Arsenite-Enhanced Procoagulant Activity through ... - ACS Publications

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Chem. Res. Toxicol. 2007, 20, 1760–1768

Arsenite-Enhanced Procoagulant Activity through Phosphatidylserine Exposure in Platelets Ok-Nam Bae,† Kyung-Min Lim,†,‡ Ji-Yoon Noh,† Seung-Min Chung,† Heon Kim,§ Choong-Ryeol Lee,| Jung-Duck Park,⊥ and Jin-Ho Chung*,† College of Pharmacy, Seoul National UniVersity, Seoul 151-742, Korea, College of Medicine, Chungbuk National UniVersity, Cheongju 361-763, Korea, Ulsan UniVersity Hospital, Ulsan 682-714, Korea, College of Medicine, Chung-Ang UniVersity, Seoul 156-756, Korea, and R&D Center, AMOREPACIFIC, Gyeonggi-do 446-729, Korea ReceiVed May 10, 2007

Numerous epidemiological studies have reported the close relationship between arsenic in drinking water and cardiovascular disease (CVD), yet the exact mechanism underlying this relationship remains unclear. We investigated whether arsenic can affect the procoagulant activity of platelets, which are essential in blood clotting, thrombus formation, and progression of CVD. While arsenite alone did not induce procoagulant activity, it significantly enhanced thrombin-induced procoagulant activity of human platelets in a concentration- and time-dependent manner. In flow cytometric analysis, arsenite potentiated phosphatidylserine (PS) exposure and microparticle (MP) formation, the major mediators of procoagulant activity. Arsenite-enhanced calcium increase and subsequent calpain activation were found to be involved in these effects, as determined by confocal microscopy and gel electrophoresis. Arsenite also inhibited flippase, an enzyme that restores PS to the inner leaflet, suggesting that PS could be retained in outer membranes after exposure. Consistent with these in vitro results, ex vivo studies revealed that PS exposure in platelets was significantly increased after acute or chronic arsenic exposure in rats. Most notably, in a rodent in vivo venous thrombosis model, arsenite indeed led to increased thrombus formation. In conclusion, arsenite-enhanced procoagulant activity in platelets by PS exposure and MP generation ultimately results in accelerated thrombus formation in vivo, suggesting that this enhanced activity is a possible contributing factor in CVD associated with chronic exposure to arsenic through drinking water. Introduction Arsenic is a widely distributed natural element in the environment, and humans are chronically exposed to arsenic through arsenic-contaminated foods or drinking water (1, 2). The predominant forms of arsenic in drinking water are inorganic arsenics, which are much more toxic than the organic forms found in contaminated foods, and inorganic arsenic and its methylated metabolites are known to be mainly responsible for the adverse health effects of arsenic to humans (1). Chronic ingestion of arsenic-contaminated drinking water was reported to cause a variety of diseases in humans, including cancers and neurological, developmental, and cardiovascular disorders (3). Millions of people are at risk for the adverse effects of arsenic exposure worldwide, and for this reason, many regulatory and public health authorities have ranked arsenic as one of the most severe environmental threats to human well-being (2). Several epidemiologic studies (4, 5) have revealed that cardiovascular mortalities were increased in arsenic-exposed populations. The prevalence of cardiovascular diseases (CVDs),1 such as hypertension, cerebrovascular diseases, atherosclerosis, * To whom correspondence should be addressed. Tel: +82-2-880-7856. Fax: +82-2-885-4157. E-mail: [email protected]. † Seoul National University. ‡ AMOREPACIFIC. § Chungbuk National University. | Ulsan University Hospital. ⊥ Chung-Ang University. 1 Abbreviations: CVD, cardiovascular disease; DMA5+, dimethylarsinic acid; FITC, fluorescein isothiocyanate; MMA5+, monomethylarsonic acid; MP, microparticle; PE, phycoerythrine; PS, phosphatidylserine.

and peripheral vascular diseases, was closely linked with chronic intake of arsenic-contaminated drinking water (6–9). Although many attempts have been made to clarify the mechanism of arsenic-induced CVD, it still remains unclear (10). Various in vitro cell systems, such as vascular smooth muscle cells, endothelial cells, and platelets, have been employed to elucidate this mechanism (11–13). Most of the studies involving these cell systems, however, have focused on cellular changes caused by arsenic, not on its role or interactions in pathological processes. In the development of various CVDs, platelets could actively participate through excessive aggregation (14, 15). More importantly, they could play a key role in the aggravation of CVDs by amplification of thrombus formation through interaction with the coagulation cascade (16). Promotion of blood coagulation by platelets, a phenomenon distinct from typical platelet aggregation, has been called procoagulant activity (17). Excessive procoagulant activity in platelets has been implicated in many CVDs, such as myocardial infarction, hypertension, atherosclerosis, and thrombus formation (18, 19). People with high risks of CVDs, such as diabetes mellitus, chronic uremia, and hyperlipidemia patients, exhibit elevated levels of platelet procoagulant activity, signifying its role in CVD development (20, 21). Platelet procoagulant activity is mediated by exposure of phosphatidylserine (PS) to outer membranes and/or generation of PS-bearing platelet microparticles (MPs) (22). These mediators are under the tight control of several enzymes, including phospholipid transporters and calpain, a calcium-dependent

10.1021/tx700159y CCC: $37.00  2007 American Chemical Society Published on Web 10/05/2007

Arsenite-Enhanced Procoagulant ActiVity in Platelets

cytoskeleton protease (23, 24). The exposed PS provides a site for assembly of coagulant enzymes, thereby leading to rapid thrombin generation and efficient blood clotting (25). Moreover, generated MPs, ranging from 0.1 to 1 µm in size, could circulate freely in the bloodstream, propagating the coagulation and transporting bioactive mediators such as arachidonic acid and tissue factors (26, 27). Recently, we demonstrated that arsenic increases the susceptibility of platelets to aggregation (12). The effects of arsenic on interactions of platelets with coagulation systems, however, have not yet been studied. In the present investigation, we have found that arsenic can enhance platelet procoagulant activity. The underlying mechanism was elucidated, and its clinical significance was explored using in vivo rat models. Here we suggest that arsenite-enhanced procoagulant activity mediated by PS exposure as well as MP generation is a possible contributing factor in the development of CVDs after chronic ingestion of arsenic.

Materials and Methods Materials. Sodium arsenite, sodium arsenate, dimethylarsinic acid (DMA5+), thrombin, citric acid, trisodium citrate, calcium ionophore A23187, bovine serum albumin (BSA), coomassie brilliant blue, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA), and fibrinogen were obtained from Sigma Chemical Co. (St. Louis, MO). Monomethylarsonic acid (MMA5+) was purchased from ChemService (West Chester, PA). Phycoerythrin (PE)-labeled monoclonal antibody against human glycoprotein Ib (anti-GP IbPE Ab), fluorescein isothiocyanate (FITC)-labeled monoclonal antibody against rat glycoprotein IIIa (anti-GP IIIa-FITC Ab), FITCand PE-labeled annexin V (annexin V-FITC and annexin V-PE, respectively), and purified annexin V were obtained from Pharmingen (San Diego, CA). Fluo-3 acetoxymethylester (fluo-3) and calcein AM were obtained from Molecular Probes (Eugene, OR). 1-Palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]caproyl]sn-glycero-3-phosphoserine (C6-NBD-PS) was obtained from Avanti Polar Lipids (Alabaster, AL). Purified human prothrombin (factor II), factor Xa, and factor Va were purchased from Haematologic Technologies, Inc. (Essex Junction, VT). Thromboplastin was obtained from Organon Teknika BRI (Rockville, MD), and all other reagents used were of the highest purity available. Animals. All of the protocols were approved by the Ethics Committee of the Animal Service Center at Seoul National University. Male Sprague-Dawley (SD) rats (Dae Han BioLink Co., Chungbuk, Korea) weighing 300–400 g were used in all experiments. Before the experiments, animals were acclimated for 1 week. Food and water were provided ad libitum. Preparation of Human Washed Platelets. With an approval from the Ethics Committee of the Health Service Center at Seoul National University, human blood was obtained from healthy male donors (18–25 years old) on the day of the experiments using acid–citrate–dextrose (ACD) as an anticoagulant in the presence of prostaglandin E1 (1 µM). Human washed platelets were prepared by differential centrifugation as previously described (12). Briefly, after isolation of platelet-rich plasma by centrifugation of blood at 150g for 15 min, platelets were pelleted by centrifugation at 500g for 10 min. The pellet was resuspended with Tyrode buffer (134 mM NaCl, 2.9 mM KCl, 1.0 mM MgCl2, 10.0 mM HEPES, 5.0 mM glucose, 12.0 mM NaHCO3, 0.34 mM Na2HPO4, and 0.3% BSA, pH 7.4) containing 1 µM prostaglandin E1 and 10% ACD. After centrifugation at 500g for 10 min, the platelets were resuspended in Tyrode buffer to a cell concentration of 3 × 108 cells/mL, and the final CaCl2 concentration was adjusted to 2 mM prior to use. Measurement of Procoagulant Activity. After incubation with arsenite or vehicle (saline), platelets were diluted to 1 × 107 cells/ mL and challenged with a subthreshold concentration of thrombin (1 unit/mL) for 5 min. An aliquot was incubated with 5 nM factor

Chem. Res. Toxicol., Vol. 20, No. 12, 2007 1761 Xa and 10 nM factor Va in Tyrode buffer containing 2 mM CaCl2 for 3 min at 37 °C. Thrombin formation was initiated by the addition of 2 µM prothrombin. Exactly 3 min after the addition of prothrombin, an aliquot of the suspension was transferred to a tube containing EDTA stop buffer (50 mM Tris HCl, 120 mM NaCl, and 2 mM EDTA, pH 7.9). Thrombin activity was determined using the chromogenic substrate S2238. The rate of thrombin formation was calculated from the change in absorbance at 405 nm using a standard calibration curve. In experiments using purified annexin V, platelets were incubated with annexin V (final concentration 2 µM) for 10 min after arsenic treatment and thrombin addition; after incubation, the prothrombinase assay was conducted as described above. Determination of PS Exposure and MP Generation. Annexin V-FITC was used as a marker for PS detection, while platelets were identified by anti-GP Ib-PE. Negative controls for annexin V binding were stained with annexin V-FITC in the presence of 4 mM EDTA. Platelets were incubated with annexin V-FITC and GP Ib-PE for 20 min and analyzed on a PAS IIIi flow cytometer (Partec, Münster, Germany) equipped with an argon laser (λex ) 488 nm). Data from 5000 events were collected and analyzed using Winlist software. MPs were identified on the basis of forward-scatter characteristics (FSC) after calibration using 1 µm standard beads. In experiments using EGTA, platelets were preincubated with EGTA (final concentration 5 mM) for 5 min and exposed to arsenic for 1 h. After thrombin (1 unit/mL) was added to initiate platelet activation, PS exposure and MP generation were examined as described above. Observation of MPs Using Confocal Microscopy. To prepare the samples for microscopic observation, platelets were immobilized as described previously (28). Platelets (3 × 107 cells/mL) suspended in HEPES buffer (10 mM HEPES, 136 mM NaCl, 5 mM glucose, 2.7 mM KCl, 2 mM MgCl2, and 0.05% BSA, pH 7.45) were attached to Laboratory-Tek coverslips (Nunc, Roskilde, Denmark) coated with fibrinogen (10 mg/mL) for 20 min. Adhered platelets were exposed to arsenite for 1 h, and then thrombin was added. After being labeled with platelet-specific GP Ib antibodies, platelets and MPs were analyzed on a confocal microscope (Leica, Wetzlar, Germany) equipped with an argon laser (λex ) 488 nm). Isolation of Generated MPs. After treatment with arsenite, platelets were challenged with thrombin (1 unit/mL) for 5 min, and then the reaction was stopped by addition of 4 mM EDTA. In order to isolate the MPs, the platelets were pelleted by centrifugation at 1000g for 10 min, and the supernatant was collected. After further centrifugation at 2000g for 10 min, the MP-containing supernatant was isolated, and an aliquot was subjected to the prothrombinase assay as described above. Procoagulant activity was expressed as the change in absorbance (∆Abs) at 405 nm because accurate counts of MPs for calculation could not be obtained. Measurement of Intracellular Calcium. Intracellular calcium levels were determined using the methods described by Heemskerk et al. (28). After preparation of immobilized platelets, fluo-3 (5 µM) was loaded in the presence of apyrase (0.2 unit/mL) for 45 min at 37 °C. The platelets were washed twice with HEPES buffer and incubated with arsenite for 1 h. Thrombin-induced calcium increases were observed using confocal microscopy and quantified in terms of changes in fluorescence intensity. Measurement of ATP Levels. ATP levels were determined using the luciferin/luciferase bioluminescence assay with perchloric acid extracts. For extraction, equal volumes of ice-cold 1 M HClO4 and arsenite-treated platelets were combined and then incubated for 30 min on ice. After the extracts were centrifuged at 12000g for 2 min, the supernatants were neutralized by the addition of 1 M K2CO3. Precipitated KClO4 was removed by centrifugation at 12000g for 10 min, and the supernatant was collected as perchloric acid extracts. After dilution with TAE buffer (100 mM Tris acetate and 2 mM EDTA, pH 7.8), samples were adapted to the luciferin/ luciferase assay in a Luminoskan luminometer (Labsystems, Franklin, MA) using an ATP assay kit (Sigma). ATP levels were

1762 Chem. Res. Toxicol., Vol. 20, No. 12, 2007 expressed as the percentage of the basal ATP level in fresh, untreated platelets. The basal ATP level was 67.7 ( 2.2 pmol/106 platelets. Assay of Cytoskeleton Proteolysis by Calpain. Platelets in BSA-free Tyrode buffer were incubated with arsenite and challenged with thrombin. After addition of EDTA to stop the reaction, an aliquot was mixed with Laemmli sample buffer (62.5 mM Tris HCl, 2% SDS, 25% glycerol, and 0.01% bromophenol blue) and incubated for 3 min at 95 °C. Platelet proteins were electrophoresed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (7% gel) and visualized by staining the gel with coomassie blue. The density of each protein was determined by TINA software (Raytest, Straubenhardt, Germany). Measurement of Flippase and Scramblase Activity. The activity of flippase was measured using the method described by Smeets et al. (29). Platelets were treated with various concentrations of arsenite in BSA-free buffer for 1 h. After the platelets were challenged with thrombin, C6-NBD-PS (1 µM) was added, and internalization of C6-NBD-PS was measured using BSA backexchange. Aliquots were collected at the indicated time and immediately placed on ice for 20 min in the presence or absence of 1% BSA. Pellets were obtained by centrifugation at 12000g for 3 min and lysed in 1% Triton X-100. The amount of internalized probe was determined by comparing the fluorescence intensities (λex ) 485 nm, λem ) 535 nm) before and after back-exchange. To determine the activity of scramblase, a dithionite assay was conducted as previously described (30). Platelets were labeled with fluorescent NBD lipid by incorporation of C6-NBD-PS (1 µM) into the inner membrane. Activity of scramblase was measured by the loss of fluorescence, which indicates exposure of the NBD lipids to the outer leaflet. Before challenge with thrombin (1 unit/mL), sodium dithionite (5 mM) was added in order to bleach the fluorescence of exposed NBD lipid. Basal fluorescence was measured after the addition of 1% Triton X-100, making all NBD groups accessible to dithionite. Detection of PS Exposure following Arsenic Administration in Rats. After SD rats were exposed to arsenic via drinking water (0, 1, 5, 10, and 25 ppm) for 4 weeks or intraperitoneal administration (0, 1, 2, and 5 mg/kg), blood was collected from the abdominal aorta using 3.8% trisodium citrate as an anticoagulant. An aliquot of blood sample was diluted 40-fold with Tyrode buffer containing 2.5 mM Gly-Pro-Arg-Pro (Calbiochem, San Diego, CA) to prevent thrombin-induced fibrin polymerization. Diluted whole blood samples were challenged with thrombin (1 unit/mL) for 5 min and stained with annexin V-PE and anti-rat GP IIIa-FITC Ab for 20 min in the dark. PS exposure was measured as described above. Rat Venous Thrombosis Model. Thrombus formation was induced by stasis combined with hypercoagulability as previously described (31). After SD rats were anesthetized with urethane (1.25 g/kg ip), the abdomen of each rat was surgically opened, and the vena cava was carefully exposed. Two loose cotton thread loops were placed 16 mm apart around the vena cava. All side branches were ligated tightly with cotton threads. Thirty minutes after intravenous injection of saline or sodium arsenite into a left femoral vein, 1000-fold diluted thromboplastin was infused for 2 min to induce thrombus formation. Stasis was initiated by tightening the two threads, first the proximal and then the distal. The abdominal cavity was provisionally closed, and blood stasis was maintained for 15 min. After the abdomen was reopened, the ligated venous segment was excised and opened longitudinally to collect the generated thrombus. Isolated thrombus was blotted to remove excess blood and immediately weighed. Statistics. We calculated the means and their standard errors for all treatment groups. The data were subjected to one-way analysis of variance followed by Duncan’s multiple range test to determine which means were significantly different from the control values. Statistical analysis was performed using SPSS software (Chicago, IL). In all cases, p < 0.05 was considered significant.

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Figure 1. Enhancement of procoagulant activity by arsenite in thrombinchallenged human platelets. Human washed platelets were treated with arsenite for 1 h and then with a subthreshold concentration of thrombin (1 unit/mL) for 5 min. Aliquots of platelets were subjected to a prothrombinase complex assay to determine platelet procoagulant activity, which was calculated from the amidolytic activity of factor IIa (thrombin) generated from factor II (prothrombin) using the chromogenic subtrate S2238. Arsenite treatment resulted in (A) concentration- and (B) time-dependent enhancement of human platelet procoagulant activity. (C) Effects of MMA5+, DMA5+, and arsenate on procoagulant activity of thrombin-challenged platelets (50 µM, 1 h). Values are reported as mean ( SE of three to five independent experiments. *p < 0.05 compared with the corresponding control.

Results To investigate whether arsenic could affect procoagulant activity, isolated human platelets were treated with various concentrations of arsenite (As3+) for 1 h, and a subthreshold level of thrombin was challenged to initiate platelet activation. While arsenite alone did not affect procoagulant activity in platelets (data not shown), it did enhance thrombin-induced procoagulant activity in a concentration- and time-dependent manner (Figure 1A,B). To elucidate whether other arsenicals could also affect agonist-induced procoagulant activity, we examined the effects of the pentavalent inorganic species arsenate (As5+) and two major metabolites of inorganic arsenic, MMA5+ and DMA5+. In contrast to arsenite, MMA5+, DMA5+, and arsenate all failed to increase platelet procoagulant activity (Figure 1C). It is well known that platelet procoagulant activity is mediated by PS exposure in the outer membrane (16). To investigate how arsenite affects platelet procoagulant activity, we examined the extent of PS exposure using flow cytometry. Treatment with arsenite substantially increased the number of PS-specific annexin V binding platelets (Figure 2A,B). When the exposed PS was blocked using purified annexin V, the procoagulant effect of arsenite was attenuated significantly, suggesting that PS exposure is critical in arsenite-enhanced procoagulant activity (Figure 2C). In addition to PS exposure, shedding of PS-bearing MPs could play a role in platelet procoagulant activity (32). Treatment with arsenite significantly increased thrombin-induced MP generation, as determined by flow cytometry (Figure 3A,B). Generation of MP by arsenite was confirmed using confocal microscopy (Figure 3C). Procoagulant activity was also found


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Figure 2. Potentiation of PS exposure by arsenite in thrombin-challenged platelets. Human platelets were treated with arsenite for 1 h and then challenged with thrombin for 5 min. Flow cytometric analysis was employed to determined PS exposure, as described in Materials and Methods. (A) Representative histograms and (B) percentages of cells expressing PS are shown. FITC signals were analyzed to determine the binding of annexin V to exposed PS on the outer membrane, and cells were considered to be positive when the fluorescence intensity was >99% of the signal from an EDTA negative control group. (C) Effect on procoagulant activity resulting from treatment with purified annexin V to block PS exposure induced by arsenite. Values are reported as mean ( SE of three to five independent experiments. *p < 0.05 compared with the corresponding control. #p < 0.05 compared with the group treated with arsenite in the absence of annexin V.

with generated MPs (Figure 3D), suggesting that arsenite could enhance procoagulant activity through MP generation as well. Since calcium influx into platelets is known to be a key mechanism of PS exposure and MP generation, the role of calcium in arsenite-induced events was investigated with EGTA, a calcium chelator. Both PS exposure and MP generation by arsenic were significantly attenuated in the presence of EGTA (5 mM), implying that intracellular calcium increase plays an important role in the effect of arsenic on platelets (Figure 4A). When the intracellular calcium level was monitored using fluo-3, a calcium-specific dye, arsenite treatment potentiated the increase of intracellular calcium elicited by thrombin addition (Figure 4B). Disregulation of calcium homeostasis could be induced by depletion of intracellular ATP. When platelets were exposed to arsenite, intracellular ATP was depleted significantly (Figure 4C), suggesting that arsenic effects might be mediated by ATP loss. In conjunction with the intracellular calcium increase, activation of calpain, a calcium-dependent protease, is known to mediate MP generation and PS exposure through degradation of cytoskeletal proteins (33). To investigate the role of calpain, procoagulant activity was measured in the presence of calpeptin, an inhibitor of calpain. Calpeptin attenuated the effect of arsenite significantly (Figure 4D), reflecting the fact that the activation of calpain plays a key role in arsenic-enhanced procoagulant activity in platelets. Arsenite-induced activation of calpain was also observed by degradation of cytoskeletal proteins (Figure 4E). Besides the calcium–calpain pathway, platelet PS exposure could be induced by disturbance of phospholipid transporters (34). Flippase (aminophospholipid translocase) maintains an asymmetry in a cell membrane by unidirectional transportation of PS to the inner leaflet (23). Activation of scramblase, on the other hand, induces rapid and nonspecific scrambling of membrane lipids across the lipid bilayer (19). In order to

examine the effect of arsenite on these enzymes, the activities of flippase and scramblase, respectively, were determined by measuring inward and outward translocation of NBD-PS, a fluorescence-tagged derivative of PS. As shown in Figure 5A, flippase activity was inhibited by arsenite treatment, suggesting that exposed PS could be retained in the outer leaflet, possibly resulting in prolonged procoagulant activity. In contrast, activation of scramblase was not observed (Figure 5B), showing that scramblase is not involved in arsenite-induced PS exposure. In order to investigate the in vivo relevance of arseniteenhanced procoagulant activity, we examined the extent of thrombin-induced PS exposure following administration of arsenite to SD rats. Prior to the in vivo studies, we studied whether the effect of arsenite on procoagulant activity in isolated rat platelets was equivalent to that in human platelets. We found that arsenite enhanced thrombin-induced PS exposure as well as procoagulant activity in rat platelets to an extent similar to that in human platelets (Figure 6A). After an acute exposure to arsenic by intraperitoneal injection, thrombin-induced PS exposure was significantly enhanced in rat platelets (Figure 6B). In addition, when rats were chronically exposed to arsenic via drinking water for 4 weeks, a statistically significant trend of increase was observed (linear regression analysis, p ) 0.042) in thrombin-challenged PS expression in platelets, suggesting that arsenic could increase platelet procoagulant activity in vivo (Figure 6C). Procoagulant activity of both platelets and platelet-derived MPs can accelerate thrombus formation, ultimately contributing to the aggravation of thrombotic CVDs (19). In an attempt to explore the in vivo significance of arsenite-enhanced procoagulant activities in platelets, the effect of arsenite on thrombus formation was investigated in a rat venous thrombosis model, where both blood coagulation and platelets are known to play key roles in thrombus formation (31, 35). Thrombus formation

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Figure 3. Arsenite-enhanced generation of MPs from thrombin-challenged platelets. After treatment with various concentrations of arsenite, thrombin (1 unit/mL) was added to human platelets. (A) Representative dot plots from flow cytometric analysis and (B) percentages of generated MPs are shown. Platelets and MPs were identified by PE-conjugated antibody against glycoprotein Ib; MPs were characterized as particles smaller than 1 µm control beads. (C) Confocal microscopy images confirmed the generation of MPs from immobilized platelets. (D) Procoagulant activity of isolated MPs was examined with the prothrombinase complex assay. Values are reported as mean ( SE of four to six independent experiments. *p < 0.05 compared with the corresponding control.

was increased in a dose-dependent manner following arsenic exposure, as measured by thrombus weight increases (Figure 7). This result implies that arsenic could enhance platelet procoagulant activity through PS exposure and/or MP generation, leading to increased thrombus formation.

Discussion In the present investigation, we have demonstrated that arsenic increases PS exposure and MP generation induced by thrombin, an endogenous agonist, resulting in enhancement of procoagulant activity in human platelets (Figure 8). These in vitro results were confirmed using in vivo animal models, in which both PS exposure and venous thrombosis were increased by acute or chronic arsenic exposure. This is the first study to demonstrate that an environmental toxicant can induce PS exposure and concomitant generation of MPs, resulting in increased procoagulant activity in platelets and ultimately contributing to the development of CVDs. Humans are exposed to arsenic mainly through drinking water, in which arsenic exists in inorganic forms at concentrations as high as milligrams per liter (1). The concentrations of arsenic in drinking water have been reported to be 3.2 mg/L in West Bengal, 3.05 mg/L in Vietnam, and 1.86 mg/L in China, corresponding to a molar concentration range of 25.23–42.72 µM (36–38). In human blood, arsenic concentrations can reach 42.1–48.1 µg/L (0.56–0.64 µM) after chronic ingestion of arsenic (39, 40). In the current investigation, arsenite induced MP generation after acute 1 h exposures at concentrations as low as 10 µM (Figure 3B), which is within an order of magnitude of the arsenic levels reported in epidemiologic

studies. The viability and integrity of freshly isolated platelets are difficult to maintain for an extended period of time, severely impeding the investigation of long-term toxicant effects. Nevertheless, arsenite exerted a significant effect on platelet function in a time- and concentration-dependent manner (Figure 1), indicating that arsenite-induced procoagulant activity of platelets could occur at much lower arsenic concentrations under real clinical conditions, where longer or chronic exposure is common. Previously, we reported that arsenite-enhanced platelet aggregation was accompanied by increases in serotonin secretion and P-selectin expression (12). However, platelet procoagulant activity, the focus of the current investigation, has been identified as a phenomenon distinct from typical platelet aggregation in several respects, such as differences in the potencies of agonists and in the time dependence of response manifestations (17). In support of this view, local anesthetics and sulfhydryl reagents such as diamide were shown to induce only procoagulant activity in platelets (41, 42). Since enhanced procoagulant activity leads to acceleration of the coagulation cascade and a further increase in formation of the coagulation product, thrombin, procoagulant activity has been suggested to work as a positive feedback mechanism in synergy with aggregation in the activation of platelets, playing an important role in hemostasis and thrombosis (15, 43). The fact that arsenite can enhance procoagulant activity as well as aggregation in platelets (12) underscores the active role of platelets in arsenite-associated CVDs. Combined with the arsenite-induced decrease of fibrinolytic activity found in a previous study (44), these procoagulant activity and proaggregatory effects of arsenite would lead to the potent


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Figure 4. Involvement of the calcium–calpain pathway in arsenite-enhanced procoagulant activity. (A) Arsenic-enhanced PS exposure and MP generation were examined after pretreatment with EGTA (5 mM) for 5 min. (B) Kinetic studies of [Ca2+]i levels were performed using confocal microscopy. After fluo-3-loaded platelets were treated with arsenite for 1 h, time-lapsed changes in fluorescence were recorded following thrombin (Thr) addition. Traces are Ca2+ responses of single platelets that are representative of 20 cells from at least four independent experiments. The inset shows the mean peak fluorescence intensity of the individual platelets. N denotes the normal control group without thrombin addition. (C) After arsenite treatment for 60 min, the ATP level was measured using the luciferin/luciferase assay. Values are expressed as the percentage of the basal ATP level from fresh platelets. (D) Effect of calpeptin (100 µg/mL, 5 min), a calpain inhibitor, on arsenic-enhanced procoagulant activity. (E) Activation of calpain was directly evaluated by measuring the degradation of cytoskeletal proteins using gel electrophoresis and coomassie blue staining. Arrowheads indicate the original cytoskeletal proteins, and arrows designate the 135 and 93 kDa proteolytic fragments of actin binding protein (ABP; 270 kDa), the 190 kDa fragment of talin (235 kDa), and the 135 kDa fragment of the heavy chain of myosin (200 kDa). A23187 was used as a positive control. The bar graph on the right shows the results of densitometric analysis of the 93 kDa fragments. Values are reported as mean ( SE of four independent experiments. *p < 0.05 compared with the corresponding control. #p < 0.05 compared with the group treated with arsenite in the absence of EGTA or calpeptin.

Figure 5. Effect of arsenite on phospholipid-transporting enzymes in platelets. (A) The effect of arsenite on flippase was examined using C6NBD-PS. After thrombin was added to arsenite-exposed platelets, NBDPS was loaded for 5 min. The amount of internalized probe was calculated by comparing the NBD fluorescence intensities associated with platelets before and after back-exchange using 1% BSA. Values are reported as mean ( SE of four independent experiments. *p < 0.05 compared with the corresponding control. (B) The activity of scramblase was investigated using a dithionate assay. Redistribution kinetics of NBD-PS was analyzed in control and arsenite-treated platelets after thrombin addition. The inset shows the loss of NBD fluorescence through activation of scramblase by A23187, a positive control. Representative tracings from one to three independent experiments are shown.

augmentation of thrombosis in arsenic-exposed populations. In fact, increased levels of coagulation factors and platelet ag-

gregation had been found in parallel with impaired fibrinolysis in arsenic-exposed humans (45, 46), indicating that derangement of normal hemostasis could play a key role in the arsenicinduced development of CVDs. Increased expression of PS on platelets and/or generation of circulating MPs could be indicative of various CVDs as well as high-risk conditions for CVDs (21, 47). For example, in patients with unstable angina, an increase of PS exposure in platelets was observed after coronary angioplasty and stent implantation (48). PS exposure and/or MP generation can be readily measured using simple whole-blood flow cytometry without the complex platelet-preparation steps required for evaluation of aggregation (49). PS exposure and MP shedding could be handy biomarkers reflecting in vivo conditions of patients that may be useful in predicting the development of CVDs (50). In this regard, it could be interesting to investigate these biomarkers in populations known or suspected to have arsenic exposure in order to explore their potential risk for CVDs. Although procoagulant activity was not increased by arsenite alone, it was greatly enhanced in the presence of both arsenite and thrombin, a representative endogenous agonist. While thrombin is known to be a potent agonist for initiating aggregation, it has been reported to be extremely weak in inducing procoagulant activity in platelets (16). Considering that


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Figure 8. Suggested mechanism of arsenite-enhanced procoagulant activity of platelets.

Figure 6. In vitro and ex vivo enhancement of procoagulant activity by arsenite in rat platelets.(A) Isolated rat platelets were exposed to arsenite for 1 h and then challenged with a subthreshold concentration of thrombin. The extents of PS exposure and procoagulant activity were analyzed using flow cytometric analysis and the prothrombinase complex assay, respectively. The effect of arsenite on platelet PS exposure was examined ex vivo after (B) acute treatment with arsenite via ip administration and (C) chronic exposure to arsenic via drinking water for 4 weeks. PS exposure was determined using whole-blood flow cytometry after stimulation by thrombin (1 unit/mL). Values are reported as mean ( SE of 4–13 independent experiments. *p < 0.05 compared with the corresponding control.

Figure 7. Increase of thrombus formation by arsenite in an in vivo rat venous thrombosis model. Arsenite increased thrombus formation in the venous thrombosis model. After iv infusion of arsenite, thromboplastin was infused to initiate thrombus formation. Stasis-induced thrombosis was induced in the vena cava, and the isolated thrombus was weighed. Values are reported as mean ( SE of five to six independent experiments. *p < 0.05 compared with the corresponding control.

platelets constantly experience various stimuli, especially from the large number of agonists at a site of clot formation or vascular injury (51), these enhancing effects of arsenite may well appear under in vivo conditions, accelerating thrombin generation and ultimately contributing to the aggravation of thrombosis. Increase of intracellular calcium levels was the major mechanism of arsenite-enhanced procoagulant activity, as confirmed by the significant attenuation of the effect of arsenic in the presence of EGTA (Figure 4A). Disruption of intracellular calcium homeostasis is known to be a key event in various effects of arsenic (52) and could be induced by abnormal

sequestering of increased calcium levels resulting from the inhibition of calcium-ATPase or intracellular ATP depletion. As shown in Figure 4C, arsenite treatment significantly induced ATP depletion, implying that arsenite-induced ATP loss could contribute in part to the impaired regulation of intracellular calcium. In addition, a previous study (53) reported that the activity of calcium-ATPase could be inhibited by arsenite in liver microsomes, suggesting that the inhibition of calciumATPase might be involved in arsenic-enhanced calcium increase in platelets. In support of this, inhibition of calcium-ATPase by certain chemicals has been reported to lead to the potentiation of agonist-induced platelet responses, including platelet procoagulant activity (29, 54). Figure 4D,E shows that arsenicenhanced calcium increase resulted in subsequent activation of calpain, which plays an important role in platelet procoagulant activity. In platelets, µ-calpain is known to be the calpain isoform responsible for the degradation of cytoskeletal proteins (55). Activation of µ-calpain requires calcium increases in the micromolar range, which could not be accomplished by thrombin alone. Actually, in our experiments, thrombin alone increased the intracellular calcium level to only 547 nM, while arsenite (50 µM) enhanced thrombin-challenged calcium increases to levels as high as 2250 nM, supporting the conclusion that arsenic-enhanced calcium increase is critical in calpain activation and the resultant enhancement of procoagulant activity. In addition to the calcium–calpain pathway, modification of intracellular thiol status by arsenite could be a possible mechanism for the enhancement of PS exposure and resultant procoagulant activity. Several thiol-depleting agents, such as diamide and N-ethylmaleimide, could lead to PS exposure, mainly through inhibition of flippase activity (17, 23). Supporting this view is the fact that arsenite, which is known to have a higher affinity for intracellular thiol than do other pentavalent arsenicals, was the only arsenic species among those tested here that was effective in enhancing procoagulant activity (Figure 1C). Although the total protein thiol level was not affected by arsenite treatment (data not shown), alteration of the specific thiol status of certain enzymes, including flippase, needs to be investigated further in order to elucidate the detailed mechanism. In summary, we have demonstrated that arsenite enhanced thrombin-induced procoagulant activity in platelets through calcium/calpain-mediated PS exposure and MP generation, resulting in increased thrombus formation. These arseniteinduced synergistic activities of platelets on coagulation systems


could be a possible contributing factor in CVD associated with chronic exposure to arsenic via drinking water. Acknowledgment. This work was supported by the NITR project of the Korea Food & Drug Administration (KFDA) and by the Eco-Technopia 21 project of the Ministry of Environment.

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