Procoagulant Effects of Isothiazolinone Biocides, Benzisothiazolinone and Octylisothiazolinone in Platelets

TL;DR

This study shows that isothiazolinone biocides BIT and OIT can trigger harmful blood clotting effects by damaging platelets, potentially increasing cardiovascular disease risk.

Contribution

The study reveals a novel procoagulant mechanism of BIT and OIT through mitochondrial dysfunction and platelet activation.

Findings

BIT and OIT deplete glutathione and increase mitochondrial ROS in platelets.

Mitochondrial dysfunction caused by BIT and OIT leads to procoagulant activity via phosphatidylserine externalization.

N-acetyl-L-cysteine reduces BIT- and OIT-induced effects, indicating a thiol-dependent mechanism.

Abstract

Isothiazolinones are commonly used biocides that are extensively used in industrial areas and household products. The extensive usage of isothiazolinones raises concerns regarding their adverse human health effects. Isothiazolinones are readily absorbed and enter circulation. However, the potential systemic effects of isothiazolinones on the circulatory system remain unclear. Here, we examined whether the isothiazolinones, benzisothiazolinone (BIT) and octylisothiazolinone (OIT) affected platelets. In isolated platelets, BIT and OIT depleted intracellular glutathione, which led to mitochondrial reactive oxygen species (ROS) accumulation. Excessive mitochondrial ROS led to mitochondrial dysfunction, altering intracellular calcium and adenosine triphosphate homeostasis. These intracellular events activated phospholipid scramblase, externalizing phosphatidylserine, thereby enhancing procoagulant activity, as evidenced by thrombin generation. Overall, OIT showed a more potent effect than BIT. Notably, supplementation with N-acetyl-L-cysteine mitigated BIT- and OIT-induced effects, suggesting a thiol-dependent mechanism. Taken together, BIT and OIT stimulated the platelet-mediated coagulation pathway, which may increase prothrombotic risk and contribute to cardiovascular disease. These results could improve our understanding of the systemic adverse effects after isothiazolinone exposure.

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1. Introduction

Isothiazolin-3-ones (isothiazolinones) are common biocides that possess potent, broad-spectrum antimicrobial activity, highly effective against bacteria, fungi, and algae [1,2]. Owing to the remarkable antimicrobial abilities, isothiazolinones are extensively used in industrial and household applications, water treatment, cosmetics and personal care products, and antifouling [1,3]. Beyond the increased use of isothiazolinones, concerns about human health have arisen [4,5,6], the potential systemic adverse effects remain poorly understood.

1,2-benzisothiazolin-3-one (benzisothiazolinone, BIT), 2-n-octyl-4-isothiazolin-3-one (octylisothiazolinone, OIT), and 4,5-dichloro-2-n-octyl-isothiazolin-3-one (dichlorooctylisothiazolinone, DCOIT) are commonly used isothiazolines that can be easily found in various commercial products such as paints, coatings, adhesives, textiles, leather, plastic, rubbers, detergents, cleaners, fabric softeners, and air fresheners [1,3]. As these isothiazolinones can be readily absorbed through various routes, assessing their toxicity and elucidating the underlying mechanisms is crucial for understanding the potential risk to human health from isothiazolinone exposure.

The electrophilic sulfenamide (S-N) bond of isothiazolinones can react with the nucleophilic thiol groups of cellular components. The biocidal effects of isothiazolinone are due to its reaction with intracellular thiol groups in key proteins, enzymes, and glutathione (GSH), blocking vital cellular functions, disrupting cellular structure, and leading to irreversible cell death [7]. It has been reported that isothiazolinones affect dehydrogenases, adenosine triphosphate (ATP) synthase, histone acetyltransferases, and tyrosine kinases. Several studies have suggested that thiol depletion by isothiazolinones could manifest toxic effects on mammalian cells, including neuronal cells [8], skin cells [9], liver cells [10], peripheral blood leukocytes [11,12], endothelial cells [13,14,15], and vascular smooth muscle cells [16].

Isothiazolinones can be absorbed through dermal, oral, and inhalation exposure from consumer and industrial products [6,17]. Systemically absorbed isothiazolinones can easily enter the bloodstream and may interact with components of the circulatory system. In this context, the toxic effects of isothiazolinones on endothelial cells, vascular smooth muscle cells, and peripheral blood leukocytes have been reported [11,12,13,14,15,16]. However, the effects of isothiazolinones on platelets have been studied only to a limited extent [18].

Platelets are prominent cells in hemostasis, playing a protective role against bleeding by forming stable plugs at the vascular injury sites through platelet activation [19]. However, excessive platelet activation could promote pathological thrombosis, contributing to the pathophysiology of cardiovascular diseases, including ischemic heart disease, stroke, hypertension, and atherosclerosis [20,21]. During platelet activation, platelets tend to aggregate and stimulate the coagulation pathway, thereby actively contributing to thrombus formation [22]. Activated platelets externalize phosphatidylserine (PS) to the outer membrane, enhancing procoagulant activity [23,24,25].

In this study, we investigated the effects of isothiazolinones on intracellular pathways in platelets and resultant procoagulant effects. We elucidated the procoagulant effects of isothiazolinones and the underlying mechanism to improve understanding of the potential adverse health effects of isothiazolinone exposure.

2. Materials and Methods

2.1. Materials

BIT, DCOIT, and OIT were obtained from Tokyo Chemical Industry (TCI, Tokyo, Japan). Ethylenediaminetetraacetic acid (EDTA), N-acetyl-L-cysteine (NAC), Triton X-100, and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 5,6-chloromethyl-2,7-dichlorodihydrofluorescein diacetate (CM-H_2_DCFDA), MitoSOX^TM^ Red, Fluo-4 acetoxymethyl ester (Fluo-4 AM), Pluronic F-127, and BCA protein assay kit were obtained from Thermo Fisher Scientific (Rockford, IL, USA). JC-1 was from Cayman Chemical (Ann Arbor, MI, USA). 1-oleoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazole-4-yl)amino]hexanoyl]-sn-glycero-3-phosphocholine (C6-NBD-PC) was purchased from Avanti Polar Lipids (Alabaster, AL, USA). Phycoerythrin hamster anti-mouse CD61 (anti-CD61-PE) was obtained from BD Biosciences (Bergen County, NJ, USA). Fluorescein isothiocyanate annexin V (annexin V-FITC) was obtained from Biolegend (San Diego, CA, USA). Purified human factor Xa and factor Va were purchased from Prolytix (Essex Junction, VT, USA). Purified human prothrombin was obtained from Enzyme Research Laboratories (South Bend, IN, USA). Chromogenic substrate for thrombin was purchased from MyBioSource (San Diego, CA, USA). Purified human thrombin was purchased from Merck Millipore (Burlington, MA, USA). All other reagents used were of the highest purity available.

2.2. Isolation of Platelets

Animal experiments were performed under approval of the Kangwon National University Animal Care and Use Committee (KW-200529-1). Male Sprague-Dawley (Envigo) rats weighing 250~300 g were obtained from Koatech (Pyeongtaek, Republic of Korea). Rats were acclimated for 1 week before use, and food and water were supplied ad libitum.

To prepare washed platelets (WP), rats were anesthetized, and whole blood was collected from the abdominal aorta and anti-coagulated with acid citrate dextrose (ACD, 85 mM trisodium citrate, 71 mM citric acid, 111 mM glucose, 1:6). Whole blood was centrifuged at 250 g for 15 min, and the top layer, platelet-rich plasma (PRP), was collected. PRP was mixed with Tyrode buffer (134 mM NaCl, 2.9 mM KCl, 1.0 mM MgCl_2_, 10 mM HEPES, 5 mM glucose, 12 mM NaHCO_3_, 0.34 mM Na_2_HPO_4_, pH 7.4) containing 1 μM prostaglandin E_1_ (PGE_1_), 0.2 U/mL apyrase, and 10% ACD. Platelets were isolated by centrifugation at 1000 g for 5 min and suspended in Tyrode buffer containing 1 μM PGE_1_, 0.2 U/mL apyrase, and 10% ACD. After centrifugation at 1000 g for 5 min, platelets were suspended in Tyrode buffer containing 0.3% BSA to adjust the cell count to 1 × 10^8^ cells. The decontamination of leukocytes and red blood cells was verified by microscopic observation. 2 mM CaCl_2_ was added before use.

2.3. Lactate Dehydrogenase (LDH) Assay

LDH leakage from platelets was measured spectrophotometrically using a commercial cytotoxicity LDH assay kit (Dojindo laboratories, Kumamoto, Japan) following the provided procedure with slight modification. Briefly, after incubation with isothiazolinone (10 and 25 μM) for 1 h, the platelet suspension was centrifuged at 12,000 g for 2 min, and the supernatant was collected for LDH assay. 100 μL of the aliquot was added to 100 μL of the working solution in the 96-well optically clear plate. The plate was incubated for 30 min at room temperature in the dark. 50 μL of the stop solution was added to each well, and the absorbance at 490 nm was measured using a microplate reader (SpectraMax i3, Molecular Devices, Sunnyvale, CA, USA). The extent of LDH leakage was expressed as the percentage of the total lysed control obtained from lysis with 1% Triton X-100. Non-cytotoxic conditions were defined as less than 10% total LDH leakage.

2.4. Measurement of Intracellular GSH Level

Intracellular GSH levels were determined using a commercial intracellular GSH assay kit (Abcam, Cambridge, United Kingdom), following the manufacturer’s protocol with minor modifications. Briefly, after treatment with isothiazolinones, platelets were incubated with the thiol green dye for 30 min at 37 °C and analyzed by flow cytometry (FACSVerse, BD Biosciences). Platelets were sorted based on size and density, gated by forward scatter (FSC) and side scatter (SSC), and FITC fluorescence intensity was measured.

2.5. Detection of Reactive Oxygen Species (ROS)

ROS generation was detected by oxidant-sensing fluorescent probes, CM-H_2_DCFDA and MitoSOX^TM^ Red. Platelets were incubated with 5 μM CM-H_2_DCFDA or MitoSOX™ Red for 30 min at 37 °C in the dark, then washed with Tyrode buffer to remove unloaded probes. After incubation with isothiazolinones, FITC fluorescence intensity for DCF and PE fluorescence intensity for MitoSOX red were measured with the flow cytometer. Platelets were sorted based on size and density, gated by FSC and SSC.

2.6. Determination of Mitochondrial Membrane Potential

Mitochondrial membrane potential was measured using the cationic fluorochrome JC-1. After treatment with isothiazolinones, platelets were incubated with 2 μg/mL JC-1 for 30 min at room temperature, then analyzed by flow cytometry. Platelets were sorted based on size and density, gated by FSC and SSC. PE and FITC fluorescence intensity were measured, and the ratio of red to green fluorescence intensity was calculated. Unstained control and positive control treated with carbonyl cyanide m-chlorophenyl hydrazone were used as fluorescence-minus-one.

2.7. Determination of Intracellular Calcium Level

Intracellular calcium levels were measured using the fluorescent calcium indicator Fluo-4 AM. Platelets were incubated with 5 μM Fluo-4 AM in the presence of 0.02% pluronic F-127 for 30 min at 37 °C in the dark, then washed with Tyrode buffer to remove excess Fluo-4 AM. Fluo-4-loaded platelets were treated with isothiazolinones and analyzed on the flow cytometer. Platelets were sorted based on size and density, gated by FSC and SSC, and FITC fluorescence intensity was recorded in the flow cytometer.

2.8. Determinaion of Intracellular ATP Level

Intracellular ATP levels were measured using the ATP detection assay kit (Cayman Chemical, Annn Arbor, MI, USA) according to the manufacturer’s instructions. In brief, platelets were centrifuged at 6000 g for 1 min at 4 °C, and the platelet pellet was lysed with ATP detection sample buffer. Intracellular ATP levels were determined by a luciferin/luciferase assay in a microplate reader and normalized to protein levels measured with the BCA protein assay kit.

2.9. Examination of Phospholipid Scramblase Activity

Phospholipid scramblase activity was examined by measuring the phosphatidylcholine (PC) translocation using fluorescent-labeled PC, C6-NBD-PC. Platelets were incubated with 0.5 μM C6-NBD-PC for 10 min at 37 °C, then diluted with Tyrode buffer containing or not containing 1% BSA and incubated on ice for 10 min [26]. Platelets were centrifuged at 5000 g for 1 min and lysed with 1% Triton X-100. The fluorescence intensity (ex. 485 nm, em. 535 nm) was measured using a microplate reader, and the values were compared before and after back extraction.

2.10. Measurement of PS Exposure

PS exposure was detected with annexin V-FITC, and platelets were identified with anti-CD61-PE. Platelets were incubated with anti-CD61-PE and annexin V-FITC for 30 min at room temperature in the dark, then analyzed by flow cytometry. Negative controls for annexin V binding were stained with annexin V-FITC in the presence of 2.5 mM EDTA. Platelets were considered positive when FITC fluorescence intensity exceeded 99% of the signal from the EDTA-negative control group. A23187-treated positive control was used for fluorescence compensation.

2.11. Determination of Procoagulant Activity

To examine procoagulant activity, thrombin generation in the presence of blood coagulation factors was determined. Platelets were incubated with 5 nM factor Xa and 10 nM factor Va in HEPES buffered saline (HBS; 21 mM HEPES, 137 mM NaCl, 5 mM KCl, 0.7 mM Na_2_HPO_4_, 5.5 mM glucose, 2 mM CaCl_2_, 0.3% BSA, pH 7.2) for 5 min at 37 °C. 2 μM prothrombin was added to initiate thrombin generation for 5 min at 37 °C. The reaction was stopped by dilution with stop buffer (50 mM Tris-HCl, 120 mM NaCl, 2 mM EDTA, pH 7.9). A chromogenic substrate for thrombin was used to determine thrombin activity by measuring absorbance at 405 nm, and the results were compared with those from active-site-titrated thrombin using the microplate reader.

2.12. Statistical Analysis

All experimental data were presented as the mean and standard error of the mean (SEM). The data were subjected to a Student’s t-test and a one-way analysis of variance followed by Dunnett’s post hoc test using IBM SPSS Statistics 29 (IBM Corp., Armonk, NY, USA). In all cases, a p-value of <0.05 was considered statistically significant.

3. Results

3.1. Intracellular GSH Depletion by Isothiazolinones in Platelets

To investigate whether isothiazolinones enhanced cytotoxicity in platelets, platelets were incubated with BIT, OIT, and DCOIT for 1 h, and LDH leakage was determined. BIT and OIT did not show a significant increase in LDH leakage up to 25 μM. However, DCOIT significantly enhanced LDH leakage in a concentration-dependent manner at 10 and 25 μM, indicating cytotoxic effects on platelets (Figure 1A). Cytotoxicity can provoke non-mechanistic, non-specific toxicities that may be revealed as artifacts in mechanism studies. Therefore, we intended to elucidate the effects of isothiazolinones on platelets under non-cytotoxic conditions, DCOIT was excluded from further experiments.

The key mechanism of the biocidal action of isothiazolinones is their reaction with the thiol groups of crucial enzymes and proteins, which disrupt vital cellular functions and lead to irreversible cell death [7]. Therefore, the effects of BIT and OIT on GSH, the most abundant intracellular thiol pool, were tested. Both BIT (0.64-fold at 25 μM) and OIT (0.43-fold at 25 μM) depleted GSH (Figure 1B). In particular, OIT reduced the GSH level to a lower level compared with BIT. Of note, the thiol donor NAC restored GSH levels to normal, which were decreased by BIT and OIT (Figure 1C).

3.2. Oxidative Stress Induced by Isothiazolinones

Depletion of GSH impairs antioxidant capacity, potentially promoting ROS accumulation and leading to oxidative stress [27]. To determine the overall cellular ROS level, the general oxidant-sensing indicator DCF fluorescence was measured. Interestingly, neither BIT nor OIT affected DCF fluorescence (Figure 2A). As mitochondria are the main source of ROS, and mitochondrial ROS is deeply related to platelet activation [28,29,30], the effects of BIT and OIT on mitochondrial ROS levels were measured by MitoSOX red fluorescence. Mitochondrial ROS were generated after BIT (1.87-fold at 25 μM) and OIT (2.50-fold at 25 μM) exposure in a concentration-dependent manner (Figure 2B). Consistent with GSH depletion, OIT showed a more potent effect on mitochondrial ROS levels than BIT. Notably, thiol supplementation with NAC totally diminished isothiazolinones-induced mitochondrial ROS generation, confirming that GSH depletion disrupted the balance between prooxidant and antioxidant systems, leading to oxidative stress.

3.3. Dysregulation of Intracellular Calcium and ATP Homeostasis by Isothiazolinone

Mitochondrial ROS generation can directly affect mitochondria, causing mitochondrial dysfunction [31,32]. To evaluate mitochondrial effects, mitochondrial membrane potential was measured using the fluorescent probe JC-1, which shifts from red to green fluorescence as mitochondrial membrane potential decreases. BIT and OIT accelerated mitochondrial dysfunction, revealed as collapse of mitochondrial membrane potential (Figure 3A).

Mitochondrial dysfunction could lead to altered intracellular calcium and ATP homeostasis, which are fundamental regulators of cellular functions [33]. The intracellular calcium level increased by BIT (2.40-fold at 25 μM) and OIT (3.27-fold at 25 μM) in a concentration-dependent manner, determined by Fluo-4 fluorescence (Figure 3B). In the same manner, the intracellular ATP level was reduced after BIT (0.57-fold at 25 μM) and OIT (0.28-fold at 25 μM) treatment (Figure 3C).

3.4. Loss of Plasma Membrane Phospholipid Asymmetry by Isothiazolinone

Alterations in intracellular calcium and ATP levels could affect transmembrane lipid transporters that transfer phospholipids between the plasma membrane leaflets, thereby maintaining or disrupting plasma membrane phospholipid asymmetry [34,35]. Especially scramblase, a calcium-dependent enzyme, scrambles phospholipids bidirectionally, breaking down phospholipid asymmetry and exposing PS. BIT and OIT significantly increased scramblase activity, as measured by phosphatidylcholine internalization (Figure 4A). Furthermore, BIT (37.4%-fold at 25 μM) and OIT (66.1% at 25 μM) consistently induced PS externalization in a concentration-dependent manner as a result of the collapse of phospholipid asymmetry (Figure 4B). Notably, PS exposure after BIT and OIT treatment was significantly restored by the thiol supplement, NAC pre-treatment, suggesting the contribution of GSH depletion to isothiazolinone-induced PS exposure (Figure 4C).

3.5. Enhanced Procoagulant Activity by Isothiazolinones

Externalized PS could serve as a site for the assembly and activation of blood coagulation factors, thereby stimulating the blood coagulation pathway [23,25]. Therefore, the effects of BIT and OIT on procoagulant activity were elucidated by converting prothrombin to thrombin in the presence of blood coagulation factors. BIT (2.56-fold at 25 μM) and OIT (6.30-fold at 25 μM) significantly induced thrombin generation in a concentration-dependent manner (Figure 5A). In consistency with other results, OIT enhanced procoagulant activity at a lower level than BIT. Moreover, pre-incubation with NAC fully suppressed isothiazolinones-induced thrombin generation, suggesting that GSH depletion is deeply involved in the platelet-mediated procoagulant activity by isothiazolinones (Figure 5B). Interestingly, DCOIT also enhanced thrombin generation, suggesting that cytotoxic events could also contribute to procoagulant activity (Figure 5C).

WP is frequently used in platelet studies to avoid interference of plasma proteins. On the other hand, WP does not fully mimic in vivo situations as plasma is removed. To confirm whether BIT and OIT exhibit procoagulant activity in a real in vivo setting, we used PRP. BIT and OIT increased PS exposure and thrombin generation in a pattern similar to that observed in WP (Figure 6).

4. Discussion

In this study, BIT and OIT, representative isothiazolinone biocides, remarkably depleted intracellular GSH, thereby inducing mitochondrial ROS accumulation and, following mitochondrial dysfunction, stimulating intracellular calcium and ATP depletion. This series of intracellular pathways activated phospholipid scramblase, altering phospholipid asymmetry and exposing PS. PS exposure led to enhanced platelet-mediated procoagulant activity.

The primary exposure route for isothiazolinones is dermal contact with isothiazolinone-containing consumer and industrial products. Based on the main exposure scenarios for isothiazolinones, numerous epidemiological and animal studies have focused on skin irritation and sensitization, revealing allergic contact dermatitis [3,4,5]. In addition, isothiazolines can be inhaled through aerosol-type products, and several studies suggested pulmonary injury by isothiazolinone exposure, raising concerns of systemic toxic effects [6,36]. After absorption, isothiazolinones enter the blood circulation and can affect the circulatory system. OIT, BIT, DCOIT, and a mixture of chloromethylisothiazolinone and methylisothiazolinone (CMIT/MIT) altered the viability of endothelial cells and induced endothelial dysfunction in the murine brain endothelial cell line bEND.3 cells [13,14,15]. CMIT/MIT impaired rat vascular smooth muscle contraction in isolated rat thoracic aorta and primary cultured vascular smooth muscle cells [16]. However, previous studies have focused on blood vessels, and the effects on blood cells, particularly platelets, have been little studied, and platelet-mediated prothrombotic effects have been overlooked. Baggaley et al. prepared a series of BIT derivatives and showed inhibitory effects on adenosine diphosphate (ADP)- and collagen-induced platelet aggregation [18]. However, the results were only primary screening experiments, lacking a mechanistic explanation. We demonstrated that isothiazolinones enhanced procoagulant activity and clarified the underlying mechanism, PS exposure derived from intracellular GSH depletion.

Isothiazolinones exhibit antimicrobial activity by interacting with thiol groups of vital intracellular components [7]. Accumulating reports suggest thiol depletion as the target of isothiazolinone-induced toxicity. In neuronal cells, MIT induced a decrease in GSH level following ERK-dependent neurotoxicity, which was restored by thiol supplementation with NAC and GSH [8]. NAC reversed oxidative stress-mediated apoptosis in keratinocytes with CMIT/MIT, and the authors speculated that cellular thiols are a potential target for CMIT/MIT [9]. In the human hepatocarcinoma cell line HepG2 cells, chlorinated isothiazolinones CMIT and DCOIT decreased total cellular GSH, resulting in necrotic morphological changes, whereas MIT and OIT did not [10]. GSH depletion has been suggested to initiate CMIT/MIT-induced cell death in human leukemia cell line HL60 cells [11,12]. Isothiazolinones also reduced GSH levels in bEnd.3 cells, altering the redox state, leading to blood–brain barrier dysfunction [13,14,15]. Particularly, OIT-mediated effects in bEnd.3 cells were reversed by NAC. CMIT/MIT altered the contractile machinery of vascular smooth muscle by depleting thiols and generating ROS. Indeed, intracellular thiol levels are key factors regulating platelet activation and cytotoxicity. Notably, several studies have suggested that thiol depletion may be an underlying mechanism of PS exposure and procoagulant activation in platelets by xenobiotics [37,38]. In line with previous studies, BIT and OIT enhanced procoagulant activity by depleting GSH and thereby triggering intracellular events. Moreover, NAC supplementation restored the isothiazolinone-induced events, supporting the pivotal role of GSH depletion.

We suggested that BIT and OIT enhanced platelet procoagulant activity through a series of intracellular events triggered by thiol depletion, thereby indirectly affecting phospholipid scrambling. Consistent with our results, thiol-depleting agents, N-ethylmaleimide and diamide, altered phospholipid scramblase and flippase via oxidative stress-mediated thiol modification, thereby exposing PS [39,40]. However, it has been suggested that the thiol-containing cysteine residue of scramblase could serve as a binding site for heavy metal ions, such as Pb^2+^ and Hg^2+^, thereby inducing phospholipid scrambling [41]. It may be speculated that isothiazolinones may directly affect phospholipid scramblase, thereby contributing to PS exposure.

We hypothesized that NAC served as a GSH precursor, increasing GSH synthesis and preserving intracellular GSH levels depleted by BIT and OIT. However, NAC may directly react with BIT and OIT, scavenging their toxic effects. Interestingly, Ettoree et al. reported that NAC pretreatment inhibited apoptotic events induced by CMIT/MIT in keratinocytes; however, NAC treatment after CMIT/MIT exposure did not show protective effects, suggesting that NAC acts as a GSH precursor rather than a CMIT/MIT scavenger [9]. Although we could speculate that NAC acts as a GSH precursor, we cannot rule out its role as an isothiazolinone scavenger.

To clarify the relevance to real-life exposure scenarios, the in vitro concentrations should be compared with human exposure levels and blood levels. However, to our best knowledge, studies on human exposure levels and blood levels of BIT and OIT are scarce. In the present study, BIT and OIT showed significant differences at 25 μM and 10 μM, which were similar to those reported in other in vitro studies. In the HepG2 cells, the EC_50_ of OIT in the LDH assay was 21 μM [10], and in the bEND.3 cells, the IC50s of BIT and OIT in the MTT assay were 74.21 and 15.78 μM [13].

Previously, Collier et al. reported that the chlorinated isothiazolinone CMIT exhibited greater growth-inhibitory and biocidal activity than BIT and MIT against Escherichia coli and Schizosaccharomyces pombe [42]. Arning et al. reported that DCOIT and CMIT altered GSH metabolism in HepG2 cells, which correlated with necrotic morphological alterations, whereas OIT and MIT did not [10]. The diverse effects on cytotoxicity were also observed in HepG2 cells, the bacterium Vibrio fischeri, and the green algae Scenedesmus vacuolatus, where DCOIT and CMIT showed superior activity [43]. Recently, Bae et al. reported that isothiazolinone displayed different toxic potencies in brain endothelial cells (DCOIT > OIT > BIT) [13]. Similarly, in the present study, the chlorinated isothiazolinone DCOIT exhibited cytotoxic effects, as measured by LDH leakage, whereas the unchlorinated isothiazolinones BIT and OIT did not. Of note, OIT showed higher potencies than BIT, consistent with results in the brain endothelium. The difference between BIT and OIT may be due to the higher lipophilicity of OIT [44], which could tend to permeate the cellular membrane more easily.

A limitation of this study is that the effects on platelet aggregation, another counterpart for prothrombotic properties of platelets, are not elucidated [45]. In a previous study, BIT derivatives showed inhibitory effects on ADP and collagen-induced platelet aggregation [18]. However, the agonists were used at high concentrations, inducing submaximal aggregation. Therefore, it remains unknown whether isothiazolinone induces platelet aggregation alone or with subminimal concentrations of agonists. Although both aggregatory and procoagulant platelets contribute to hemostasis and thrombosis, they possess distinct properties and mechanisms, including the state of integrin αIIbβ3, sustained cytosolic calcium levels, and mitochondrial involvement [46]. The effects of isothiazolinones and their underlying mechanisms on platelet aggregation should be investigated further.

Red blood cells, white blood cells, and endothelial cells also contribute to the coagulation pathway and thrombosis by exposing PS [47,48,49,50]. OIT stimulated apoptotic events in endothelial cells, including PS exposure and caspase-3 activation [14], which may manifest as endothelial cell-mediated procoagulant activation. It would be meaningful to clarify the procoagulant effects of isothiazolinone on red blood cells, white blood cells, and endothelial cells in future studies.

Murine platelets have been used as a valid model for evaluating platelet aggregation, as they share many properties with human platelets [51,52]. However, in several cases, distinct responses were observed between murine and human platelets, casting doubt on the validity of using murine platelets [53,54,55,56]. Therefore, the lack of human platelet experiments remains a limitation of the present study.

5. Conclusions

We demonstrate that BIT and OIT can promote procoagulant pathway PS exposure in rat platelets, initiated by GSH depletion and followed by mitochondrial ROS accumulation, mitochondrial dysfunction, elevation of intracellular calcium levels, and a decrease in intracellular ATP levels, thereby altering phospholipid scramblase to disrupt membrane phospholipid asymmetry. Notably, NAC supplementation attenuated this series of events, confirming the pivotal role of thiol depletion in the procoagulant effects of platelets induced by BIT and OIT. These events may contribute to prothrombotic capacity, providing an important clue for improving understanding of systemic adverse health effects by isothiazolinone exposures, especially thrombotic and cardiovascular risks.