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Erythrocyte deformability and nitric oxide mobilization under pannexin-1 and PKC dependence
Publication . Herdade, A. S. Silva; Freitas, T.; Almeida, J. Pedro; Saldanha, Carlota
The erythrocyte adenosine triphosphate (ATP) is utilised for protein phosphorylation and exported through the pannexin 1 hemichannel (Px1) in the microcirculation. The physiological stimuli for ATP release are dependent of blood shear rate level and of the tissue oxygen content. The deoxygenated and oxygenated states of haemoglobin are respectively bound and unbound to N terminal domain of the protein band 3 of the erythrocyte membrane in dependence of its degree of phosphorylation. The protein tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP) contribute to the phosphorylation degree of band 3 and are modulated by protein kinase C (PKC). Chelerythrine (Che) is a competitive inhibitor of ATP for PKC and a negative modulator of erythrocyte deformability. The aim of this study was to assess the mobilization of nitric oxide (NO) in erythrocyte in absence and presence of Che and Px1 inhibitor (carbenoxolone). Erythrocyte deformability was evaluated in presence of carbenoxolone (Carb). Regarding the effects observed in the erythrocyte by presence of Che or Carb, the values of efflux of NO and the concentration of nitrosogluthatione are similar and with no changes in relation to their absence. Px1inhibition by Carb 10 μM ameliorates the erythrocyte deformability at a shear force of 0.6 and 1.2 Pa. The PKC inhibitor shows similar effects to the Carb on the mobilization of nitric oxide in erythrocyte. The blockage of ATP release by Carb from erythrocytes suggests a possible benefit to develop in ischemia reperfusion or in inflammatory response where will be needed to rescue the excess of NO present and ameliorate the red blood cell deformability at low shear rates.
An in vitro study on the modulation of the neutrophil adhesive behavior by soluble fibrinogen
Publication . Almeida, V. Vitorino de; Calado, A.; Herdade, A. S. Silva; Rosário, H. S.; Saldanha, Carlota
Fibrinogen constitutes an important plasma glycoprotein involved in hemostasis and in inflammation. Previously, we have shown that at physiological concentrations, soluble fibrinogen is able to modulate the pattern of neutrophil activation. This led us to propose that under these conditions, fibrinogen could as well interfere with the adhesive behaviour of circulating neutrophils which is of utmost importance in their recruitment to the vascular wall during inflammatory processes. To address our working hypothesis, in vitro adhesion assays were here performed in a flow chamber by using primary cultures of human umbilical vein endothelial cells (HUVEC) and neutrophils isolated from peripheral venous blood of healthy human donors. In the presence of a physiological concentration of soluble fibrinogen (300 mg/dL), we observed that despite the number of neutrophils rolling on an activated endothelium was not affected, their rolling velocity was increased in comparison to that of non-activated neutrophils. Consequently as expected, the number of fibrinogen-treated neutrophils adhering to activated HUVEC monolayers was significantly diminished. Overall, we have here demonstrated that at least in vitro, soluble fibrinogen under physiological concentrations is able to modulate the interaction of neutrophils with the vascular endothelium. In vivo studies will enable us in the future to study the physiological relevance of these findings and further to understand the mechanisms underlying this function.
Signal transduction pathways in erythrocyte nitric oxide metabolism under high fibrinogen levels
Publication . Saldanha, Carlota; Freitas, T.; Almeida, J. P. Lopes de; Herdade, A. Silva
Previous studies show that the fibrinogen molecule modulates the metabolism of nitric oxide (NO) in erythrocyte. The in vitro induced hiperfibrinogenemia interferes in the metabolism of the NO in the erythrocyte
in dependence of the phosphorylation degree of the band 3. The soluble form of fibrinogen binds into CD47
protein present in the erythrocyte membrane. The soluble thrombomodulin is an inflammatory marker that
binds to the erythrocyte CD47 in a site with a sequence peptide known as 4N1K. A study done in vitro
shows that when hiperfibrinogenemia was induced in the presence of the peptide 4N1K agonist of CD47
it were observed variations in the efflux of NO from erythrocyte and an increase in the concentrations of
GSNO, peroxinitrite, nitrite and nitrate of the erythrocytes. The aim of this work was to study the influence
of the peptide 4N1K, on the metabolism of NO in the erythrocyte under high fibrinogen concentration and
in the presence of inhibitors of the status of phosphorylation of protein band 3. In this in vitro study, whole
blood samples were harvested from healthy subjects and NO, peroxynitrite, nitrite, nitrate and S-nitroglutathione (GSNO) were determined in presence of 4N1K, calpeptine, Syk inhibitor and under high fibrinogen concentrations. The results obtained in erythrocytes under high fibrinogen levels when 4N1K is
present with the Syk inhibitor or with calpeptine, showed in relation to the control samples increased significant
concentrations of efflux of NO and of peroxynitrite, nitrite, nitrate and GSNO. In conclusion it was
verified that in the in vitro model of hiperfibrinogenemia the peptide 4N1K, agonist of CD47, induces mobilization of NO in the erythrocyte in dependence of the status of phosphorylation of protein band 3.
Fibrinogen modulates leukocyte recruitment in vivo during the acute inflammatory response
Publication . Almeida, V. Vitorino de; Herdade, A. Silva; Calado, A.; Rosário, H. S.; Saldanha, Carlota
Besides playing an important role in blood hemostases, fibrinogen also regulates leukocyte function in inflammation. Our previous in vitro studies showed that the adhesive behaviour of the neutrophil is modulated by soluble fibrinogen when present at a physiological concentration. This led us to propose that this plasma glycoprotein might further influence leukocyte recruitment in vivo and thus contribute to the inflammatory response. To address this in vivo, leukocyte recruitment was here investigated under acute inflammatory conditions in the absence of soluble fibrinogen in the blood circulation. For such, intravital microscopy on mesentery post-capillary venules was performed on homozygous fibrinogen α chain-deficient mice ((α-/-) mice). Acute inflammatory states were induced by perfusing platelet activating factor (PAF) over the exposed tissue. As control animals, two groups of mice expressing soluble fibrinogen in circulation were used, namely, C57BL/6 wild type animals and heterozygous fibrinogen α chain-deficient mice ((α+/-) mice). Under acute inflammatory conditions, an abnormal pattern of recruitment was observed for leukocytes in homozygous (α-/-) mice in comparison to both control groups. In fact, the former exhibited a significantly decreased number of rolling leukocytes that nevertheless, migrated with increased rolling velocities when compared to leukocytes from control animals. Consistently, homozygous mice further displayed a diminished number of adherent leukocytes than the other groups. Altogether our observations led us to conclude that leukocyte recruitment in homozygous (α-/-) mice is compromised what strongly suggests a role for soluble fibrinogen in leukocyte recruitment in inflammation.
Fibrinogen signalling in erythrocyte nitric oxide mobilization in presence of PI3-K and adenylyl cyclase inhibitors
Publication . Herdade, Ana S. Silva; Freitas, Teresa; Almeida, José P.; Saldanha, Carlota
Soluble form of fibrinogen (Fib) and the peptide 4N1K are ligands of erythrocyte membrane CD47. Fibrinogen
reinforces the ability of erythrocyte to scavenger nitric oxide (NO). Hiperfibrinogenemia increased NO efflux
from erythrocyte in dependence of band 3 phosphorylation which is abolished by the presence of 4N1K. Herein
we study in vitro the effect of high fibrinogen levels, on the NO efflux from erythrocytes and on its mobilization
under influence of phosphoinositide-3 kinase (PI3-K) and adenylyl cyclase (AC) inhibitors in presence of 4N1K.
Erythrocyte NO efflux, peroxynitrite, nitrite, nitrate and S-nitrosoglutathione (GSNO) were determined in blood
samples in presence of 4N1K, wortmannin (WORT, PI3-K inhibitor) and MDL (AC inhibitor) under high
fibrinogen concentrations. 4N1K with WORT and high fibrinogen levels induce, in relation to Fib plus WORT
samples no variations on the erythrocyte NO efflux, decreased peroxynitrite, increased of nitrite, nitrate and
GSNO concentrations. When 4N1K is present with MDL and high fibrinogen levels show, in relation to
fibrinogen plus MDL samples increased erythrocyte NO efflux and nitrite, nitrate and GSNO concentrations. In
conclusion, under high Fib levels and 4N1K the erythrocytes show: preservation of NO and impaired peroxynitrite in presence of PI3K inhibition; increased efflux of NO at lower levels of cAMP resulting from adenylyl cyclase inhibition.
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Fundação para a Ciência e a Tecnologia
Programa de financiamento
3599-PPCDT
Número da atribuição
PTDC/SAU-OSM/73449/2006