Structural mechanisms for self-activation of protease-activated receptor 4 by tethered ligand

Protease-activated receptor 4 (PAR4) mediates platelet activation during thrombosis and represents a promising target for antiplatelet therapies. This receptor is activated when serine proteases cleave its N terminus, exposing a self-activating tethered ligand. Here, we report the cryo-electron microscopy (cryo-EM) structure of self-activated PAR4 in complex with G_q heterotrimers. The tethered ligand binds a shallow pocket, forming limited interactions with the transmembrane bundle and adopting a lateral binding pattern distinct from other PAR subtypes. It activates PAR4 by propagating conformational rearrangements to conserved micro-switches, triggering helix 8 rotation. G_q signaling is uniquely modulated by shallow engagement of the wavy hook, which disrupts its interaction with the DRY motif. Molecular docking demonstrates that the selective agonist PAR4-AP enhances activation through additional hydrophobic contacts and a stabilizing salt bridge, revealing the determinants of subtype selectivity. These findings provide mechanistic insights into PAR4 signaling, laying a foundation for the discovery of PAR4-targeted antithrombotic drugs.