PO032 | INHIBITION OF PLATELET ACTIVATION BY SMALL MOLECULE SYNTHETIC AGONISTS OF THE BILE ACID RECEPTOR: A NOVEL APPROACH TO ANTIPLATELET THERAPY

L. Bury¹, E. Falcinelli¹, V. Fazzi¹, C. Tili¹, P. Gresele¹, S. Fiorucci² | ¹Department of Medicine and Surgery, Section of Internal and Cardiovascular Medicine; ²Section of Gastroenterology, University of Perugia, Italy

Background and Aims. Bile acid receptors regulate lipid, glucose and energy metabolism especially in the liver, but they also play a key role as signaling molecules in other processes such as inflammation and immune modulation. Platelets express the two main bile acid receptors: the farnesoid X receptor (FXR) and the Takeda G-protein- coupled receptor 5 (TGR5) and it has been reported that bile acids inhibit platelet activation in vitro and in vivo (PMID:27758768, PMID: 38968449). Indeed deoxycholic acid (DCA), a natural bile acid, acts as a down-regulator of platelet activation acting on TGR5. Moreover, circulating DCA is decreased in patients with coronary artery disease (CAD), a condition associated with increased platelet activation (PMID: 40217125), suggesting that TGR5 signaling may act as a negative modulator for platelets in vivo. Our aim was to investigate the effects of two small molecule synthetic agonists of TGR5, BAR501 and BAR502, currently in clinical development for the treatment of chronic inflammatory bowel diseases (IBD), on platelet function in order to explore whether they may represent potential innovative therapeutic candidates for cardiovascular disease.
Methods. Platelet rich plasma (PRP) from healthy controls was pre-incubated with DCA and the synthetic TGR5 agonists BAR501 and BAR502 (1 to 100 nM) for 30 minutes, then platelet activation was triggered by convulxin (CVX, 5 ng/ml), ADP (5 M) or TRAP-6 (20 M), and assessed by the measurement of P-selectin expression and αIIbβ3 activation by flow cytometry and platelet aggregation by light transmission aggregometry. Platelet spreading on fibrinogen was assessed by immunofluorescence. To investigate on the signaling involved, intracellular Ca²⁺ mobilization was assessed by flow cytometry.
Results. αIIbβ3 activation in response to CVX, ADP and TRAP-6 was reduced by pre-incubation with the synthetic TGR5 agonists BAR501 and BAR502 as well as with the natural bile acid DCA (Figure 1 A-C). Also P-selectin expression in response to CVX was decreased by DCA, BAR501 and BAR502 (Figure 1 D). Accordingly, BAR501 and BAR502, at the dose of 1 nM, significantly reduced platelet aggregation in response to ADP, collagen and TRAP-6 (Figure 1 E-G). All these molecules also decreased platelet spreading on fibrinogen (Figure 1 H). Ca²⁺ mobilization was reduced upon incubation with BAR501 and BAR502 (Figure 1 I).
Conclusions. Activation of TGR5 by physiological concentrations of DCA or by the two synthetic agonists BAR501 and BAR502 at their EC50 markedly suppresses key platelet responses, including platelet activation, adhesion and aggregation. The mechanism of action involves the decreased intracellular Ca²⁺ increase upon agonist stimulation. Our findings highlight TGR5 as a novel pharmacological target for platelet inhibition and support the development of synthetic agonists, such as BAR501 and BAR502, as potential antiplatelet strategies for cardiovascular disease.