Rafael P. Morganti

Vardenafil, but not sildenafil or tadalafil, has calcium-channel blocking activity in rabbit isolated pulmonary artery and human washed platelets

Phosphodiesterase type‐5 (PDE5) inhibitors constitute a novel and important therapeutic option for the treatment of pulmonary hypertension. The effects of the PDE5 inhibitors sildenafil, tadalafil and vardenafil on rabbit isolated pulmonary artery ring preparations and on intracellular Ca2+ concentration of thrombin‐stimulated human platelets were investigated.

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.

Modulation of the pharmacological effects of enzymatically-active PLA2 by BTL-2, an isolectin isolated from the Bryothamnion triquetrum red alga

BackgroundAn interaction between lectins from marine algae and PLA2 from rattlesnake was suggested some years ago. We, herein, studied the effects elicited by a small isolectin (BTL-2), isolated from Bryothamnion triquetrum, on the pharmacological and biological activities of a PLA2 isolated from rattlesnake venom (Crotalus durissus cascavella), to better understand the enzymatic and pharmacological mechanisms of the PLA2 and its complex.ResultsThis PLA2 consisted of 122 amino acids (approximate molecular mass of 14 kDa), its pI was estimated to be 8.3, and its amino acid sequence shared a high degree of similarity with that of other neurotoxic and enzymatically-active PLA2s. BTL-2 had a molecular mass estimated in approximately 9 kDa and was characterized as a basic protein. In addition, BTL-2 did not exhibit any enzymatic activity.The PLA2 and BTL-2 formed a stable heterodimer with a molecular mass of approximately 24–26 kDa, estimated by molecular exclusion HPLC. In the presence of BTL-2, we observed a significant increase in PLA2 activity, 23% higher than that of PLA2 alone. BTL-2 demonstrated an inhibition of 98% in the growth of the Gram-positive bacterial strain, Clavibacter michiganensis michiganensis (Cmm), but only 9.8% inhibition of the Gram-negative bacterial strain, Xanthomonas axonopodis pv passiflorae (Xap). PLA2 decreased bacterial growth by 27.3% and 98.5% for Xap and Cmm, respectively, while incubating these two proteins with PLA2-BTL-2 inhibited their growths by 36.2% for Xap and 98.5% for Cmm.PLA2 significantly induced platelet aggregation in washed platelets, whereas BTL-2 did not induce significant platelet aggregation in any assay. However, BTL-2 significantly inhibited platelet aggregation induced by PLA2. In addition, PLA2 exhibited strong oedematogenic activity, which was decreased in the presence of BTL-2. BTL-2 alone did not induce oedema and did not decrease or abolish the oedema induced by the 48/80 compound.ConclusionThe unexpected results observed for the PLA2-BTL-2 complex strongly suggest that the pharmacological activity of this PLA2 is not solely dependent on the presence of enzymatic activity, and that other pharmacological regions may also be involved. In addition, we describe for the first time an interaction between two different molecules, which form a stable complex with significant changes in their original biological action. This opens new possibilities for understanding the function and action of crude venom, an extremely complex mixture of different molecules.

EFFECT OF BAY 41-2272 IN THE PULMONARY HYPERTENSION INDUCED BY HEPARIN–PROTAMINE COMPLEX IN ANAESTHETIZED DOGS

1 BAY 41‐2272 is a potent activator of the nitric oxide‐independent site of soluble guanylate cyclase and has been recently introduced as a new therapeutic agent to treat chronic pulmonary hypertension (PH) in neonatal sheep. Because the in vivo heparin–protamine interaction may lead to severe PH, the aim of the present study was to evaluate the effects of BAY 41‐2272 in the PH induced by heparin–protamine interaction in anaesthetized dogs. 2 Sixteen male dogs (10 mongrel dogs and six Beagles) were anaesthetized and instrumented for acquisition of mean arterial blood pressure (MABP), mean pulmonary arterial pressure (MPAP), heart rate (HR), pulmonary capillary wedge pressure (PCWP), cardiac index (CI) and indices of systemic and pulmonary vascular resistance (ISVR and IPVR, respectively). Plasma cGMP levels and Spo2 were evaluated. 3 Intravenous administration of heparin (500 IU/kg) followed 3 min later by protamine (10 mg/kg) caused marked PH, as evaluated by the increase in MPAP, PCWP and IPVR. This was accompanied by a significant fall in MABP and a transient increase in HR. Infusion of BAY 41‐2272 (10 µg/kg per h, starting 10 min before heparin administration) augmented plasma cGMP levels and slightly and significantly increased HR and CI, without affecting the other cardiovascular parameters. The elevation in IPVR, MPAP and PCWP triggered by the heparin–protamine interaction was significantly reduced in animals exposed to BAY 41‐2272. 4 In vehicle‐treated dogs, the Spo2 values decreased significantly at the peak of the PH and this was significantly attenuated by treatment with BAY 41‐2272. In addition, BAY 41‐2272 (10 µmol/L) had no effect on the activated partial thromboplastin time of citrated plasma after the addition of heparin–protamine. 5 In conclusion, BAY 41‐2272 was effective in reducing canine PH induced in vivo by the heparin–protamine interaction, thus indicating its potential in the treatment of this type of disorder.

Mechanisms of relaxant activity of the nitric oxide-independent soluble guanylyl cyclase stimulator BAY 41-2272 in rat tracheal smooth muscle.

The soluble guanylyl cyclase is expressed in airway smooth muscle, and agents that stimulate this enzyme activity cause airway smooth muscle relaxation and bronchodilation. The compound 5-Cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine (BAY 41-2272) is a potent nitric oxide (NO)-independent soluble guanylyl cyclase stimulator, but little is known about its effects in airway smooth muscle. Therefore, this study aimed to investigate the mechanisms underlying the relaxations of rat tracheal smooth muscle induced by BAY 41-2272. Tracheal rings were mounted in 10-ml organ baths for isometric force recording. BAY 41-2272 concentration-dependently relaxed carbachol-precontracted tracheal rings (pEC(50)=6.68+/-0.14). Prior incubation with the NO synthesis inhibitor l-NAME (100 microM) or the soluble guanylyl cyclase inhibitor ODQ (10 microM) caused significant rightward shifts in the concentration-response curves to BAY 41-2272. Sodium nitroprusside caused concentration-dependent relaxations, which were greatly potentiated by BAY 41-2272 and completely inhibited by ODQ. In addition, BAY 41-2272 shifted to the right the tracheal contractile responses to either carbachol (0.01-1 microM) or electrical field stimulation (EFS, 1-32 Hz). BAY 41-2272 (1 microM) also caused a marked rightward shift and decreased the maximal contractile responses to extracellular CaCl2, and such effect was not modified by pretreatment with ODQ. In addition, BAY 41-2272 (up to 1 microM) significantly increased the cGMP levels, and that was abolished by ODQ. Our results indicate that BAY 41-2272 causes cGMP-dependent rat tracheal smooth muscle relaxations in a synergistic fashion with exogenous NO. BAY 41-2272 has also an additional mechanism independently of soluble guanylyl cyclase activation possibly involving Ca(2+) entry blockade.

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.

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.

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.

Platelet adhesion and intracellular calcium levels in antigen-challenged rats

There is considerable evidence that platelet activation occurs in allergic airways diseases. In this study we aimed to investigate platelet adhesion to immobilized fibrinogen and intracellular calcium levels in a rat model of allergic inflammation. Male Wistar rats were challenged with ovalbumin (OVA). At 30 min to 24h after OVA-challenge, assays of platelet adhesion to immobilized fibrinogen and intracellular calcium levels using fura 2-AM loaded platelets were performed. The serum levels of IgE were approximately 5-fold greater in OVA-sensitized rats. A marked eosinophil influx in bronchoalveolar lavage (BAL) fluid of OVA-challenged rats at 24h after OVA-challenge was also seen. OVA-challenge resulted in a marked thrombocytopenia, as observed within 12h after OVA-challenge. The agonists ADP (0.5-50 microM) and thrombin (30-100 mU/ml) concentration-dependently increased platelet adhesion to immobilized fibrinogen. At an early time after OVA-challenge (30 min), platelets exhibited greater platelet adhesion compared with the non-sensitized group, whereas at a late time (24h) they exhibited lower platelet adhesion to both agonists. Moreover, at 30 min after OVA-challenge, intracellular calcium levels to ADP (20 microM) and thrombin (100 mU/ml)-activated platelets were greater compared with non-challenged rats. As opposed, at 24h after OVA challenge, a lower intracellular calcium level to ADP- and thrombin-activated platelets was observed. In conclusion, OVA-challenge in rats promotes a biphasic response in platelet adhesion consisting of an increased adhesion and intracellular calcium levels at an early phase (30 min), which progress to a reduction in adhesion and intracellular calcium levels at a late time (24h) after antigen challenge.