---
title: "Isothiocyanate mixture inhibits platelet signaling and mitochondrial metabolism to reduce thrombos"
id: "frontiers-in-immunology-6-a-defined-isothiocyanate-mixture-suppresses-platelet-signaling-and-metabolism"
canonical_url: "https://medichelpline.com/clinical-feed/frontiers-in-immunology-6-a-defined-isothiocyanate-mixture-suppresses-platelet-signaling-and-metabolism"
content_type: "clinical_feed_article"
specialty: "Infectious Disease"
source_name: "Frontiers in Immunology"
source_url: "https://www.frontiersin.org/articles/10.3389/fimmu.2026.1944710"
published_at: "2026-09-30T00:00:00.000Z"
evidence_level: "Journal Feed"
license: "CC-BY-NC-4.0 / Informational Use"
---
# Isothiocyanate mixture inhibits platelet signaling and mitochondrial metabolism to reduce thrombos
## Provenance & Clinical Metadata
- **Canonical URL:** https://medichelpline.com/clinical-feed/frontiers-in-immunology-6-a-defined-isothiocyanate-mixture-suppresses-platelet-signaling-and-metabolism
- **Specialty:** [Infectious Disease](https://medichelpline.com/clinical-feed/infectious-disease.md)
- **Primary Source:** Frontiers in Immunology
- **Source URL:** [Original Journal Publication](https://www.frontiersin.org/articles/10.3389/fimmu.2026.1944710)
- **Published At:** 2026-09-30T00:00:00.000Z
- **Evidence Rating:** Journal Feed
## Executive GIST (TL;DR)
- The study evaluated a defined **isothiocyanate (ITC) mixture** composed of allyl- (AITC), benzyl- (BITC) and phenethyl-isothiocyanate (PEITC) on human platelets ex vivo and in a murine carrageenan-induced thrombosis model. - The ITC mixture proportions (37.9% AITC, 50.0% BITC, 12.1% PEITC) match the ANGOCIN® clinical preparation; in vitro concentration used was 845 µM total (320 µM AITC, 422.5 µM BITC, 102.5 µM PEITC) prepared in DMSO with matched vehicle controls. - In aggregometry, the ITC mixture broadly inhibited platelet aggregation induced by multiple agonists (TRAP-6, convulxin, U46619, arachidonic acid, ADP, PMA, FcγRIIa crosslinking). - Kinome and phosphorylation profiling showed reduced peptide phosphorylation across multiple platelet signaling pathways, including attenuation of **AKT** phosphorylation and upstream kinase signaling. - The ITC mixture reduced intracellular Ca2+ mobilization and the activation-associated oxidant signal; markers of oxidative stress included glutathione oxidation and increased protein S-glutathionylation. - Mitochondrial effects were observed: loss of mitochondrial membrane potential, impaired mitochondrial respiration by respirometry, and ultrastructural changes on electron microscopy consistent with mitochondrial dysfunction. - When added shortly after agonist stimulation, the ITC mixture destabilized early forming platelet aggregates (aggregometry reversal), while granule secretion, once initiated, was irreversible under the tested conditions. - In vivo, administration of the ITC mixture before or after carrageenan challenge reduced thrombus formation and tissue injury, decreased tail-necrosis length, and prolonged bleeding times to an extent similar to a heparin reference group in this model. - Cytotoxicity assays (Annexin V, LDH) indicated that the selected pre-treatment interval and dosing prevented overt platelet membrane damage; details and full cytotoxicity data are reported in the Supplementary Figures. - The authors conclude the ITC mixture functions as a dual regulator of **platelet signaling** and **mitochondrial metabolism**, providing mechanistic rationale for further translational evaluation in thrombotic disease.
## Clinical Analysis & Structured Key Points
Frontiers | A defined isothiocyanate mixture suppresses platelet signaling and metabolism to limit thrombosis in vivo ORIGINAL RESEARCH article Front. Immunol. , 30 September 2026 Sec. Inflammation Volume 17 - 2026 | https://doi.org/10.3389/fimmu.2026.1944710 Published in Frontiers in Immunology Inflammation 7 impact factor 11.3 citescore Editor & Reviewers Edited by A B Alexander Brill Reviewed by R G ROCIO GOMEZ K D Karthik Dhanabalan Outline Figures and Tables Figure 1 View in article Figure 2 View in article Figure 3 View in article Figure 4 View in article Figure 5 View in article Figure 6 View in article Table 1 Experimental treatment groups in the carrageenan-induced tail thrombosis model. View in article Table 2 Histopathological summary of organ injury in mice with carrageenan-induced thrombosis following treatment with ITC mixture or heparin. View in article ORIGINAL RESEARCH article Front. Immunol. , 30 September 2026 Sec. Inflammation Volume 17 - 2026 | https://doi.org/10.3389/fimmu.2026.1944710 A defined isothiocyanate mixture suppresses platelet signaling and metabolism to limit thrombosis in vivo J Y Jan Yağız Buchholz 1 † M S Maria Shoykhet 1 † A C Anthea C. Darius 1 T W Tobias Weiser 1 E L Erika Lynn Roberts 1,2 M K Mohammad Khosravi 3 J P Julie Petry 1 S Z Seyedeh Zahra Mousavi 1 B M Babak Mohammadian 3 L A Lina Adams 1 K H Khouloud Hachani 1 C E Carola Eberhagen 4 O A Omid Azimzadeh 5 H Z Hans Zischka 4,6 G M Gabriele Multhoff 2 A B Ali Bashiri Dezfouli 1 ‡ * +8 more B W Barbara Wollenberg 1 ‡ * 1. Department of Otorhinolaryngology, TUM School of Medicine and Health, Technical University of Munich (TUM), Munich, Germany 2. Central Institute for Translational Cancer Research, Technical University of Munich (TranslaTUM), Klinikum rechts der Isar, Munich, Germany 3. Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran 4. Institute of Molecular Toxicology and Pharmacology, Helmholtz Center Munich, German Research Center for Environmental Health, Neuherberg, Germany 5. Section Radiation Biology, Federal Office for Radiation Protection (BfS), Neuherberg, Germany 6. Institute of Toxicology and Environmental Hygiene, Technical University of Munich School of Medicine and Health, Munich, Germany See more Article metrics View details Abstract Pathological platelet activation drives thrombosis and is a major contributor to morbidity and mortality. Current antiplatelet therapies reduce thrombotic risk but target a limited range of signaling pathways, have minimal impact on established platelet aggregates, and are frequently associated with bleeding complications. These drawbacks have prompted interest in alternative antiplatelet strategies. Isothiocyanates (ITCs), phytochemicals from cruciferous plants, have been reported to inhibit platelet function; however, their mechanisms and translational relevance remain incompletely defined. The effects of a defined mixture of allyl-, benzyl-, and phenethyl-isothiocyanate on platelet function were evaluated using ex vivo human platelets and an in vivo murine model of carrageenan-induced thrombosis. Platelet aggregation, intracellular signaling, calcium mobilization, redox balance, and mitochondrial function were assessed using functional assays, kinome profiling, flow cytometry, respirometry, and electron microscopy. The ITC mixture was administered before or after the carrageenan challenge. The ITC mixture inhibited platelet activation and aggregation induced by multiple agonists. It reduced peptide phosphorylation across multiple platelet signaling pathways, reduced intracellular calcium mobilization and the activation-associated oxidant signal, and impaired mitochondrial function. When the ITC mixture was added shortly after activation, aggregometry signals consistent with destabilization of early platelet aggregates were observed whereas granule secretion was irreversible once initiated. In vivo , ITC mixture administration before or after the carrageenan challenge reduced thrombus formation and tissue injury, prolonged bleeding times, and reduced tail-necrosis length to a similar extent as the heparin reference group in this model. These findings identify the ITC mixture as a modulator of platelet signaling and metabolism, provide mechanistic insight into ITC-mediated antithrombotic effects, and support further evaluation of ITC-based interventions in thrombotic disease. Graphical Abstract The graphical abstract illustrates the multimodal antiplatelet mechanism of the ITC mixture. Platelet activation is initiated by diverse agonists acting through G-protein-coupled and immunoreceptor pathways, including ADP (P2Y 12 ) the PAR1/PAR4-activating peptide TRAP-6, U46619 (thromboxane A 2 receptor), convulxin (GPVI), IV.3-mediated FcγRIIa crosslinking, arachidonic acid (COX-1-dependent) and PMA (direct PKC activation), which collectively converge to amplify platelet signaling. Serotonin is depicted as a dense-granule release product quantified in this study, and not as an agonist applied to platelets; thrombin receptor signaling was addressed using TRAP-6 rather than thrombin itself. Centrally, the ITC mixture impairs mitochondrial oxidative phosphorylation and disrupts redox homeostasis, leading to altered redox homeostasis and activation-associated oxidant signaling, thereby limiting the energetic support required for sustained platelet activation. Consequently, granule secretion and surface expression of activation markers (CD62P, CD63, CD40L) are reduced, resulting in diminished integrin activation, impaired fibrinogen binding, and attenuated platelet aggregation. Created in BioRender. Hachani, K (2026). https://BioRender.com/9exdvj3 . 1 Introduction Platelets are anucleate cell fragments derived from bone marrow megakaryocytes and are central regulators of vascular hemostasis and thrombosis ( 1 ). Under physiological conditions, platelets remain in a quiescent, non-adhesive state maintained by endothelial-derived mediators such as prostacyclin (PGI 2 ), nitric oxide (NO), and a tightly regulated mitochondrial energy program ( 2 – 4 ). Following vascular injury, platelets are rapidly activated by extracellular agonists, including collagen and adenosine diphosphate (ADP), and, in immune-mediated contexts, by immune complexes engaging platelet surface receptors. These stimuli trigger intracellular signaling cascades involving phosphoinositide 3-kinase/protein kinase B (PI3K/AKT), protein kinase C (PKC), and related kinase pathways, which coordinate cytoskeletal remodeling, integrin activation, granule secretion, and firm adhesion ( 5 – 8 ). Upon activation, platelets release cytokines and pro-thrombotic mediators, establishing positive feedback loops that further amplify platelet recruitment and activation ( 9 , 10 ). Although this process is essential for physiological hemostasis, dysregulated platelet activation contributes to pathological thrombosis, a major cause of myocardial infarction, stroke, pulmonary embolism, and multi-organ failure ( 11 – 14 ). Despite the widespread clinical use of antithrombotic drugs to combat such complications, major limitations persist ( 15 – 17 ). Most clinically used agents target a single receptor or enzymatic pathway, such as cyclooxygenase inhibition by aspirin or P2Y 12 blockade by ADP receptor antagonists, leaving alternative activation routes intact. As a result, residual platelet reactivity and thrombotic risk remain in many patients. Moreover, most of these agents suppress platelet activation or aggregation but do not actively dissolve pre-existing platelet aggregates, largely restricting true thrombolysis to plasminogen activators ( 17 – 20 ). In addition, inter-individual variability in platelet reactivity and incomplete platelet inhibition contribute to residual thrombotic risk even in treated patients ( 15 ). These shortcomings support the need for novel antithrombotic strategies that more broadly suppress platelet activation and, ideally, act on aggregates that have already begun to form, while maintaining an acceptable safety profile. Platelet activation is an energetically demanding and tightly regulated process that integrates intracellular signaling, calcium mobilization, redox balance, and mitochondrial metabolism to culminate in the formation of a stable hemostatic plug ( 14 , 21 ). Reactive oxygen species (ROS) play a critical role as secondary messengers in this activation, particularly downstream of collagen stimulation, where oxidative modification of signaling proteins amplifies platelet responses ( 22 , 23 ). Mitochondria integrate these energetic and redox processes by supplying the ATP required for granule secretion and cytoskeletal remodeling, while simultaneously regulating ROS generation through the electron transport chain ( 21 ). However, excessive oxidative stress may paradoxically impair platelet function and facilitate receptor shedding via mechanisms dependent on p38 mitogen-activated protein kinase (p38 MAPK) ( 23 ). These observations highlight platelet metabolism and redox regulation as attractive but underexplored targets for antithrombotic intervention. Due to their pleiotropic biological activities and generally favorable safety profiles, naturally occurring phytochemicals have emerged as promising candidates for antithrombotic intervention. Among these, isothiocyanates (ITCs), bioactive compounds derived from glucosinolates found in cruciferous plants such as mustard seed and horseradish root (allyl-ITC), garden cress and papaya seed (benzyl-ITC), watercress (phenethyl-ITC), and broccoli and broccoli sprouts (sulforaphane), in which the parent glucosinolates are hydrolyzed to the corresponding ITCs by myrosinase upon tissue damage during chopping, chewing or food processing, have long been used in phytomedicine for their antimicrobial, anti-inflammatory, and cytoprotective properties ( 24 , 25 ). Clinically, ITCs are utilized in a herbal formulation known as ANGOCIN ® Anti-Infekt N (Angocin) ( 26 ), which contains benzyl isothiocyanate (BITC), allyl isothiocyanate (AITC), and phenethyl isothiocyanate (PEITC), and is prescribed for respiratory and urinary tract infections ( 27 , 28 ). Beyond their antimicrobial activity, ITCs exert potent anti-inflammatory effects through inhibition of NF-κB and NLRP3 inflammasome signaling in immune cells ( 29 – 32 ). ITCs are also well recognized for their ability to modulate mitochondrial function and cellular redox balance. In cancer cells, PEITC and BITC induce apoptosis via ROS-dependent mitochondrial membrane depolarization, cytochrome c release, and caspase activation ( 33 – 36 ). In contrast, ITCs exert antioxidant and cytoprotective effects in endothelial cells and astrocytes by enhancing Nrf2-dependent defense systems and suppressing inflammatory signaling ( 37 , 38 ). These context-dependent redox effects, together with the capacity of ITCs to regulate inflammatory signaling and mitochondrial metabolism, intersect with several core regulators of platelet activation, including ROS signaling, calcium mobilization, PI3K/AKT signaling, and mitochondrial bioenergetics. Consistent with this conceptual overlap, several ITCs have been reported to inhibit platelet aggregation. Sulforaphane (SFN) suppresses platelet activation and coagulation factor activity and protects against cerebral thrombotic dysfunction in vivo ( 39 , 40 ), while AITC and isopropyl ITC inhibit platelet aggregation and thrombus formation in experimental models ( 41 – 43 ). Nevertheless, the molecular mechanisms underlying ITC-mediated platelet inhibition remain incompletely understood, especially regarding effects on platelet redox balance, calcium signaling, and mitochondrial function. Moreover, although ITCs are commonly administered clinically as defined combinations, no previous study has comprehensively examined whether ITCs exert synergistic or mechanistically distinct effects on platelet activation and thrombosis. In the present study, we investigated the effects of a defined mixture of AITC, BITC, and PEITC on platelet activation and aggregation in ex vivo human platelets and in an in vivo murine thrombosis model. We show that the ITC mixture inhibits platelet aggregation, partially suppresses platelet activation and degranulation, and destabilizes early, already-forming platelet aggregates. Mechanistically, ITC treatment attenuates key activation pathways, including AKT phosphorylation and upstream kinase signaling, reduces intracellular Ca 2+ mobilization, and reduces the activation-associated oxidant signal. In parallel, the ITC mixture is associated with oxidative stress and mitochondrial dysfunction in platelets, as reflected by glutathione oxidation, increased protein S-glutathionylation, loss of mitochondrial membrane potential, and impaired mitochondrial respiration. In vivo , administration of the ITC mixture before or after the carrageenan challenge reduced thrombus formation and tissue injury, with reductions in the measured thrombosis endpoint of a similar magnitude to those observed in the heparin reference group, and a similar prolongation of bleeding time. These findings identify this ITC mixture as a dual regulator of platelet signaling and metabolic support systems and reveal a previously unrecognized mechanism by which this ITC mixture suppresses platelet-driven thrombosis. This work establishes a mechanistic foundation for the development of ITC-based antithrombotic strategies and provides a framework for future translational studies aimed at evaluating their therapeutic potential in thrombotic disease. 2 Materials and methods 2.1 Platelet preparation and treatments Human platelet studies were approved by the Ethics Committee of the Technical University of Munich (approval numbers 2020-474_2-S-NP and 2022-211-S-NP). All blood donors provided written informed consent. Platelets were obtained from unrelated healthy volunteers of mixed gender, aged 25–35 years. All donors were of self-reported good health with no acute infection or inflammatory condition at the time of donation, and had not taken any medication known to affect platelet function (including non-steroidal anti-inflammatory drugs) for at least 10 days before blood sampling. All donors were non-smokers and none reported regular alcohol consumption. Ethnicity and ancestry were not systematically recorded. All donors were recruited in Munich, Germany. The number of independent donors contributing to each analysis is reported in Supplementary Table S1 ; these panel-level values are numbers of independent biological replicates and do not represent the total number of unique donors across the study. 2.1.1 Platelet isolation Platelet isolation was performed as previously described in established platelet isolation protocols ( 44 , 45 ). Peripheral blood from healthy donors was collected in 3.2% sodium citrate tubes and processed within 2 hours of venipuncture. Platelet-rich plasma (PRP) was generated by centrifugation at 150 × g for 20 minutes at room temperature (RT, ~22 °C). To prevent inadvertent activation, PRP was supplemented with prostacyclin (PGI 2 , 400 nM) and centrifuged at 800 × g for 10 min. The resulting platelet pellet was gently resuspended in Ca 2+ -free Tyrode buffer (pH 7.4) ( 46 ). Platelet counts and purity (CD41 + /CD45 - ) were assessed by flow cytometry. 2.1.2 ITC mixture preparation The ITC mixture consisted of 37.9% allyl-ITC (AITC; #377430, Sigma-Aldrich), 50.0% benzyl-ITC (BITC; #252492, Sigma-Aldrich), and 12.1% phenethyl-ITC (PEITC; #253731, Sigma-Aldrich). The relative proportions of the three compounds were fixed a priori to reflect the relative isothiocyanate content of the clinical preparation ANGOCIN ® Anti-Infekt N ( 26 , 47 ), and were not optimized for antiplatelet activity in the present study. A concentrated stock solution of the ITC mixture was prepared in dimethyl sulfoxide (DMSO) and subsequently diluted into assay buffers for experimental use. Control samples received matched DMSO concentrations for in vitro experiments (final < 0.2% v/v). In every experiment reported, the control condition was the corresponding dilution buffer carrying the matched DMSO concentration. Inhibitory effects of DMSO on human platelet adhesion and aggregation have been described at concentrations of 0.5% v/v and above ( 48 ), i.e. more than twofold above the maximum concentration reached in the present study. For in vitro experiments, the mixture was applied at a total concentration of 845 µM (320 µM AITC, 422.5 µM BITC, 102.5 µM PEITC), a dose selected from dose-response experiments demonstrating complete inhibition without cytotoxicity. 2.1.3 Platelet agonists and ITC timing protocols Platelets were stimulated with the following agonists at their final concentrations: TRAP-6 (25 µM; #3497, Tocris), convulxin (CVX; 125 ng/mL; #119-02, Pentapharm), U46619 (1 µM; #16450, Cayman), arachidonic acid (AA; 100 µM; #23401, Sigma-Aldrich), ADP (100 µM; #A2754, Sigma-Aldrich), and PMA (1 µM; #P1585, Sigma-Aldrich). For FcγRIIa-mediated activation, platelets were incubated with the monoclonal antibody IV.3 (383 ng/mL), generously provided by Ronald Taylor (University of Virginia), and cross-linked using anti-Fc Fab (15 µg/mL; #115-006-062, Jackson ImmunoResearch). To assess the timing-dependent effects of the ITC mixture, two experimental paradigms were employed. For pre-treatment experiments, platelets were incubated with the ITCs for 5 minutes prior to agonist stimulation. For post-activation (reversal) protocols, platelets were first activated with the indicated agonist, after which the ITC mixture was added 25 or 300 s post-stimulation, as specified in the corresponding figure legends. These post-activation addition experiments were designed to evaluate whether the ITC mixture can modulate ongoing platelet activation under experimental conditions. 2.2 Functional platelet assays 2.2.1 Light transmission aggregometry and cytotoxicity Platelet aggregation was assessed by light transmission aggregometry using the Born method on an APACT 4S Plus aggregometer (DiaSys) ( 49 ). Platelets were adjusted to 2 × 10 8 platelets/mL and stirred at 1000 rpm at 37 °C. Aggregation was initiated by agonist addition and monitored immediately for up to 10 min, after which maximal aggregation and area under the curve (AUC) were quantified. Cytotoxicity was evaluated using Annexin V-APC staining to detect phosphatidylserine exposure after 5 minutes of exposure to the ITC mixture, i.e. the pre-treatment interval used in the aggregation experiments. Plasma-membrane integrity was assessed in parallel by lactate dehydrogenase (LDH) release, measured across the full range of exposure intervals used in this study (300, 900 and 1800 s). The assay is described in the Supplementary and the results are shown in Supplementary Figure S1 , Supporting Information . 2.2.2 Flow cytometry Flow cytometric analysis was performed on a CytoFLEX (Beckman Coulter). At least 20,000 events were collected per sample. Platelets were identified by FSC/SSC profile and CD41 + /CD45 - gating. Activation markers included CD62P-BV510 (P-selectin; #304936, Biolegend), CD63-BV421 (#353029, Biolegend), and CD154-PE/Cy7 (CD40L; #310831, Biolegend) as previously described ( 50 ). Integrin activation was assessed using PAC-1-FITC (#362804, Biolegend). Fibrinogen binding was measured using Alexa Fluor 488-conjugated fibrinogen (#F13191, Thermo Fisher). Apoptosis was detected with Annexin V-APC (#640932, Biolegend). Compensation beads (BD CompBeads, #552843) were used to prepare single-stain compensation controls. 2.2.3 Release assays α-granule release was quantified by CCL5 ELISA (#440804, Biolegend). Dense-granule release was measured by ATP quantification (#G924, Promega) and serotonin ELISA (#DEE8900, Demeditec). Supernatants were collected immediately after stimulation, centrifuged, a
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