Joanna Malinowska

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.

Comparison of the effect of homocysteine in the reduced form, its thiolactone and protein homocysteinylation on hemostatic properties of plasma.

Mechanisms involved in the relationship between hyperhomocysteinemia and hemostatic process are still unclear. In the literature there are few papers describing studies on the effects of homocysteine (Hcys) on proteins that participate in blood coagulation and fibrinolysis in human. The aim of our study was to establish and compare the influence of a reduced form of Hcys (at final doses of 0.01 - 1 mM) and the most reactive form of Hcys - its cyclic thioester, homocysteine thiolactone (HTL, 0.1 - 1 μM) on the clot formation (using whole human plasma and purified fibrinogen) and the fibrin lysis. Moreover, the aim of our study was to explain the effect of plasma protein modifications (S- and N-homocysteinylation) on selected parameters of hemostasis. We observed that HTL, like its precursor, a reduced form of Hcys stimulated polymerization of fibrinogen, but this process was not dose-dependent. In the presence of HTL (at the lowest tested concentration - 0.1μM) the increase was about 55%. Our present results also demonstrated that Hcys in the reduced form (0.01 - 1 mM) and HTL at lower doses than Hcys (0.1 - 1 μM) reduced the fibrin lysis in whole human plasma. Our results reported that HTL, like the reduced form of Hcys (at concentrations corresponding to concentrations in plasma during hyperhomocysteinemia) induced modifications of hemostatic plasma proteins, and the consequence of these modifications may be alteration in protein structure associated with changes of hemostatic functions.

Response of blood platelets to resveratrol during a model of hyperhomocysteinemia

Resveratrol (3,4’,5-trihydroxystilben), a phenolic antioxidant synthesized in grapes and other plants, and also present in wine, has been suggested to help prevent cardiovascular events. In this study the influence of resveratrol on platelet aggregation during a model of hyperhomocysteinemia was investigated. We induced hyperhomocysteinemia using a reduced form of Hcys (final dose, 0.1 mM) and the most reactive form of Hcys, its cyclic thioester, homocysteine thiolactone (HTL, 1 µM). The aim of our study in vitro was also to investigate superoxide anion radical () generation after incubation of platelets with Hcys, HTL, and resveratrol. We have observed that HTL, like its precursor Hcys, stimulated the generation of in platelets and caused an augmentation of platelet aggregation induced by the strong physiological agonist thrombin. Our results in vitro also demonstrated that resveratrol reduced the toxic action of Hcys and HTL on blood platelet aggregation and superoxide anion radical production in platelets, suggesting its potential protective effects on hemostasis are negatively influenced by homocysteine and its derivatives.

Comparison of the effect of homocysteine and its thiolactone on the fibrinolytic system using human plasma and purified plasminogen

L-Homocysteine (Hcys) occurs in human blood in free (i.e. as reduced form—about 100 nM) and protein bound form. Increased concentration of Hcys in the blood is called hyperhomocysteinemia (about [15 lM). Mechanisms involved in the relationship between hyperhomocysteinemia and haemostatic process are still unclear and sometimes controversial. In the literature there are few papers describing studies on the effects of Hcys on proteins that participate in blood coagulation and fibrinolysis in human. The most reactive form of Hcys is its cyclic thioester— homocysteine thiolactone (HTL; formed through editing mechanisms with methionyl t-RNA synthetase), which represents up to 0.29% of plasma total homocysteine, and the Hcysor tiolactone-induced modifications of haemostatic proteins (N-homocysteinylation or S-homocysteinylation) seem to be the main mechanism of biotoxicity of these compounds. Fibrinogen, plasminogen (Plg) and other haemostatic proteins can be also covalently modified by Hcys and HTL [1, 2]. The aim of our study was to establish and compare the effect of a reduced form of L-Hcys (C95%, Sigma Chemical Company, St. Louis, MO, USA) and its cyclic thioester (C99%, Sigma Chemical Company, St. Louis, MO, USA) on the fibrinolytic system (using human plasma or purified plasminogen). Blood samples were taken from 12 healthy volunteers (aged between 26 and 32 years; mean 29.7 ± 2.5) without cardiovascular disorders, allergy and lipid or carbohydrate metabolism disorders, untreated with drugs. Healthy subjects did not use addictive substances and antioxidant supplementation, their diet was balanced (meat and vegetables), lived in similar socio-economic conditions. Subjects with significant medical illness were excluded. They were no smokers. Human blood was collected at 3.2% citrate (used to 12 mM final concentration) and immediately centrifuged (20009g, 15 min) to get plasma. Plg was isolated by affinity chromatography on Lysine–Sepharose [3]. The natural concentration of total Hcys in plasma was 10.6 ± 3.8 lM. The endogenous concentration of reduced form of Hcys and HTL was about 100 ± 11.2 and 0–35 nM, respectively. The classical technique HPLC has been used to analysis Hcys or HTL in human plasma. The HPLC analysis was performed with a Hewlett-Packard 1100 Series system according to Glowacki et al. [4] and Bald et al. [5]. The concentration of Plg in plasma was 2 ± 0.3 lM. Samples of human Plg (2 lM) were exposed to the reduced form of L-homocysteine (at a final concentration between 10 and 100 lM); or to homocysteine thiolactone (at a final concentration between 0.1 and 1 lM) in the presence of 100 mM potassium phosphate buffer, pH 7.4, for 30 min at 37 C. Human plasma was exposed also to the reduced form of L-homocysteine (at a final concentration between 10 and 100 lM); or to homocysteine thiolactone (at a final concentration between 0.1 and 1 lM) for 30 min at 37 C. The tested concentrations of Hcys or HTL correspond to levels found in human plasma during hyperhomocysteinemia in vivo. Plasmin activity and Plg activation were estimated by the hydrolysis of chromogenic substrate by streptokinase (SK) or by tissue plasminogen activator (tPA); assays were performed at room temperature in 96well polystyrene flat-bottom plates. The absorbance measurements were performed in a microplate reader (Bio-Rad Microplate Reader, model 550) at 415 nm. No activity of generated plasmin was detected in the absence of SK or tPA. We also studied changes of proteolytic activities of J. Kolodziejczyk J. Malinowska P. Nowak B. Olas (&) Department of General Biochemistry, Institute of Biochemistry, University of Łodź, Banacha 12/16, 90-237 Lodz, Poland e-mail: olasb@biol.uni.lodz.pl

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

Chemotherapy modulates the biological activity of breast cancer patients plasma: The protective properties of black chokeberry extract

In breast cancer patients (before and during anti-cancer therapy) oxidative/nitrative damage to various molecules is observed. Furthermore, anti-cancer treatments may also influence the hemostatic properties of blood platelets and plasma. The aim of our study was to assess the effect of oxidative/nitrative stress (estimated by measurements of the levels of carbonyl groups and 3-nitrotyrosine in proteins--ELISA and C-ELISA methods, respectively; lipid peroxidation and total antioxidant level--TAS) on the selected parameters of hemostatic activity of plasma (the process of fibrin polymerization and lysis) collected from breast cancer patients after surgery and after various phases of chemotherapy (doxorubicin and cyclophosphamide). Subsequently, we also evaluated the level of oxidative/nitrative stress and hemostatic activity in plasma from these patients in the presence of the commercial extract of Aronia melanocarpa (Aronox®) in vitro. Patients were hospitalized in Department of Oncological Surgery and Department of Chemotherapy in Medical University of Lodz, Poland. We observed increased levels of biomarkers of oxidative/nitrative stress in plasma from patients with breast cancer (before or after surgery and after various phases of chemotherapy) in comparison to healthy group. Our further experiments demonstrated the hemostatic activity of plasma from the investigated patients differs from hemostatic properties of plasma obtained from healthy volunteers. We also recognize the existence of a relationship between oxidative stress (measured by the level of carbonyl groups) and changes of hemostasis in breast cancer patients after I and IV phases of chemotherapy. Moreover, the obtained results showed that the commercial extract from A. melanocarpa berries significantly reduced, in in vitro system, the oxidative/nitrative stress and hemostasis changes in plasma from breast cancer patients, after surgery and different phases of chemotherapy. Considering the data presented in this study, we suggest that the oxidative/nitrative stress in plasma obtained from breast cancer patients (not only before or after the surgery, but also after various phases of doxorubicin and cyclophosphamide chemotherapy) may induce changes of hemostatic activity, which may contribute to thrombosis in these patients. Our results also suggest that the commercial extract of A. melanocarpa may be regarded as a promising new source of bioactive antioxidant natural compounds for breast cancer patients.

Homocysteine and its thiolactone may promote apoptotic events in blood platelets in vitro.

The actions of homocysteine and its major metabolite, cyclic thioester, homocysteine thiolactone on endothelial cells, blood platelets, plasmatic fibrinogen and plasminogen – the important major components of haemostasis, regulating the flowing properties of blood – are complex and sometimes controversial. Homocysteine (Hcys) can promote apoptosis in endothelial cells, but the role of Hcys and its thiolactone in the apoptotic process in blood platelets is unknown. In order to study the appearance of apoptosis in platelets after treatment with the reduced form of Hcys or its thiolactone different markers were chosen: annexin V binding (phosphatidylserine exposure), platelet microparticle formation, mitochondrial membrane depolarization and αIIbβ3 expression in vitro. Apoptotic events and platelet activation were measured by a flow cytometer. In gel-filtered platelets treated with different concentrations of the reduced form of Hcys (25, 50 and 100 µM, 10 min) a significant increase of phosphatidylserine exposure (about 37% at the highest concentration, p < 0.001) and platelet microparticle formation were observed. Homocysteine caused also a dose-dependent depolarization of mitochondrial potential. The same apoptotic markers appeared in HTL-treated platelets (0.2 and 1 µM). Moreover, resveratrol (25 µM), a well known antioxidant, distinctly reduced the level of apoptotic markers. The obtained results indicate that Hcys and its thiolactone may promote in vitro apoptotic events in human gel-filtered platelets.

Phenolic fractions from Trifolium pallidum and Trifolium scabrum aerial parts in human plasma protect against changes induced by hyperhomocysteinemia in vitro

Elevated concentration of homocysteine (Hcy) in human plasma, defined as hyperhomocysteinemia has been correlated with some diseases, such as cardiovascular, neurodegenerative, and kidney disorders. Homocysteine occurs in human plasma in several forms, including the most reactive form of Hcy - its cyclic thioester - homocysteine thiolactone (HTL), which represents up to 0.29% of plasma total Hcy. It is suggested that Hcy and HTL may also act as oxidants, but various polyphenolic antioxidants are able to inhibit the oxidative damage induced by Hcy or HTL. The aim of our present study was to investigate in vitro oxidative changes in human plasma induced by the model of hyperhomocysteinemia in the presence of the phenolic fractions from selected clovers (Trifolium pallidum and Trifolium scabrum aerial parts). Hyperhomocysteinemia was stimulated by a reduced form of Hcy (final dose 100 μM) or HTL (final dose 1 μM). The aim of our study was also to explain the effect of the phenolic fractions on the coagulation activity of human plasma treated with Hcy and its thiolactone. Tested phenolic fractions significantly inhibited the oxidative stress (measured by the total antioxidant level - TAS) in plasma treated with Hcy or HTL. The phenolic fractions from T. pallidum and T. scabrum also caused a distinct reduction of plasma lipid peroxidation (measured by the level of thiobarbituric acid reactive substance) induced by the model of hyperhomocysteinemia. Moreover, tested fractions modulated the coagulation properties of plasma treated with homocysteine and its thiolactone. It seems that antioxidative activities of the phenolic fractions from T. pallidum and T. scabrum aerial parts may be responsible for their medicinal properties during hyperhomocysteinemia.

Extracts from Trifolium pallidum and Trifolium scabrum aerial parts as modulators of blood platelet adhesion and aggregation.

A growing number of reports indicate that some species of clover (Trifolium) may have remarkable medical importance; however, the effects of these plants on blood platelets and hemostasis are inadequately recognized. This work was designed to study the effects of Trifolium pallidum and Trifolium scabrum extracts on the functions of human blood platelets in vitro. Platelet suspensions were preincubated with extracts from aerial parts of T. pallidum (phenolic fraction and clovamide fraction) and T. scabrum (phenolic fraction) at the final concentrations of 12.5, 25, and 50 µg/ml. Then, for platelet activation thrombin (0.1 U/ml), thrombin receptor activating peptide (TRAP; 20 µM), or adenosine diphosphate (ADP; 1 µM) were used. The effects of Trifolium extracts on adhesion of blood platelets to fibrinogen and collagen were determined by enzyme-linked immunosorbent assay (ELISA) method. Platelet aggregation was monitored on a dual-channel Chronolog aggregometer. In these studies, we also compared the action of tested plant extracts with the effects of another antiplatelet plant-derived compound – resveratrol (3,4′,5-trihydroxystilbene). The performed assays demonstrated that the tested extracts might influence the platelet functions in vitro. The inhibitory, concentration-dependent effects of all tested extracts on adhesion of thrombin-stimulated platelets to collagen was found. Both extracts from T. pallidum and from T. scabrum reduced the thrombin-induced platelet adhesion to fibrinogen. Furthermore, in the presence of all three extracts, the platelet aggregation induced by thrombin was slightly inhibited. Our results also indicate that the tested plant extracts (at the highest concentrations used of 50 µg/ml), similar to purified resveratrol, inhibit selected steps of platelet activation stimulated by both proteolytic (thrombin) and nonproteolytic agonists (TRAP or ADP). In the comparative studies, T. pallidum and T. scabrum extracts was not found to be more effective antiaggregatory factor, than resveratrol. Extracts from T. pallidum and T. scabrum aerial parts reveal antiplatelet properties: the antiadhesive effect was similar to that of the reference compound resveratrol, whereas the antiaggregant effect was less marked. The results obtained suggest that these plants may be a promising source of natural compounds, valuable in the prevention of the enhanced activity of blood platelets in numerous cardiovascular diseases, observed in menopausal or postmenopausal women.

Homocysteine and its thiolactone-mediated modification of fibrinogen affect blood platelet adhesion

Homocysteine (Hcys) and homocysteine thiolactone (HTL) concentrations in organism are correlated with a number of serious pathologies. In the literature, there are few papers describing studies on the effects of homocysteine on proteins that participate in blood coagulation and fibrinolysis in human. However, mechanisms involved in the relationship between hyperhomocysteinemia and hemostatic process are still unclear. The role of N- or S-homocysteinylation (induced by Hcys and its derivatives) of different hemostatic proteins, including fibrinogen is also still poorly known. The aim of this study was to establish the functional changes of the fibrinogen molecule induced by Hcys (at final doses of 10–100 µM) and the most reactive form of Hcys – its cyclic thioester, homocysteine thiolactone (0.1–1 µM), and to examine the effects of these changes on the capability of fibrinogen to interact with human blood platelets (by measuring the platelet adhesion). Our present results demonstrated that Hcys-treated fibrinogen in comparison with native molecule had a distinct capability to mediate platelet adhesion. Both, unstimulated and thrombin-activated platelets showed a reduced ability to adhere to Hcys-mediated fibrinogen. HTL (at all tested concentrations) had similar properties when we used thrombin-activated platelets. In conclusion, the results reported in this study could be useful for a better understanding of changes in hemostasis during hyperhomocysteinemia.