The proteome analysis of rat platelet with nano-liquid chromatography-matrix-assisted laser desorption/ionization time-of-flight mass spectrometry technique

Recently, the proteomic analysis has become an ideal tool to study the structure and function of platelets. We proposed a nano-liquid chromatography (nano-LC) technique coupled off-line with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) for rat platelet proteome analysis. In this study, we attempted to analyze the rat platelet proteome in two different subcellular fractions: cytosol and membrane. Platelet-rich plasma was collected from healthy rats. The platelet samples were extracted with Subcellular Proteome Extraction Kit to collect subcellular compartments. For further investigations, platelet lysate, cytosol and membrane fractions were used. Enzymatic digestion of proteins was performed using Filter Aided Sample Preparation method with trypsin as a proteolytic enzyme. Tryptic peptides were analyzed using nano-LC-MALDI-TOF/TOF-MS. Platelet proteins identification was performed using the Mascot engine. We identified 238 proteins in the platelet lysate, 210 in the cytosol, and 148 in the membrane fraction. Among them, 45 were unique for platelet lysate, 55 for cytosol, and 34 for the membrane fraction. The gene ontology analysis showed that there were differences in the proteome of cytosol and membrane fractions related to the molecular functions, i.e. coagulative activity. Our results may suggest that the membrane or cytosol location of the proteins with coagulative activity may be responsible for the acute or delayed platelet response to an agonist. The nano-LC-MALDI-TOF/TOF-MS method can be used for identifying proteins of subcellular fraction in rat platelets

Aldosterone Increases Vascular Permeability in Rat Skin

The aim of this study was to evaluate the effect of acute aldosterone (ALDO) administration on the vascular permeability of skin. ALDO was injected intradermally into rats, and vascular permeability was measured. Eplerenone (EPL), a selective mineralocorticoid receptor (MR) antagonist, was used. Skin biopsies were carried out for immunohistochemical (IHC) staining, and polymerase chain reactions were performed to analyze the expression of MR, 11β-hydroxysteroid dehydrogenase type 2, von Willebrand factor (vWF), vascular endothelial growth factor (VEGF), and zonula occludens 1. Our study showed the presence of MR in the rat skin vasculature for the first time. It was found that ALDO injection resulted in a more than 30% increase in vascular permeability and enhanced the endothelial exocytosis of vWF. The effect of ALDO diminished after EPL administration. An accumulation of vWF and a reduction in VEGF IHC staining were observed following chronic EPL administration. No effect of ALDO or EPL on the mRNA expression of the studied genes or skin structure was observed. The results suggest that ALDO increases vascular permeability in the skin via an MR-dependent mechanism. This effect of ALDO on skin microcirculation may have important therapeutic implications for diseases characterized by increased levels of ALDO and coexisting skin microangiopathy

Hyperglycemia Potentiates Prothrombotic Effect of Aldosterone in a Rat Arterial Thrombosis Model

We investigated the role of aldosterone (ALDO) in the development of arterial thrombosis in streptozotocin-induced diabetic rats. To evaluate the effect of endogenous ALDO, the rats underwent adrenalectomy (ADX). ADX reduced the development of arterial thrombosis. A 1 h infusion of ALDO (30 μg/kg/h) enhanced thrombosis in adrenalectomized rats, while this effect was potentiated in diabetic rats. ALDO shortened bleeding time, increased plasma levels of tissue factor (TF) and plasminogen activator inhibitor, decreased plasma level of nitric oxide (NO) metabolites, and increased oxidative stress. Moreover, 2 h incubation of human umbilical vein endothelial cells (HUVECs) with ALDO (10−7 M) disrupted hemostatic balance in endothelial cells in normoglycemia (glucose 5.5 mM), and this effect was more pronounced in hyperglycemia (glucose 30 mM). We demonstrated that the acute ALDO infusion enhances arterial thrombosis in rats and hyperglycemia potentiates this prothrombotic effect. The mechanism of ALDO action was partially mediated by mineralocorticoid (MR) and glucocorticoid (GR) receptors and related to impact of the hormone on primary hemostasis, TF-dependent coagulation cascade, fibrinolysis, NO bioavailability, and oxidative stress balance. Our in vitro study confirmed that ALDO induces prothrombotic phenotype in the endothelium, particularly under hyperglycemic conditions

Angiotensin-converting enzyme inhibitors attenuate propofol-induced pro-oxidative and antifibrinolytic effect in human endothelial cells

Introduction: The aim of this study was to investigate the effects of plasma and tissue angiotensin-converting enzyme inhibitors (ACE-Is) against propofol-induced endothelial dysfunction and to elucidate the involved mechanisms in vitro. Materials and methods: We examined the effects of propofol (50 μM), quinaprilat and enalaprilat (10 −5 M) on fibrinolysis (t-PA, PAI-1, TAFI antigen levels), oxidative stress parameters (H 2 O 2 and MDA antigen levels and SOD and NADPH oxidase mRNA levels) and nitric oxide bioavailability (NO 2 /NO 3 concentration and NOS expression at the level of mRNA) in human umbilical vein endothelial cells (HUVECs). Results: We found that both ACE-Is promoted similar endothelial fibrinolytic properties and decreased oxidative stress in vitro. Propofol alone increased the release of antifibrinolytic and pro-oxidative factors from the endothelium and increased mRNA iNOS expression. We also found that the incubation of HUVECs in the presence of propofol following ACE-Is pre-incubation caused weakness of the antifibrinolytic and pro-oxidative potential of propofol and this effect was similar after both ACE-Is. Conclusions: This observation suggests that the studied ACE-Is exerted protective effects against endothelial cell dysfunction caused by propofol, independently of hemodynamics

Antithrombotic Potential of Tormentil Extract in Animal Models

Potentilla species that have been investigated so far display pharmacological activity mainly due to the presence of polyphenols. Recently, it was shown that polyphenol-rich extract from rhizome of Potentilla erecta (tormentil extract) affects the metabolism of arachidonic acid and exerts both anti-inflammatory and anti-oxidant activities, suggesting a possible effect on thrombosis. Accordingly, the aim of the study was to evaluate the effect of tormentil extract on haemostasis in a rat model of thrombosis. Lyophilized water-methanol extract from P. erecta rhizome was administrated per os for 14 days in doses of 100, 200, and 400 mg/kg in a volume of 2 mL/kg in a 5% water solution of gummi arabici (VEH). In the in vivo experiment an electrically induced carotid artery thrombosis model with blood flow monitoring was used in Wistar rats. Collected blood samples were analyzed ex vivo functionally and biochemically for changes in haemostasis. Tormentil extract (400 mg/kg) significantly decreased thrombus weight and prolonged the time to carotid artery occlusion and bleeding time without changes in the blood pressure. In the ex vivo experiment tormentil extract (400 mg/kg) reduced thromboxane production and decreased t-PA activity, while total t-PA concentration, as well as total PAI-1 concentration and PAI-1 activity remained unchanged. Furthermore, tormentil extract (400 mg/kg) decreased bradykinin concentration and shortened the time to reach maximal optical density during fibrin generation. Prothrombin time, activated partial thromboplastin time, QUICK index, fibrinogen level, and collagen-induced aggregation remained unchanged. To investigate the involvement of platelets in the antithrombotic effect of tormentil, the extract was administrated per os for 2 days to mice and irreversible platelets activation after ferric chloride induced thrombosis was evaluated under intravital conditions using confocal microscopy system. In this model tormentil extract (400 mg/kg) significantly reduced platelet activation at the same extent as acetylsalicylic acid. Taken together, we have shown for the first time that tormentil extract inhibits arterial thrombosis in platelet- and endothelial-dependent mechanisms without hemodynamic changes. Further studies on the detailed mechanism of action of tormentil extract toward fibrinolysis and the kinin system should be carried out