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Changes of blood platelet adhesion to collagen and fibrinogen induced by homocysteine and its thiolactone
Clinical Biochemistry 45 (2012) 1225–1228
Contents lists available at SciVerse ScienceDirect
Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Changes of blood platelet adhesion to collagen and fibrinogen induced by homocysteine and its thiolactone Joanna Malinowska ⁎, Małgorzata Tomczynska, Beata Olas Department of General Biochemistry, University of Łódź, Pomorska 141/3, 90‐236 Łódź, Poland
a r t i c l e
i n f o
Article history: Received 6 April 2012 Received in revised form 10 May 2012 Accepted 12 May 2012 Available online 23 May 2012 Keywords: Homocysteine Homocysteine thiolactone Collagen Fibrinogen Platelets
a b s t r a c t Background: Mechanisms of homocysteine (Hcy) and its derivatives contribution to thrombosis are complex and are only partly recognized. The available data suggest that the prothrombic properties of homocysteine and its thiolactone (HTL) are not only a result of the changes in coagulation process, fibrinolysis, or endothelial dysfunction, but also the dysfunction of blood platelets. Objective and methods: The present work was designed to study the effects of Hcy and HTL on one of the step of platelet activation — the platelet adhesion to collagen and fibrinogen in vitro. Platelet suspensions were preincubated with Hcy and HTL, at the final concentrations of 10, 25, 50 and 100 μM, and 0.1, 0.2 and 1 μM, respectively. Then, for platelet activation thrombin (0.1 U/mL) or TRAP (20 μM), were used. Results: The performed assays demonstrated that Hcy (at high tested concentrations: 50 and 100 μM) and its thiolactone (at all used concentrations: 0.1, 0.2 and 1 μM) stimulated the adhesion of thrombin- or TRAP- activated platelets to collagen and fibrinogen. Moreover, the exposure of blood platelets to HTL (even at lower concentrations than Hcy) resulted to a stronger modulatory effect on the platelet adhesion than when blood platelets were treated with Hcy. Conclusion: In conclusion, the results obtained in this study demonstrate that Hcy and its thiolactone may affect adhesive properties of blood platelets. © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction Homocysteine (Hcy) is a sulfur-containing amino acid derived from methionine. Approximately 80% of total plasma Hcy is protein bound, and only a small amount exists as a free reduced Hcy (about 0.1 μM). The majority of the unbound fraction of Hcy is oxidized, and forms dimers (homocystine) or mixed disulfides consisting of cysteine and Hcy (Table 1) [1–3]. Genetic (e.g. cystathionine β–synthase deficiency) or nutritional (e.g. diet poor in B vitamin) deficiencies of Hcy metabolism lead to hyperhomocysteinemia, which is a risk factor for cardiovascular disorders, including atherosclerosis and thrombogenesis. The mechanisms underlying homocysteine-induced effects have been intensively investigated over the last two decades. Hcy can induce oxidative stress promoting oxidant injury to vascular and blood cells, including blood platelets. The effects of elevated concentrations of circulating homocysteine on the vascular wall, platelet functions and coagulation factors promote the development of a pro-coagulant state [4]. However, the molecular basis for different steps of blood platelet activation during hyperhomocysteinemia is not well known, and is sometimes controversial. Authors very often do not write what form of Hcy
⁎ Corresponding author. E-mail address: [email protected] (J. Malinowska).
was studied. Our earlier reports demonstrated that Hcy (10–100 μM) alone does not induce the platelet aggregation, but increases the platelet aggregation (measured by turbidimetry and flow cytometry) stimulated by different agonists (thrombin, ADP or collagen) [5,6]. Moreover, our earlier results found that the most reactive form of homocysteine, which modifies hemostasis (including biological properties of blood platelets) may be homocysteine thiolactone (HTL; 0.1–1 μM) [7,8]. In all organisms Hcy is metabolized to the thioester–homocysteine thiolactone in an error-editing reaction in protein biosynthesis when Hcy is selected in place of methionine by methionyl-tRNA synthetase. HTL levels (≥100 nM) are elevated in hyperhomocysteinemic humans. HTL, known to be cytotoxic in experimental animals and cell cultures, is detrimental mostly because of its ability to form isopeptide bonds with protein lysine residues [9–12]. N-Hcy-proteins, including hemostatic proteins — fibrinogen may be linked to human pathology such as atherosclerosis [13]. The aim of our study was to determine the effects of Hcy and its thiolactone on washed human platelet adhesion to the important adhesive protein — collagen and fibrinogen in vitro. We have used 1) TRAP—peptide which is “tethered” ligand domain of thrombin receptor, and begins with the sequence Ser-Phe-Leu-Leu-Arg-Asn (SFLLRN) and 2) thrombin to test the influence of Hcy and HTL on the platelet adhesion. Hyperhomocysteinemia was stimulated by using the reduced form of Hcy (at final doses of 10–100 μM) and HTL (at final doses of 0.1–1 μM).
0009-9120/$ – see front matter © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2012.05.017
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J. Malinowska et al. / Clinical Biochemistry 45 (2012) 1225–1228
Table 1 Different forms of homocysteine present in human blood in normal individuals. *Total level of homocysteine—the term “total homocysteine” describes the pool of homocysteine released by reduction of all disulphide bonds in the sample ([24–26], modified). The form of Hcy
The concentration in human blood
Homocysteine thiolactone (HTL) Protein N-linked homocysteine: N-Hcy-hemoglobin N-(Hcy-S-S-Cys)-albumin Protein S-linked homocysteine—S-Hcy-albumin Homocystine (Hcy-S-S-Hcy) and combined with cysteine to from mixed disulfides (Hcy-S-S-Cys) Free reduced Hcy
0–35 nM About 15.5 μM: 12.7 μM 2.8 μM About 7.3 μM (⁎) About 2 μM (⁎) About 0.1 μM (⁎)
The measurement of platelet adhesion to collagen and fibrinogen Adhesion of platelets to collagen and fibrinogen was determined according to Tuszynski and Murphy [19] as described earlier [20]. The absorbance of control platelets (without Hcy or HTL) was expressed as 100%. Data analysis 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 Hcy or HTL) was performed with ANOVA test and POST Hoc test (Bonferroni). The statistically significant differences were also assessed by applying the paired Student's t test and the significance level was p b 0.05. In order to eliminate uncertain data, the Q-Dixon test was performed.
Materials and methods Results Materials Reduced form of D, L-homocysteine, D, L-homocysteine thiolactone, collagen type I and thrombin were purchased from Sigma (St Louis, MO). Thrombin receptor activating peptide (TRAP) SFLLRN was obtained from Sigma (St Louis, MO) and it was stored at −32 °C. TRAP was diluted with 0.15 M NaCl to desired concentrations just before the experiments. Fibrinogen isolated from pooled citrated human plasma by the cold ethanol precipitation technique followed by ammonium sulfate fractionation at 26% saturation at 4 °C, according to Doolittle [14]. Its concentration was determined spectrophotometrically at 280 nm using an extinction coefficient 1.55 for 1 mg/mL solution.
Isolation of plasma and blood platelets Human blood was taken from 5 healthy volunteers aged 23 to 36 (average: 25; SD = 6.3 years) not taking any medications or addictive substances (including tobacco, alcohol and aspirin or any other antiplatelet drugs) and keeping a balanced diet (meat and vegetables), with similar socio-economic background, using no antioxidant supplementation. The endogenous concentration of total Hcy in plasma was 12.5 ± 2.7 μM (five experiments done in triplicate). The endogenous concentration of reduced form of Hcy and HTL was about 98.9 ± 10.8 nM (five experiments done in triplicate) and 0–35 nM (five experiments done in triplicate), respectively. The classical technique HPLC has been used for the determination of Hcy [15] or HTL [16] in human plasma. The HPLC analysis was performed with a Hewlett-Packard 1100 Series system according to Głowacki et al. [16] and Bald et al. [15]. 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 [17]. The final platelet concentration was 3×108 platelets/mL (five experiments done in triplicate). The platelets were counted by the photometric method according to Walkowiak et al. [18]. Washed human platelet suspensions in the modified Tyrode's Ca+ 2/Mg+ 2 free buffer (127 mM NaCl, 2.7 mM KCl, 0.5 mM NaH2PO4, 12 mM NaHCO3, 5 mM HEPES, 5.6 mM glucose, pH 7.4) were exposed (15 min, 37 °C) to: -
D, L-homocysteine D, L-homocysteine
at a final concentration 10–100 μM thiolactone at a final concentration 0.1–1 μM.
To the control platelet samples, the modified Tyrode's Ca + 2/Mg+ 2 free buffer (127 mM NaCl, 2.7 mM KCl, 0.5 mM NaH2PO4, 12 mM NaHCO3, 5 mM HEPES, 5.6 mM glucose, pH 7.4) was added in the place of Hcy or HTL. The protocol was passed by the Committee for Research on Human Subjects of the University of Lodz number KBBN-UŁ/I/5/2011.
Our comparative studies demonstrated that the reduced homocysteine (at final concentrations of 10–100 μM) and its thiolactone (at final concentrations of 0.1–1 μM) modulate the platelet adhesion to collagen and fibrinogen (Fig. 1A and B). First, we observed that the adhesion to collagen of resting platelets incubated with Hcy was changed, but this process was not statistically significant (p> 0.05) (Fig. 1A). We noticed the same process when we measured the adhesion of thrombin- or TRAP-activated platelets in the presence of Hcy at low concentrations −10 and 25 μM (p> 0.05) (Fig. 1A). Higher concentrations of Hcy (50 and 100 μM) significantly increased the adhesion of thrombin- or TRAP-activated platelets to collagen (pb 0.05) (Fig. 1A). Moreover, our results (in the model system—in vitro) showed that the adhesion to collagen of blood platelets incubated with HTL was changed (Fig. 1A). Incubation of platelets with HTL (at all tested concentrations: 0.1–1 μM) had stimulatory effects on the adhesion of resting platelets (pb 0.05) and the adhesion of thrombin- or TRAP- activated platelets to collagen (pb 0.01). The stimulatory effects of HTL appears to be concentration‐dependent in all adhesion tests (Fig. 1A). Maximum stimulatory effect of HTL was observed when platelets were treated with the highest concentration of HTL — 1 μM (the increase of platelet adhesion was about 19.5% (for resting platelets) and about 56.5% (for thrombinactivated platelets)) (Fig. 1A). Homocysteine (at high concentrations — 50 and 100 μM) and its thiolactone (at all tested concentrations of 0.1– 1 μM) also stimulated the platelet adhesion to fibrinogen (Fig. 1B). Table 2 reports a synergistic action of Hcy and its thiolactone (at the highest tested concentrations) on modulation of blood platelet adhesion to collagen or fibrinogen. Discussion Platelet adhesion to the injured vessel wall is one of the important steps in the hemostatic function. Therefore, analysis of the platelet adhesion is of great importance. Various methods have been developed for detection and quantifying of the number of adherent cells. The method of using bicinchoninic acid to measure the total cellular proteins of adherent cells is rapid and can be used both quantitatively and qualitatively to detect adherent blood platelets. In this technique, the wells are washed, and adherent blood platelets are solubilized with bicinchoninic acid protein assay reagent. In our experiments we used this technique. To eliminate the numerous plasma factors that may interfere with tested thiols (Hcy and its thiolactone) in a different way, we studied the adhesion of cells to collagen and fibrinogen in the system containing washed blood platelets, possibly highlighting the role of glycoprotein expression on platelet surface. This study provides the first evidence reporting that Hcy (at all tested concentrations) had no effect on the adhesion of resting platelets to collagen. Our results showed a significant increase in adhesion of
J. Malinowska et al. / Clinical Biochemistry 45 (2012) 1225–1228
A
HTL
Hcy
B HTL Hcy
Fig. 1. The effects of homocysteine (10, 25, 50 and 100 μM, 15 min, 37 °C) and its thiolactone (0.1, 0.2 and 1 μM, 15 min, 37 °C) on the adhesion of resting platelets or thrombin‐ or TRAP-activated platelets to collagen (A) and fibrinogen (B). Data represent means± SD of five experiments done in quadruplicate. The effect of four different concentrations of Hcy (10, 25, 50 and 100 μM) vs control was not statistically significant according ANOVA test and Post Hoc test –p > 0.05 (for the adhesion of resting platelets). The effect of two different concentrations of Hcy (10, 25 μM) vs control was not statistically significant according ANOVA test and Post Hoc test –p > 0.05 (for the adhesion of thrombin- or TRAP- activated platelets). The effect of two different concentrations of Hcy (50 and 100 μM) vs control was statistically significant according ANOVA test and Post Hoc test –p b 0.05 (for the adhesion of thrombin- or TRAP-activated platelets). The effect of three different concentrations of HTL (0.1, 0.2 and 1 μM) vs control was statistically significant according ANOVA test and Post Hoc test – b 0.05 (for the adhesion of resting platelets), p b 0.01 (for the adhesion of thrombin- or TRAP-activated platelets to collagen).
thrombin-activated platelets to collagen, when platelets were treated with a high concentration of Hcy (50 and 100 μM), but not with low concentration of Hcy (10 and 25 μM). The obtained results
Table 2 The effects of homocysteine (100 μM, 15 min, 37 °C) and its thiolactone (1 μM, 15 min, 37 °C) on the adhesion of resting platelets or thrombin‐ or TRAP-activated platelets to collagen and fibrinogen. Data represent means ± SD of five experiments done in quadruplicate. The effects were statistically significant according to the Student's t test (*‐p > 0.05—Hcy‐treated platelets versus control; ** ‐ p b 0.001—Hcy and/or HTL‐treated platelets versus control). Platelet adhesion to collagen [%] Resting platelets
Thrombin‐activated platelets
TRAP‐activated platelets
Control
100
100
100
Hcy HTL Hcy + HTL
104 ± 7.2* 119.5 ± 3.8** 125.4 ± 5.7**
121.5 ± 5.1** 156.5 ± 13.4** 167.2 ± 10.4**
117.9 ± 4.9** 139.8 ± 9.8** 149.9 ± 7.7**
to fibrinogen [%] 107.5 ± 8.2* 118.9 ± 3.8** 123.7 ± 5.2**
125.5 ± 6.1** 136.6 ± 13.4** 144.7 ± 12.9**
119.9 ± 7.9** 129.8 ± 9.8** 145.8 ± 10.4**
Platelet adhesion Hcy HTL Hcy + HTL
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are consistent with the literature. Luo et al. [21] investigated the effect of Hcy on the platelet adhesion to collagen under different flow conditions. They observed the same process under the low shear rate model (100/s), but the platelet adhesion to collagen remained unchanged under a high shear rate model (1600/s) after Hcy treatment. Moreover, the present study for the first time, indicates that homocysteine (50 and 100 μM) stimulated blood platelet adhesion to fibrinogen induced not only by proteolytic (thrombin), but also non-proteolytic agonist—TRAP. However, the stimulatory action of Hcy is weaker when TRAP as the agonist is used. Our earlier results showed not only the reduced form of Hcy, but HTL augmented blood platelet activation (induced by collagen) measured by flow cytometry (for platelet aggregates and microparticle formation in the whole blood) [8]. An important finding in this study is that homocysteine thiolactone also modulates the platelet adhesion to collagen and fibrinogen. McGarrigle et al. [22] found that the circulating Hcy can modulate the activation state of the platelet integrin αIIbβ3. Because, we studied the platelet adhesion in the system containing washed platelets (under static conditions), we may also suppose that Hcy and its thiolactone may modulate the expression of the platelet integrin αIIbβ3. However, mechanism of these tested thiols on the platelet activation is complex and still unclear. Results of Luo et al. [21] suggest that by enhancing tyrosine phosphorylation whole cellular protein, especially tyrosine phosphorylation of signaling molecules in GP VI and α2β1 integrin pathway, such as Src kinase and phospholipaseCγ2 (PLCγ2), Hcy may potentiate platelet response to collagen. It seems that tested thiols may also interact with thrombin receptors on the platelet membrane. It is known that Hcy and its thiolactone modify platelet proteins, e. g. modify thiol groups, which are the important for the platelet activation [6]. The consequence of this modification may be alteration in the protein structure (e. g. platelet receptors) associated with changes of various steps of platelet activation, including adhesion. Moreover, experiments presented earlier showed that Hcy and HTL caused the changes in the level of reactive oxygen species [5], which may be responsible for the modification of platelet reactivity induced by these thiols, because blood platelets, in analogy to other circulating blood cells, generate reactive oxygen/ nitrogen species that may behave as second messengers and may regulate platelet functions. In clinical and experimental studies, a homocysteine-mediated oxidative stress has been also shown to trigger platelet activation, in turn leading to a tendency of thrombosis, in patients with severe hyperhomocysteinemia [23]. In our comparative studies, HTL was found to be more effective modulator of platelet adhesion than homocysteine. Therefore, homocysteine thiolactone (even at lower concentration than Hcy) may be an additional factor, leading to the significant increase of cardiovascular complications risk. Moreover, in the experiments presented here, we have shown that a combination of Hcy and HTL had even greater modulation action on the platelet adhesion than any of tested thiol compound alone. On the basis of presented observations in vitro, the combination Hcy and HTL may explain the pleiotropic effects of these molecules. In conclusion, our present results indicate that Hcy and its thiolactone display a multiple effect of blood platelet activation, and may serve different strategies of Hcy/HTL action in blood platelets. They may modulate the signal transduction in different and sometimes in opposite ways. Moreover, our earlier results [7,8] demonstrated that, during hyperhomocysteinemia (induced by Hcy and/or its thiolactone) adhesion of platelets are impaired not only due to alteration of platelet function (changes of platelet membrane, signaling pathway), but due to the modification of adhesive protein—fibrinogen. Collagen as important component of extracellular matrix, which has lysine residues may be also modified during hyperhomocysteinemia in a process called N-homocysteinylation, so we suppose that interaction of modified
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adhesive proteins, including collagen with platelet receptors may cause impaired adhesion. Studies are in progress in our laboratory to characterize changes in collagen induced by different forms of Hcy and their role in the platelet activation. Conflict of interest None to declare. Acknowledgment This work was supported by grant (No 0365/B/PO1/2011/40) from National Science Centre (Poland). References [1] Mansoor MA, Svardal AM, Ueland PM. Determination of the in vivo redox status of cysteine, cysteinylglycine, homocysteine, and glutathione in human plasma. Anal Biochem 1992;200:218–29. [2] D'Angelo A, Selhub J. Homocysteine and thrombotic disease. Blood 1997;90:1–11. [3] Ramakrishnan S, Sulochana KN, Lakshmi S, Selvi R, Angayarkanni K. Biochemistry of homocysteine in health and diseases. Indian J Biochem Biophys 2006;43:275–83. [4] Dionisio N, Jardin I, Salido GM, Rosado JA. Homocysteine, intracellular signaling and thrombotic disorders. Curr Med Chem 2010;17:3109–19. [5] Olas B, Kedzierska M, Wachowicz B. Comparative studies on homocysteine and its metabolite—homocysteine thiolactone action in blood platelets in vitro. Platelets 2008;19:520–7. [6] Olas B, Kolodziejczyk J, Kedzierska M, Rywaniak J, Wachowicz B. Modification of human blood platelet induced by homocysteine and its thiolactone in vitro. Thromb Res 2009;24:689–94. [7] Malinowska J, Olas B. Homocysteine and its thiolactone-mediated modification of fibrinogen affects blood platelet adhesion. Platelets 2011;1–4. [8] Malinowska J, Olas B. Analysis of biological properties of selected elements of haemostasis after treatment with the oxidized form of homocysteine in vitro. Platelets 2011;22:629–32. [9] Jakubowski H. Molecular basis of homocysteine toxicity in humans. Cell Mol Life 2004;61:470–87.
[10] Jakubowski H. The molecular basis of homocysteine thiolactone-mediated vascular disease. Clin Chem Lab Med 2007;12:1704–16. [11] Glowacki R, Bald E, Jakubowski H. Identification of origin of Nε-homocysteinyl-lysine isopeptide in humans and mice. Amino Acids 2010;39:1563–9. [12] Jakubowski H, Glowacki R. Chemical biology of homocysteine thiolactone and related metabolites. Adv Clin Chem 2011;55:81–103. [13] Jakubowski H, Boers GH, Strauss KA. Mutations in cystathionine beta-synthase or methylenetetrahydrofolate reductase gene increase N-homocysteinylated protein levels in humans. FASEB J 2008;22:4071–6. [14] Doolittle RF, Schubert D, Schwartz SA. Amino acid sequence studies on artiodactyl fibrinopeptides I Dromedary camel, mule deer, and cape buffalo. Arch Biochem Biophys 1967;118:456–67. [15] Bald E, Chwatko G, Glowacki R, Kusmierek K. Analysis of plasma thiols by high-performance liquid chromatography with ultraviolet detection. J Chromatogr 2004;1032:109–15. [16] Glowacki R, Bald E, Jakubowski H. An on-column derivatization method for the determination of homocysteine-thiolactone and protein N-linked homocysteine. Amino Acids 2011;41:187–94. [17] Wachowicz B, Kustroń J. Effect of cisplatin on lipid peroxidation in pig blood platelets. Cytobios 1992;70:41–7. [18] Walkowiak B, Michalak E, Koziołkiewicz W, Cierniewski CS. Rapid photometric method for estimation of platelet count in blood plasma or platelet suspension. Thromb Res 1989;56:763–6. [19] Tuszyński GP, Murphy A. Spectrophotometric quantitation of anchorage‐dependent cell numbers using the bicinchoninic acid protein assay reagent. Anal Biochem 1990;184:189–91. [20] Olas B, Mielicki W, Wachowicz B, Krajewski T. Cancer procoagulant (CP) stimulates platelet adhesion. Thromb Res 1999;94:199–203. [21] Luo F, Liu X, Wang S, Chen H. Effect of homocysteine on platelet activation induced by collagen. Nutrition 2006;22:69–75. [22] McGarrigle SA, O'Neill S, Walsh GM, Moran N, Groham IM, Cooney MT, et al. Integrin αIIbβ3 exists in an activated state in subjects with elevated plasma homocysteine levels. Platelets 2011;22:63–71. [23] Di Minno MND, Tremoli E, Coppola A, Lupoli R, Di Minno GD. Homocysteine and arteria thrombosis: challenge and opportunity. Thromb Haemost 2010;103:942–61. [24] Perła-Kajan J, Twardowski T, Jakubowski H. Mechanisms of homocysteine toxicity in humans. Amino Acids 2007;32:561–72. [25] Zimny J. Mechanisms that protect against homocysteine toxicity. Prog Biochem 2008;54:91–6. [26] Manolescu BN, Oprea E, Farcasanu IC, Berteanu M, Cercasov C. Homocysteine and vitamin therapy in stroke prevention and treatment: a review. Acta Biochim Pol 2010;57:467–77.
Contents lists available at SciVerse ScienceDirect
Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Changes of blood platelet adhesion to collagen and fibrinogen induced by homocysteine and its thiolactone Joanna Malinowska ⁎, Małgorzata Tomczynska, Beata Olas Department of General Biochemistry, University of Łódź, Pomorska 141/3, 90‐236 Łódź, Poland
a r t i c l e
i n f o
Article history: Received 6 April 2012 Received in revised form 10 May 2012 Accepted 12 May 2012 Available online 23 May 2012 Keywords: Homocysteine Homocysteine thiolactone Collagen Fibrinogen Platelets
a b s t r a c t Background: Mechanisms of homocysteine (Hcy) and its derivatives contribution to thrombosis are complex and are only partly recognized. The available data suggest that the prothrombic properties of homocysteine and its thiolactone (HTL) are not only a result of the changes in coagulation process, fibrinolysis, or endothelial dysfunction, but also the dysfunction of blood platelets. Objective and methods: The present work was designed to study the effects of Hcy and HTL on one of the step of platelet activation — the platelet adhesion to collagen and fibrinogen in vitro. Platelet suspensions were preincubated with Hcy and HTL, at the final concentrations of 10, 25, 50 and 100 μM, and 0.1, 0.2 and 1 μM, respectively. Then, for platelet activation thrombin (0.1 U/mL) or TRAP (20 μM), were used. Results: The performed assays demonstrated that Hcy (at high tested concentrations: 50 and 100 μM) and its thiolactone (at all used concentrations: 0.1, 0.2 and 1 μM) stimulated the adhesion of thrombin- or TRAP- activated platelets to collagen and fibrinogen. Moreover, the exposure of blood platelets to HTL (even at lower concentrations than Hcy) resulted to a stronger modulatory effect on the platelet adhesion than when blood platelets were treated with Hcy. Conclusion: In conclusion, the results obtained in this study demonstrate that Hcy and its thiolactone may affect adhesive properties of blood platelets. © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction Homocysteine (Hcy) is a sulfur-containing amino acid derived from methionine. Approximately 80% of total plasma Hcy is protein bound, and only a small amount exists as a free reduced Hcy (about 0.1 μM). The majority of the unbound fraction of Hcy is oxidized, and forms dimers (homocystine) or mixed disulfides consisting of cysteine and Hcy (Table 1) [1–3]. Genetic (e.g. cystathionine β–synthase deficiency) or nutritional (e.g. diet poor in B vitamin) deficiencies of Hcy metabolism lead to hyperhomocysteinemia, which is a risk factor for cardiovascular disorders, including atherosclerosis and thrombogenesis. The mechanisms underlying homocysteine-induced effects have been intensively investigated over the last two decades. Hcy can induce oxidative stress promoting oxidant injury to vascular and blood cells, including blood platelets. The effects of elevated concentrations of circulating homocysteine on the vascular wall, platelet functions and coagulation factors promote the development of a pro-coagulant state [4]. However, the molecular basis for different steps of blood platelet activation during hyperhomocysteinemia is not well known, and is sometimes controversial. Authors very often do not write what form of Hcy
⁎ Corresponding author. E-mail address: [email protected] (J. Malinowska).
was studied. Our earlier reports demonstrated that Hcy (10–100 μM) alone does not induce the platelet aggregation, but increases the platelet aggregation (measured by turbidimetry and flow cytometry) stimulated by different agonists (thrombin, ADP or collagen) [5,6]. Moreover, our earlier results found that the most reactive form of homocysteine, which modifies hemostasis (including biological properties of blood platelets) may be homocysteine thiolactone (HTL; 0.1–1 μM) [7,8]. In all organisms Hcy is metabolized to the thioester–homocysteine thiolactone in an error-editing reaction in protein biosynthesis when Hcy is selected in place of methionine by methionyl-tRNA synthetase. HTL levels (≥100 nM) are elevated in hyperhomocysteinemic humans. HTL, known to be cytotoxic in experimental animals and cell cultures, is detrimental mostly because of its ability to form isopeptide bonds with protein lysine residues [9–12]. N-Hcy-proteins, including hemostatic proteins — fibrinogen may be linked to human pathology such as atherosclerosis [13]. The aim of our study was to determine the effects of Hcy and its thiolactone on washed human platelet adhesion to the important adhesive protein — collagen and fibrinogen in vitro. We have used 1) TRAP—peptide which is “tethered” ligand domain of thrombin receptor, and begins with the sequence Ser-Phe-Leu-Leu-Arg-Asn (SFLLRN) and 2) thrombin to test the influence of Hcy and HTL on the platelet adhesion. Hyperhomocysteinemia was stimulated by using the reduced form of Hcy (at final doses of 10–100 μM) and HTL (at final doses of 0.1–1 μM).
0009-9120/$ – see front matter © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2012.05.017
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J. Malinowska et al. / Clinical Biochemistry 45 (2012) 1225–1228
Table 1 Different forms of homocysteine present in human blood in normal individuals. *Total level of homocysteine—the term “total homocysteine” describes the pool of homocysteine released by reduction of all disulphide bonds in the sample ([24–26], modified). The form of Hcy
The concentration in human blood
Homocysteine thiolactone (HTL) Protein N-linked homocysteine: N-Hcy-hemoglobin N-(Hcy-S-S-Cys)-albumin Protein S-linked homocysteine—S-Hcy-albumin Homocystine (Hcy-S-S-Hcy) and combined with cysteine to from mixed disulfides (Hcy-S-S-Cys) Free reduced Hcy
0–35 nM About 15.5 μM: 12.7 μM 2.8 μM About 7.3 μM (⁎) About 2 μM (⁎) About 0.1 μM (⁎)
The measurement of platelet adhesion to collagen and fibrinogen Adhesion of platelets to collagen and fibrinogen was determined according to Tuszynski and Murphy [19] as described earlier [20]. The absorbance of control platelets (without Hcy or HTL) was expressed as 100%. Data analysis 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 Hcy or HTL) was performed with ANOVA test and POST Hoc test (Bonferroni). The statistically significant differences were also assessed by applying the paired Student's t test and the significance level was p b 0.05. In order to eliminate uncertain data, the Q-Dixon test was performed.
Materials and methods Results Materials Reduced form of D, L-homocysteine, D, L-homocysteine thiolactone, collagen type I and thrombin were purchased from Sigma (St Louis, MO). Thrombin receptor activating peptide (TRAP) SFLLRN was obtained from Sigma (St Louis, MO) and it was stored at −32 °C. TRAP was diluted with 0.15 M NaCl to desired concentrations just before the experiments. Fibrinogen isolated from pooled citrated human plasma by the cold ethanol precipitation technique followed by ammonium sulfate fractionation at 26% saturation at 4 °C, according to Doolittle [14]. Its concentration was determined spectrophotometrically at 280 nm using an extinction coefficient 1.55 for 1 mg/mL solution.
Isolation of plasma and blood platelets Human blood was taken from 5 healthy volunteers aged 23 to 36 (average: 25; SD = 6.3 years) not taking any medications or addictive substances (including tobacco, alcohol and aspirin or any other antiplatelet drugs) and keeping a balanced diet (meat and vegetables), with similar socio-economic background, using no antioxidant supplementation. The endogenous concentration of total Hcy in plasma was 12.5 ± 2.7 μM (five experiments done in triplicate). The endogenous concentration of reduced form of Hcy and HTL was about 98.9 ± 10.8 nM (five experiments done in triplicate) and 0–35 nM (five experiments done in triplicate), respectively. The classical technique HPLC has been used for the determination of Hcy [15] or HTL [16] in human plasma. The HPLC analysis was performed with a Hewlett-Packard 1100 Series system according to Głowacki et al. [16] and Bald et al. [15]. 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 [17]. The final platelet concentration was 3×108 platelets/mL (five experiments done in triplicate). The platelets were counted by the photometric method according to Walkowiak et al. [18]. Washed human platelet suspensions in the modified Tyrode's Ca+ 2/Mg+ 2 free buffer (127 mM NaCl, 2.7 mM KCl, 0.5 mM NaH2PO4, 12 mM NaHCO3, 5 mM HEPES, 5.6 mM glucose, pH 7.4) were exposed (15 min, 37 °C) to: -
D, L-homocysteine D, L-homocysteine
at a final concentration 10–100 μM thiolactone at a final concentration 0.1–1 μM.
To the control platelet samples, the modified Tyrode's Ca + 2/Mg+ 2 free buffer (127 mM NaCl, 2.7 mM KCl, 0.5 mM NaH2PO4, 12 mM NaHCO3, 5 mM HEPES, 5.6 mM glucose, pH 7.4) was added in the place of Hcy or HTL. The protocol was passed by the Committee for Research on Human Subjects of the University of Lodz number KBBN-UŁ/I/5/2011.
Our comparative studies demonstrated that the reduced homocysteine (at final concentrations of 10–100 μM) and its thiolactone (at final concentrations of 0.1–1 μM) modulate the platelet adhesion to collagen and fibrinogen (Fig. 1A and B). First, we observed that the adhesion to collagen of resting platelets incubated with Hcy was changed, but this process was not statistically significant (p> 0.05) (Fig. 1A). We noticed the same process when we measured the adhesion of thrombin- or TRAP-activated platelets in the presence of Hcy at low concentrations −10 and 25 μM (p> 0.05) (Fig. 1A). Higher concentrations of Hcy (50 and 100 μM) significantly increased the adhesion of thrombin- or TRAP-activated platelets to collagen (pb 0.05) (Fig. 1A). Moreover, our results (in the model system—in vitro) showed that the adhesion to collagen of blood platelets incubated with HTL was changed (Fig. 1A). Incubation of platelets with HTL (at all tested concentrations: 0.1–1 μM) had stimulatory effects on the adhesion of resting platelets (pb 0.05) and the adhesion of thrombin- or TRAP- activated platelets to collagen (pb 0.01). The stimulatory effects of HTL appears to be concentration‐dependent in all adhesion tests (Fig. 1A). Maximum stimulatory effect of HTL was observed when platelets were treated with the highest concentration of HTL — 1 μM (the increase of platelet adhesion was about 19.5% (for resting platelets) and about 56.5% (for thrombinactivated platelets)) (Fig. 1A). Homocysteine (at high concentrations — 50 and 100 μM) and its thiolactone (at all tested concentrations of 0.1– 1 μM) also stimulated the platelet adhesion to fibrinogen (Fig. 1B). Table 2 reports a synergistic action of Hcy and its thiolactone (at the highest tested concentrations) on modulation of blood platelet adhesion to collagen or fibrinogen. Discussion Platelet adhesion to the injured vessel wall is one of the important steps in the hemostatic function. Therefore, analysis of the platelet adhesion is of great importance. Various methods have been developed for detection and quantifying of the number of adherent cells. The method of using bicinchoninic acid to measure the total cellular proteins of adherent cells is rapid and can be used both quantitatively and qualitatively to detect adherent blood platelets. In this technique, the wells are washed, and adherent blood platelets are solubilized with bicinchoninic acid protein assay reagent. In our experiments we used this technique. To eliminate the numerous plasma factors that may interfere with tested thiols (Hcy and its thiolactone) in a different way, we studied the adhesion of cells to collagen and fibrinogen in the system containing washed blood platelets, possibly highlighting the role of glycoprotein expression on platelet surface. This study provides the first evidence reporting that Hcy (at all tested concentrations) had no effect on the adhesion of resting platelets to collagen. Our results showed a significant increase in adhesion of
J. Malinowska et al. / Clinical Biochemistry 45 (2012) 1225–1228
A
HTL
Hcy
B HTL Hcy
Fig. 1. The effects of homocysteine (10, 25, 50 and 100 μM, 15 min, 37 °C) and its thiolactone (0.1, 0.2 and 1 μM, 15 min, 37 °C) on the adhesion of resting platelets or thrombin‐ or TRAP-activated platelets to collagen (A) and fibrinogen (B). Data represent means± SD of five experiments done in quadruplicate. The effect of four different concentrations of Hcy (10, 25, 50 and 100 μM) vs control was not statistically significant according ANOVA test and Post Hoc test –p > 0.05 (for the adhesion of resting platelets). The effect of two different concentrations of Hcy (10, 25 μM) vs control was not statistically significant according ANOVA test and Post Hoc test –p > 0.05 (for the adhesion of thrombin- or TRAP- activated platelets). The effect of two different concentrations of Hcy (50 and 100 μM) vs control was statistically significant according ANOVA test and Post Hoc test –p b 0.05 (for the adhesion of thrombin- or TRAP-activated platelets). The effect of three different concentrations of HTL (0.1, 0.2 and 1 μM) vs control was statistically significant according ANOVA test and Post Hoc test – b 0.05 (for the adhesion of resting platelets), p b 0.01 (for the adhesion of thrombin- or TRAP-activated platelets to collagen).
thrombin-activated platelets to collagen, when platelets were treated with a high concentration of Hcy (50 and 100 μM), but not with low concentration of Hcy (10 and 25 μM). The obtained results
Table 2 The effects of homocysteine (100 μM, 15 min, 37 °C) and its thiolactone (1 μM, 15 min, 37 °C) on the adhesion of resting platelets or thrombin‐ or TRAP-activated platelets to collagen and fibrinogen. Data represent means ± SD of five experiments done in quadruplicate. The effects were statistically significant according to the Student's t test (*‐p > 0.05—Hcy‐treated platelets versus control; ** ‐ p b 0.001—Hcy and/or HTL‐treated platelets versus control). Platelet adhesion to collagen [%] Resting platelets
Thrombin‐activated platelets
TRAP‐activated platelets
Control
100
100
100
Hcy HTL Hcy + HTL
104 ± 7.2* 119.5 ± 3.8** 125.4 ± 5.7**
121.5 ± 5.1** 156.5 ± 13.4** 167.2 ± 10.4**
117.9 ± 4.9** 139.8 ± 9.8** 149.9 ± 7.7**
to fibrinogen [%] 107.5 ± 8.2* 118.9 ± 3.8** 123.7 ± 5.2**
125.5 ± 6.1** 136.6 ± 13.4** 144.7 ± 12.9**
119.9 ± 7.9** 129.8 ± 9.8** 145.8 ± 10.4**
Platelet adhesion Hcy HTL Hcy + HTL
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are consistent with the literature. Luo et al. [21] investigated the effect of Hcy on the platelet adhesion to collagen under different flow conditions. They observed the same process under the low shear rate model (100/s), but the platelet adhesion to collagen remained unchanged under a high shear rate model (1600/s) after Hcy treatment. Moreover, the present study for the first time, indicates that homocysteine (50 and 100 μM) stimulated blood platelet adhesion to fibrinogen induced not only by proteolytic (thrombin), but also non-proteolytic agonist—TRAP. However, the stimulatory action of Hcy is weaker when TRAP as the agonist is used. Our earlier results showed not only the reduced form of Hcy, but HTL augmented blood platelet activation (induced by collagen) measured by flow cytometry (for platelet aggregates and microparticle formation in the whole blood) [8]. An important finding in this study is that homocysteine thiolactone also modulates the platelet adhesion to collagen and fibrinogen. McGarrigle et al. [22] found that the circulating Hcy can modulate the activation state of the platelet integrin αIIbβ3. Because, we studied the platelet adhesion in the system containing washed platelets (under static conditions), we may also suppose that Hcy and its thiolactone may modulate the expression of the platelet integrin αIIbβ3. However, mechanism of these tested thiols on the platelet activation is complex and still unclear. Results of Luo et al. [21] suggest that by enhancing tyrosine phosphorylation whole cellular protein, especially tyrosine phosphorylation of signaling molecules in GP VI and α2β1 integrin pathway, such as Src kinase and phospholipaseCγ2 (PLCγ2), Hcy may potentiate platelet response to collagen. It seems that tested thiols may also interact with thrombin receptors on the platelet membrane. It is known that Hcy and its thiolactone modify platelet proteins, e. g. modify thiol groups, which are the important for the platelet activation [6]. The consequence of this modification may be alteration in the protein structure (e. g. platelet receptors) associated with changes of various steps of platelet activation, including adhesion. Moreover, experiments presented earlier showed that Hcy and HTL caused the changes in the level of reactive oxygen species [5], which may be responsible for the modification of platelet reactivity induced by these thiols, because blood platelets, in analogy to other circulating blood cells, generate reactive oxygen/ nitrogen species that may behave as second messengers and may regulate platelet functions. In clinical and experimental studies, a homocysteine-mediated oxidative stress has been also shown to trigger platelet activation, in turn leading to a tendency of thrombosis, in patients with severe hyperhomocysteinemia [23]. In our comparative studies, HTL was found to be more effective modulator of platelet adhesion than homocysteine. Therefore, homocysteine thiolactone (even at lower concentration than Hcy) may be an additional factor, leading to the significant increase of cardiovascular complications risk. Moreover, in the experiments presented here, we have shown that a combination of Hcy and HTL had even greater modulation action on the platelet adhesion than any of tested thiol compound alone. On the basis of presented observations in vitro, the combination Hcy and HTL may explain the pleiotropic effects of these molecules. In conclusion, our present results indicate that Hcy and its thiolactone display a multiple effect of blood platelet activation, and may serve different strategies of Hcy/HTL action in blood platelets. They may modulate the signal transduction in different and sometimes in opposite ways. Moreover, our earlier results [7,8] demonstrated that, during hyperhomocysteinemia (induced by Hcy and/or its thiolactone) adhesion of platelets are impaired not only due to alteration of platelet function (changes of platelet membrane, signaling pathway), but due to the modification of adhesive protein—fibrinogen. Collagen as important component of extracellular matrix, which has lysine residues may be also modified during hyperhomocysteinemia in a process called N-homocysteinylation, so we suppose that interaction of modified
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adhesive proteins, including collagen with platelet receptors may cause impaired adhesion. Studies are in progress in our laboratory to characterize changes in collagen induced by different forms of Hcy and their role in the platelet activation. Conflict of interest None to declare. Acknowledgment This work was supported by grant (No 0365/B/PO1/2011/40) from National Science Centre (Poland). References [1] Mansoor MA, Svardal AM, Ueland PM. Determination of the in vivo redox status of cysteine, cysteinylglycine, homocysteine, and glutathione in human plasma. Anal Biochem 1992;200:218–29. [2] D'Angelo A, Selhub J. Homocysteine and thrombotic disease. Blood 1997;90:1–11. [3] Ramakrishnan S, Sulochana KN, Lakshmi S, Selvi R, Angayarkanni K. Biochemistry of homocysteine in health and diseases. Indian J Biochem Biophys 2006;43:275–83. [4] Dionisio N, Jardin I, Salido GM, Rosado JA. Homocysteine, intracellular signaling and thrombotic disorders. Curr Med Chem 2010;17:3109–19. [5] Olas B, Kedzierska M, Wachowicz B. Comparative studies on homocysteine and its metabolite—homocysteine thiolactone action in blood platelets in vitro. Platelets 2008;19:520–7. [6] Olas B, Kolodziejczyk J, Kedzierska M, Rywaniak J, Wachowicz B. Modification of human blood platelet induced by homocysteine and its thiolactone in vitro. Thromb Res 2009;24:689–94. [7] Malinowska J, Olas B. Homocysteine and its thiolactone-mediated modification of fibrinogen affects blood platelet adhesion. Platelets 2011;1–4. [8] Malinowska J, Olas B. Analysis of biological properties of selected elements of haemostasis after treatment with the oxidized form of homocysteine in vitro. Platelets 2011;22:629–32. [9] Jakubowski H. Molecular basis of homocysteine toxicity in humans. Cell Mol Life 2004;61:470–87.
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