Background and aims:
The gut microbiome and its metabolites have emerged as important regulators of cardiometabolic health. In type 2 diabetes, alterations in microbial composition are associated with changes in the production of bioactive metabolites. Imidazole propionate (ImP), a microbial metabolite derived from histidine metabolism, is elevated in patients with type 2 diabetes and coronary artery disease and has been linked to insulin resistance, vascular dysfunction, and accelerated atherosclerosis in vivo. Clinical data further associate increased circulating ImP levels with adverse cardiovascular outcomes and earlier recurrent events, suggesting a role in thrombotic processes. Given the central contribution of platelet hyperreactivity to atherothrombosis, we investigated whether ImP directly modulates platelet function and prothrombotic signaling.
Methods and results:
Platelet-rich plasma from healthy donors was pre-incubated with control (0.9% NaCl) or ImP (10 nM, 100 nM) for 30 min. Platelet aggregation was assessed by light transmission aggregometry following stimulation with submaximal concentrations of established platelet agonists, including ADP (4 μM), collagen (0.5 μg/ml), epinephrine (5 μM), and TRAP-6 (8 μM). Platelet activation was further evaluated by flow cytometric analysis of P-selectin (CD62P) expression and PAC-1 binding, and intracellular Ca²⁺ dynamics were measured in Fluo-4-loaded platelets to assess agonist-induced calcium mobilization following pre-treatment with control or ImP (100 nM). To investigate the underlying molecular mechanisms, kinase activity array and Western blotting were performed in washed platelets treated with and without ImP following agonist stimulation with ADP. Our results demonstrate that pre-treatment with ImP significantly increases platelet aggregation across all agonists tested, indicating a broad potentiation of platelet reactivity. This was accompanied by elevated surface expression of CD62P and enhanced PAC-1 binding on platelets, indicating both increased α-granule secretion and αIIbβ₃ integrin activation. In addition, ImP significantly amplified intracellular Ca²⁺ mobilization upon ADP stimulation compared to control, while at basal condition Ca²⁺ levels remained unchanged, suggesting a selective effect on agonist-dependent signaling. Mechanistically, kinase profiling identified ERK1/2 activation as a key downstream signaling pathway, which was confirmed by increased ERK1/2-phosphorylation in ImP-treated platelets.
Conclusion:
In summary, ImP promotes platelet hyperreactivity under ex vivo condition by enhancing platelet aggregation, -activation, and agonist-induced intracellular Ca²⁺ signaling. These effects are associated with increased ERK1/2 activation, implicating MAPK-dependent pathways in ImP-mediated platelet reactivity. Together, these findings identify ImP as a microbiota-derived modulator of platelet function that may contribute to atherothrombotic processes, suggesting that targeting ImP production may represent a novel therapeutic strategy to reduce thrombotic risk.
