Tethered ligand–mediated PAR4 activation model. (A) Before thrombin activation, the tethered ligand is buried and already docks close to the LBS of PAR4. The interaction between the tethered ligand and its binding site is blocked by ECL3 (orange line). Thrombin cleavage triggers a structural rearrangement of ECL3 that opens the accessibility between the tethered ligand and the binding site. The tethered ligand drops directly into the LBS (shown in red) and the Gly48 interacts with Thr153 to further initiate downstream signaling, which requires G-protein complex to recruit and dock at ICL2 of PAR4. (B) The computational modeling of the thrombin-activated PAR4 model, in which the ECL3 adapted in an “out” position to provide space for the tethered ligand to interact with the binding site further support the working model of the PAR4 activation mechanism. The tethered ligand is green, the LBS is red, and the ECL3 is blue.