Cardelli

doi:10.1371/journal.pone.0137901

Dermot Cox, M Elisa Lopes-Pires + Show 5 more

Open Access

https://doi.org/10.1371/journal.pone.0137901

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Journal: PloS one	Publication Date: Sep 16, 2015
Citations: 15	License type: CC BY 4.0

Affiliation: Universidade Estadual de Campinas

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.

Highlights

Platelets are important blood elements responsible for homeostasis maintenance and formation of pathological thrombus
We have demonstrated that protein kinase C (PKC) and AKT mediate the reduction of ADPinduced rat platelet aggregation after LPS-injection by upregulating upstream the soluble guanylyl cyclase (sGC)/cGMP/
We have showed that Src family kinases (SFKs) are not involved in the inhibitory effect of LPS on platelet aggregation

Summary

Introduction

Platelets are important blood elements responsible for homeostasis maintenance and formation of pathological thrombus. Stimulation of P2Y1 receptor leads to increase in cytosolic Ca++ concentration and PKC activation [2]. PKC in turn increases platelet secretion [3,4] and activates fibrinogen receptor (integrin αIIbβ3) that mediates the outside-in signaling, triggering a series of intracellular events that lead to platelet spreading, stabilization of platelet aggregate and cytoskeletal reorganization [5,6]. Stimulation of P2Y12 receptors activates PI3K, which is important for sustained αIIbβ integrin activation [2,7]. Most of the effects of NO in platelets are mediated by activation of soluble guanylyl cyclase (sGC) and cGMP formation, which activates cGMP-dependent protein kinase (PKG) leading to inhibition of platelet aggregation through phosphorylation of different targets [10,11]

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