Thrombin is a potent activator of human platelets, playing a critical role in hemostasis and thrombosis. This study investigates the roles of three protease-activated receptors (PARs) – PAR1, PAR3, and PAR4 – in thrombin-induced platelet activation. The results show that PAR1 and PAR4 are functionally expressed in human platelets and are responsible for most, if not all, thrombin signaling in these cells. PAR1 mediates platelet responses at low thrombin concentrations and is essential for rapid and robust responses, even at high concentrations. In contrast, PAR4 only mediates activation at high thrombin concentrations. PAR3, while present in mouse platelets, is not detected in human platelets and does not contribute to thrombin signaling in human platelets. The study demonstrates that both PAR1 and PAR4 are necessary for thrombin-induced platelet aggregation and secretion. Inhibition of either receptor significantly reduces platelet activation, with simultaneous inhibition of both receptors nearly abolishing thrombin signaling. PAR1 and PAR4 are also shown to be distinct receptors with different roles in thrombin signaling. PAR1 has a hirudin-like domain that binds thrombin's fibrinogen-binding exosite, while PAR4 lacks this domain. These findings suggest that PAR1 and PAR4 are critical targets for developing antithrombotic agents. The study also highlights the importance of understanding the roles of different receptors in platelet activation for the development of effective therapies for thrombotic disorders.Thrombin is a potent activator of human platelets, playing a critical role in hemostasis and thrombosis. This study investigates the roles of three protease-activated receptors (PARs) – PAR1, PAR3, and PAR4 – in thrombin-induced platelet activation. The results show that PAR1 and PAR4 are functionally expressed in human platelets and are responsible for most, if not all, thrombin signaling in these cells. PAR1 mediates platelet responses at low thrombin concentrations and is essential for rapid and robust responses, even at high concentrations. In contrast, PAR4 only mediates activation at high thrombin concentrations. PAR3, while present in mouse platelets, is not detected in human platelets and does not contribute to thrombin signaling in human platelets. The study demonstrates that both PAR1 and PAR4 are necessary for thrombin-induced platelet aggregation and secretion. Inhibition of either receptor significantly reduces platelet activation, with simultaneous inhibition of both receptors nearly abolishing thrombin signaling. PAR1 and PAR4 are also shown to be distinct receptors with different roles in thrombin signaling. PAR1 has a hirudin-like domain that binds thrombin's fibrinogen-binding exosite, while PAR4 lacks this domain. These findings suggest that PAR1 and PAR4 are critical targets for developing antithrombotic agents. The study also highlights the importance of understanding the roles of different receptors in platelet activation for the development of effective therapies for thrombotic disorders.