Abstract | Background: Platelet activation is a tightly regulated physiological process influenced by a range of internal and external factors. One such factor is physical exercise, which is widely recognized for its beneficial role in both the prevention and management of various cardiovascular diseases (CVDs). However, depending on the intensity, duration, and type of exercise, platelet activation may become excessive or dysregulated. In such cases, exercise may contribute to the pathogenesis of CVDs or exacerbate thrombotic risk in individuals with pre-existing cardiovascular conditions. Aims: This study aimed to investigate the molecular mechanisms underlying platelet activation potentiated by the combined effect of adenosine diphosphate (ADP) and arginine vasopressin (AVP), both known to be released during physical exercise. In addition, the study evaluated the inhibitory efficacy of the P2Y₁₂ receptor antagonist cangrelor under these conditions. Methods: Fresh whole blood samples were stimulated with low concentrations of ADP (0.1 µM), AVP (0.1 µM), or a combination of both agonists to evaluate platelet activation. To investigate the inhibitory effect of cangrelor, the P2Y₁₂ receptor antagonist was added at a concentration of 10 µM to each sample. Platelet activation was quantified by flow cytometry based on the surface expression of CD62P (P-selectin). The extent of platelet activation was assessed following stimulation with either single or combined agonists, and the inhibitory effectiveness of cangrelor was compared across all conditions. Results: Stimulation with low concentrations of individual agonists did not result in strong platelet activation. Compared to the baseline condition without agonist stimulation (8.0% ± 4.7%), the addition of 0.1 µM ADP induced a modest increase in activation (29.9% ± 7.5%, P = 0.0178). Similarly, 0.1 µM AVP led to a modest activation response (34.7% ± 15.8%, P = 0.0059). In contrast, co-stimulation with ADP and AVP produced a pronounced synergistic effect, resulting in significantly elevated platelet activation (67.9% ± 7.9%, P < 0.0001) compared to the no-agonist condition. The addition of 10 µM cangrelor effectively abolished platelet activation induced by 0.1 µM ADP (5.1% ± 3.7%, P = 0.0090) as well as that induced by 0.1 µM AVP (10.1% ± 6.1%, P = 0.0090). Furthermore, in the presence of 10 µM cangrelor, P-selectin (CD62P) expression was significantly reduced (13.2% ± 7.6%, P < 0.0001) relative to the synergistic activation observed with combined agonist stimulation. |
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