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Study of guanylyl cyclase via soluble - cGMP in platelet dysfunction in rats with sepsis

Grant number: 14/26592-4
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): May 01, 2015
Effective date (End): October 31, 2015
Field of knowledge:Health Sciences - Pharmacy
Principal Investigator:Edson Antunes
Grantee:Nieli de Aguiar Geraldi
Home Institution: Faculdade de Ciências Médicas (FCM). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil

Abstract

One of the earliest events in sepsis is the reductions in circulating number of leukocytes and platelets. The number of platelets retained in peripheral organs and the degree of platelet activation positively correlate with sepsis severity and multiple organ failure. Sepsis is characterized by uncontrolled release of different inflammatory mediators, including nitric oxide (NO). The soluble guanylyl cyclase (sGC) acts as the main intracellular receptor for NO, and comprises four subunits, namely alpha 1, alpha 2, beta 1 e beta 2 (catalytic domain) and prosthetic (Fe2+) domain. Binding of Fe2+ promotes the formation of hemenitrosil, leading to an increase in sGC activity, thus elevating the levels of cGMP. The NO-sGC-cGMP is impaired by reduction of NO bioavailability and/or by heme-oxidation group (Fe3+), making the enzyme insensitive to NO. Recent evidence shows that oxidative stress affects the sGC redox state, reducing the affinity of this enzyme to NO. In sepsis, there is accumulation of both reactive-oxygen (ROS) and -nitrogen species (RNS), as well as reductions of antioxidant mechanisms. In the last decade, the so-called "stimulators" and "activators" of sGC emerged as important drugs for the treatment of cardiovascular diseases, including pulmonary hypertension (ex. Riociguat, Cinaciguat). These drugs have also been valuable tools to understanding the defects in sGC-cGMP signaling pathway. BAY 41-2272 is a prototypal as sGC stimulator. It stimulates sGC by an NO-independent mechanism, but rather dependent on heme-reduced sGC state (Fe2+). The prototypal for sGC activators include BAY 58-2667 e BAY 60-2770 that also exert its actions in an NO-independent mechanism, but prefer the heme-oxidation (Fe3+) state of sGC. Activators of sGC like BAY 60-2770 are also able to rescue the protein levels of alpha 1 / beta 1 subunits in platelets undergoing oxidation. Taken into consideration that LPS-induced sepsis increases the intraplatelet ROS production and that ROS accumulation degrades alpha 1 / beta 1 sGC subunits, we hypothesized that lower protein levels of sGC contributes to platelet dysfunction in sepsis. Therefore, this project aimed to examine the sGC-cGMP signaling pathway in platelets from LPS-induced sepsis in rats, quantifying the platelet aggregation, the protein levels of alpha 1 / beta 1 sGC (Western Blotting) and cGMP levels in washed platelets. We also aim to study the effects of activators (BAY 60-2770, BAY 58-2667) and stimulators (BAY 41-2272) in the functional and molecular assays above-described.