Protease Activated Receptor-4 (PAR4) Uses Anionic Residues to Interact with α-Thrombin in the Absence or Presence of PAR1.

Thrombin activates protease activated receptor 1 (PAR1) faster than protease activated receptor 4 (PAR4) due to a hirudin-like sequence in the exodomain of PAR1 that binds thrombin’s exosite I. However, recombinant exodomain studies indicate that PAR4 does have extended contacts with α-thrombin that influence PAR4’s kinetics of cleavage. In the current report, the role of an anionic cluster (Asp⁵⁷, Asp⁵⁹, Glu⁶², Asp⁶⁵) in the exodomain of PAR4 is examined for its influence on cleavage and activation of PAR4 on cells in the absence or presence of PAR1. α-Thrombin induces wild type PAR4 (PAR4-wt) calcium flux with an EC₅₀ of 110 nM whereas mutation of the four anionic residues (PAR4-AAAA) increases the EC₅₀ to 641 nM. In contrast, PAR4-wt and PAR4-AAAA are activated by γ-thrombin with a similar EC₅₀ (588 nM and 449 nM, respectively, p = 0.48), indicating a role for α-thrombin’s exosite I in PAR4 activation. Coexpression of PAR1, lowered the EC₅₀ of cleavage 10 fold for both PAR4-wt from 321 to 26 nM and PAR4-AAAA from 2.2 μM to 360 nM, respectively. Individual point mutations at Asp⁵⁷, Asp⁵⁹, Glu⁶² or Asp⁶⁵ show that PAR4-D57A is activated by α-thrombin with the same EC₅₀ as PAR4-wt (140 nM) whereas PAR4-D59A is the same as PAR4-AAAA (699 nM). Glu⁶² and Asp⁶⁵ contribute to α-thrombin recognition, but to a lesser extent. The current report shows that PAR4 uses its anionic cluster to interact with α-thrombin and that this interaction is important even in the presence of PAR1.