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.

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