Free Radical Research, December 2014; 48(12): 1443–1453 © 2014 Informa UK, Ltd. ISSN 1071-5762 print/ISSN 1029-2470 online DOI: 10.3109/10715762.2014.960866
ORIGINAL ARTICLE
Clinically relevant HOCl concentrations reduce clot retraction rate via the inhibition of energy production in platelet mitochondria T. Misztal, T. Rusak & M. Tomasiak
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Department of Physical Chemistry, Medical University of Bialystok, Bialystok, Poland Abstract Using porcine blood, we examined the impact of hypochlorite, product of activated inflammatory cells, on clot retraction (CR), an important step of hemostasis. We found that, in vitro, HOCl is able to reduce CR rate and enlarge final clot size in whole blood (t.c. 100 mM), plateletrich plasma (PRP) threshold concentration (t.c. 50 mM), and an artificial system (washed platelets and fibrinogen) (t.c. 25 nM). Combination of low HOCl and peroxynitrite concentrations resulted in synergistic inhibition of CR by these stressors. Concentrations of HOCl completely inhibiting CR failed to affect the kinetics of coagulation measured in PRP and in platelet-free plasma. Concentrations of HOCl reducing CR rate in PRP augmented production of lactate, inhibited consumption of oxygen by platelets, and decreased total adenosine triphosphate (ATP) content in PRP-derived clots. In an artificial system, concentrations of HOCl resulting in inhibition of CR (25–100 nM) reduced mitochondrial transmembrane potential and did not affect actin polymerization in thrombin-stimulated platelets. These concentrations of HOCl failed to affect the adhesion of washed platelets to fibrinogen and to evoke sustained calcium signal, thus excluding stressor action on glycoprotein IIb/IIIa receptors. Exogenously added Mg-ATP almost completely recovered HOCl-mediated retardation of CR. Concentrations of HOCl higher than those affecting CR reduced thromboelastometric variables (maximum clot firmness and a angle). We conclude that low clinically relevant HOCl concentrations may evoke the inhibition of CR via the reduction of platelet contractility resulted from malfunction of platelet mitochondria. At the inflammatory conditions, CR may be the predominant HOCl target. Keywords: platelets, mitochondria, hypochlorous acid, peroxynitrite, clot retraction
Introduction At sites of inflammation, activated phagocytes (neutrophils and macrophages) generate large quantities of hydrogen peroxide and release an enzyme myeloperoxidase (MPO) stored in their granules [1–2]. The majority of hydrogen peroxide generated during the phagocytes’ respiratory burst is subsequently used to oxidize chloride anion (Cl) to hypochlorous acid (HOCl) in a reaction catalyzed by secreted myeloperoxidase [3]. Hypochlorous acid and its conjugate base (OCl) are potent cytotoxic oxidants and chlorinating agents possessing microbicidal and viricidal activities, and also contribute to inflammatory injury in host tissues [3–4]. In response to activation, MPO is released both into phagocytic vesicles (phagosomes) and the extracellular space (plasma or interstitial matrix) [3,5]. In healthy subjects, plasma MPO concentration varies from 18 to 39 ng/ml [6–7], but in some clinical conditions it can rise to 55 [6] or even 287 ng/ml [8]. The primary physiological role of HOCl is to kill pathogens but its overproduction, as observed in prolonged, acute, or malcontrolled inflammatory state, may result in upregulation of hemostasis. In fact, HOCl has been proposed to play a role in the pathogenic mechanism of atherosclerosis [9], respiratory distress syndrome, reperfusion injury and rheumatoid arthritis [2,10–14], glomerulonephritis [11],
and chronic renal failure [15–16], that is, clinical conditions often associated with hemostatic abnormalities. The exact mechanism(s) linking hemostasis abnormalities with HOCl overproduction are not fully understood. HOCl has also been reported to oxidize in vitro fibrinogen and clotting factors V, VIII, and X [17] and to inhibit aggregation of rabbit platelets [18]. It should be stressed, however, that its action on platelet aggregation and on clotting factors was observed only at millimolar concentrations (1.5–5 mM); that is, at concentrations which are less likely to be present in the blood stream. Recently, we have proposed that peroxynitrite (ONOO), a reactive nitrogen species produced by activated inflammatory cells, may affect hemostasis through the reduction of platelet contractility resulting from inhibition of energy production in platelet mitochondria [19–20]. Platelet contractility is crucial for the stability of the primary platelet aggregate connected to an injured vessel wall and for the retraction of a platelet–fibrin clot [21]. This novel sensitive mechanism is very likely to operate in vivo, since mitochondria have been reported to be very sensitive to low (nanomolar–micromolar) concentrations of reactive oxygen, nitrogen, and chlorine species produced by activated inflammatory cells [22–27]. HOCl is likely to affect platelet mitochondria. Recent studies indicate that low micromolar concentrations of
Correspondence: Prof. Marian Tomasiak, Department of Physical Chemistry, Medical University of Bialystok, Kilinskiego 1, 15-089 Bialystok, Poland. Tel: 48-85-748-57-14. Fax: 48-85-748-54-16. E-mail: [email protected] (Received date: 7 April 2014; Accepted date: 29 August 2014; Published online: 17 October 2014)
1444 T. Misztal et al.
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HOCl, that is, concentrations much lower than those reported to modify clotting factors and inhibit platelet aggregation, can suppress mitochondrial energy production in cultured human endothelial cells [28], cultured human liver cells, and in isolated rat liver mitochondria [29]. The effect of HOCl on clot retraction (CR) and the functionality of platelet mitochondria has never been studied before. CR has been reported to have a great impact not only on the mechanical properties of thrombus (stability and elasticity), but also on its lysis [19,30–31]. Consequently, these studies were undertaken to establish whether HOCl may affect hemostasis through the inhibition of CR. Another aim was to assess the possible mechanism of HOCl action on platelet mitochondria.
factor, the initial 10 ml of blood was discarded. Platelet number in the blood of studied animals varied from 4.6 108/ml to 6.5 108/ml. Platelet number in plateletrich plasma (PRP) was adjusted to 4 108/ml with autologous plasma before each experiment. Synthesis of ONOO
Materials and methods
Peroxynitrite was synthesized by the reaction of acidified H2O2 (1.4 M) with NaNO2 (1.2 M) in a quenched flow reactor [33]. The excess of H2O2 was removed by passing 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 (e302 1670 M1cm1) [32]. Typically used ONOO concentrations did not cause an increase in pH of the samples.
Chemicals
Platelet preparation
Hypochlorite used was in the form of sodium hypochlorite (NaOCl). Throughout this article, we use the term hypochlorite (pKa, 7.46) to refer to approximately 50% ionized mixture of HOCl and ClO species that exists at physiological pH (referred in this article as HOCl). The concentration was determined prior to each experiment by measuring the absorbance at 290 nm (e292 350 M 1cm 1) [32]. Recombinant tissue plasminogen activator (Actylise) was purchased from Boehringer Ingelheim GmbH (Ingelheim, Germany). Chrono-lume (luciferin–luciferase mix) was purchased from Chrono-log (Havertown, PA, U.S.A.). Tetramethylrhodamine methyl ester (TMRM) was purchased from Invitrogen (Carlsbad, CA, U.S.A.). Collagen (fibrillar, from equine tendon) was from Hormon Chemie (Munich, Germany). Other chemicals were from Sigma Chemical Co (St. Louis, MO, U.S.A.).
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 using 1 M citric acid and the suspension was centrifuged at 1500 g for 20 min to obtain a pellet which was resuspended in a Ca2-free Tyrode-Hepes (T-H) buffer (152 mM NaCl, 2.8 mM KCl, 8.9 mM NaHCO3, 0.8 mM KH2PO4, 0.8 mM MgCl2, 5.6 mM glucose, apyrase (2 U/ml), 10 mM ethylene glycol tetraacetic acid (EGTA), bovine serum albumin (BSA) (3.5 mg/ml), and 10 mM Hepes; pH, 6.5; osmolarity of 340 mOsm). The platelets were washed once with the abovedescribed buffer and finally suspended in the same buffer with the exception that, in the final suspension medium, apyrase and EGTA were omitted and pH was adjusted to 7.4. In some experiments, the platelets suspension was next passed through a chromatographic column filled with Sepharose 2B using T-H buffer as an eluent. The platelet concentration was standardized to 4 108 cells/ml by dilution with T-H buffer. Platelet number was determined using a Coulter® Hematology Analyzer (Beckman Coulter Inc., U.S.A.).
Animals A total of 50 domestic pigs (breed: Polish Large White) of both sexes, approximately 9 months old, with a mean weight of 90–100 kg, were used in the experiments. The animals were raised on local farms under normal agricultural husbandry conditions. Pigs were fed the grower chow diet consisting of grain (rye, barley, wheat, and maize) and soybean. The animals for this study were selected at random from available litters. Prior to blood collection, the pigs were starved for at least 12 h. The study protocol and procedures were approved by the Ethics Committee at the Medical University of Białystok. Blood collection Blood collection was performed in a local slaughterhouse (PMB, Bialystok, Poland). Forty milliliters of blood was withdrawn by direct carotid catheterization and collected into 3.8% (w/v) sodium citrate, one volume per nine volumes of blood. To avoid contamination of blood by tissue
Measurement of kinetics of CR Measurement of the kinetics of CR 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 T-H 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 CR was initiated by gently mixing the suspension The kinetics of CR in artificial system consisting of washed platelets, bovine fibrinogen (2 mg/ml of final conc.), and thrombin from bovine plasma (1 U/ml final conc.) was evaluated by the method described in detail [34]. Pictures were taken for 1 h at 10-min intervals and after 120 min using a digital camera. Quantification of retraction was performed by
Hypochlorite and hemostasis 1445
assessment of clot area using the Motic Images Plus 2.0 ML software, and data were processed using MS 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 (area t 0 area t ) 100 (relative clot volume) (area t ) 0
In experiments, where exogenous Mg-ATP was used, washed platelets were permeabilized with saponin, typically 5 mg per each 108 platelets.
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Thromboelastometric (ROTEM) analyses
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 protein and neutralized with ice-cold 6 M KOH/0.5 M morpholine sulfonic acid. ATP content was determined in neutralized cellular extracts by the luciferase–luciferin assay [38]. Measurement of the mitochondrial membrane potential in porcine platelets The mitochondrial membrane potential (ΔYm) was determined in TMRM-loaded platelets using the dequench mode of measurement, essentially as described [20]. One measure of ΔYm fall was a rise in TMRM fluorescence.
ROTEM technology is described elsewhere [35]. Thromboelastometric measurements were performed using ROTEM system (Tem International GmbH, Manheim, Germany). We measured the following parameters: clotting time (CT)—time from start of measurement to the beginning of the fibrin polymerization process; alpha angle (a)—the angle showing the dynamics of clot formation; and maximum clot firmness (MCF)—a parameter reflecting the strength of the formed clot. All ROTEM measurements were performed by the same experienced operator as described [20].
Assay of platelet adhesion
Determination of fibrin polymerization profiles
Cytosolic free Ca2 ([Ca2]cyt) was determined in Fura2-loaded platelets essentially as described by Pollock et al. [40]. PRP was incubated with 3 mM (final conc.) Fura-2/ AM and Pluronic-137 (0.005% final conc.) for 45 min at room temperature in the dark with gentle agitation. Then, 1 mM prostaglandin E1 was added and platelets were separated from PRP as described above, and resuspended at 1.5–1.7 109/ml in a Ca2-free T-H buffer. Aliquots of these suspensions were than transferred to a cuvette, containing 2 ml of T-H buffer containing 1 mM Ca2 (final conc.). Measurement of Fura-2 fluorescence in stirred platelet suspension was performed at 37°C using a Hitachi F-7000 spectrofluorometer (Hitachi Corp., Japan). Monochromator settings were 339 nm for excitation and 500 nm for emission. The Fura-2 responses were calibrated to obtain [Ca2]cyt as described by Siffert et al. [41].
The kinetics of fibrin formation and plasma clot lysis was evaluated by the turbidimetric method described in details by Carter et al. [36]. 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). Measurement of lactate production in clotting PRP Aliquots of clotting suspensions prepared as described above (in the section “Measurement of kinetics of CR”) 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. Lactate was measured in the deproteinized and neutralized extract by the lactate dehydrogenase (LDH) assay [37]. Measurement of the respiration rate Oxygen consumption was measured polarographically with a Clark-type oxygen electrode (model YSI 5300A, YSI Life Sciences, U.S.A.), in a 1-ml closed vessel (YSI sample micro chamber) at 37°C. Measurement of ATP content in retracted clots Clots derived from standardized (4 108 cells/ml) PRP samples formed during 1-h incubation at 37°C were carefully transferred using plastic Pasteur pipette to
Platelet adhesion was quantified by measuring the acid phosphatase activity of adherent cells, as described by Bellavite et al. [39]. Collagen was used 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. Measurement of cytosolic free Ca2
Platelet membrane integrity test The extent of platelet lysis following incubation with HOCl was estimated in PRP by measuring the activity of LDH, lost from the cells into the suspending fluid [37]. Determination of actin polymerization in platelets Determination of F-actin content in platelets was conducted as described in essence [42]. To determine total actin content in platelets, whole cell lysates (pellet supernatant) were used. Data analysis Data reported in this paper are the mean ( S.D.) of the number of determinations indicated (n). Statistical
1446 T. Misztal et al. analysis was performed by Student’s t-test and elaboration of experimental data by the use of Slide Write plus (Advanced Graphics Software, Inc. Carlsbad, CA, U.S.A.).
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Results Figure 1 shows the results of experiments in which we compared the effect of increasing HOCl concentrations on CR measured in whole blood (A), PRP (B), and a reconstituted system consisting of washed platelets and purified bovine fibrinogen (D). As is seen, treatment of whole blood, PRP, and washed platelets with increasing HOCl concentrations resulted in a dose-dependent inhibition of CR rate with an IC50 value of 360 mM (whole blood), 250 mM (PRP), and 100 nM (reconstituted system), respectively. Figure 1C presents the results of experiments conducted to establish how simultaneous incubation of PRP with HOCl and ONOO may affect CR. As it is seen,
cumulative action of both stressors resulted in synergistic inhibition of CR. The effect was significant, since combination of 50 mM HOCl and 50 mM ONOO produced about a 60% inhibition of CR. Figure 2 demonstrates the effect of increasing concentrations (50–500 mM) of HOCl on the final clot volume following retraction, measured in PRP. As can be seen, HOCl augmented clot volume in a dose-dependent fashion (A). The effect was substantial, since 500 mM HOCl produced about a 250% rise in clot volume (panel B). Experiments shown in Figure 3 were performed to establish whether HOCl affects total ATP content in clots derived from PRP. As is seen, HOCl (100–1000 mM) reduced ATP content dose-dependently. The effect was significant, since 1000 mM HOCl produced about a 30% reduction of total ATP content. Experiments shown in Figure 4A were performed to establish whether HOCl affects glycolytic energy production in platelets (in PRP). Production of lactate was the measure of the glycolysis rate. As can be seen, HOCl
Figure 1. Effect of increasing hypochlorite and peroxynitrite concentrations on the kinetics of clot retraction. Aliquots (1 ml) of whole blood (A), standardized PRP (4 108 cells/ml) (B, C) or washed platelets (4 108 cells/ml) (D) were incubated in polypropylene tubes at 37°C for 2 min without (control) and with HOCl or HOCl ONOO added (in 1 min intervals) to the final concentrations as indicated. Immediately after incubation, samples (0.4 ml) of cell suspensions were transferred to glass tubes and clot retraction was initiated by the addition of calcium chloride (A–C) or thrombin (D). Further details in Materials and Methods. Values are maximal retraction obtained at indicated time interval and are expressed as means S.D. The results of one representative experiment (out of five, each in duplicate) are presented. *p 0.05, **p 0.01, ***p 0.001.
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Figure 2. Effect of hypochlorite on a final clot volume after retraction. Aliquots (1 ml) of standardized PRP (4 108 cells/ml) were incubated in polypropylene tubes at 37°C for 2 min without (control) and with HOCl added to the final concentrations (in mM) 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. After 2 h of retraction, clot volume was recorded using a digital camera (A) and final clot volume (B) was calculated as in “Measurement of kinetics of clot retraction” section. The results of one representative experiment (out of five, each in duplicate) are shown. *p 0.05, **p 0.01, ***p 0.001.
Figure 3. Effect of hypochlorite on total ATP content in clots. Total intracellular ATP level was determined by luciferine-luciferase chemiluminescent assay. Further details as in Methods. Presented data are means ( S.D.) of one representative (n 6) experiment performed on a single standardized PRP (4 108 cells/ml) sample. Control value varied from 2.96 to 3.23 nanomoles/108 cells. *p 0.05, ***p 0.001.
Hypochlorite and hemostasis 1447
(100–500 mM) augmented lactate production in a dosedependent manner. The effect was substantial—treatment with 500 mM HOCl resulted in about a 300% stimulation of lactate production. Figure 4B shows the results of experiments conducted to establish whether HOCl may affect platelet mitochondria functions measured as a rate of oxygen consumption in platelet suspension. As is shown, treatment of platelets (in PRP) with 50–500 mM HOCl resulted in a dose-dependent inhibition of oxygen consumption. The effect was considerable, since 500 mM HOCl reduced oxygen consumption by platelets about 55%. Experiments shown in Figure 4C were performed to establish whether HOCl may affect ΔYm—a prerequisite for ATP synthesis via the oxidative phosphorylation. As can be seen, treatment of washed platelets with HOCl (25–100 nM) resulted in a dose-dependent, rapid rise in TMRM fluorescence, which corresponded with a reduction of ΔYm. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) was used as a positive control, evoking a total fall in ΔYm. To assess whether inhibitory action of HOCl on CR may be related with the attenuation of cellular energy production, we studied the effect of exogenously added MgATP on the HOCl-evoked inhibition of CR. Experiments were performed on washed platelets permeabilized by saponin. Saponin treatment did not affect kinetics of CR markedly. As is seen in Figure 4D, exogenously added Mg-ATP almost completely recovered HOCl-mediated retardation of CR. Experiments shown in Table I were performed to assess the effect of HOCl (100–1000 mM) on the kinetics of platelet–fibrin clot formation measured in PRP by means of rotational thromboelastometry. We measured CT, dynamics of clot formation (a angle) and MCF. As is shown, HOCl was able to affect CT variable significantly only at high concentrations (500–1000 mM). The CT was prolonged by about 16 and 50% by 500 and 1000 mM HOCl, respectively. Alpha angle was reduced by HOCl (500–1000 mM) by 14 and 20%, respectively; while MCF variable was diminished by 500–1000 mM HOCl by about 10 and 20%, respectively. Experiments shown in Table II were performed to establish whether HOCl (100–1000 mM) may affect the kinetics of clot formation measured in plasma depleted from platelets by means of the turbidimetric method. Clotting was triggered by thrombin. We measured lag time of clotting (Lagc), maximal fibrin concentration (MaxAbsc), and fibrin polymerization rate (PR). As is shown, HOCl did affect the measured variables only at millimolar concentration (1 mM). Lagc was prolonged by about 36%, MaxAbsc was reduced by about 32% and PR was decreased by about 27%. To assess whether HOCl action on CR may be related with malfunction of platelet GPIIb/IIIa receptors, the effect of HOCl on washed platelet adhesion to a fibrinogen-coated surface was studied—a phenomenon intrinsically connected with the presence of activated GPIIb/IIIa. As can be seen, HOCl (25–100 mM) reduced platelet adhe-
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1448 T. Misztal et al.
Figure 4. Effect of hypochlorite on platelet energy metabolism. Aliquots (1 ml) of standardized PRP (4 108 cells/ml) were incubated in polypropylene tubes at 37°C for 2 min without (control) or with HOCl 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 min incubation. Lactate production in control varied from 3.76 to 4.2 mmoles min 1 1011 cells. The results of 6 independent experiments (each performed in duplicate, n 12) are presented. p 0.05; ** p 0.01, ***p 0.001 (A). Aliquots (1 ml) of standardized PRP (4 108 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 Methods. Results of 6 independent experiments (each performed in duplicate, n 12) are presented Oxygen consumption in the control varied from 360 to 430 nanomoles min 1 1011 cells. HOCl per se (up to 2 mM) did not alter oxygen concentration in cell-free plasma. *p 0.05, **p 0.01, ***p 0.001 (B). The traces show changes of fluorescence of TMRM-loaded platelets following addition of 10 mM CCCP (panel A) or HOCl (panel B) to the final concentrations as indicated. Further details as in Methods. The results of one representative experiment (out of six) are presented. (C) Aliquots (1 ml) of washed platelets (4 108 cells/ml) were incubated in polypropylene tubes at 37°C for 2 min with stirring with indicated HOCl concentrations. Then, platelets were permeabilized with saponin (20 mg/ml final conc.) for 2 min and the suspensions were supplemented with Mg-ATP (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) and Mg-ATP (5 mM final conc.). CR was initiated by the addition of thrombin (1 U/ml final conc.). Further details in Methods. *p 0.01 vs sample without ATP (D).
sion in a dose-dependent manner (Figure 5). Tirofiban (250 mg/ml), a GPIIb/IIIa antagonist, reduced platelet adhesion by about 85%.
Experiments shown in Figure 6 were performed to assess whether HOCl alone or in combination with thrombin is able to evoke strong and sustained calcium signal.
Table I. Effect of hypochlorite on the kinetics of coagulation in PRP.
Table II. Effect of hypochlorite on the variables of fibrin polymerization profile induced by thrombin.
Addition
Addition
None (control) HOCl 100 mM HOCl 250 mM HOCl 500 mM HOCl 1000 mM
CT (sec)
a (degrees)
MCF (mm)
357 37 359 44 376 28 418 41* 541 34***
72 2 72 3 67 2 62 3** 58 3**
82 2 82 3 82 3 73 3* 65 2**
*p 0.05; **p 0.01; ***p 0.001.
None (control) HOCl 100 mM HOCl 250 mM HOCl 500 mM HOCl 1000 mM *p 0.05.
Lagc [s] 260 25 255 35 270 30 290 45 355 40*
MaxAbsC [abs] PR [abs/s 10 3] 1.04 0.08 1.03 0.13 1.01 0.06 0.97 0.07 0.72 0.16*
2.96 0.18 2.97 0.22 2.94 0.15 2.69 0.09 2.12 0.18*
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Hypochlorite and hemostasis 1449
Figure 5. Effect of hypochlorite on the adhesion of washed platelets to fibrinogen coated surfaces. Aliquots (1 ml) of washed platelets (4 108 cells/ml) were incubated in polypropylene tubes at 37°C for 2 min without (control) or with Tirofiban or HOCl added to the final concentration as indicated. After that, 50 ml 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 mg/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 Methods. The data represent the mean SD of six experiments, each performed in triplicate on a separate platelet preparation. *p 0.05; **p 0.01, ***p 0.005.
As is seen, HOCl, at concentrations 100–1000 mM, produced dose-dependent, rapid, and sustained rise in [Ca2]cyt (panel A). [Ca2]cyt raises were moderate to strong. 200 mM HOCl produced calcium signal similar to that evoked by 0.2 U/ml of thrombin. Preincubation of platelets with low micromolar HOCl (10–50 mM) resulted in a reduction of thrombin-evoked calcium signal (panel B). Combination of HOCl concentrations producing calcium signal similar to that evoked by 0.2 U/ml of thrombin (200 mM HOCl) with 0.2 U/ml of thrombin resulted in generation of calcium signal lower than that evoked by thrombin alone (lack of synergism, panel C). To assess whether HOCl affects platelet contractility through the inhibition of functional contractile apparatus formation, we measured its effect on the F-actin content in gel-filtrated platelets activated by thrombin. As can be seen in Figure 7, unlike cytochalasin B (actin polymerization blocker), HOCl (up to 250 nM) failed to affect F-actin formation kinetics. HOCl, up to the concentration of 100 mM, failed to affect platelet membrane integrity measured in artificial system as LDH release, while the threshold cytotoxic concentration of HOCl in PRP was 500 mM (Table III). Discussion For the first time, the results presented here show that relatively low HOCl concentrations affect the kinetics of clot shrinking. Our study revealed that HOCl strongly reduces the rate of retraction and augments the final clot
Figure 6. Effect of hypochlorite on cytosolic calcium concentration in porcine platelets. Fura-2-loaded platelets suspended in the medium containing 1 mM Ca2 were treated with HOCl alone (panel A) or with combination of HOCl plus thrombin (panels B–C) added to final concentrations as indicated. Changes in [Ca2]cyt were measured as described in Methods. Each trace is representative of at least six determinations performed in three different preparations.
volume at concentrations much lower (micromolar) than those previously reported (millimolar) to affect clotting factors [17], and lower than those inhibiting aggregation and secretion of porcine platelets [17–18]. This makes the possibility of HOCl action on CR in an in vivo condition much more likely. The susceptibility of CR to HOCl depends on the composition of the microenvironment in which this oxidant acts. Thus, the CR in an artificial system, consisting of washed platelets, fibrinogen, and thrombin, was inhibited by low nanomolar concentrations of HOCl, whereas in PRP and whole blood, CR was retarded by low and high micromolar concentrations, respectively (Figure 1A, B and D). The lower susceptibility of CR to HOCl in PRP and in whole blood is most likely associated with the rise in the number of potential targets of this oxidant. These observations also indicate that HOCl has its targets in plasma, in erythrocytes, and in platelets; these targets dif-
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1450 T. Misztal et al.
Figure 7. Effect of hypochlorite and cytochalasin B on the F-actin content in activated platelets. Gel filtered platelets (4 108 cells/ml) were incubated in polypropylene tubes at 37°C for 10 min with stirring, without (control) or with HOCl 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. Tritoninsoluble fraction was separated by 10% SDS-PAGE. The amount of actin in cytoskeletal fraction relative to total was quantified by densitometric analysis. Further details in Methods. Values represent means SD of 3 independent experiments (each in duplicate). (Δ) – control, () – HOCl (25 nM), () – HOCl (250 nM), () – cytochalasin B (20 mM). The longer incubation (up to 60 min) did not change F-actin content. *p 0.01.
fer in their susceptibility to stressor action. It is also evident that the most susceptible HOCl target in blood is confined to platelets. What could the mechanism underlying HOCl action on CR be? Platelet-dependent CR is a complex process in which several steps can be distinguished: the initial production of large quantities of thrombin (on the platelet surface), simultaneous formation of a loose fibrin network and activation of the platelet GPIIb/IIIa receptors (inside–out signaling), fibrin(ogen) binding with activated GPIIb/IIIa receptors, transmission of the contractile forces generated by the platelet actin–myosin cytoskeleton to connecting fibrin fibers around platelets (contraction), and conversion of a diffuse fibrin network into a dense platelet–fibrin clot (shrinking) [19,43–45]. Table III. Effect of hypochlorite on platelet integrity measured in PRP. LDH activity (% of total) Addition None (control) HOCl 50 mM HOCl 100 mM HOCl 200 mM HOCl 300 mM HOCl 500 mM
PLT
PRP
5.9 0.6 6.3 0.5 7.8 0.8* 8.1 1.1* 8.4 1.5** 9.1 1.3***
3.3 0.4 3.4 0.6 3.6 0.9 3.8 0.7 4.4 1 4.9 0.8*
*p 0.05; **p 0.01; ***p 0.001.
Therefore, the reduction of CR rate (CRR) reported here, at least potentially, may result from HOCl action on any of the above-mentioned steps. The results presented here strongly indicate that the effect of HOCl on CR is unlikely to be related to fibrin network formation. This is because the susceptibility of CR to HOCl action (measured in PRP) was markedly higher than that of clot formation evaluated by rotational thromboelastometry. Specifically, the effective HOCl concentrations modulating CR and clot formation in PRP were 50 and 500 mM, respectively (compare Figure 1B with Table I). What is more, CRR measured in an artificial system was reduced by 25 nM HOCl (Figure 1D). Likewise, direct HOCl action on clotting factors is also excluded, especially at low stressor concentrations ( 300 mM), since its effect on fibrin strand formation in plasma depleted of platelets was observed at concentrations above 1000 mM, that is, much higher than those for reducing CRR in PRP (compare Figure 1B with Table II). This is consistent with the observation of Stief et al., who demonstrated that inactivation of fibrinogen, factor V, factor VIII, and factor X, in human plasma was observed in the presence of 2–3 mM concentrations of HOCl [17]. Similarly, the high susceptibility of CR to HOCl action cannot be explained by its effect on platelet GPIIb/IIIa receptors. This is because adhesion of activated platelets to fibrinogen-coated surfaces (a phenomenon intrinsically dependent on the presence of activated integrin receptors) was reduced by oxidant concentrations much higher than those affecting CR. Particularly, the effective HOCl concentrations reducing CRR in an artificial system and the adhesion of washed platelets were 25 nM and 25 mM, respectively (compare Figure 1B with Figure 5). Extremely impaired platelet mitochondria, that is, mitochondria with mitochondrial permeability transition pore (MPTP) formed in a high conductance state, have been proposed to support calpain 1-evoked inactivation of GPIIb/IIIa receptors in platelets stimulated by very strong agonists, for example, a combination of high thrombin concentrations convulxin [46–49]. One can expect that, at least potentially, HOCl-uncoupled mitochondria may inactivate platelet GPIIb/IIIa receptors in a similar way. The involvement of high-conductance MPTP in HOClevoked retardation of CR is however highly unlikely, at least in the presence of lower (nanomolar) concentrations of stressor. This is because HOCl, at concentrations up to 100 mM, failed to evoke a high and sustained rise in intracellular calcium concentrations, a prerequisite for MPTP formation [46–48], in washed platelets suspended in a calcium-containing medium (Figure 6A). What is more, HOCl was able to reduce thrombin-evoked calcium signal (Figure 6B and C), indicating a lack of synergistic effect. Generation of strong and sustained calcium signal in washed platelets treated with higher (above 100 mM) stressor concentrations is most likely due to the loss of plasma membrane integrity, manifested by the LDH release (compare Figure 6 and Table III). It is, therefore, likely that inhibition of CR by low HOCl concentrations is associated with dysfunction of
Hypochlorite and hemostasis 1451
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the platelet contractile apparatus, rather than with abnormalities in fibrin formation or platelet GPIIb/IIIa function. Dysfunction of the platelet contractile apparatus is likely to suppress CR. This is based on the observations that the blocking of actin polymerization (a prerequisite for formation of a functional contractile apparatus) by cytochalasin B resulted in a significant reduction of CRR [20–21]. However, the direct action of HOCl on components of contractile apparatus is rather unlikely since, as we demonstrate here, the stressor concentrations reducing CR in an artificial system failed to affect the kinetics of F-actin formation in activated washed platelets (Figure 7). Reduced platelet contractility may also result from inefficient energy production. CR is an energy-requiring process, in which ATP hydrolysis is crucial for the contraction of platelet actomyosin [50]. It is now well accepted that the glycolytic pathway and mitochondrial oxidative phosphorylation are substantial for effective CR [20–21,50]. The results presented here indicate that the inhibitory effect of HOCl on CR is closely related to decreased platelet contractility resulting from inefficient energy production. Consistent with this is the observation that concentrations of HOCl affecting CRR decrease the total ATP contents in clots derived from PRP (Figure 3), and the finding that exogenously added Mg-ATP is able to abolish the inhibitory effect of HOCl on CR (Figure 4D). The results presented here also show that concentrations of HOCl affecting CRR reduce platelet oxygen consumption and augment lactate production, indicating the occurrence of the Pasteur effect (Figure 4A and B). Since in platelets, the glycolytic pathway and mitochondrial respiration are tightly functionally connected [51], this can be interpreted to mean that the stimulatory effect of HOCl on glycolysis in platelets may be related to the impairment of mitochondria. This is likely to be true since, as reported here, in porcine platelets HOCl is able not only to reduce mitochondrial oxygen consumption, but also to induce the loss of ΔYm (Figure 4C), which may result in diminishing the chemiosmotic potential, a prerequisite for ATP synthesis via oxidative phosphorylation. This is in agreement with several previous reports indicating that HOCl is able to evoke the dysfunction of mitochondria in various cell types [28–29]. In summary, these data clearly indicate that HOCl may affect in vitro CR through the inhibition of energy production in platelet mitochondria. To sum up, it is now clear those concentrations of HOCl much lower than those which alter clotting factors activity may reduce the shrinking of platelet–fibrin clots through the inhibition of energy production in platelet mitochondria. It is now well documented that activated inflammatory cells produce large quantities of substances used to kill pathogens. The group of such compounds comprises not only hypochlorous acid but also peroxynitrite (ONOO), formed in a very rapid reaction from NO and O2., produced by activated inflammatory cells [22]. Recently, we reported that physiologically relevant ONOO concentrations may inhibit CR, and accelerate
its lysis through the inhibition of platelet mitochondrial energy production [20]. It is, therefore, highly likely that both HOCl and ONOO may synergistically act on clot in vivo. The results presented here confirm such a possibility (Figure 1C). How can this be relevant to the in vivo situation? A recognized indicator of in vivo HOCl presence is elevated 3-chlorotyrosine [16]. Enhanced production of 3-chlorotyrosine has been reported in numerous clinical conditions associated with an inflammatory state including asthma [52], atherosclerosis [9], and inflammatory vasculitis [53]. The results presented here indicate that in platelets suspended in plasma, low micromolar concentrations of HOCl are able to suppress mitochondrial energy production. In washed platelets, these concentrations were much lower, reaching values of about 25 nM. Activated neutrophils were reported to form 150–425 mM/h of HOCl [54], whereas local concentrations of stressor in inflamed tissue are estimated to be as high as 5 mM [3,17]. At concentrations within the reported range generated by activated neutrophils (20–400 mM), HOCl induced a rapid loss of ∆Ym in human fetal liver and in hepatoma HepG2 cells [29]. It is, therefore, clear that pathophysiologically relevant HOCl concentrations, associated with an abnormal or inefficiently controlled inflammatory response, are likely to suppress mitochondrial energy production in platelets. Our data relate to clinical realities in two ways. First, abnormal CR may result in the formation of thrombi that are less mechanically stable and prone to embolization, and second, weekly shrinked (more loose) clots are expected to be more susceptible to fibrinolysis. Collectively, we conclude that clinically relevant HOCl concentrations associated with an inflammatory state may inhibit CR through the inhibition of platelet mitochondrial energy production. Declaration of interest The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper. This research was supported by Medical University of Bialystok [124-01500 F]. References [1] Eiserich JP, Hristova M, Cross CE, Jones AD, Freeman BA ,Halliwell B, van der Vliet A. Formation of nitric oxidederived inflammatory oxidants by myeloperoxidase in neutrophils. Nature 1998;391:393–397. [2] Nauseef WM. Insights into myeloperoxidase biosynthesis from its inherited deficiency. J Mol Med (Berl) 1998;76: 661–668. [3] Weiss SJ. Tissue destruction by neutrophils. N Engl J Med 1989 9;320:365–376. [4] Pullar JM, Vissers MC, Winterbourn CC. Living with a killer: the effects of hypochlorous acid on mammalian cells. IUBMB Life 2000;50:259–266.
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[27] Levi M, van der Poll T, Buller HR. Bidirectional relation between inflammation and coagulation. Circulation 2004;109:2698–2704. [28] Sugiyama S, Kugiyama K, Aikawa M, Nakamura S, Ogawa H, Libby P. Hypochlorous acid, a macrophage product, induces endothelial apoptosis and tissue factor expression: involvement of myeloperoxidase-mediated oxidant in plaque erosion and thrombogenesis. Arterioscler Thromb Vasc Biol 2004;24:1309–1314. [29] Whiteman M, Rose P, Siau JL, Cheung NS, Tan GS, Halliwell B, Armstrong JS. Hypochlorous acid-mediated mitochondrial dysfunction and apoptosis in human hepatoma HepG2 and human fetal liver cells: role of mitochondrial permeability transition. Free Radic Biol Med 2005;38:1571–1584. [30] Collet JP, Montalescot G, Lesty C, Weisel JW. A structural and dynamic investigation of the facilitating effect of glycoprotein IIb/IIIa inhibitors in dissolving platelet-rich clots. Circ Res 2002;90:428–434. [31] Kunitada S, Fitzgerald GA, Fitzgerald DJ. Inhibition of clot lysis and decreased binding of tissue-type plasminogen activator as a consequence of clot retraction. Blood 1992; 79:1420–1427. [32] Whiteman M, Szabó C, Halliwell B. Modulation of peroxynitrite- and hypochlorous acid-induced inactivation of a1antiproteinase by mercaptoethylguanidine. Br J Pharmacol 1999;126:1646–1652. [33] Koppenol WH, Kissner R, Beckman JS. Syntheses of peroxynitrite: to go with the flow or on solid grounds? Methods Enzymol 1996;269:296–302. [34] Osdoit S, Rosa J-P. Fibrin clot retraction by human platelets correlates with aIIbb33 integrin-dependent protein tyrosine dephosphorylation. J Biol Chem 2001;273: 6703–6710. [35] Luddington R. Thrombelastography/thromboelastometry. Clin Lab Haematol 2005;27:81–90. [36] Carter AM, Cymbalista CM, Spector TD, Grant PJ. Heritability of clot formation, morphology, and lysis: the EuroCLOT study. Arterioscler Thromb Vasc Biol 2007; 27:2783–2789. [37] Gutmann I, Wahlefeld AW. L-()Lactate determination with lactate dehydrogenase and NAD. In: Bergmeyer HU (ed.). Methods of enzymatic analysis. Weiheim: VCH Velagsgesellschaft mbH; 1985. pp. 1464–1468. [38] Holmsen HE, Storm E, Day HJ. Determination of ATP and ADP in blood platelets: a modification of the firefly luciferase assay for plasma. Anal Biochem 1976;46:481–502. [39] Bellavite P, Andrioli G, Guzzo P, Arigliano P, Chirumbolo S, Manzato F, Santonastaso C. A colorimetric method for the measurement of platelet adhesion in microtiter plates. Anal Biochem 1994;216:444–450. [40] Pollock WK, Rink TJ, Irvine RP. Liberation of [3H]arachidonic acid and changes in cytoplasmic free calcium in fura-2loaded human platelets stimulated by ionomycin and collagen. Biochem J 1986;235:869–877. [41] Siffert W, Siffert G, Scheid P, Akkerman JW. Activation of Na/H exchange and Ca2 mobilization start simultaneously in thrombin-stimulated platelets. Evidence that platelet shape change disturbs early rises of BCECF fluorescence which causes an underestimation of actual cytosolic alkalinization. Biochem J 1989;258:521–527. [42] Jackson SP, Schoenwaelder SM, Yuan Y, Rabinowitz I, Salem HH, Mitchell CA. Adhesion receptor activation of phosphatidylinositol 3-kinase. Von Willebrand factor stimulates the cytoskeletal association and activation of phosphatidylinositol 3-kinase and pp60c-src in human platelets. J Biol Chem 1994;269:27093–27099. [43] Schoenwaelder SM, Ono A, Nesbitt WS, Lim J, Jarman K, Jackson SP. Phosphoinositide 3-Kinase p110b regulates integrin aIIbb3 avidity and the cellular transmission of contractile forces. J Biol Chem 2010;285:2886–2896.
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ORIGINAL ARTICLE
Clinically relevant HOCl concentrations reduce clot retraction rate via the inhibition of energy production in platelet mitochondria T. Misztal, T. Rusak & M. Tomasiak
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Department of Physical Chemistry, Medical University of Bialystok, Bialystok, Poland Abstract Using porcine blood, we examined the impact of hypochlorite, product of activated inflammatory cells, on clot retraction (CR), an important step of hemostasis. We found that, in vitro, HOCl is able to reduce CR rate and enlarge final clot size in whole blood (t.c. 100 mM), plateletrich plasma (PRP) threshold concentration (t.c. 50 mM), and an artificial system (washed platelets and fibrinogen) (t.c. 25 nM). Combination of low HOCl and peroxynitrite concentrations resulted in synergistic inhibition of CR by these stressors. Concentrations of HOCl completely inhibiting CR failed to affect the kinetics of coagulation measured in PRP and in platelet-free plasma. Concentrations of HOCl reducing CR rate in PRP augmented production of lactate, inhibited consumption of oxygen by platelets, and decreased total adenosine triphosphate (ATP) content in PRP-derived clots. In an artificial system, concentrations of HOCl resulting in inhibition of CR (25–100 nM) reduced mitochondrial transmembrane potential and did not affect actin polymerization in thrombin-stimulated platelets. These concentrations of HOCl failed to affect the adhesion of washed platelets to fibrinogen and to evoke sustained calcium signal, thus excluding stressor action on glycoprotein IIb/IIIa receptors. Exogenously added Mg-ATP almost completely recovered HOCl-mediated retardation of CR. Concentrations of HOCl higher than those affecting CR reduced thromboelastometric variables (maximum clot firmness and a angle). We conclude that low clinically relevant HOCl concentrations may evoke the inhibition of CR via the reduction of platelet contractility resulted from malfunction of platelet mitochondria. At the inflammatory conditions, CR may be the predominant HOCl target. Keywords: platelets, mitochondria, hypochlorous acid, peroxynitrite, clot retraction
Introduction At sites of inflammation, activated phagocytes (neutrophils and macrophages) generate large quantities of hydrogen peroxide and release an enzyme myeloperoxidase (MPO) stored in their granules [1–2]. The majority of hydrogen peroxide generated during the phagocytes’ respiratory burst is subsequently used to oxidize chloride anion (Cl) to hypochlorous acid (HOCl) in a reaction catalyzed by secreted myeloperoxidase [3]. Hypochlorous acid and its conjugate base (OCl) are potent cytotoxic oxidants and chlorinating agents possessing microbicidal and viricidal activities, and also contribute to inflammatory injury in host tissues [3–4]. In response to activation, MPO is released both into phagocytic vesicles (phagosomes) and the extracellular space (plasma or interstitial matrix) [3,5]. In healthy subjects, plasma MPO concentration varies from 18 to 39 ng/ml [6–7], but in some clinical conditions it can rise to 55 [6] or even 287 ng/ml [8]. The primary physiological role of HOCl is to kill pathogens but its overproduction, as observed in prolonged, acute, or malcontrolled inflammatory state, may result in upregulation of hemostasis. In fact, HOCl has been proposed to play a role in the pathogenic mechanism of atherosclerosis [9], respiratory distress syndrome, reperfusion injury and rheumatoid arthritis [2,10–14], glomerulonephritis [11],
and chronic renal failure [15–16], that is, clinical conditions often associated with hemostatic abnormalities. The exact mechanism(s) linking hemostasis abnormalities with HOCl overproduction are not fully understood. HOCl has also been reported to oxidize in vitro fibrinogen and clotting factors V, VIII, and X [17] and to inhibit aggregation of rabbit platelets [18]. It should be stressed, however, that its action on platelet aggregation and on clotting factors was observed only at millimolar concentrations (1.5–5 mM); that is, at concentrations which are less likely to be present in the blood stream. Recently, we have proposed that peroxynitrite (ONOO), a reactive nitrogen species produced by activated inflammatory cells, may affect hemostasis through the reduction of platelet contractility resulting from inhibition of energy production in platelet mitochondria [19–20]. Platelet contractility is crucial for the stability of the primary platelet aggregate connected to an injured vessel wall and for the retraction of a platelet–fibrin clot [21]. This novel sensitive mechanism is very likely to operate in vivo, since mitochondria have been reported to be very sensitive to low (nanomolar–micromolar) concentrations of reactive oxygen, nitrogen, and chlorine species produced by activated inflammatory cells [22–27]. HOCl is likely to affect platelet mitochondria. Recent studies indicate that low micromolar concentrations of
Correspondence: Prof. Marian Tomasiak, Department of Physical Chemistry, Medical University of Bialystok, Kilinskiego 1, 15-089 Bialystok, Poland. Tel: 48-85-748-57-14. Fax: 48-85-748-54-16. E-mail: [email protected] (Received date: 7 April 2014; Accepted date: 29 August 2014; Published online: 17 October 2014)
1444 T. Misztal et al.
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HOCl, that is, concentrations much lower than those reported to modify clotting factors and inhibit platelet aggregation, can suppress mitochondrial energy production in cultured human endothelial cells [28], cultured human liver cells, and in isolated rat liver mitochondria [29]. The effect of HOCl on clot retraction (CR) and the functionality of platelet mitochondria has never been studied before. CR has been reported to have a great impact not only on the mechanical properties of thrombus (stability and elasticity), but also on its lysis [19,30–31]. Consequently, these studies were undertaken to establish whether HOCl may affect hemostasis through the inhibition of CR. Another aim was to assess the possible mechanism of HOCl action on platelet mitochondria.
factor, the initial 10 ml of blood was discarded. Platelet number in the blood of studied animals varied from 4.6 108/ml to 6.5 108/ml. Platelet number in plateletrich plasma (PRP) was adjusted to 4 108/ml with autologous plasma before each experiment. Synthesis of ONOO
Materials and methods
Peroxynitrite was synthesized by the reaction of acidified H2O2 (1.4 M) with NaNO2 (1.2 M) in a quenched flow reactor [33]. The excess of H2O2 was removed by passing 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 (e302 1670 M1cm1) [32]. Typically used ONOO concentrations did not cause an increase in pH of the samples.
Chemicals
Platelet preparation
Hypochlorite used was in the form of sodium hypochlorite (NaOCl). Throughout this article, we use the term hypochlorite (pKa, 7.46) to refer to approximately 50% ionized mixture of HOCl and ClO species that exists at physiological pH (referred in this article as HOCl). The concentration was determined prior to each experiment by measuring the absorbance at 290 nm (e292 350 M 1cm 1) [32]. Recombinant tissue plasminogen activator (Actylise) was purchased from Boehringer Ingelheim GmbH (Ingelheim, Germany). Chrono-lume (luciferin–luciferase mix) was purchased from Chrono-log (Havertown, PA, U.S.A.). Tetramethylrhodamine methyl ester (TMRM) was purchased from Invitrogen (Carlsbad, CA, U.S.A.). Collagen (fibrillar, from equine tendon) was from Hormon Chemie (Munich, Germany). Other chemicals were from Sigma Chemical Co (St. Louis, MO, U.S.A.).
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 using 1 M citric acid and the suspension was centrifuged at 1500 g for 20 min to obtain a pellet which was resuspended in a Ca2-free Tyrode-Hepes (T-H) buffer (152 mM NaCl, 2.8 mM KCl, 8.9 mM NaHCO3, 0.8 mM KH2PO4, 0.8 mM MgCl2, 5.6 mM glucose, apyrase (2 U/ml), 10 mM ethylene glycol tetraacetic acid (EGTA), bovine serum albumin (BSA) (3.5 mg/ml), and 10 mM Hepes; pH, 6.5; osmolarity of 340 mOsm). The platelets were washed once with the abovedescribed buffer and finally suspended in the same buffer with the exception that, in the final suspension medium, apyrase and EGTA were omitted and pH was adjusted to 7.4. In some experiments, the platelets suspension was next passed through a chromatographic column filled with Sepharose 2B using T-H buffer as an eluent. The platelet concentration was standardized to 4 108 cells/ml by dilution with T-H buffer. Platelet number was determined using a Coulter® Hematology Analyzer (Beckman Coulter Inc., U.S.A.).
Animals A total of 50 domestic pigs (breed: Polish Large White) of both sexes, approximately 9 months old, with a mean weight of 90–100 kg, were used in the experiments. The animals were raised on local farms under normal agricultural husbandry conditions. Pigs were fed the grower chow diet consisting of grain (rye, barley, wheat, and maize) and soybean. The animals for this study were selected at random from available litters. Prior to blood collection, the pigs were starved for at least 12 h. The study protocol and procedures were approved by the Ethics Committee at the Medical University of Białystok. Blood collection Blood collection was performed in a local slaughterhouse (PMB, Bialystok, Poland). Forty milliliters of blood was withdrawn by direct carotid catheterization and collected into 3.8% (w/v) sodium citrate, one volume per nine volumes of blood. To avoid contamination of blood by tissue
Measurement of kinetics of CR Measurement of the kinetics of CR 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 T-H 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 CR was initiated by gently mixing the suspension The kinetics of CR in artificial system consisting of washed platelets, bovine fibrinogen (2 mg/ml of final conc.), and thrombin from bovine plasma (1 U/ml final conc.) was evaluated by the method described in detail [34]. Pictures were taken for 1 h at 10-min intervals and after 120 min using a digital camera. Quantification of retraction was performed by
Hypochlorite and hemostasis 1445
assessment of clot area using the Motic Images Plus 2.0 ML software, and data were processed using MS 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 (area t 0 area t ) 100 (relative clot volume) (area t ) 0
In experiments, where exogenous Mg-ATP was used, washed platelets were permeabilized with saponin, typically 5 mg per each 108 platelets.
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Thromboelastometric (ROTEM) analyses
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 protein and neutralized with ice-cold 6 M KOH/0.5 M morpholine sulfonic acid. ATP content was determined in neutralized cellular extracts by the luciferase–luciferin assay [38]. Measurement of the mitochondrial membrane potential in porcine platelets The mitochondrial membrane potential (ΔYm) was determined in TMRM-loaded platelets using the dequench mode of measurement, essentially as described [20]. One measure of ΔYm fall was a rise in TMRM fluorescence.
ROTEM technology is described elsewhere [35]. Thromboelastometric measurements were performed using ROTEM system (Tem International GmbH, Manheim, Germany). We measured the following parameters: clotting time (CT)—time from start of measurement to the beginning of the fibrin polymerization process; alpha angle (a)—the angle showing the dynamics of clot formation; and maximum clot firmness (MCF)—a parameter reflecting the strength of the formed clot. All ROTEM measurements were performed by the same experienced operator as described [20].
Assay of platelet adhesion
Determination of fibrin polymerization profiles
Cytosolic free Ca2 ([Ca2]cyt) was determined in Fura2-loaded platelets essentially as described by Pollock et al. [40]. PRP was incubated with 3 mM (final conc.) Fura-2/ AM and Pluronic-137 (0.005% final conc.) for 45 min at room temperature in the dark with gentle agitation. Then, 1 mM prostaglandin E1 was added and platelets were separated from PRP as described above, and resuspended at 1.5–1.7 109/ml in a Ca2-free T-H buffer. Aliquots of these suspensions were than transferred to a cuvette, containing 2 ml of T-H buffer containing 1 mM Ca2 (final conc.). Measurement of Fura-2 fluorescence in stirred platelet suspension was performed at 37°C using a Hitachi F-7000 spectrofluorometer (Hitachi Corp., Japan). Monochromator settings were 339 nm for excitation and 500 nm for emission. The Fura-2 responses were calibrated to obtain [Ca2]cyt as described by Siffert et al. [41].
The kinetics of fibrin formation and plasma clot lysis was evaluated by the turbidimetric method described in details by Carter et al. [36]. 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). Measurement of lactate production in clotting PRP Aliquots of clotting suspensions prepared as described above (in the section “Measurement of kinetics of CR”) 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. Lactate was measured in the deproteinized and neutralized extract by the lactate dehydrogenase (LDH) assay [37]. Measurement of the respiration rate Oxygen consumption was measured polarographically with a Clark-type oxygen electrode (model YSI 5300A, YSI Life Sciences, U.S.A.), in a 1-ml closed vessel (YSI sample micro chamber) at 37°C. Measurement of ATP content in retracted clots Clots derived from standardized (4 108 cells/ml) PRP samples formed during 1-h incubation at 37°C were carefully transferred using plastic Pasteur pipette to
Platelet adhesion was quantified by measuring the acid phosphatase activity of adherent cells, as described by Bellavite et al. [39]. Collagen was used 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. Measurement of cytosolic free Ca2
Platelet membrane integrity test The extent of platelet lysis following incubation with HOCl was estimated in PRP by measuring the activity of LDH, lost from the cells into the suspending fluid [37]. Determination of actin polymerization in platelets Determination of F-actin content in platelets was conducted as described in essence [42]. To determine total actin content in platelets, whole cell lysates (pellet supernatant) were used. Data analysis Data reported in this paper are the mean ( S.D.) of the number of determinations indicated (n). Statistical
1446 T. Misztal et al. analysis was performed by Student’s t-test and elaboration of experimental data by the use of Slide Write plus (Advanced Graphics Software, Inc. Carlsbad, CA, U.S.A.).
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Results Figure 1 shows the results of experiments in which we compared the effect of increasing HOCl concentrations on CR measured in whole blood (A), PRP (B), and a reconstituted system consisting of washed platelets and purified bovine fibrinogen (D). As is seen, treatment of whole blood, PRP, and washed platelets with increasing HOCl concentrations resulted in a dose-dependent inhibition of CR rate with an IC50 value of 360 mM (whole blood), 250 mM (PRP), and 100 nM (reconstituted system), respectively. Figure 1C presents the results of experiments conducted to establish how simultaneous incubation of PRP with HOCl and ONOO may affect CR. As it is seen,
cumulative action of both stressors resulted in synergistic inhibition of CR. The effect was significant, since combination of 50 mM HOCl and 50 mM ONOO produced about a 60% inhibition of CR. Figure 2 demonstrates the effect of increasing concentrations (50–500 mM) of HOCl on the final clot volume following retraction, measured in PRP. As can be seen, HOCl augmented clot volume in a dose-dependent fashion (A). The effect was substantial, since 500 mM HOCl produced about a 250% rise in clot volume (panel B). Experiments shown in Figure 3 were performed to establish whether HOCl affects total ATP content in clots derived from PRP. As is seen, HOCl (100–1000 mM) reduced ATP content dose-dependently. The effect was significant, since 1000 mM HOCl produced about a 30% reduction of total ATP content. Experiments shown in Figure 4A were performed to establish whether HOCl affects glycolytic energy production in platelets (in PRP). Production of lactate was the measure of the glycolysis rate. As can be seen, HOCl
Figure 1. Effect of increasing hypochlorite and peroxynitrite concentrations on the kinetics of clot retraction. Aliquots (1 ml) of whole blood (A), standardized PRP (4 108 cells/ml) (B, C) or washed platelets (4 108 cells/ml) (D) were incubated in polypropylene tubes at 37°C for 2 min without (control) and with HOCl or HOCl ONOO added (in 1 min intervals) to the final concentrations as indicated. Immediately after incubation, samples (0.4 ml) of cell suspensions were transferred to glass tubes and clot retraction was initiated by the addition of calcium chloride (A–C) or thrombin (D). Further details in Materials and Methods. Values are maximal retraction obtained at indicated time interval and are expressed as means S.D. The results of one representative experiment (out of five, each in duplicate) are presented. *p 0.05, **p 0.01, ***p 0.001.
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Figure 2. Effect of hypochlorite on a final clot volume after retraction. Aliquots (1 ml) of standardized PRP (4 108 cells/ml) were incubated in polypropylene tubes at 37°C for 2 min without (control) and with HOCl added to the final concentrations (in mM) 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. After 2 h of retraction, clot volume was recorded using a digital camera (A) and final clot volume (B) was calculated as in “Measurement of kinetics of clot retraction” section. The results of one representative experiment (out of five, each in duplicate) are shown. *p 0.05, **p 0.01, ***p 0.001.
Figure 3. Effect of hypochlorite on total ATP content in clots. Total intracellular ATP level was determined by luciferine-luciferase chemiluminescent assay. Further details as in Methods. Presented data are means ( S.D.) of one representative (n 6) experiment performed on a single standardized PRP (4 108 cells/ml) sample. Control value varied from 2.96 to 3.23 nanomoles/108 cells. *p 0.05, ***p 0.001.
Hypochlorite and hemostasis 1447
(100–500 mM) augmented lactate production in a dosedependent manner. The effect was substantial—treatment with 500 mM HOCl resulted in about a 300% stimulation of lactate production. Figure 4B shows the results of experiments conducted to establish whether HOCl may affect platelet mitochondria functions measured as a rate of oxygen consumption in platelet suspension. As is shown, treatment of platelets (in PRP) with 50–500 mM HOCl resulted in a dose-dependent inhibition of oxygen consumption. The effect was considerable, since 500 mM HOCl reduced oxygen consumption by platelets about 55%. Experiments shown in Figure 4C were performed to establish whether HOCl may affect ΔYm—a prerequisite for ATP synthesis via the oxidative phosphorylation. As can be seen, treatment of washed platelets with HOCl (25–100 nM) resulted in a dose-dependent, rapid rise in TMRM fluorescence, which corresponded with a reduction of ΔYm. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) was used as a positive control, evoking a total fall in ΔYm. To assess whether inhibitory action of HOCl on CR may be related with the attenuation of cellular energy production, we studied the effect of exogenously added MgATP on the HOCl-evoked inhibition of CR. Experiments were performed on washed platelets permeabilized by saponin. Saponin treatment did not affect kinetics of CR markedly. As is seen in Figure 4D, exogenously added Mg-ATP almost completely recovered HOCl-mediated retardation of CR. Experiments shown in Table I were performed to assess the effect of HOCl (100–1000 mM) on the kinetics of platelet–fibrin clot formation measured in PRP by means of rotational thromboelastometry. We measured CT, dynamics of clot formation (a angle) and MCF. As is shown, HOCl was able to affect CT variable significantly only at high concentrations (500–1000 mM). The CT was prolonged by about 16 and 50% by 500 and 1000 mM HOCl, respectively. Alpha angle was reduced by HOCl (500–1000 mM) by 14 and 20%, respectively; while MCF variable was diminished by 500–1000 mM HOCl by about 10 and 20%, respectively. Experiments shown in Table II were performed to establish whether HOCl (100–1000 mM) may affect the kinetics of clot formation measured in plasma depleted from platelets by means of the turbidimetric method. Clotting was triggered by thrombin. We measured lag time of clotting (Lagc), maximal fibrin concentration (MaxAbsc), and fibrin polymerization rate (PR). As is shown, HOCl did affect the measured variables only at millimolar concentration (1 mM). Lagc was prolonged by about 36%, MaxAbsc was reduced by about 32% and PR was decreased by about 27%. To assess whether HOCl action on CR may be related with malfunction of platelet GPIIb/IIIa receptors, the effect of HOCl on washed platelet adhesion to a fibrinogen-coated surface was studied—a phenomenon intrinsically connected with the presence of activated GPIIb/IIIa. As can be seen, HOCl (25–100 mM) reduced platelet adhe-
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1448 T. Misztal et al.
Figure 4. Effect of hypochlorite on platelet energy metabolism. Aliquots (1 ml) of standardized PRP (4 108 cells/ml) were incubated in polypropylene tubes at 37°C for 2 min without (control) or with HOCl 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 min incubation. Lactate production in control varied from 3.76 to 4.2 mmoles min 1 1011 cells. The results of 6 independent experiments (each performed in duplicate, n 12) are presented. p 0.05; ** p 0.01, ***p 0.001 (A). Aliquots (1 ml) of standardized PRP (4 108 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 Methods. Results of 6 independent experiments (each performed in duplicate, n 12) are presented Oxygen consumption in the control varied from 360 to 430 nanomoles min 1 1011 cells. HOCl per se (up to 2 mM) did not alter oxygen concentration in cell-free plasma. *p 0.05, **p 0.01, ***p 0.001 (B). The traces show changes of fluorescence of TMRM-loaded platelets following addition of 10 mM CCCP (panel A) or HOCl (panel B) to the final concentrations as indicated. Further details as in Methods. The results of one representative experiment (out of six) are presented. (C) Aliquots (1 ml) of washed platelets (4 108 cells/ml) were incubated in polypropylene tubes at 37°C for 2 min with stirring with indicated HOCl concentrations. Then, platelets were permeabilized with saponin (20 mg/ml final conc.) for 2 min and the suspensions were supplemented with Mg-ATP (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) and Mg-ATP (5 mM final conc.). CR was initiated by the addition of thrombin (1 U/ml final conc.). Further details in Methods. *p 0.01 vs sample without ATP (D).
sion in a dose-dependent manner (Figure 5). Tirofiban (250 mg/ml), a GPIIb/IIIa antagonist, reduced platelet adhesion by about 85%.
Experiments shown in Figure 6 were performed to assess whether HOCl alone or in combination with thrombin is able to evoke strong and sustained calcium signal.
Table I. Effect of hypochlorite on the kinetics of coagulation in PRP.
Table II. Effect of hypochlorite on the variables of fibrin polymerization profile induced by thrombin.
Addition
Addition
None (control) HOCl 100 mM HOCl 250 mM HOCl 500 mM HOCl 1000 mM
CT (sec)
a (degrees)
MCF (mm)
357 37 359 44 376 28 418 41* 541 34***
72 2 72 3 67 2 62 3** 58 3**
82 2 82 3 82 3 73 3* 65 2**
*p 0.05; **p 0.01; ***p 0.001.
None (control) HOCl 100 mM HOCl 250 mM HOCl 500 mM HOCl 1000 mM *p 0.05.
Lagc [s] 260 25 255 35 270 30 290 45 355 40*
MaxAbsC [abs] PR [abs/s 10 3] 1.04 0.08 1.03 0.13 1.01 0.06 0.97 0.07 0.72 0.16*
2.96 0.18 2.97 0.22 2.94 0.15 2.69 0.09 2.12 0.18*
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Hypochlorite and hemostasis 1449
Figure 5. Effect of hypochlorite on the adhesion of washed platelets to fibrinogen coated surfaces. Aliquots (1 ml) of washed platelets (4 108 cells/ml) were incubated in polypropylene tubes at 37°C for 2 min without (control) or with Tirofiban or HOCl added to the final concentration as indicated. After that, 50 ml 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 mg/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 Methods. The data represent the mean SD of six experiments, each performed in triplicate on a separate platelet preparation. *p 0.05; **p 0.01, ***p 0.005.
As is seen, HOCl, at concentrations 100–1000 mM, produced dose-dependent, rapid, and sustained rise in [Ca2]cyt (panel A). [Ca2]cyt raises were moderate to strong. 200 mM HOCl produced calcium signal similar to that evoked by 0.2 U/ml of thrombin. Preincubation of platelets with low micromolar HOCl (10–50 mM) resulted in a reduction of thrombin-evoked calcium signal (panel B). Combination of HOCl concentrations producing calcium signal similar to that evoked by 0.2 U/ml of thrombin (200 mM HOCl) with 0.2 U/ml of thrombin resulted in generation of calcium signal lower than that evoked by thrombin alone (lack of synergism, panel C). To assess whether HOCl affects platelet contractility through the inhibition of functional contractile apparatus formation, we measured its effect on the F-actin content in gel-filtrated platelets activated by thrombin. As can be seen in Figure 7, unlike cytochalasin B (actin polymerization blocker), HOCl (up to 250 nM) failed to affect F-actin formation kinetics. HOCl, up to the concentration of 100 mM, failed to affect platelet membrane integrity measured in artificial system as LDH release, while the threshold cytotoxic concentration of HOCl in PRP was 500 mM (Table III). Discussion For the first time, the results presented here show that relatively low HOCl concentrations affect the kinetics of clot shrinking. Our study revealed that HOCl strongly reduces the rate of retraction and augments the final clot
Figure 6. Effect of hypochlorite on cytosolic calcium concentration in porcine platelets. Fura-2-loaded platelets suspended in the medium containing 1 mM Ca2 were treated with HOCl alone (panel A) or with combination of HOCl plus thrombin (panels B–C) added to final concentrations as indicated. Changes in [Ca2]cyt were measured as described in Methods. Each trace is representative of at least six determinations performed in three different preparations.
volume at concentrations much lower (micromolar) than those previously reported (millimolar) to affect clotting factors [17], and lower than those inhibiting aggregation and secretion of porcine platelets [17–18]. This makes the possibility of HOCl action on CR in an in vivo condition much more likely. The susceptibility of CR to HOCl depends on the composition of the microenvironment in which this oxidant acts. Thus, the CR in an artificial system, consisting of washed platelets, fibrinogen, and thrombin, was inhibited by low nanomolar concentrations of HOCl, whereas in PRP and whole blood, CR was retarded by low and high micromolar concentrations, respectively (Figure 1A, B and D). The lower susceptibility of CR to HOCl in PRP and in whole blood is most likely associated with the rise in the number of potential targets of this oxidant. These observations also indicate that HOCl has its targets in plasma, in erythrocytes, and in platelets; these targets dif-
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1450 T. Misztal et al.
Figure 7. Effect of hypochlorite and cytochalasin B on the F-actin content in activated platelets. Gel filtered platelets (4 108 cells/ml) were incubated in polypropylene tubes at 37°C for 10 min with stirring, without (control) or with HOCl 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. Tritoninsoluble fraction was separated by 10% SDS-PAGE. The amount of actin in cytoskeletal fraction relative to total was quantified by densitometric analysis. Further details in Methods. Values represent means SD of 3 independent experiments (each in duplicate). (Δ) – control, () – HOCl (25 nM), () – HOCl (250 nM), () – cytochalasin B (20 mM). The longer incubation (up to 60 min) did not change F-actin content. *p 0.01.
fer in their susceptibility to stressor action. It is also evident that the most susceptible HOCl target in blood is confined to platelets. What could the mechanism underlying HOCl action on CR be? Platelet-dependent CR is a complex process in which several steps can be distinguished: the initial production of large quantities of thrombin (on the platelet surface), simultaneous formation of a loose fibrin network and activation of the platelet GPIIb/IIIa receptors (inside–out signaling), fibrin(ogen) binding with activated GPIIb/IIIa receptors, transmission of the contractile forces generated by the platelet actin–myosin cytoskeleton to connecting fibrin fibers around platelets (contraction), and conversion of a diffuse fibrin network into a dense platelet–fibrin clot (shrinking) [19,43–45]. Table III. Effect of hypochlorite on platelet integrity measured in PRP. LDH activity (% of total) Addition None (control) HOCl 50 mM HOCl 100 mM HOCl 200 mM HOCl 300 mM HOCl 500 mM
PLT
PRP
5.9 0.6 6.3 0.5 7.8 0.8* 8.1 1.1* 8.4 1.5** 9.1 1.3***
3.3 0.4 3.4 0.6 3.6 0.9 3.8 0.7 4.4 1 4.9 0.8*
*p 0.05; **p 0.01; ***p 0.001.
Therefore, the reduction of CR rate (CRR) reported here, at least potentially, may result from HOCl action on any of the above-mentioned steps. The results presented here strongly indicate that the effect of HOCl on CR is unlikely to be related to fibrin network formation. This is because the susceptibility of CR to HOCl action (measured in PRP) was markedly higher than that of clot formation evaluated by rotational thromboelastometry. Specifically, the effective HOCl concentrations modulating CR and clot formation in PRP were 50 and 500 mM, respectively (compare Figure 1B with Table I). What is more, CRR measured in an artificial system was reduced by 25 nM HOCl (Figure 1D). Likewise, direct HOCl action on clotting factors is also excluded, especially at low stressor concentrations ( 300 mM), since its effect on fibrin strand formation in plasma depleted of platelets was observed at concentrations above 1000 mM, that is, much higher than those for reducing CRR in PRP (compare Figure 1B with Table II). This is consistent with the observation of Stief et al., who demonstrated that inactivation of fibrinogen, factor V, factor VIII, and factor X, in human plasma was observed in the presence of 2–3 mM concentrations of HOCl [17]. Similarly, the high susceptibility of CR to HOCl action cannot be explained by its effect on platelet GPIIb/IIIa receptors. This is because adhesion of activated platelets to fibrinogen-coated surfaces (a phenomenon intrinsically dependent on the presence of activated integrin receptors) was reduced by oxidant concentrations much higher than those affecting CR. Particularly, the effective HOCl concentrations reducing CRR in an artificial system and the adhesion of washed platelets were 25 nM and 25 mM, respectively (compare Figure 1B with Figure 5). Extremely impaired platelet mitochondria, that is, mitochondria with mitochondrial permeability transition pore (MPTP) formed in a high conductance state, have been proposed to support calpain 1-evoked inactivation of GPIIb/IIIa receptors in platelets stimulated by very strong agonists, for example, a combination of high thrombin concentrations convulxin [46–49]. One can expect that, at least potentially, HOCl-uncoupled mitochondria may inactivate platelet GPIIb/IIIa receptors in a similar way. The involvement of high-conductance MPTP in HOClevoked retardation of CR is however highly unlikely, at least in the presence of lower (nanomolar) concentrations of stressor. This is because HOCl, at concentrations up to 100 mM, failed to evoke a high and sustained rise in intracellular calcium concentrations, a prerequisite for MPTP formation [46–48], in washed platelets suspended in a calcium-containing medium (Figure 6A). What is more, HOCl was able to reduce thrombin-evoked calcium signal (Figure 6B and C), indicating a lack of synergistic effect. Generation of strong and sustained calcium signal in washed platelets treated with higher (above 100 mM) stressor concentrations is most likely due to the loss of plasma membrane integrity, manifested by the LDH release (compare Figure 6 and Table III). It is, therefore, likely that inhibition of CR by low HOCl concentrations is associated with dysfunction of
Hypochlorite and hemostasis 1451
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the platelet contractile apparatus, rather than with abnormalities in fibrin formation or platelet GPIIb/IIIa function. Dysfunction of the platelet contractile apparatus is likely to suppress CR. This is based on the observations that the blocking of actin polymerization (a prerequisite for formation of a functional contractile apparatus) by cytochalasin B resulted in a significant reduction of CRR [20–21]. However, the direct action of HOCl on components of contractile apparatus is rather unlikely since, as we demonstrate here, the stressor concentrations reducing CR in an artificial system failed to affect the kinetics of F-actin formation in activated washed platelets (Figure 7). Reduced platelet contractility may also result from inefficient energy production. CR is an energy-requiring process, in which ATP hydrolysis is crucial for the contraction of platelet actomyosin [50]. It is now well accepted that the glycolytic pathway and mitochondrial oxidative phosphorylation are substantial for effective CR [20–21,50]. The results presented here indicate that the inhibitory effect of HOCl on CR is closely related to decreased platelet contractility resulting from inefficient energy production. Consistent with this is the observation that concentrations of HOCl affecting CRR decrease the total ATP contents in clots derived from PRP (Figure 3), and the finding that exogenously added Mg-ATP is able to abolish the inhibitory effect of HOCl on CR (Figure 4D). The results presented here also show that concentrations of HOCl affecting CRR reduce platelet oxygen consumption and augment lactate production, indicating the occurrence of the Pasteur effect (Figure 4A and B). Since in platelets, the glycolytic pathway and mitochondrial respiration are tightly functionally connected [51], this can be interpreted to mean that the stimulatory effect of HOCl on glycolysis in platelets may be related to the impairment of mitochondria. This is likely to be true since, as reported here, in porcine platelets HOCl is able not only to reduce mitochondrial oxygen consumption, but also to induce the loss of ΔYm (Figure 4C), which may result in diminishing the chemiosmotic potential, a prerequisite for ATP synthesis via oxidative phosphorylation. This is in agreement with several previous reports indicating that HOCl is able to evoke the dysfunction of mitochondria in various cell types [28–29]. In summary, these data clearly indicate that HOCl may affect in vitro CR through the inhibition of energy production in platelet mitochondria. To sum up, it is now clear those concentrations of HOCl much lower than those which alter clotting factors activity may reduce the shrinking of platelet–fibrin clots through the inhibition of energy production in platelet mitochondria. It is now well documented that activated inflammatory cells produce large quantities of substances used to kill pathogens. The group of such compounds comprises not only hypochlorous acid but also peroxynitrite (ONOO), formed in a very rapid reaction from NO and O2., produced by activated inflammatory cells [22]. Recently, we reported that physiologically relevant ONOO concentrations may inhibit CR, and accelerate
its lysis through the inhibition of platelet mitochondrial energy production [20]. It is, therefore, highly likely that both HOCl and ONOO may synergistically act on clot in vivo. The results presented here confirm such a possibility (Figure 1C). How can this be relevant to the in vivo situation? A recognized indicator of in vivo HOCl presence is elevated 3-chlorotyrosine [16]. Enhanced production of 3-chlorotyrosine has been reported in numerous clinical conditions associated with an inflammatory state including asthma [52], atherosclerosis [9], and inflammatory vasculitis [53]. The results presented here indicate that in platelets suspended in plasma, low micromolar concentrations of HOCl are able to suppress mitochondrial energy production. In washed platelets, these concentrations were much lower, reaching values of about 25 nM. Activated neutrophils were reported to form 150–425 mM/h of HOCl [54], whereas local concentrations of stressor in inflamed tissue are estimated to be as high as 5 mM [3,17]. At concentrations within the reported range generated by activated neutrophils (20–400 mM), HOCl induced a rapid loss of ∆Ym in human fetal liver and in hepatoma HepG2 cells [29]. It is, therefore, clear that pathophysiologically relevant HOCl concentrations, associated with an abnormal or inefficiently controlled inflammatory response, are likely to suppress mitochondrial energy production in platelets. Our data relate to clinical realities in two ways. First, abnormal CR may result in the formation of thrombi that are less mechanically stable and prone to embolization, and second, weekly shrinked (more loose) clots are expected to be more susceptible to fibrinolysis. Collectively, we conclude that clinically relevant HOCl concentrations associated with an inflammatory state may inhibit CR through the inhibition of platelet mitochondrial energy production. Declaration of interest The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper. This research was supported by Medical University of Bialystok [124-01500 F]. References [1] Eiserich JP, Hristova M, Cross CE, Jones AD, Freeman BA ,Halliwell B, van der Vliet A. Formation of nitric oxidederived inflammatory oxidants by myeloperoxidase in neutrophils. Nature 1998;391:393–397. [2] Nauseef WM. Insights into myeloperoxidase biosynthesis from its inherited deficiency. J Mol Med (Berl) 1998;76: 661–668. [3] Weiss SJ. Tissue destruction by neutrophils. N Engl J Med 1989 9;320:365–376. [4] Pullar JM, Vissers MC, Winterbourn CC. Living with a killer: the effects of hypochlorous acid on mammalian cells. IUBMB Life 2000;50:259–266.
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