Distinct Thromboxane A₂-Dependent Pathways Regulate Arachidonic Acid-Triggered VASP Phosphorylation at Ser239 and Ser157 in Human Platelets: Real-Time Visualization Reveals Superior Antithrombotic Efficacy by Targeting Thromboxane A₂ Signaling over Cyclooxygenase Inhibition

Platelets, as anucleate blood cells, play a pivotal role in the pathogenesis of cardiovascular diseases (CVDs), making antiplatelet therapy essential for preventing thrombotic events such as myocardial infarction. Thromboxane A₂ (TXA₂) is a key pro-aggregatory mediator that drives platelet activation. Phosphorylation of vasodilator-stimulated phosphoprotein (VASP) at Ser157 and Ser239 serves as a marker of cyclic nucleotide-mediated inhibitory signaling. The crosstalk between TXA₂ signaling and site-specific VASP phosphorylation in arachidonic acid (AA)-stimulated human platelets remains unclear and requires further investigation. In this study, AA at 60 µM induced maximal platelet activation, as evidenced by ultrastructural changes and increased P-selectin expression. Picotamide, a thromboxane synthase (TXS) inhibitor, effectively reversed AA-induced alterations, including ultrastructural remodeling, P-selectin expression, TXA₂ production, adenosine triphosphate (ATP)-release, mobilization of [Ca²⁺]ᵢ, and Integrin α_IIbβ₃ activation. Importantly, picotamide's inhibition of platelet aggregation was unaffected by adenylate or Guanylate Cyclase inhibitors, suggesting a mechanism independent of cyclic nucleotide signaling. AA selectively increased VASP phosphorylation at Ser239, but not Ser157. While picotamide alone had no effect, its sequential administration with AA significantly enhanced Ser157 phosphorylation without altering Ser239 levels. These findings suggest that AA differentially regulates VASP phosphorylation sites via distinct mechanisms: Ser239 via a TXA₂-independent pathway associated with inhibitory signaling, and Ser157 via a TXA₂-dependent pathway linked to platelet activation. Finally, picotamide demonstrated superior antithrombotic efficacy compared to aspirin at an equivalent dose, as evidenced by real-time intravital imaging of thrombotic platelet plug formation in vivo. These results highlight TXS inhibition as a promising strategy for modulating platelet activation and thrombosis.