Peroxynitrite may affect clot retraction in human blood through the inhibition of platelet mitochondrial energy production

Thrombosis Research 133 (2014) 402–411

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Peroxynitrite may affect clot retraction in human blood through the inhibition of platelet mitochondrial energy production Tomasz Misztal, Tomasz Rusak, Marian Tomasiak ⁎ Department of Physical Chemistry, Medical University of Bialystok, Kilinskiego 1, 15-089 Bialystok, Poland

a r t i c l e

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Article history: Received 3 October 2013 Received in revised form 12 November 2013 Accepted 16 December 2013 Available online 19 December 2013 Keywords: Clot retraction Energy metabolism Mitochondria Platelets Peroxynitrite Inflammation

a b s t r a c t Peroxynitrite (ONOO-) contributes to hemostasis abnormalities associated with inflammatory states by a poorly understood mechanism. Here we show that ONOO- may affect clot retraction (CR), an important step in hemostasis, by reducing contractility of human platelets resulting from the inhibition of mitochondrial energy production. Reduced CR may result in thromboembolic and hemorrhage events. The results show that in human blood, in vitro, physiologically relevant ONOO- concentrations reduce clot retraction rate and enlarge final clot size. The stressor was more effective in reconstituted system consisting of washed platelets and fibrinogen, (IC50 = 25 nM) than in platelet rich plasma (IC50 = 75 μM) or in whole blood (IC50 = 120 μM), indicating that its effect depends on the number of targets. Retardation of CR by lower concentrations of ONOO- resulted in reduction of platelet energy production due to impairment of mitochondria but not from tyrosine nitration or inhibition of actin polymerization. In washed platelets nanomolar ONOO- concentrations produced a drop of the mitochondrial transmembrane potential (ΔΨm) explaining high sensitivity of CR (a large consumer of platelet energy) to stressor. Thromboelastometry measurements showed that ONOO- may diminish clot stability and elasticity through the reduction of platelet contractility. Our findings suggest that in humans ONOO-- altered platelet mitochondria represent a new link between inflammation and hemostasis. © 2014 Elsevier Ltd. All rights reserved.

Introduction In response to inflammation, mammalian immune cells form high quantities of NO and superoxide which are used to kill pathogens [1]. Neither superoxide nor NO is toxic in vivo to host cells because there are efficient means to minimize their accumulation [1–3]. However, when both superoxide and NO are synthesized within a few cell diameters of each other – a condition likely to take place in the blood of subjects with an inflammatory state – they will rapidly combine to form peroxynitrite (ONOO-) [1,4,5]. Due to the extremely low stability of ONOO- in aqueous buffers (half-life ~1 s) its distribution in blood is nonhomogenous. Thus, under inflammatory conditions, the highest amounts of ONOO- are expected to be present in close proximity to stimulated macrophages and dysfunctional endothelial cells, where the local concentrations of a stressor were estimated to reach about 1 mM [6] and 0.5 μM respectively [7]. The cytotoxic action of ONOO- is related to its ability to peroxidate lipids, alter DNA structure, and oxidize protein sulfhydryls and nitrate tyrosine residues in a variety of proteins [1,2,5,8].

Abbreviations: CRR, clot retraction rate; ROTEM, rotational thromboelastometry; CT, clotting time; MCF, maximum clot firmness; PRP, platelet-rich plasma; ODQ, H-[1,2,4] Oxadiazolo[4,3-a]quinoxalin-1-one; CCCP, Carbonyl cyanide 3-chlorophenylhydrazone; TMRM, tetramethylrhodamine methyl ester. ⁎ Corresponding author. Tel.: +48 85 748 57 14; fax: +48 85 748 54 16. E-mail address: [email protected] (M. Tomasiak). 0049-3848/$ – see front matter © 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.thromres.2013.12.016

Accumulating evidence support the view that local formation of peroxynitrite from superoxide and NO, generated by activated inflammatory cells in the vicinity of the vascular endothelium, represents a crucial pathogenic mechanism in serious hemostasis abnormalities associated with myocardial infarction, diabetes, stroke, sepsis, hypercholesterolaemia and atherosclerosis [1,5,9]. The detailed mechanism(s) by which peroxynitrite affects hemostasis remain unclear. So far performed studies indicate that relatively high (micromolar– milimolar) concentration of this stressor can affect clotting factors (mainly through the nitration of tyrosine) and platelets [10–14]. However, numerous experimental studies on the effect of peroxynitrite on cellular energetics have established that much lower (nanomolar) concentrations of ONOO- can suppress mitochondrial energy production. Notably, ONOO- readily inactivates mitochondrial enzymes involved in oxidative metabolism, alter mitochondrial calcium homeostasis, reduce mitochondrial transmembrane potential (ΔΨm), and promote the opening of the permeability transition pore [1,15,16]. In porcine platelets, peroxynitrite has been reported to inactivate mitochondrial enzymes involved in oxidative metabolism [14]. In patients with septic shock (a clinical condition characterized by impaired hemostasis) a strong positive correlation between decreasing platelet mitochondrial functionality and disease severity was reported [17]. Similarly, more recent studies also demonstrate that in platelets from patients with SIRS (Systemic Inflammatory Response Syndrome), clinical condition associated with abnormal peroxynitrite production and impaired hemostasis [1], decrease of ΔΨm significantly correlates

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with the severity of a disease [18]. It is therefore possible that at pathological conditions ONOO-, formed in close proximity to activated inflammatory cells, may affect hemostasis through the inhibition of platelet energy production. Such a hypothesis seems likely since one of the important steps in hemostasis – clot retraction – critically depends on platelet energy production [19,20]. Clot retraction (CR) is defined as the slow shrinking of a freshly formed platelet aggregate (attached to an injured blood vessel wall) reinforced by the fibrin strands connecting neighboring cells [21–23]. Platelet-fibrin clot suppresses bleeding and serves as a temporary extracellular matrix in the wound area [23]. The physiological role of CR is to reduce the clot volume which facilitates recanalization of an occluded, by thrombus, blood vessel. Faster vessel recanalization results in shortening the time of ischemia of neighboring tissue. A properly retracted clot is strongly connected with the vessel wall and mechanically more stable [21]. An unstable clot, after detaching, may cause thromboembolic events. In vivo, clot retraction may thus regulate the size and stability of forming thrombi, and its abnormalities may contribute to both the pathogenesis of thromboembolic events and a tendency toward bleeding. Consequently, we initiated studies to establish whether peroxynitrite can affect hemostasis through the inhibition of CR resulted from impaired platelet mitochondria. Our preliminary studies performed on a porcine blood model confirmed such a possibility [20]. We now report, for the first time, that in human blood physiologically relevant peroxynitrite concentrations may inhibit retraction and stability of the clot due to inhibition of energy production in platelet mitochondria via a mechanism not necessarily related to tyrosine nitration. Materials and methods Chemicals Polyclonal rabbit anti-3-nitrotyrosine antibody and monoclonal HRP-conjugated goat anti-rabbit IgG were purchased Santa Cruz Biotech (Santa Cruz, CA, U.S.A.) Enhanced luminescence Super Signal West Pico Substrate was from Thermo Fisher Scientific (Waltham, MA, U.S.A.). Chrono-lume (luciferin-luciferase mix) was purchased by Chrono-log (Havertown, PA, U.S.A.). Tetramethylrhodamine methyl ester (TMRM) was purchased by Invitrogen (Carlsbad, CA, U.S.A.). Collagen (fibrillar, from equine tendon) was from Hormon Chemie (Munich, Germany). Tirofiban (Aggrastat) was from Merck Sharp & Dohme Idea Inc. (Glattbrugg, Switzerland). Other chemicals were from Sigma Chemical Co (St. Louis, MO, U.S.A). Blood collection and platelet preparation Venous blood was collected from healthy volunteers with minimum trauma and stasis via a 21-gauge needle (0.8 × 40 mm) into 10 ml polypropylene tubes containing 1 ml of 130 mM trisodium citrate. All procedures were conducted in accordance with the principles of Declaration of Helsinki and the study was approved by the local Ethics Committee on human research. Platelet rich plasma (PRP) was obtained by centrifugation of whole blood at 200 ×g for 20 min. To prepare washed platelets, PRP was acidified to pH 6.5 with 1 M citric acid, supplemented with apyrase (2 U/ml) and 1 μM PGE1, and centrifuged at 1500 ×g for 20 min to obtain a pellet which was resuspended in a Ca2 +-free Tyrode-Hepes buffer (152 mM NaCl, 2.8 mM KCl, 8.9 mM NaHCO3, 0.8 mM KH2PO4, 0.8 mM MgCl2, 5.6 mM glucose, 0.2% BSA and 10 mM Hepes, pH 7.4, osmolarity of 300 mOsm). To some experiments, the platelets suspension was next passed through a chromatographic column filled with Sepharose 2B using Tyrode-Hepes buffer as an eluent. Collected platelets were standardized to 2 × 108 cells/ml by dilution with Tyrode-Hepes buffer. Platelet number was determined using Coulter® Hematology Analyzer (Beckman Coulter, Fullerton, CA).

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Synthesis of ONOOPeroxynitrite was synthesized by the reaction of acidified H2O2 (1.4 M) with NaNO2 (1.2 M) in a quenched flow reactor [24]. The excess of H2O2 was removed by passage the product over column filled with a granular manganese oxide (IV). Stock solutions containing at least 200 mM ONOO- were collected and stored at -70 °C. The concentration was determined prior to each experiment by measuring the absorbance at 302 nm (ε302 = 1670 M-1 cm-1). Typically used ONOO- concentrations did not caused an increase of pH of the samples. Control experiments were carried out with decomposed ONOO-, obtained by allowing the compound to decay in 0.5 M phosphate buffer (pH 7.4) at 25 °C for 30 min. Measurement of kinetics of clot retraction Measurement of the kinetics of clot retraction in PRP and whole blood were performed in non-siliconized glass tubes (12 × 75 mm) containing a cushion of polymerized polyacrylamide, 6% (w/v), at the bottom to avoid clot adherence. Prior to measurements tubes were rinsed extensively with Tyrode–Hepes buffer. Aliquots (0.4 ml) of whole blood or PRP were added to 3.1 ml of T-H buffer (pH 7.4), containing 2.5 mM CaCl2, preheated to 37 °C, and clot retraction was initiated by gently mixing of the suspension The kinetics of clot retraction in artificial system consisting of washed platelets, bovine fibrinogen (2 mg/ml final conc.) and thrombin from human plasma (1 U/ml final conc.) was evaluated by the method described in details by Osdoit and Rosa [25]. Pictures were taken for one hour at 10 min intervals and after 120 min using a digital camera. Quantification of retraction was performed by assessment of clot area by use of the Motic Images Plus 2.0 ML (Motic, China) software, and data were processed using Microsoft Excel 11. Clot surface areas were plotted as percentage of maximal retraction (i.e. volume of platelet suspension). Data were expressed as follows: percentage of retraction (relative clot volume) = ((area t0 − area t)/area t0) × 100. In experiments, when exogenous ATP was used, washed platelets were permeabilized with saponin, typically 5 μg per each 108 platelets. This concentration was reported to enable aggregation of platelets in the presence of exogenous inositol triphosphate (which has similar molar weight to ATP) [26]. This concentration of saponin also provided aggregation of washed platelets in the presence of extracellular calcium (1 mM). Measurement of ATP content in retracted clots Clots derived from standardized (2 × 108 cells/ml) PRP samples formed during one hour incubation at 37 °C were carefully transferred with plastic Pasteur pipette to 3 volumes of ice cold 6% (w/v) perchloric acid, sonicated and left at 0 °C for 20 min. The extracts were centrifuged to remove proteins and neutralized with ice cold 6 M KOH/0.5 M morpholine sulphonic acid. ATP content was determined in neutralized cellular extracts by the luciferase-luciferin assay [27]. Measurement of the respiration rate Oxygen consumption was measured polarographically with a Clarktype oxygen electrode, (model YSI 5300A, YSI Life Sciences, U.S.A.), in a closed vessel (YSI sample micro chamber) of 1 ml at 37 °C. Measurement of lactate production in clotting PRP Aliquots of clotting suspensions prepared as described above (in the section “Measurement of kinetics of clot retraction”) were incubated at 37 °C in glass tubes. Incubation was started by the addition of glucose to the final concentration of 10 mM and was carried out for 60 min. It was stopped by the addition of 3 volumes of cold 6% (w/v) perchloric acid.

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Lactate was measured in the sonicated, deproteinized and neutralized extract by the LDH assay [28]. Measurement of the mitochondrial transmembrane potential in platelets The mitochondrial transmembrane potential (ΔΨm) was determined fluorometrically in TMRM-loaded platelets using the dequench mode of measurement, essentially as described before [20]. In this conditions rise in fluorescence reflects the decrease of ΔΨm. We found that dequench mode is more reliable and reproductive approach for ΔΨm measurement in intact platelets than non-quenching.

and incubated with stirring at 37 °C. Following activation platelets were lysed with 1 volume of lysis buffer (200 m M Tris-HCl, pH 7.4, 10% Triton X-100, 10 mM EGTA, 20 mM EDTA, protease inhibitor cocktail) per 9 volumes of sample. Lysis was performed at 4 °C for 60 min with gentle agitation. Detergent-insoluble (cytoskeletal) fraction was pelleted (14 000 ×g, 15 min), rinsed (3 times) with lysis buffer and suspended in 1/5 of original volume. After addition of Laemmli buffer with SDS and β-mercaptoethanol, samples were boiled for 5 min and SDS-PAGE was performed using a 10% separating gel. Dynamics of F-actin content changes was evaluated by optical densitometry. To determine total actin content in platelets, whole cell lysates (pellet + supernatant) were treated with Laemmli reducing buffer.

Thromboelastometric (ROTEM) analyses Platelet membrane integrity test Thromboelastometric measurements were performed using ROTEM system (Tem International GmbH, Munich, Germany) as described before in [20]. We measured the CT (clotting time) and MCF (maximal clot firmness) parameters.

The extent of platelet lysis following incubation with ONOO− was estimated in PRP by measuring the activity of lactate dehydrogenase (LDH), lost from the cells into the suspending fluid [28].

Determination of fibrin polymerization in plasma

Data analysis

The kinetics of fibrin formation in plasma was evaluated by the turbidimetric method described in details by Carter et al. [29]. The following variables were determined from the turbidimetric clotting assay curve: lag time (LagC), which represents the time at which sufficient protofibrils have formed to enable lateral aggregation; maximum absorbance (MaxAbsC) which reflects the degree of fibrin cross linking and fibrin polymerization rate (PR).

Data reported in this paper are the mean (±S.D.) of the number of determinations indicated (n). Statistical analysis was performed by Student's test and elaboration of experimental data by the use of Slide Write plus (Advanced Graphics Software, Inc. Carlsbad, CA, USA.). Results ONOO--related inhibition of clot retraction

Assay of platelet adhesion Platelet adhesion was quantified by measure the acid phosphatase activity of adherent cells, as described by Bellavite et al. [30]. We used collagen as a stimulator of platelet adhesion. The percentage of adherent cells was calculated on the basis of a standard curve obtained with a defined number of platelets. Immunobloting of 3-nitrotyrosine Peroxynitrite was added to the stirred suspension of gel-filtered platelet (2 × 108 cells/ml). Following ten minutes incubation at 37 °C with stirring, platelets were lysed with ice-cold RIPA buffer (10 mM NaH2PO4, pH 7.0, 150 mM NaCI, 2 mM EDTA, 1% sodium deoxycholate (w/v), 0.1% SDS (w/v)) supplemented with a cocktail of protease inhibitors (Sigma). Lysates were mixed with reducing Laemmli buffer, boiled for 5 min and SDS-polyacrylamide gel electrophoresis was performed using a 10% separating gel. The proteins were electrophoretically transferred to nitrocellulose membranes (Hybond ECL, Amersham Pharmacia Biotech, Amersham, UK). After 1-hour saturation at 4 °C in a TBST buffer containing 5% fat-free milk, membranes were incubated at 4 °C overnight with anti-3-nitrotyrosine antibody (1:1000 dilution). After six washing steps, blots were incubated for 1 hour with the secondary antibody conjugated with HRP at 1:10000 dilution. After washing, immunoreactive signals were measured using chemiluminescence reagent (Super Signal West Pico Substrate). Chemiluminescence was recorded after 6 sec exposure to the substrate, using UVP BioSpectrum Imaging System (UVP, Upland, CA, U.S.A.). Signals were quantified by optical densitometry. Determination of actin polymerization in platelets Determination of F-actin content in platelets was conducted as described in [31]. Briefly, aliquots (500 μl) of gel-filtered platelets (2 × 108 cells/ml) were incubated for 10 min with cytochalasin B (20 μM final conc.) or with ONOO- (10-100 nM final conc.) at 37 °C. Then, samples were supplemented with thrombin (1 U/ml final conc.)

Fig. 1 shows the results of experiments in which we compared the effect of increasing ONOO- concentrations on clot retraction measured in whole blood (panel A), PRP (panel B) and in a reconstituted system consisting of washed platelets and purified bovine fibrinogen (panel C). As is seen, the treatment of whole blood, PRP, and washed platelets, with increasing ONOO- concentrations resulted in a dose-dependent inhibition of clot retraction rate with an IC50 value of 120 μM (whole blood), 75 μM (PRP), and 25 nM (reconstituted system) respectively. As can be seen, ONOO- augmented clot volume in a dose-dependent manner (panel D). The effect was considerable, since 300 μM ONOO- produced about a 360% rise in clot volume (panel E). Literature data indicate that ONOO- could affect CR through the NO released from peroxynitrite-nitrosylated compounds [1,32–34]. Thus produced NO is expected to reduce platelet contractility via a mechanism related to NO-stimulated soluble guanylate cyclase (sGC). The results presented here show that an artificially-evoked rise in platelet cGMP produces retardation of CR (Fig. 2). At the same time, inhibition of sGC by ODQ in the absence of ONOO- did not affect CR (data not shown), and blockage of cyclase prior to addition of ONOO- did not alter stressor action on CR (Fig. 3). To compare the effect of ONOO- on various cellular responses we used the bolus addition of a native stressor. Under physiological conditions ONOO- is produced constantly and may exert its effect for a long period of time. Bolus addition may thus not properly mimic the in vivo situation. To resolve this dilemma, we used SIN-1 as a sustained source of ONOO- – although this approach suffers from the imprecise evaluation of the amount of ONOO- actually present in the medium [35]. As can bee seen in Fig. 4, SIN-1 (1 mM) produced time-dependent inhibition of CR. After 60-minutes of preincubation with SIN-1, CR was reduced by about 50%. Retardation of clot retraction by actin polymerization blocker and inhibitors of energy production Fig. 5A demonstrates that blocking of actin polymerization by cytochalasin B reduced CRR in a dose-dependent manner. The effect

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Fig. 1. Effect of increasing ONOO- concentrations on the kinetics of clot retraction and a final clot volume after retraction. Aliquots (1 ml) of whole blood (A), standardized PRP (2 × 108 cells/ml) (B) or washed platelets (2 × 108 cells/ml) (C) were incubated in polypropylene tubes at 37 °C for 2 minutes without (control) or with ONOO- added to the final concentrations as indicated. Immediately after incubation, samples (0.4 ml) of cell suspensions were transferred to preheated (37 °C) glass tubes and clot retraction was initiated by the addition of calcium chloride (A, B) or thrombin (C). Further details in Materials and Methods. After 2 hours of retraction, clot volume was recorded using a digital camera (D) and final clot volume (E) was calculated as in “Measurement of kinetics of clot retraction” section.Values are maximal retraction obtained at indicated time interval and are expressed as means ± S.D. Additions of decomposed ONOO- added to the final concentrations of 500 μM (A), 300 μM (B) or 100 nM (C) were without any effect on the kinetics of clot retraction. Results of 3-5 independent experiments (each performed in duplicate) are shown. * p b 0.05, ** p b 0.01, *** p b 0.001.

was considerable, since 20 μM cytochalasin produced about an 80% inhibition of clot retraction. Experiments shown in Fig. 5 (panels B and C) were performed to establish which source of cellular energy production – glycolysis or mitochondrial respiration – is critical for clot retraction. As is seen, the blocking of mitochondrial energy production by cyanide (2 mM) or

CCCP (uncoupler, 50 μM) reduced clot retraction by about 20 (panel B) and 45% (panel C) respectively. Inhibition of solely glycolytic energy production by 2-deoxy-D-glucose (10 mM) failed to affect CRR (data not shown). Simultaneous inhibition of glycolysis (by 2-deoxy-Dglucose, 10 mM) and mitochondrial respiration (by KCN, 2 mM) produced about a 75% inhibition of CR (synergism, panel B).

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Fig. 2. Effect of 8-Br-cGMP, a stable analog of cGMP on the kinetics of clot retraction. Aliquots (1 ml) of standardized PRP (2 × 108 cells/ml) were incubated in polypropylene tubes at 37 °C for 15 minutes without (control) or with indicated concentrations of 8Br-cGMP. Immediately after incubation, samples (0.4 ml) of cell suspensions were transferred to preheated (37 °C) glass tubes and clot retraction was initiated by the addition of calcium chloride. Further details in Materials and Methods. Results of 3-5 independent experiments (each performed in duplicate) are shown. * p b 0.05 vs. control, ** p b 0.01 vs. control.

Inhibition of platelet energy production by ONOOTable 1 illustrates the results of experiments which were performed to estimate whether ONOO- affects total ATP content in clots derived from PRP. As can be seen, ONOO- (50-300 μM) reduced ATP content in a dose-dependent manner. The degree of this inhibition (300 μM ONOO-) was similar to that observed in the presence of cyanide, CCCP, and the combination of 2-deoxy-D-glucose and cyanide. The experiments shown in Fig. 6 (panel A) were performed to establish whether ONOO- affects glycolytic energy production. One measure of the glycolysis rate was lactate production. As can be seen, ONOO- (10-100 μM) augmented lactate production dose-dependently. The effect was substantial, since treatment with 100 μM ONOO- resulted in 200% stimulation.

Fig. 4. Kinetics of clot retraction in the presence of SIN-1. Aliquots (1 ml) of standardized PRP (2 × 108 cells/ml) were incubated in polypropylene tubes at 37 °C for indicated times without (control) and with SIN-1 (1 mM final conc.). Immediately after incubation, samples (0.4 ml) of cell suspensions were transferred to preheated (37 °C) glass tubes and clot retraction was initiated by the addition of calcium chloride. Further details in Materials and Methods. Results of 3-5 independent experiments (each performed in duplicate) are shown. * p b 0.05, ** p b 0.01, *** p b 0.001.

Fig. 6 (panel B) shows that ONOO- (10-100 μM) inhibited oxygen consumption by unstimulated platelets (in PRP) in a dose-dependent manner. The effect was substantial, since treatment with 100 μM ONOO- resulted in a 60% reduction of oxygen consumption. The experiments shown in Fig. 6 (panel C) were performed to establish whether ONOO- treatment can reduce transmembrane potential (ΔΨm) in platelet mitochondria. The measure of ΔΨm collapse was a rise in the fluorescence of washed platelets loaded with TMRM. As is seen, ONOO- (5-30 nM) reduced ΔΨm. The effect was dose dependent. The degree of ΔΨm reduction in the presence of 30 nM ONOO- was similar to that observed following the addition of CCCP (a mitochondrial uncoupler).

Abolition of ONOO--evoked inhibition of clot retraction by exogenous MgATP To assess whether ONOO- action on CR can be related to the diminution of cellular energy production, we studied the effect of exogenously added MgATP on the ONOO--evoked inhibition of platelet-fibrin clot shrinking. Experiments were performed on platelets permeabilized by saponin. Saponin treatment did not affect the kinetics of CR distinctly (not shown). As is seen in Fig. 6 (panel D), exogenously added MgATP almost completely abolished the inhibitory effect of ONOO- on CR.

Retardation of platelet-fibrin clot formation by ONOO-, energy production inhibitors, or actin polymerization blocker

Fig. 3. Effect of ODQ on the ONOO--evoked inhibition of clot retraction. Aliquots (1 ml) of standardized PRP (2 × 108 cells/ml) were incubated in polypropylene tubes at 37 °C for 10 minutes without (control) and with ODQ (20 μM final conc.). Then, ONOO- was added to the final concentrations as indicated. Following 2 minutes incubation, samples (0.4 ml) of cell suspensions were transferred to preheated (37 °C) glass tubes and clot retraction was initiated by the addition of calcium chloride. Further details in Materials and Methods. Results of 3-5 independent experiments (each performed in duplicate) are shown.

Experiments shown in Table 2 were performed to compare the effects of ONOO- (75-300 μM), blockers of energy production in mitochondria (KCN, CCCP), inhibitor of glycolysis (2-deoxy-D-glucose), and actin polymerization (cytochalasin B) on the kinetics of plateletfibrin clot formation in PRP. Kinetics measurements were performed by means of rotational thromboelastometry. We measured clotting time (CT) and maximum clot firmness (MCF). As is shown, none of the studied compounds (excluding 200-300 μM ONOO-) was able to significantly affect the CT variable. Treatment with ONOO- (200 and 300 μM) resulted in prolongation of CT by about 11 and 16% respectively. The MCF variable was diminished by: 100-300 μM ONOO- (6-13%), KCN (9%), 2-deoxy-D-glucose/KCN (25%), CCCP (10%), and by cytochalasin B (12%).

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Table 1 Effect of peroxynitrite and inhibitors of mitochondrial and/or glycolytic energy production on total ATP content in clots derived from PRP. Addition

ATP content (% of control)

None (control) ONOO- 50 μM ONOO- 100 μM ONOO- 200 μM ONOO- 300 μM ONOO- 3 × 100 μM Dec. ONOODeoxyGlc 10 mM KCN 2 mM KCN 2 mM + DeoxyGlc 10 mM CCCP 50 μM

100 ± 4.9 91.8 ± 3.6 84.6 ± 4.6** 78.8 ± 4.2** 73.4 ± 4.7*** 68.2 ± 3.1*** 99 ± 4 97.1 ± 2.2 84.3 ± 3.7** 69.5 ± 5.3 *** 77.2 ± 2.9**

Aliquots (1 ml) of standardized PRP (2 × 108 cells/ml) were incubated in polypropylene tubes at 37 °C for 10 minutes without (control) or with substances added to final concentrations as indicated. PRP clots were prepared as in Methods section. Total intracellular ATP level was determined by the luciferine-luciferase chemiluminescent assay. Presented data are means (±S.D.) of one representative (n = 6) experiment performed on single PRP sample. Control value varied from 3.58 to 4.16 nanomoles/108 cells. Decomposed ONOO- was an equivalent of 300 μM of native stressor. **p b 0.01; ***p b 0.001.

plasma depleted from platelets by means of the turbidimetric method. Clotting was triggered by thrombin. We measured the lag time of clotting (Lagc), maximal fibrin concentration (MaxAbsc), and fibrin polymerization rate (PR). As is shown, ONOO- at concentrations below 300 μM failed to affect all the measured variables. Treatment of plasma with 500 μM ONOO- resulted in a 25% rise in Lagc, a 16% reduction of MaxAbsc, and a 20% decrease in PR. ONOO--related inhibition of platelet GPIIb/IIIa receptors To assess whether ONOO- action on clot retraction may be related to the malfunction of GPIIb/IIIa receptors, we compared the effect of ONOO(50-300 μΜ) and tirofiban – an antagonist of GPIIb/IIIa receptors – on the adhesion of washed platelets to fibrinogen-coated surfaces. Active GPIIb/IIIa receptors are critical for adhesion. As can be seen, only high (cytotoxic, 100-300 μΜ) concentrations of ONOO- significantly reduced platelet adhesion (Fig. 7). Lower concentrations (10 nM-25 μΜ) were without any effect (not shown). Tirofiban alone (250 μg/ml) reduced platelet adhesion by about 85%. Resistance of F-actin formation to ONOO- treatment To assess whether ONOO- affects platelet contractility through the inhibition of actomyosin formation, we measured its effect on the F-actin content in gel-filtrated platelets activated by thrombin. As is shown in Fig. 8, unlike cytocholasin B (an actin polymerization blocker), ONOO- (up to 100 nM) failed to affect the kinetics of F-actin formation. Increased 3-nitrotyrosine immunoreactivity in platelet proteins following ONOO- treatment Fig. 5. Effect of glycolytic and mitochondrial energy production inhibitors and cytochalasin B on the kinetics of clot retraction in PRP. Aliquots (1 ml) of standardized PRP (2 × 108 cells/ml) were incubated in polypropylene tubes at 37 °C for 10 minutes without (control) or with cytochalasin B (A), KCN/2-D-deoxyglucose (B) or CCCP (C) added to the final concentrations as indicated. Immediately after incubation, samples (0.4 ml) of cell suspensions were transferred to preheated (37 °C) glass tubes and clot retraction was initiated by the addition of calcium chloride. Further details in Materials and Methods. Values are maximal retraction obtained at indicated time interval and are expressed as means ± S.D. Results of 3-5 independent experiments (each performed in duplicate) are shown. p b 0.05, ** p b 0.01, *** p b 0.001.

To determine whether the inhibitory action of ONOO- on clot retraction may result from tyrosine nitration, we measured 3-nitrotyrosine immunoreactivity in homogenates of gel-filtered platelets treated with ONOO- (10 nM-100 μΜ). As is seen in Fig. 9, nitration of tyrosine residues was observed following treatment with ONOO- at concentrations from 100 nM to 100 μM. Lower concentrations (below 100 nM) failed to nitrate tyrosine residues. Platelet membrane integrity in the presence of ONOO-

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Alteration of kinetics of fibrin clot formation by ONOO

Experiments shown in Table 3 were performed to establish whether ONOO- (75-500 μM) affects the kinetics of clot formation measured in

Table 4 demonstrates the results of experiments in which we studied the effect of ONOO- on the integrity of plasma membrane in platelets suspended in whole blood, in plasma (PRP), and in an artificial medium

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Fig. 6. Effect of ONOO- on platelet energy metabolism. Panel A: effect of ONOO- on platelet glycolytic activity. Aliquots (1 ml) of standardized PRP (2 × 108 cells/ml) were incubated in polypropylene tubes at 37 °C for 2 min without (control) or with ONOO- added to the final concentrations as indicated. CR was initiated as described in Materials and Methods. Lactate production was measured in clotting PRP after 60 minutes incubation. Lactate production in control varied from 6.93 to 7.36 μmoles × min-1 × 1011 cells. In non-clotting PRP, lactate production varied from 2.78 to 3.33 μmoles × min-1 × 1011 cells. Decomposed ONOO- did not increase lactate production by platelets. The numbers are means ± S.D. (n = 12) of one representative (out of six) experiment performed in duplicate on a single platelet preparation. ** p b 0.01, *** p b 0.001. Panel B: effect of ONOO- on oxygen consumption in PRP. Aliquots (1 ml) of standardized PRP (2 × 108 cells/ml) were added to the thermostated (37 °C) vessel; measurements were started after 2 min preincubation and were carried out for 10 min. Additions to the measuring system were done 3 min after starting the recording of oxygen consumption. No exogenous glucose was added. Further details in Materials and Methods. Results of 6 independent experiments (each performed in duplicate, n = 12) are presented. Oxygen consumption in the control varied from 33 to 39 microliters O2 × min-1 × 1011 cells. Peroxynitrite per se (up to 2 mM) did not alter oxygen concentration in cell-free plasma. Decomposed ONOO- did not alter oxygen consumption by platelets. * p b 0.05,** p b 0.01, *** p b 0.001. Panel C: effect of ONOO- and CCCP on the platelet mitochondrial membrane potential (ΔΨm). The traces show changes of fluorescence of TMRM-loaded platelets following addition of 10 μM CCCP or ONOO- to the final concentrations as indicated. Further details in Materials and Methods. The results of one representative experiment (out of five) are presented. Panel D: effect of exogenous MgATP on the ONOO--evoked inhibition of clot retraction. Aliquots (1 ml) of washed platelets (2 × 108 cells/ml) were incubated in polypropylene tubes at 37oC for 2 min with stirring with indicated ONOO- concentrations. Then, platelets were permeabilized with saponin (10 μg/ml final conc.) for 2 min and the suspensions were supplemented with MgATP (5 mM final conc.). After 5 min of incubation, aliquots (0.4 ml) of platelets were transferred to preheated (37 °C) glass tubes containing 3.1 ml of Ca2+free Tyrode-Hepes buffer (supplemented with bovine fibrinogen at concentration 2 mg/ml). CR was initiated by the addition of thrombin (1 U/ml final conc.). Further details in Materials and Methods. * p b 0.01 vs. sample without ATP.

Discussion

(washed platelets). The measure of plasma membrane integrity was LDH release. As can be seen, threshold concentrations of ONOO- producing significant LDH release (cytotoxic effect) were 50 μM (washed platelets), 300 μM (PRP), and 500 μM (whole blood).

This is the first report that evaluates the effect of ONOO- on a retraction of platelet-fibrin clot in human blood. Our study revealed that

Table 2 Kinetics of PRP coagulation in the presence of ONOO-, cytocholasin B, and inhibitors of mitochondrial and glycolytic energy production. Addition

Clotting Time (sec)

Maximum Clot Firmness (mm)

None (control) ONOO- 75 μM ONOO- 100 μM ONOO- 200 μM ONOO- 300 μM Dec. ONOODeoxyGlc 10 mM KCN 2 mM KCN 2 mM + DeoxyGlc 10 mM CCCP 50 μM Cytochalasin B

429 436 455 485 501 436 436 422 456 436 440

70 69 66 63 61 71 70 64 53 63 62

± ± ± ± ± ± ± ± ± ± ±

34 25 28 41* 24* 40 29 26 37 33 24

± ± ± ± ± ± ± ± ± ± ±

2 4 3 3* 2** 3 3 3* 3*** 4* 2**

Aliquots (1 ml) of standardized PRP (2 × 108 cells/ml) were incubated in polypropylene tubes at 37 °C for 10 minutes without (control) or with substances added to final concentrations as indicated. All results are the averaged data from at least three assays performed on different PRP samples (n = 12). Decomposed ONOO- was an equivalent of 300 μM of native stressor. p b 0.05, ** p b 0.01, *** p b 0.001.

T. Misztal et al. / Thrombosis Research 133 (2014) 402–411

409

Table 3 Effect of ONOO- on the variables of fibrin polymerization profile. Addition

Lagc [sec]

MaxAbsC [au]

PR [au/sec × 10-4]

None (control) ONOO- 75 μM ONOO- 150 μM ONOO- 300 μM ONOO- 500 μM Decomposed ONOO-

310 316 325 345 390 323

0.33 ± 0.04 0.33 ± 0.06 0.311 ± 0.05 0.31 ± 0.07 0.278 ± 0.1** 0.322 ± 0.05

2.46 2.44 2.41 2.39 1.97 2.44

± ± ± ± ± ±

30 35 28 45 45** 20

± ± ± ± ± ±

0.17 0.2 0.16 0.13 0.12** 0.16

Aliquots (1 ml) of standardized PRP (2 × 108 cells/ml) were incubated in polypropylene tubes at 37 °C for 2 minutes without (control) or with ONOO- added to the final concentrations as indicated. Then, samples were centrifuged (5 min, 11 000 ×g) and supernatants were collected for turbidimetric analyses of fibrin polymerization profile. All results are the averaged data from at least three assays performed on different PRP samples (n = 12). Clotting was triggered by the addition of thrombin (to final conc. 0.01U/ml). Decomposed ONOO- was an equivalent of 500 μM of native stressor. ** p b 0.01.

ONOO- is able to strongly reduce both the rate of retraction and the final clot volume at concentrations much lower (nanomolar) than those previously reported (micromolar) to affect clotting factors and platelets (Fig. 1, [10–13,20]). This make the possibility of ONOO- action on CR at in vivo condition much more likely. The susceptibility of clot retraction to peroxynitrite depends on the composition of the medium in which this stressor acts. Thus, the CR in an artificial system, consisting of washed platelets, fibrinogen and thrombin, was inhibited by low nanomolar concentrations of ONOO-, whereas in PRP and whole blood, CR was retarded by low and high micromolar concentrations respectively (Fig. 1 A, B and C). The lower susceptibility of CR to ONOO- in PRP and in whole blood is most likely associated with the rise in the number of potential targets of this stressor [36]. Taking this in mind, our studies on the susceptibility of clot retraction and clot formation to ONOO- treatment were performed in PRP i.e. in systems containing a similar quantity of potential targets. Moreover, PRP acceptably mimics the natural microenvironment in which ONOO- is expected to be formed under conditions of an inflammatory state: an erythrocyte–poor and platelet–rich blood layer close to the vascular endothelium that forms in laminar flowing blood [5]. What could be a cause of the high susceptibility of CR to ONOOtreatment? Recent studies point to several steps in platelet-dependent clot retraction upon stimulation of coagulation: the initial production

Fig. 7. Effect of ONOO- on platelet adhesion to fibrinogen coated surfaces. Aliquots (1 ml) of washed platelets (2 × 108 cells/ml) were incubated in polypropylene tubes at 37 °C for 2 min without (control, C) or with ONOO- added to the final concentration as indicated. After that, 50 μl of the samples were transferred to fibrinogen coated wells of a microtiter plate. Adhesion was initiated by the addition of threshold concentration of collagen (15-20 μg/ml final conc.). The extent of platelet adhesion was measured 60 min after the addition of the agonist and the maximum extent of adhesion evoked by collagen was taken as 100%. Further details in Materials and Methods. The data represent the mean ± SD of six experiments, each performed in triplicate on a separate platelet preparation. Decomposed ONOO- did not affect platelet adhesion. * p b 0.05; ** p b0.01, *** p b 0.005.

Fig. 8. Effect of ONOO- and cytochalasin B on the F-actin content in activated platelets. Gel filtered platelets (2 × 108 cells/ml) were incubated in polypropylene tubes at 37 °C for 10 min with stirring, without (control) or with ONOO- or cytochalasin B added to the final concentrations as indicated. Then, platelets were activated by thrombin (1 U/ml final conc.). At the indicated time intervals, aliquots of suspension were transferred to lysis buffer. Triton-insoluble (cytoskeletal) fractions were separated by 10% SDS-PAGE. The amount of actin in cytoskeletal fraction relative to total was quantified by densitometric analysis. Further details in Materials and Methods. Values represent means ± SD of 3 independent experiments. (Δ) – control, (▲) – ONOO- (10 nM), (■) – ONOO- (100 nM), (♦) –– cytochalasin B (20 μM). The longer incubation (up to 60 min) did not change F-actin content. * p b 0.01.

of a large quantities of thrombin (on the platelet surface), simultaneous formation of a diffuse fibrin network and transformation of the platelet GPIIb/IIIa receptors in the active conformation, platelet fibrin(ogen) binding and integrin outside-in signaling, transmission of the contractile forces generated in the platelet actin– myosin cytoskeleton to connecting fibrin fibers around platelets (contraction) and conversion of a diffuse fibrin network into a small and dense platelet–fibrin clot (shrinking) [21,22,37,38]. Therefore, the reduction of CR rate reported here, at least potentially may result from the action of ONOO- on any of the above mentioned steps. To assess the ONOO- action on platelet-fibrin clot formation we used rotational thromboelastometry. This approach provides information both about the velocity of thrombin production (reflecting the activity of clotting factors and platelet procoagulant activity) and the rate of fibrin network formation. The results presented here strongly indicate that ONOO- effect on CR can not be related to fibrin network formation. This is because the kinetics of clot formation measured in PRP demonstrates that susceptibility of clot retraction to the studied stressor is much higher than that of clot formation. Specifically, the effective ONOO- concentration modulating CRR and clot formation in PRP were

Fig. 9. Effect of ONOO- on the 3-nitrotyrosine immunoreactivity in platelet proteins. Gel-filtered platelets (2 × 108 cells/ml) were incubated in polypropylene tubes at 37 °C for 10 minutes with stirring, without (control, 0) or with indicated ONOO- concentrations (in μM). The amount of 3-nitrotyrosine was determined using immunobloting with an enhanced chemiluminescent detection system. Further details in Materials and Methods. Decomposed ONOO- did not cause any detectable rise in 3-nitrotyrosine levels (not shown). The results of one representative experiment (out of four) are presented.

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Table 4 Effect of ONOO- on the plasma membrane integrity. Addition

None (control) ONOO- 50 μM ONOO- 100 μM ONOO- 200 μM ONOO- 300 μM ONOO- 500 μM Dec. ONOO-

LDH activity (% of total) Whole blood

PRP

4.6 4.6 4.8 5.0 5.1 5.9 4.4

3.1 3.0 3.3 3.7 4.6 7.7 3.3

± ± ± ± ± ± ±

0.6 0.9 0.7 1.1 1.0 1.4 * 0.9

± ± ± ± ± ± ±

Washed platelets 0.5 0.8 0.6 1.3 1.0 * 1.7 ** 0.9

2.2 ± 0.5 3.8 ± 0.8 * 6.2 ± 0.7 ** 8.1 ± 0.9 ** 9.7 ± 1.3 *** 14.4 ± 1.6 *** 2.3 ± 0.6

Aliquots (1 ml) of whole blood (WB), standardized PRP (2 × 108 cells/ml) or washed platelets (WP, 2 × 108 cells/ml) were incubated at 37 °C for 5 min without (control) and with ONOO- or decomposed ONOO- added to the final concentration as indicated. Lactate dehydrogenase (LDH) activity was measured (n = 6) in the supernatants, after centrifugation (20 min, 200 ×g and next 5 min, 11 000 ×g for WB samples or 5 min, 11 000 ×g in the case of PRP and WP). LDH release (measure of cell integrity) was expressed as a percentage of LDH activity in the supernatant relative to total LDH activity (after addition of Triton X-100 to final concentration of 0.2 %). Decomposed ONOO- was an equivalent of 500 μM of native stressor. * p b 0.05; ** p b 0.01, *** p b 0.001.

2.5 and 200 μM respectively (compare Fig. 1B with Table 2). What is more, CRR measured in an artificial system can be affected by 2.5 nM ONOO- (Fig. 1C) which is a concentration more than 1000 times lower than that affecting fibrinogen clotting (10 μM) measured in experimental systems containing comparable amounts of potential ONOO- targets [13]. Likewise, direct ONOO- action on clotting factors is also unlikely, especially at low stressor concentrations (b300 μM), since its effect on fibrin strand formation in plasma depleted of platelets was observed at concentrations above 300 μM, i.e. much higher than those reducing CR in PRP (compare Fig. 1B with Table 3). To sum up, CR is much more sensitive to ONOO- than plasma clot formation, and the inhibitory effect of low ONOO- concentrations on CR is not a consequence of altered fibrin network formation. ONOO- action on platelet GPIIb/IIIa receptors can also be excluded, since adhesion of activated platelets to the fibrinogen coated surfaces (a phenomenon intrinsically dependent on the presence of activated integrin receptors) was reduced by stressor concentrations much higher than those affecting clot retraction. Notably, the effective ONOO- concentrations modulating CRR in an artificial system and the adhesion of washed platelets were 2.5 nM and 75 μM respectively (compare Fig. 1C with Fig. 7). Consequently, our results indicate that inhibition of CR by low ONOO- concentrations is associated with dysfunction of the platelet contractile apparatus rather than with abnormalities in fibrin formation or GPIIb/IIIa function. Malfunction of contractile apparatus suppress CR since cytochalasin B-evoked reduction in actin polymerization resulted in a significant reduction of CR rate (Fig. 5A). There is also literature report indicating that in activated bovine platelets nitration of profilin, an actin binding protein involved in a regulation of actin polimerization, may be mediated by peroxynitrite [39]. This points to the possibility that ONOO- may directly affect platelet contractility which may consequence in a suppression of CR. This is however not the case since, as we show here, the concentrations of ONOO- strongly reducing CR failed to affect kinetics of F-actin formation in activated platelets (Fig. 8). Results presented here indicate that a nitrosative effect of low ONOO- concentrations on the components of platelet contractile apparatus is also unlikely. This is because peroxynitrite–evoked nitration of platelet proteins was observed at concentrations started from 100 nM of stressor, and 3-nitrotyrosine was not found in major platelet contractile proteins, i.e. in actin and myosin (Fig. 9). Contribution of guanylate cyclase-related mechanisms in the ONOOinhibitory action on CR, especially at lower (b30 μM) concentrations is also excluded. First, because in the case of washed platelets, ONOOaffects CR at the concentrations ~1000 times lower than those reported to produce a rise in cellular cGMP level (compare Fig. 1C and [34]) and

second, because inhibition of CR by ONOO- was not abolished following blocking of platelet guanylate cyclase by ODQ (Fig. 3). Reduced platelet contractility may also be related to inefficient energy production [19,20]. Clot retraction is an energy requiring process, in which ATP hydrolysis is crucial for the contraction of platelet actomyosin. It has been reported that glycolysis and mitochondrial oxidative phosphorylation are substantial for effective clot retraction [19]. Consistent with this is our observation that, in clotting PRP, oxygen consumption was distinctly higher than in non-clotting PRP [20]. Our study also shows that uncoupling of platelet mitochondria by CCCP or blocking the respiratory chain by cyanide inhibits retraction of the plateletfibrin clots (Fig. 5B and C), indicating that oxidative phosphorylation is crucial for optimal efficiency of this process. The results presented here indicate, that inhibitory action of ONOOon clot retraction is closely related to decreased platelet contractility resulted from inefficient energy production. This is based mainly on the observation that exogenous MgATP abolished inhibition of clot retraction mediated by peroxynitrite-treated semipermeabilized platelets (Fig. 6D). The results presented here also show that concentrations of ONOO- affecting CRR decreased the total ATP content in clots derived from PRP (Table 1). This was accompanied by augmented lactate production and reduced platelet oxygen consumption, indicating the occurrence of a phenomenon known as the Pasteur Effect (Fig. 6A and B). This mechanism permits a compensation of the decreasing mitochondrial ATP production by increasing the rate of glycolysis. An interesting observation is that lactate production by platelets during contraction of PRP-clots is about 2.5-times higher in comparison to resting platelets (Fig. 6). Since in platelets glycolysis and mitochondrial respiration are tightly functionally connected [14,40], this may indicate that the stimulatory effect of ONOO- on glycolysis in platelets may be related to impairment of mitochondria. This is likely to be true since, as reported here, ONOO- is able not only to diminish mitochondrial oxygen consumption, but also to reduce the mitochondrial transmembrane potential (ΔΨm) (Fig. 6C), a prerequisite for ATP synthesis via oxidative phosphorylation. This data clearly indicates that ONOO- may affect in vitro CR through the inhibition of energy production in platelet mitochondria. How can this be relevant to the in vivo situation? Serious bacterial infections, such as sepsis, are associated with the rise of ONOO- concentrations in blood, where it can be produced in concentrations up to 50–100 μM per minute [41]. The results presented here indicate that, in vitro, exposure of PRP to low micromolar (IC50 = 75 μM) concentrations of ONOO- added as a single pulse (bolus) produce retardation of mitochondrial energy production and clot retraction (Figs. 1B, 6A-C, and Table 1). In vivo, where ONOO- release occurs continuously, concentrations of stressor affecting CR are expected to be even lower. This view is based on the literature [e.g. 42] and our observation that exposure of PRP to slowly released ONOO- (by adding 1 mM SIN-1) results in a 50% inhibition of CR after 60 min. incubation (Fig. 4). Based on available information on the kinetics of SIN-1 decomposition [35], the expected peak ONOO- concentration in our experimental system amounts to approximately 14 μM, which is a figure about 5 times lower than in the case of a native stressor producing a similar degree of CR inhibition (compare Figs. 1B and 4). It is therefore likely that amounts of ONOO- delivered by activated inflammatory cells are high enough to affect CR and platelet mitochondrial energy production. This notion is confirmed by the observations of Alvarez et al. who have reported that, following administration of endotoxin to rats, steady state concentrations of peroxynitrite in the heart and in diaphragm mitochondria rose from 8 to 12 nM and from 21 to 49 nM respectively [41]. The results presented here show that in washed platelets bolus addition of 5 nM (Figs. 1C and 6C) concentration of ONOO- is able to suppress mitochondrial energy production and inhibit clot retraction, strongly indicating that amounts of ONOO- formed in vivo near activated inflammatory cells have the capacity to produce dysfunction of platelet mitochondria. This is further supported by the

T. Misztal et al. / Thrombosis Research 133 (2014) 402–411

recent observations of Yamakawa et al. who have reported that, in patients with SIRS, the increased clinical severity of the disease correlated positively with the dysfunction of platelet mitochondria (measured as an alteration in ΔΨm value) [18]. A recognized indicator of in vivo ONOO- presence is elevated 3nitrotyrosine immunoreactivity [1,2,8] – a phenomenon reported in numerous clinical conditions associated with an inflammatory state (reviewed in [1]). Results presented here show that, at least in vitro, ONOO--evoked nitration of tyrosine residues takes place when the stressor concentrations are at least 20 times higher than those affecting mitochondrial transmembrane potential in washed platelets (compare Figs. 6C and 9). This indicates that dysfunction of platelet mitochondria may in vivo occur at concentrations of ONOO- lower than those required for nitration of tyrosine residues in numerous proteins and that low stressor concentrations may exert an effect on hemostasis by mechanisms not related to nitrotyrosine formation. Altogether, it is concluded that in humans, physiologically relevant ONOO- concentrations, associated with an abnormal or inefficiently controlled inflammatory response, are likely to suppress mitochondrial energy production in platelets, which may result in malfunction of the contractile apparatus and retardation of clot retraction. ONOO--altered platelet mitochondria may thus constitute a new link between inflammation and impaired hemostasis. Funding This research was supported by European Union grant [UDAPOKL.08.02.01-20-069/11-00] and by Medical University of Bialystok [133-01525 F]. Conflict of Interest Statement We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. References [1] Pacher P, Beckman JS, Liaudet L. Nitric oxide and peroxynitrite in health and disease. Physiol Rev 2007;87:315–424. [2] Beckman JS. Oxidative damage and tyrosine nitration from peroxynitrite. Chem Res Toxicol 1996;9:836–44. [3] Beckman JS. The physiological and pathological chemistry of nitric oxide. In: Lancaster JR, editor. Nitric Oxide: Principles and Actions. Orlando: Academic; 1996. p. 1–82. [4] Huie RE, Padmaja S. The reaction rate of nitric oxide with superoxide. Free Radic Res Commun 1993;18:195–9. [5] Dickhout JG, Basseri S, Austin RC. Macrophage function and its impact on atherosclerotic lesion composition, progression, and stability: the good, the bad, and the ugly. Arterioscler Thromb Vasc Biol 2008;28:1413–5. [6] Ischiropoulos H, Zhu L, Beckman JS. Peroxynitrite formation from macrophagederived nitric oxide. Arch Biochem Biophys 1992;298:446–51. [7] Corbalan JJ, Medina C, Jacoby A, Malinski T, Radomski MW. Amorphous silica nanoparticles trigger nitric oxide/peroxynitrite imbalance in human endothelial cells: inflammatory and cytotoxic effects. Int J Nanomedicine 2011;6:2821–35. [8] Sabetkar M, Low SY, Naseem KM, Bruckdorfer KR. The nitration of proteins in platelets: significance in platelet function. Free Radic Biol Med 2002;33:728–36. [9] Zeerleder S, Hack CE, Wuillemin WA. Disseminated intravascular coagulation in sepsis. Chest 2005;128:2864–75. [10] Nowak P, Wachowicz B. Peroxynitrite-mediated modification of fibrinogen affects platelet aggregation and adhesion. Platelets 2002;13:292–9. [11] Wachowicz B, Rywaniak JZ, Nowak P. Apoptotic markers in human blood platelets treated with peroxynitrite. Platelets 2008;19:624–35. [12] Lupidi G, Angeletii M, Eleuteri AM, Tacconi L, Coletta M, Fioretti E. Peroxynitritemediated oxidation of fibrinogen inhibits clot formation. FEBS Lett 1999;462:236–40. [13] Nowak P, Zbikowska HM, Ponczek M, Kołodziejczyk J, Wachowicz B. Different vulnerability of fibrinogen subunits to oxidative/nitrative modifications induced by peroxynitrite: functional consequences. Thromb Res 2007;121:163–74.

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