Abstract Body: Introduction
Arterial thrombosis driven by platelet activation is the central pathological mechanism underlying myocardial infarction and ischemic stroke. Elevation of intraplatelet cyclic adenosine monophosphate (cAMP) is a critical endogenous mechanism that restrains platelet activation. Although the adenosine A_2A receptor is a dominant regulator of this pathway in platelets, the clinical application of existing agonists is hindered by limited potency and poor pharmacokinetics.

Hypothesis
We assessed the hypothesis that a novel high affinity adenosine A_2A receptor agonist inhibits platelet activation via the cAMP–protein kinase A (PKA) pathway and prevents arterial thrombosis in vivo without impairing physiological hemostasis.

Methods
Human platelet-rich plasma and washed platelets were used to evaluate aggregation, cAMP levels, and calcium mobilization. Binding affinity was determined by radioligand assays (n = 3 to 4). In vivo antithrombotic efficacy was assessed using a ferric-chloride induced carotid artery thrombosis model, and hemostasis was evaluated by tail bleeding assays in mice (n = 6 per group).

Results
The lead agonist bound the human A_2A receptor with high affinity (K_i = 7.4 ± 0.8 nM) and inhibited adenosine diphosphate-induced human platelet aggregation with a half maximal inhibitory concentration (IC₅₀) of 11.8 ± 4.3 nM. This potency was superior to that of the P2Y₁₂ receptor inhibitor ticagrelor (IC₅₀ = 21.5 ± 9.8 nM). Treatment with 10 nM of the agonist increased intraplatelet cAMP levels by 204%, corresponding to an increase from 2.5 ± 0.1 to 5.1 ± 0.3 pmol/10⁸ platelets (n = 3). This signaling induced PKA-dependent phosphorylation of vasodilator stimulated phosphoprotein and inositol trisphosphate receptor and significantly attenuated calcium mobilization. Consequently, integrin α_IIbβ₃ activation and fibrinogen binding were suppressed. Oral administration of 10 mg/kg of the agonist significantly delayed carotid artery occlusion compared with vehicle (16.6 ± 3.3 vs. 7.6 ± 0.5 min, n = 6, p = 0.0005) without prolonging bleeding time. The oral bioavailability was 5.3% (n = 4).

Conclusions
In conclusion, this novel adenosine A_2A receptor agonist suppresses platelet activation through cAMP–PKA signaling and prevents arterial thrombosis without compromising hemostasis, supporting its potential as a next-generation antithrombotic agent with an improved safety profile.