Sisi Marcondes

Nitration of succinyl-CoA:3-oxoacid CoA-transferase in rats after endotoxin administration.

The tyrosine nitration of proteins has been observed in diverse inflammatory conditions and has been linked to the presence of reactive nitrogen species. From many in vitro experiments, it is apparent that tyrosine nitration may alter the function of proteins. A limited number of experiments under in vivo conditions also demonstrate that protein nitration is associated with altered cellular processes. To understand the association of protein nitration with the pathogenic mechanism of the disease, it is essential to identify specific protein targets of nitration with in vivo or intact tissue models. Using anti-nitrotyrosine antibodies, we demonstrated the accumulation of nitrotyrosine in a 52-kDa protein in rat kidney after lipopolysaccharide treatment. The 52-kDa protein was purified and identified with partial sequence as succinyl-CoA:3-oxoacid CoA-transferase (SCOT; EC 2.8.3.5). Western blot analysis revealed that the nitration of this mitochondrial enzyme increased in the kidneys and hearts of lipopolysaccharide-treated rats, whereas its catalytic activity decreased. These data suggest that tyrosine nitration may be a mechanism for the inhibition of SCOT activity in inflammatory conditions. SCOT is a key enzyme for ketone body utilization. Thus, tyrosine nitration of the enzyme with sepsis or inflammation may explain the altered metabolism of ketone bodies present in these disorders.

Activation of Haem-Oxidized Soluble Guanylyl Cyclase with BAY 60-2770 in Human Platelets Lead to Overstimulation of the Cyclic GMP Signaling Pathway

Background and Aims Nitric oxide-independent soluble guanylyl cyclase (sGC) activators reactivate the haem-oxidized enzyme in vascular diseases. This study was undertaken to investigate the anti-platelet mechanisms of the haem-independent sGC activator BAY 60-2770 in human washed platelets. The hypothesis that sGC oxidation potentiates the anti-platelet activities of BAY 60-2770 has been tested. Methods Human washed platelet aggregation and adhesion assays, as well as flow cytometry for αIIbβ3 integrin activation and Western blot for α1 and β1 sGC subunits were performed. Intracellular calcium levels were monitored in platelets loaded with a fluorogenic calcium-binding dye (FluoForte). Results BAY 60-2770 (0.001–10 µM) produced significant inhibition of collagen (2 µg/ml)- and thrombin (0.1 U/ml)-induced platelet aggregation that was markedly potentiated by the sGC inhibitor ODQ (10 µM). In fibrinogen-coated plates, BAY 60-2770 significantly inhibited platelet adhesion, an effect potentiated by ODQ. BAY 60-2770 increased the cGMP levels and reduced the intracellular Ca2+ levels, both of which were potentiated by ODQ. The cell-permeable cGMP analogue 8-Br-cGMP (100 µM) inhibited platelet aggregation and Ca2+ levels in an ODQ-insensitive manner. The cAMP levels remained unchanged by BAY 60-2770. Collagen- and thrombin-induced αIIbβ3 activation was markedly inhibited by BAY 60-2770 that was further inhibited by ODQ. The effects of sodium nitroprusside (3 µM) were all prevented by ODQ. Incubation with ODQ (10 µM) significantly reduced the protein levels of α1 and β1 sGC subunits, which were prevented by BAY 60-2770. Conclusion The inhibitory effects of BAY 60-2770 on aggregation, adhesion, intracellular Ca2+ levels and αIIbβ3 activation are all potentiated in haem-oxidizing conditions. BAY 60-2770 prevents ODQ-induced decrease in sGC protein levels. BAY 60-2770 could be of therapeutic interest in cardiovascular diseases associated with thrombotic complications.

Platelet hyperaggregability in high-fat fed rats: A role for intraplatelet reactive-oxygen species production

BackgroundAdiposity greatly increases the risk of atherothrombotic events, a pathological condition where a chronic state of oxidative stress is reported to play a major role. This study aimed to investigate the involvement of (NO)-soluble guanylyl cyclase (sGC) signaling pathway in the platelet dysfunction from high fat-fed (HFF) rats.MethodsMale Wistar rats were fed for 10 weeks with standard chow (SCD) or high-fat diet (HFD). ADP (10 μM)- and thrombin (100 mU/ml)-induced washed platelet aggregation were evaluated. Measurement of intracellular levels of ROS levels was carried out using flow cytometry. Cyclic GMP levels were evaluated using ELISA kits.ResultsHigh-fat fed rats exhibited significant increases in body weight, epididymal fat, fasting glucose levels and glucose intolerance compared with SCD group. Platelet aggregation induced by ADP (n = 8) and thrombin from HFD rats (n = 8) were significantly greater (P < 0.05) compared with SCD group. Platelet activation with ADP increased by 54% the intraplatelet ROS production in HFD group, as measured by flow cytometry (n = 6). N-acetylcysteine (NAC; 1 mM) and PEG-catalase (1000 U/ml) fully prevented the increased ROS production and platelet hyperaggregability in HFD group. The NO donors sodium nitroprusside (SNP; 10 μM) and SNAP (10 μM), as well as the NO-independent soluble guanylyl cyclase stimulator BAY 41-2272 (10 μM) inhibited the platelet aggregation in HFD group with lower efficacy (P < 0.05) compared with SCD group. The cGMP levels in response to these agents were also markedly lower in HFD group (P < 0.05). The prostacyclin analogue iloprost (1 μM) reduced platelet aggregation in HFD and SCD rats in a similar fashion (n = 4).ConclusionsMetabolic abnormalities as consequence of HFD cause platelet hyperaggregability involving enhanced intraplatelet ROS production and decreased NO bioavailability that appear to be accompanied by potential defects in the prosthetic haem group of soluble guanylyl cyclase.

Role of chondroitin 4‐sulphate as a receptor for polycation induced human platelet aggregation

1 Proteoglycans provide negatively charged sites on the surface of platelets, leukocytes and endothelial cells. Since chondroitin 4‐sulphate is the main proteoglycan present on the platelet surface, the role of this molecule in mediating the activation of human platelets by polylysine was studied. 2 Platelets were desensitized with phorbol 12‐myristate 13‐acetate (PMA, 10 nM) 5 min before the addition of polylysine to platelet‐rich plasma (PRP). Changes in the intracellular Ca2+ concentration were measured in fura2‐am (2 μm) loaded platelets and protein phosphorylation was assessed by autoradiography of the electrophoretic profile obtained from [32P]‐phosphate labelled platelets. The release of dense granule contents was measured in [14C]‐5‐hydroxytryptamine loaded platelets and the synthesis of thromboxane (TXA2) was assessed by radioimmunoassay. Surface chondroitin 4‐sulphate proteoglycan was degraded by incubating platelets with different concentrations of chondroitinase AC (3 min, 37°C). The amount of chondroitin 4‐sulphate remaining in the platelets was then quantified after proteolysis and agarose gel electrophoresis. 3 The addition of PMA to PRP before polylysine inhibited the aggregation by 88 ± 18% (n = 3). Staurosporine (1 μm, 5 min) prevented the PMA‐induced inhibition. Chondroitinase AC (4 pu ml−1 to 400 μu ml−1, 3 min) abolished the polylysine‐induced aggregation in PRP but caused only a discrete inhibition of ADP‐induced aggregation. The concentration of chrondroitin 4‐sulphate in PRP (0.96 ± 0.2 μg/108 platelets, n = 3) and in washed platelets (WP; 0.35 ± 0.1 μg/108 platelets, n = 3) was significantly reduced following incubation with chondroitinase AC (PRP = 0.63 ± 0.1 μg/108 platelets and WP = 0.08 ± 0.06 μg/108 platelets). 4 Washed platelets had a significantly lower concentration of chondroitin 4‐sulphate than platelets in PRP. The addition of polylysine to WP induced a rapid increase in light transmission which was not accompanied by TXA2 synthesis or the release of dense granule contents. This effect was not inhibited by sodium nitroprusside (SNP), iloprost, EDTA or the peptide RGDS. This event was accompanied by the discrete phosphorylation of plekstrin and myosin light chain, which were inhibited by staurosporine (10 μm, 10 min). The hydrolysis of platelet surface chondroitin 4‐sulphate strongly reduced the polylysine‐induced phosphorylation. 5 Our results indicate that polylysine activates platelets through a specific receptor which could be the proteoglycan chondroitin 4‐sulphate present on the platelet membrane.

Mechanisms underlying the inhibitory effects of lipopolysaccharide on human platelet adhesion

Alterations in platelet aggregation in septic conditions are well established. However, little is known about the effects of lipopolysaccharide (LPS) on platelet adhesion. We have therefore investigated the effects of LPS in human platelet adhesion, using an in vitro model of platelet adhesion to fibrinogen-coated wells. Microtiter plates were coated with human fibrinogen, after which washed platelets (6 × 108 platelets/ml) were allowed to adhere. Adherent platelets were quantified through measurement of acid phosphatase activity. Calcium mobilization in Fura2-AM-loaded platelets was monitored with a spectrofluorimeter. Platelet flow cytometry in thrombin-stimulated platelets was performed using monoclonal mouse anti-platelet GPIIb/IIIa antibody (PAC-1). Prior incubation of washed platelets with LPS (0.01–300 µg/ml) for 5 to 60 min concentration- and time-dependently inhibited non-activated platelet adhesion. In thrombin-activated (50 mU/ml) platelets, LPS inhibited the adhesion to a significantly lesser extent than non-activated platelets. Cyclohexamide, superoxide dismutase polyethylene glycol (PEG-SOD) or catalase polyethylene glycol did not affect the LPS responses. No alterations in cyclic GMP levels were seen after platelet incubation with LPS, except with the highest concentration employed (300 µg/ml) where an increase of 36% (P < 0.05) was observed. Thrombin increased by 7.5-fold the internal Ca2+ platelet levels, an effect markedly inhibited by LPS. Thrombin induced concentration-dependent platelet GPIIb/IIIa activation, but LPS failed to affect the activation state of this membrane glycoprotein. In conclusion, LPS inhibits human platelet adhesion to fibrinogen by mechanisms involving blockade of external Ca2+, independently of cGMP generation and activation of GPIIb/IIIa complex.

Lipopolysaccharide treatment reduces rat platelet aggregation independent of intracellular reactive-oxygen species generation

High production of reactive-oxygen species (ROS) by blood cells is involved in damage of the vascular endothelium and multiple organ dysfunction in sepsis. However, little is known about the intraplatelet ROS production in sepsis and its consequences on platelet reactivity. In this study, we evaluated whether the treatment of rats with lipopolysaccharide (LPS) affects platelet aggregation through intraplatelet ROS generation. Rats were injected with LPS (1 mg/kg, i.p.), and at 2 to 72 h thereafter, adenosine diphosphate (ADP) (3–10 µM) induced platelet aggregation was evaluated. Production of ROS in platelets was measured by flow cytometry using 2′,7′-dichlorofluorescein diacetate (DCFH-DA). Treatment of rats with LPS time-dependently inhibited ADP-induced platelet aggregation within 72 h. The inhibitory effect of LPS on platelet aggregation was further increased when the platelets were incubated with polyethylene glycol-superoxide dismutase (PEG-SOD; 30 U/mL), polyethylene glycol-catalase (PEG-CAT; 1000 U/mL) or the NADPH oxidase inhibitor diphenyleneiodonium (DPI; 10 µM). The ROS production in non-stimulated platelets did not differ between control and LPS-treated rats. However, in ADP-activated platelets, generation of ROS was increased by 3.0- and 7.0-fold, as evaluated at 8 and 48 h after LPS injection, respectively. This increased ROS production was significantly reduced when platelets were incubated in vitro with DPI, PEG-SOD or PEG-CAT. In contrast, treatment of rats with N-acetylcysteine (150 mg/kg, i.p.) significantly reduced the inhibitory effect of LPS on platelet aggregation, and prevented the increased ROS production by in vivo LPS. Our results indicate that the increased intraplatelet ROS production does not contribute to the inhibitory effect of LPS on platelet aggregation; however, the maintenance of redox balance in LPS-treated rats is fundamental to restore the normal platelet response in these animals.

The role of superoxide anion in the inhibitory effect of SIN-1 in thrombin-activated human platelet adhesion.

Reactive oxygen species have an important role in the control of platelet activity. Superoxide anion (O(2)(-)) is a free radical that can be converted into other reactive oxygen species such as peroxynitrite (ONOO(-)) that is formed from the reaction between O(2)(-) and nitric oxide (NO). There are conflicting data on ONOO(-) effects in platelets because it presents pro- or anti-aggregatory actions. 3-morpholinosydnonimine (SIN-1) co-generates NO and O(2)(-), yielding ONOO(-). Therefore, the present study aimed to investigate the mechanisms involved in the inhibition of human platelet adhesion by SIN-1. Microtiter plates were coated with human fibrinogen, after which washed platelets (6 x 10(8)platelets/ml) were added to adhere. Exposure of non-activated and thrombin-activated platelets to SIN-1 (0.001-100 microM) concentration-dependently inhibited adhesion, which was accompanied by marked increases in the cyclic GMP levels. In non-activated platelets, the soluble guanylate cyclase inhibitor ODQ prevented the SIN-1-induced cGMP elevations and adhesion inhibition. In thrombin-activated platelets, ODQ fully prevented the SIN-1-induced cGMP elevations, but only partly prevented the adhesion inhibition. The O(2)(-) and ONOO(-) scavengers superoxide dismutase (SOD) and -(-)epigallocatechin gallate, respectively, had minimal effects in non-activated platelets. The inhibition of activated platelets by SIN-1 was reversed by SOD and partly reduced by ECG. Western blot analysis of SIN-1-treated platelets showed a single 105 kDa-nitrated band. Nanospray LC-MS-MS identified the protein containing 3-nitrotyrosine residues as human alpha-actinin-1-cytoskeletal isoform. Our data show that platelet adhesion inhibition by SIN-1 in activated platelets involves cGMP-independent mechanism through O(2)(-) generation. Superoxide anion signaling pathway includes ONOO(-) formation and alpha-actinin nitration.

Role of cyclic GMP on inhibition by nitric oxide donors of human eosinophil chemotaxis in vitro.

This study was designed to investigate the effects of the nitric oxide (NO) donors sodium nitroprusside (SNP), 3‐morpholinosydnonimine (SIN‐1) and S‐nitroso‐N‐acetylpenicillamine (SNAP) on N‐formyl‐L‐methionyl‐L‐leucyl‐phenylalanine (fMLP, 1 × 10−7 M)‐induced human eosinophil chemotaxis, cyclic guanosine‐3′,5′‐monophosphate (cGMP) levels, protein nitration and cytotoxicity. Human eosinophils were exposed to SNP, SIN‐1 and SNAP (0.001–1.0 mM) for either short (10 min) or prolonged (90 min) time periods. Exposition of eosinophils with these NO donors significantly inhibited the eosinophil chemotaxis irrespective of whether cells were exposed to these agents for 10 or 90 min. No marked differences were detected among them regarding the profile of chemotaxis inhibition. Exposition of eosinophils to SNP, SIN‐1 and SNAP (0.001–1.0 mM) markedly elevated the cGMP levels above basal levels, but the 90‐min exposition resulted in significantly higher levels compared with the 10‐min protocols (5.3±0.6 and 2.6±0.2 nM 1.5 × 106 cells−1, respectively). The cGMP levels achieved with SNAP were greater than SNP and SIN‐1. The NO donors did not induce cell toxicity in any experimental condition used. Additionally, eosinophils exposed to SNP, SIN‐1 and SNAP (1.0 mM each) either for 10 or 90 min did not show any tyrosine nitration in conditions where a strong nitration of bovine serum albumin was observed. Our findings show that inhibitory effects of fMLP‐induced human eosinophil chemotaxis by NO donors at short or prolonged exposition time were accompanied by significant elevations of cGMP levels. However, additional elevations of cGMP levels do not change the functional profile (chemotaxis inhibition) of stimulated eosinophils.

Platelets induce human umbilical vein endothelial cell proliferation through P-selectin

We studied whether platelets could participate in the endothelial cell monolayer regeneration in the case of a vessel damage. Incorporation of [3H]-thymidine into the DNA of human umbilical vein endothelial cells (HUVECs) was measured after 48 h of co-incubation with platelets. The effect of platelets was compared to that of platelet-free supernatants from thrombin-activated platelets that had secreted their active granule constituents. Platelets dose-dependently induced HUVEC proliferation. Platelets preactivated by thrombin induced similar proliferation as did unactivated platelets (proliferation factor = 7 - 8), indicating that preactivation of platelets was not required. Platelets fixed with paraformaldehyde had no effect, suggesting that the platelet mitogenic effect required a mobile, alive membrane. Ketanserine and suramin reduced by at most 30 % the platelet-induced proliferation; supernatants of thrombin-activated platelets caused only minor proliferation (proliferation factor = 2), suggesting that secreted 5-hydroxytryptamine and growth factors poorly contributed to the proliferative effect. When the co-incubation was performed in the presence of an anti P-selectin antibody, the platelet-induced HUVEC proliferation was inhibited. The results suggest that platelet adhesion participate in the control of the endothelial regeneration and that platelet P-selectin is a molecular determinant of the proliferative signal.

Reactive oxygen and nitrogen species modulate the ex-vivo effects of LPS on platelet adhesion to fibrinogen.

AIMS Excessive production of nitric oxide (NO) and reactive oxygen species (ROS) in sepsis modulates different cell functions. Since the sepsis severity is associated with the degree of platelet activation, we decided to investigate the role of systemic generation of NO and ROS in modulating the platelet adhesion of lipopolysaccharide (LPS)-treated rats. MAIN METHODS Platelet adhesion was evaluated using fibrinogen-coated 96-well microtiter plates. Cyclic GMP levels were measured using enzyme immunoassay kit. KEY FINDINGS Treatment of rats with LPS significantly increased spontaneous platelet adhesion, but reduced the thrombin-activated platelet adhesion when compared with control rats. Chronic treatment of rats with the NO synthase inhibitor L-NAME (20 mg/rat/day, 7 days) prior to LPS injection normalized the increased adhesion in non-activated platelets, but failed to affect the adhesion in thrombin-activated platelets. The cGMP levels were modified neither in non-activated nor in thrombin-activated platelets of LPS-treated rats when compared with control rats. The incubation of non-activated platelets with the O2- scavenger PEG-SOD reversed the stimulatory effect of LPS on spontaneous adhesion, but had no effect in stimulated-platelet adhesion of non-treated or LPS-treated groups. Moreover, pretreatment of rats with the antioxidant N-acetylcysteine (NAC; 150 mg/kg) prevented the increase of non-activated platelet adhesion, and significantly reduced the inhibitory effect of LPS on thrombin-stimulated adhesion. SIGNIFICANCE Our findings suggest that in LPS-treated rats, NO plays an important modulatory role only in non-stimulated platelet adhesion through cGMP-independent mechanisms, while ROS, directly or by affecting the redox state of the animals, modulates both non-activated and thrombin-activated platelet adhesion.