Pathological platelet activation underlies arterial and venous thrombosis and contributes substantially to morbidity and mortality. Current antiplatelet agents often target single receptors or enzymes, leave alternative activation routes intact, and do not reliably dissolve nascent platelet aggregates. The study interrogated a defined isothiocyanate (ITC) mixture—a clinically used combination in the herbal formulation ANGOCIN®—for its capacity to modulate platelet activation, redox balance, and mitochondrial metabolism as a broader antithrombotic strategy.
The ITC mixture tested contained fixed proportions of allyl-ITC (AITC, 37.9%), benzyl-ITC (BITC, 50.0%), and phenethyl-ITC (PEITC, 12.1%) to mirror the relative isothiocyanate content of the ANGOCIN® preparation. Stocks were prepared in DMSO and diluted such that the final DMSO concentration in assays was below 0.2% v/v. For in vitro experiments the total ITC concentration was 845 µM (320 µM AITC, 422.5 µM BITC, 102.5 µM PEITC), a dose selected from dose–response experiments that achieved complete inhibition of aggregation without cytotoxicity in the pre-treatment interval.
Platelets were isolated from healthy adult donors (age 25–35 years) under ethics approval. Platelet-rich plasma was prepared and washed with prostacyclin to limit inadvertent activation, then resuspended in Ca2+-free Tyrode buffer. Two timing paradigms were employed: a 5-minute pre-treatment with the ITC mixture before agonist stimulation, and post-activation (reversal) protocols in which the ITC mixture was added 25 or 300 seconds after agonist challenge to assess effects on ongoing aggregation.
Light-transmission aggregometry showed that the ITC mixture inhibited aggregation induced by a panel of agonists: TRAP-6 (PAR-activating peptide), convulxin (GPVI agonist), U46619 (thromboxane receptor agonist), arachidonic acid (COX-1 dependent), ADP, PMA (direct PKC activation), and FcγRIIa-mediated activation via IV.3 antibody cross-linking. Maximal aggregation and area under the curve (AUC) were reduced compared with matched DMSO vehicle controls. When the ITC mixture was added after agonist stimulation at early time points, aggregometry traces were consistent with destabilization of newly forming platelet aggregates; conversely, secretion-associated responses appeared irreversible once granule release had occurred.
Kinome profiling and targeted phosphorylation analyses demonstrated a reduction in peptide phosphorylation across multiple platelet signaling pathways, including attenuation of AKT phosphorylation and upstream kinase activities. Flow cytometry revealed decreased surface expression of activation markers (CD62P, CD63, CD40L), reduced integrin activation (PAC-1 binding), and diminished fibrinogen binding. Intracellular calcium mobilization in response to agonists was reduced after ITC treatment. The study also documented a reduction in the activation-associated oxidant signal alongside evidence of altered redox homeostasis, including glutathione oxidation and increased protein S-glutathionylation.
The ITC mixture impaired mitochondrial function in platelets. Evidence included loss of mitochondrial membrane potential, impaired mitochondrial respiration measured by respirometry, and electron microscopy findings consistent with mitochondrial alterations. These mitochondrial effects align with the observed energetic and redox disturbances and provide a mechanistic link between disrupted bioenergetics and the failure to sustain platelet activation.
Pre-treatment with the ITC mixture prevented activation and aggregation across tested agonists. In post-activation protocols, early addition of the ITC mixture (25–300 s post-stimulation) destabilized nascent aggregates as detected by aggregometry, indicating an ability to interfere with early aggregate stabilization. However, granule secretion, once initiated, was not reversible under the experimental conditions used, indicating a boundary to reversal of activation-dependent processes.
In a murine model of carrageenan-induced tail thrombosis, administration of the ITC mixture either before or after the carrageenan challenge reduced thrombus formation and tissue injury. The reductions in the primary thrombosis endpoint and in tail necrosis length were reported to be of similar magnitude to those observed in a heparin reference group. Bleeding times were prolonged in ITC-treated animals, again comparable to heparin in this model. Histopathological summaries and experimental treatment group details are reported in the article’s tables and supplementary materials.
Cytotoxicity assays included Annexin V staining for phosphatidylserine exposure and LDH release across exposure intervals; the selected in vitro pre-treatment interval and dosing were reported to avoid overt membrane damage or cytotoxicity. In vivo, ITC-treated mice showed prolonged bleeding times similar to heparin-treated controls in the carrageenan model. Full numerical cytotoxicity data and supplementary figures are cited in the article’s supporting information.
The data identify the tested ITC mixture as a multimodal modulator of platelet function that converges on both signaling networks (reduced kinase activity, Ca2+ mobilization, activation markers) and metabolic support systems (oxidative stress, mitochondrial dysfunction). This dual mechanism plausibly explains both inhibition of aggregation and the capacity to destabilize early aggregates. In vivo antithrombotic efficacy in the carrageenan model, with effects comparable to heparin on measured endpoints, supports further evaluation of ITC-based interventions in thrombotic disease. The study provides mechanistic groundwork but does not itself establish clinical efficacy or safety in humans; additional translational and dose-finding studies are needed as noted by the authors.