A defined isothiocyanate mixture suppresses platelet signaling and metabolism to limit thrombosis in vivo.

The ITC mixture inhibited platelet activation and aggregation induced by multiple agonists ex vivo.

• The mixture contained allyl-, benzyl-, and phenethyl-isothiocyanate in a defined formulation.
• Platelet aggregation was assessed using aggregometry with multiple activating agonists.
• Inhibition was observed across a range of stimuli, suggesting broad-spectrum antiplatelet activity rather than pathway-specific inhibition.
• Effects were evaluated using ex vivo human platelets.

The ITC mixture reduced peptide phosphorylation across multiple platelet signaling pathways as assessed by kinome profiling.

• Kinome profiling was used to evaluate intracellular signaling changes.
• Phosphorylation was reduced across multiple pathways, not limited to a single signaling axis.
• This broad suppression of kinase activity suggests the ITC mixture acts at multiple nodes within platelet signal transduction.
• The breadth of pathway inhibition contrasts with current antiplatelet therapies that target a limited range of signaling pathways.

The ITC mixture reduced intracellular calcium mobilization in platelets.

• Calcium mobilization was assessed using flow cytometry.
• Intracellular calcium is a key second messenger in platelet activation.
• Reduction in calcium signaling is consistent with the observed inhibition of downstream platelet activation events.
• This finding was evaluated in ex vivo human platelets.

The ITC mixture reduced the activation-associated oxidant signal in platelets.

• Redox balance was assessed using flow cytometry.
• The activation-associated oxidant signal was reduced following ITC mixture treatment.
• This finding indicates the ITC mixture influences reactive oxygen species dynamics during platelet activation.
• Isothiocyanates are known to have redox-modulating properties, consistent with this observation.

The ITC mixture impaired mitochondrial function in platelets.

• Mitochondrial function was assessed using respirometry and electron microscopy.
• Impairment of mitochondrial function suggests the ITC mixture affects platelet energy metabolism.
• Platelet metabolism is increasingly recognized as important for sustained activation and aggregate stability.
• The combination of respirometry and electron microscopy provided both functional and structural evidence of mitochondrial effects.

When added shortly after platelet activation, the ITC mixture produced aggregometry signals consistent with destabilization of early platelet aggregates, but granule secretion was irreversible once initiated.

• Aggregometry signals consistent with destabilization were observed when the ITC mixture was added after activation.
• Granule secretion, once initiated, was not reversed by ITC mixture treatment.
• This suggests the ITC mixture can partially disrupt early aggregates but cannot undo all activation-associated events.
• The finding has implications for the potential therapeutic window of ITC-based interventions.

In vivo administration of the ITC mixture before or after carrageenan challenge reduced thrombus formation and tissue injury.

• A murine model of carrageenan-induced thrombosis was used for in vivo evaluation.
• The ITC mixture was administered both as a pre-treatment and as a post-challenge treatment.
• Both pre- and post-challenge administration reduced thrombus formation and tissue injury.
• Reduction in thrombus formation and tissue injury was observed regardless of the timing of ITC administration relative to the thrombotic challenge.

The ITC mixture prolonged bleeding times and reduced tail-necrosis length to a similar extent as the heparin reference group in the carrageenan-induced thrombosis model.

• Bleeding times were prolonged in ITC mixture-treated animals, indicating a systemic anticoagulant or antiplatelet effect.
• Tail-necrosis length was reduced, providing a measure of thrombus-related tissue injury.
• The magnitude of these effects was comparable to heparin, the reference anticoagulant used in this model.
• The carrageenan-induced thrombosis model is an established in vivo model for evaluating antithrombotic interventions.

Current antiplatelet therapies have a limited range of targeted signaling pathways, have minimal impact on established platelet aggregates, and are frequently associated with bleeding complications.

• This limitation of existing therapies is cited as the motivation for investigating alternative antiplatelet strategies.
• The ITC mixture was evaluated as a potential alternative approach addressing these drawbacks.
• The paper notes that ITC-mediated mechanisms and translational relevance were previously incompletely defined.
• The broad mechanistic profile of the ITC mixture is contrasted with the narrower targets of current therapies.