Agnieszka Morel

Melatonin Redox Activity. Its Potential Clinical Applications in Neurodegenerative Disorders

Neurodegeneration is the hallmark of many chronic progressive neurogical disorders characterized by specific clinical, morphological and biochemical features. Central nervous system is very sensitive to oxidative stress, which is considered as a key factor of neurodegenerative disorders. Therefore, many therapeutical strategies are focused on molecules with redox activity to re-establish the equilibrium between pro and antioxidants. Due to the fact that melatonin readily crosses the blood- brain-barrier, concomitant with its safety profile at the highest dosages makes this dietary supplement very useful in possible clinical application in neurodegeneration. Melatonin is currently marketed in several countries as a dietary supplement with no prescription. Clinical trials have shown different effectiveness of melatonin supplementation in several disorders, including neurodegenerative disorders. Melatonin has unique biochemical properties such as scavenging of hydroxyl, carbonate, alkoxyl, peroxyl and aryl cation radicals and stimulation of activities main antioxidative enzymes (glutathione peroxidase, superoxide dismutase etc.). Moreover, it can suppress nitric oxide synthase. The present paper highlighted the potential clinical role of melatonin in main neurodegenerative diseases including Alzheimer disease, Parkinson disease, amylotrophic lateral sclerosis and multiple sclerosis. Moreover, in this review the main molecular aspects of melatonin in brain cell protection and survival mechanisms were discussed. Therefore, melatonin is regarded as a potential therapeutical agent in clinical application in neurodegenerative disorders, but this findings needs to be confirmed by the larger, more well-designed clinical trials.

Isoprostanes and Neuroprostanes as Biomarkers of Oxidative Stress in Neurodegenerative Diseases

Accumulating data shows that oxidative stress plays a crucial role in neurodegenerative disorders. The literature data indicate that in vivo or postmortem cerebrospinal fluid and brain tissue levels of F2-isoprostanes (F2-IsoPs) especially F4-neuroprotanes (F4-NPs) are significantly increased in some neurodegenerative diseases: multiple sclerosis, Alzheimers disease, Huntingtons disease, and Creutzfeldt-Jakob disease. Central nervous system is the most metabolically active organ of the body characterized by high requirement for oxygen and relatively low antioxidative activity, what makes neurons and glia highly susceptible to destruction by reactive oxygen/nitrogen species and neurodegeneration. The discovery of F2-IsoPs and F4-NPs as markers of lipid peroxidation caused by the free radicals has opened up new areas of investigation regarding the role of oxidative stress in the pathogenesis of human neurodegenerative diseases. This review focuses on the relationship between F2-IsoPs and F4-NPs as biomarkers of oxidative stress and neurodegenerative diseases. We summarize the knowledge of these novel biomarkers of oxidative stress and the advantages of monitoring their formation to better define the involvement of oxidative stress in neurological diseases.

Antioxidative properties of hydrogen sulfide may involve in its antiadhesive action on blood platelets.

BACKGROUND Hydrogen sulfide (H(2)S) is a signaling molecule in different systems, including the cardiovascular system. However, mechanisms involved in the relationship between the action of H(2)S and hemostasis process are still unclear. OBJECTIVE AND METHODS The present work was designed to study the effects of hydrogen sulfide on adhesion of blood platelets in vitro. Platelet suspensions were preincubated (5-30 min) with NaHS as a hydrogen sulfide donor at the final concentrations of 0.00001-10 mM. Then, for platelet activation thrombin (0.1 U/mL) or TRAP, peptide with the sequence Ser-Phe-Leu-Leu-Arg-Asn (SFLLRN; 20 μM) was used. We also measured the effects of H(2)S on superoxide anion radicals (O(2)(-•)) production in blood platelets. RESULTS We observed that adhesion to collagen and to fibrinogen of resting platelets preincubated with NaHS was changed, and this process was statistically significant (for 0.00001-5mM NaHS, p<0.05; 10 mM, p<0.01). The inhibitory effect of NaHS on adhesion of thrombin - or TRAP - stimulated platelets to collagen was found (for 0.00001 and 0.0001 mM NaHS, p<0.05; 0.001-1 mM NaHS, p<0.01; 5 and 10 mM NaHS, p<0.001). Hydrogen sulfide reduced also the thrombin- or TRAP-induced platelet adhesion to fibrinogen (for 0.00001 and 0.0001 mM NaHS, p<0.05; 0.001-1 mM NaHS, p<0.01; 5 and 10 mM NaHS, p<0.001). Moreover, H(2)S caused a dose-dependent reduction of O(2)(-•) produced in platelets (p<0.05). CONCLUSION The results obtained that the antioxidative activity of H(2)S may involve in its antiadhesive properties on blood platelets.

Hydrogen sulfide changes adhesive properties of fibrinogen and collagen in vitro.

Hydrogen sulfide (H2S) is a well-known toxic gas that is synthesized from two amino acids: cysteine (Cys) and homocysteine (Hcy). H2S, like other organic gases–nitric oxide (NO ) or carbon monoxide (CO) – is a signaling molecule in the cardiovascular system. Some studies have shown that H2S is a therapeutic agent in cardiovascular diseases [1–3], but the mechanisms involved in the relationship between the action of H2S and hemostasis process are still unclear. The main aim of this study was to establish the functional changes of two hemostatic proteins (collagen and fibrinogen) induced by H2S, and also to examine the effects of these changes on the capability of fibrinogen and collagen to interact with human blood platelets (by measuring the platelet adhesion) in vitro. Thrombin, collagen type I, bovine serum albumin (BSA), and bicinchoninic acid (BCA) solution were purchased from Sigma (St Louis, MO). Sodium hydrosulfide (NaHS), which has been well established as a reliable H2S donor [4, 5], was from Sigma (St Louis, MO). Fibrinogen isolated from pooled citrated human plasma by the cold ethanol precipitation technique was followed by ammonium sulfate fractionation at 26% saturation at 4 C, according to Doolittle [6]. Its concentration was determined spectrophotometrically at 280 nm using an extinction coefficient 1.55 for 1 mg/ml solution. The concentration of fibrinogen in plasma was 2 0.2 mg/ml. The concentration of purified fibrinogen in the reaction system was also about 2 mg/ml. The concentration of collagen type I was 40 mg/ml collagen (dissolved in 0.05% CH3COOH). The reaction was initiated by placing a small drop of NaHS, on the side of the tube containing the fibrinogen solution or collagen solution (the incubation time 5, 15, and 30 minutes, 37 C). We have been using NaHS at the final concentrations of 0.00001–10 mM. The physiological concentration of H2S in plasma and in tissues is about 50 mM; its physiological level in the brain is up to three-fold higher than in plasma. Human blood was taken from healthy volunteers aged 23–32 (average: 24; SD1⁄4 5.5 years) not taking any medications or addictive substances (including tobacco, alcohol, and aspirin or any other anti-platelet drugs) and keeping a balanced diet (meat and vegetables), with similar socio-economic background, using no antioxidant supplementation. Human blood was collected into ACD solution (citric acid/citrate/ dextrose; 5:1 v/v) and platelets were isolated by differential centrifugation of blood, as described by Wachowicz and Kustroń [7]. The final platelet concentration was 3 10 platelets/ml. The platelets were counted by the photometric method, according to Walkowiak et al. [8]. Adhesion of blood platelets to native fibrinogen or native collagen and fibrinogen or collagen treated with NaHS was determined according to Tuszynski and Murphy [9]. The absorbance of control platelets (with native fibrinogen or native collagen) was expressed as 100%. All the values in this study were expressed as means SD. The statistical analysis (to calculate the differences among the effect of different concentration of NaHS) was performed with an ANOVA test and POST Hoc test (Bonferoni). In order to eliminate uncertain data, the Q-Dixon test was performed. As shown in Table I, both resting as well as thrombinactivated blood platelets demonstrate a reduced ability to adhere to NaHS-treated collagen and NaHS-treated fibrinogen in vitro. The distinct inhibitory effect on the platelet adhesion to modified adhesive proteins (collagen and fibrinogen) was observed when collagen or fibrinogen was treated with NaHS, even at the lowest concentration of NaHS (0.00001 mM) (Table 1). The inhibitory properties of NaHS appears to be concentration-dependent for both adhesive proteins. Moreover, we observed the time-dependent (5, 15, and 30 minutes) inhibition of adhesive properties of proteins either by NaHS (0.01 and 10 mM) (for collagen–p< 0.01 (for resting platelets), p< 0.01 (for stimulated platelets); for fibrinogen– p< 0.02 (for resting platelets), p< 0.001 (for stimulated platelets)) (Table I). Our initial results have also shown that, the adhesion to collagen and to fibrinogen of platelet preincubated with NaHS was reduced in vitro [10]. Moreover, H2S caused the reduction of anion radicals generation in these cells [10]. Other latest experiments have demonstrated the inhibitory properties of H2S on the platelet aggregation in vitro. The total inhibition of platelet aggregation (stimulated by various agonists: collagen, arachidonic acid and ADP) was observed at

Relationship between the Increased Haemostatic Properties of Blood Platelets and Oxidative Stress Level in Multiple Sclerosis Patients with the Secondary Progressive Stage.

Multiple sclerosis (MS) is the autoimmune disease of the central nervous system with complex pathogenesis, different clinical courses and recurrent neurological relapses and/or progression. Despite various scientific papers that focused on early stage of MS, our study targets selective group of late stage secondary progressive MS patients. The presented work is concerned with the reactivity of blood platelets in primary hemostasis in SP MS patients. 50 SP MS patients and 50 healthy volunteers (never diagnosed with MS or other chronic diseases) were examined to evaluate the biological activity of blood platelets (adhesion, aggregation), especially their response to the most important physiological agonists (thrombin, ADP, and collagen) and the effect of oxidative stress on platelet activity. We found that the blood platelets from SP MS patients were significantly more sensitive to all used agonists in comparison with control group. Moreover, the platelet hemostatic function was advanced in patients suffering from SP MS and positively correlated with increased production of O2 −∙ in these cells, as well as with Expanded Disability Status Scale. We postulate that the increased oxidative stress in blood platelets in SP MS may be primarily responsible for the altered haemostatic properties of blood platelets.

Protective action of proanthocyanidin fraction from Medemia argun nuts against oxidative/nitrative damages of blood platelet and plasma components

The oxidative/nitrative stress induced by different factors plays an important role in the pathogenesis of various disorders, including cardiovascular diseases and cancer. Proanthocyanidins have antioxidative properties and may protect biomolecules (lipids, DNA, and proteins) exposed to reactive oxygen and nitrogen species, including peroxynitrite (ONOO−). The effects of proanthocyanidin fraction from Medemia argun nuts on oxidative/nitrative protein damages (determined by such parameters as level of thiol groups, carbonyl groups, and nitrotyrosine residues) and on the amount of glutathione (as an important component of redox status; using HPLC) in human blood platelets and plasma after treatment with peroxynitrite were studied in vitro. The preincubation of blood platelets and plasma with proanthocyanidin fraction from M. argun nuts (0.5–50 µg/ml) reduced the formation of 3-nitrotyrosine, diminished oxidation of thiol groups, and decreased the level of carbonyl groups in proteins caused by 100 µM peroxynitrite. An action of tested plant fraction and ONOO− evoked a significant increase of GSH in platelets and plasma in comparison with platelets and plasma treated with ONOO− only. The proanthocyanidin fraction from M. argun nuts can be useful as a protecting factor against oxidative/nitrative stress associated with different diseases (cancer, cardiovascular, and neurodegenerative diseases) and proanthocyanidins of M. argun nuts may be promising antioxidants.

Flow cytometric analysis reveals the high levels of platelet activation parameters in circulation of multiple sclerosis patients

The epidemiological studies confirm an increased risk of cardiovascular disease in multiple sclerosis, especially prothrombotic events directly associated with abnormal platelet activity. The aim of our study was to investigate the level of blood platelet activation in the circulation of patients with chronic phase of multiple sclerosis (SP MS) and their reactivity in response to typical platelets’ physiological agonists. We examined 85 SP MS patients diagnosed according to the revised McDonald’s criteria and 50 healthy volunteers as a control group. The platelet activation and reactivity were assessed using flow cytometry analysis of the following: P-selectin expression (CD62P), activation of GP IIb/IIIa complex (PAC-1 binding), and formation of platelet microparticles (PMPs) and platelet aggregates (PA) in agonist-stimulated (ADP, collagen) and unstimulated whole blood samples. Furthermore, we measured the level of soluble P-selectin (sP-selectin) in plasma using ELISA method, to evaluate the in vivo level of platelet activation, both in healthy and SP MS subjects. We found a statistically significant increase in P-selectin expression, GP IIb/IIIa activation, and formation of PMPs and PA, as well as in unstimulated and agonist-stimulated (ADP, collagen) platelets in whole blood samples from patients with SP MS in comparison to the control group. We also determined the higher sP-selectin level in plasma of SP MS subjects than in the control group. Based on the obtained results, we might conclude that during the course of SP MS platelets are chronically activated and display hyperreactivity to physiological agonists, such as ADP or collagen.

Evaluation of polyphenolic fraction isolated from aerial parts of Tribulus pterocarpus on biological properties of blood platelets in vitro.

The antiplatelet and antioxidative activity of polyphenolic fraction isolated from aerial parts of Tribulus pterocarpus in blood platelets stimulated by thrombin was studied. Thrombin as a strong physiological agonist induces the enzymatic peroxidation of endogenous arachidonic acid, the formation of different reactive oxygen species, including superoxide anion radicals (·) and the platelet aggregation. Therefore, the aim of our study was to assess if the polyphenolic fraction from aerial parts of T. pterocarpus may change the biological properties of blood platelets activated by thrombin. We used cytochrome c reduction method to test the ability of this fraction to change · generation in platelets. Arachidonic acid metabolism was measured by the level of thiobarbituric acid reactive substances (TBARS) and by the production of 8-epi-prostaglandin (8-EPI) F2. Moreover, we determined the effects of the fraction on blood platelet aggregation induced by thrombin. We observed that the polyphenolic fraction from T. pterocarpus reduced ·, 8-EPI and TBARS production in these cells. The ability of the fraction to decrease the · generation in blood platelets supports the importance of free radicals in platelet functions, including aggregation process. This study may suggest that the tested plant fraction might be a good candidate for protecting blood platelets against changes of their biological functions, which may be associated with the pathogenesis of different cardiovascular disorders.

Comparative antiadhesive properties of crude extract and phenolic fraction isolated from aerial parts of Tribulus pterocarpus during severe hyperhomocysteinemia.

The phenolic fraction and the crude extract from Tribulus pterocarpus have different biological activity, including antiplatelet-antiadhesive properties. Since it is demonstrated that hyperhomocysteinemia may act as stimulator of blood platelet activation (platelet adhesion, aggregation, and secretion), but various antiplatelet compounds are able to reduce hyperactivation of blood platelets induced by hyperhomocysteinemia. The aim of our present experiments was to investigate in vitro one of the step in platelet activation process - platelet adhesion to collagen induced by the model of severe hyperhomocyateinemia in the presence of the phenolic fraction and the crude extract from T. pterocarpus. Severe hyperhomocysteinemia was induced by reduced form of Hcy in the concentrations 0.1mM and 1mM, or using HTL in the concentrations 0.1, 0.5 and 1 μM. Adhesion of blood platelets to collagen was determined according to Tuszynski and Murphy. We observed that the phenolic fraction and the crude extract from T. pterocarpus have the inhibitory effect on platelet adhesion during severe hyperhomocysteinemia. The action of tested phenolic and crude extract was concentration-dependent, but the phenolic fraction was stronger antiadhesive action than the crude extract. We suggest that T. pterocarpus may be good source of antiplatelet compounds during hyperhomocysteinemia.