Substrate-Assisted Catalysis of the PAR1 Thrombin Receptor

Abstract

Platelet activation and aggregation are mediated by thrombin cleavage of the exodomain of the PAR1 receptor. The specificity of thrombin for PAR1 is enhanced by binding to a hirudin-like region (Hir) located in the receptor exodomain. Here, we examine the mechanism of thrombin-PAR1 recognition and cleavage by steadystate kinetic measurements using soluble PAR1 N-terminal exodomains. We determined that the primary role of the PAR1 Hir sequence is to reduce the kinetic barriers to formation of the docked thrombin-PAR1 complex rather than to form high affinity ground-state interactions. In addition, the exosite I-bound Hir motif facilitates the productive interaction of the PAR1 38 LDPR/SFL 44 sequence with the active site of thrombin. This locking process is the most energetically unfavorable step of the overall reaction. The subsequent irreversible steps of peptide bond cleavage are rapid and allosterically enhanced by the presence of the docked Hir sequence. Furthermore, the C-terminal exodomain product of thrombin cleavage, corresponding to the activated receptor, binds tightly to thrombin. This would suggest that an additional role of the Hir sequence in the thrombin-activated receptor is to sequester thrombin to the platelet surface and modulate cleavage of other platelet receptors such as the PAR4 thrombin receptor, which lacks a functional Hir sequence. Cellular responses to thrombin are regulated by a novel class of seven transmembrane protease-activated receptors (PARs). 1 PAR1 plays an important role in platelet activation and blood coagulation and has been implicated in the pathological processes leading to heart disease and stroke (1). PAR1 and PAR4 mediate platelet aggregation in humans, whereas PAR3 and PAR4 are responsible for platelet aggregation in mice (2-4). PAR1 and PAR4 have markedly different kinetics of activation by thrombin that contribute to their distinct roles in signaling in human platelets (5). PAR2 is a trypsin/tryptase receptor that is not cleaved by thrombin (6, 7 (Hir), that resembles the C-tail of the leech anti-coagulant protein, hirudin. The PAR1 Hir sequence interacts with exosite I of thrombin as indicated by x-ray structural analysis (10). Mutagenesis studies indicate that the presence of the Hir sequence in the PAR1 exodomain confers significant enhancements in the efficiency of thrombin cleavage (11-13). In analogy to the dual interactions of hirudin and serpins with thrombin (14, 15), it is possible that the Hir sequence may increase the probability that productive orientations occur at the active site by anchoring the LDPR region and by an induced fit mechanism. Thus, PAR4, which lacks a functional Hir sequence (16), is activated 20-to 70-fold slower by thrombin than PAR1 on human platelets (5).An unusual feature of thrombin-PAR interactions is that stoichiometric amounts of thrombin, rather than catalytic amounts, are required for platelet activation (5). This raises the possibility that the low turnover of thrombin could be due to tight binding and a slow off-rate fro...

N-Linked Glycosylation of Protease-activated Receptor-1 Second Extracellular Loop

“…Interestingly, an N-linked glycosylation site present in the N terminus of PAR2 appears to regulate signaling by the serine protease tryptase but not trypsin or synthetic peptide agonist (16,37), suggesting that glycosylation of PARs at the N terminus can dictate protease specificity. Thrombin binds to and cleaves PAR1 with exquisite specificity and recognizes both the N-terminal LDPR2S cleavage site and an acidic region C-terminal to the cleavage site termed the hirudin-like sequence (46). Our results indicate that PAR1 is glycosylated at the N terminus, but the actual site of glycosylation remains to be determined.…”

Binding of Exosite Ligands to Human Thrombin

“…Exosite I binds fibrinogen (Fbg) 1 (3), fibrin I and II (3,4), the 12-residue carboxyl-terminal hirudin 54 -65 sequence (5,6), thrombomodulin (7), the thrombin receptor (8,9), and an acidic sequence on the serpin, heparin cofactor II (10,11). Exosite II binds heparin and other glycosaminoglycans (2,12,13), prothrombin activation fragment 2 (F2) (14), the chondroitin sulfate moiety of thrombomodulin (15,16), the leech peptide hemadin (17), and an exosite II-specific human monoclonal antibody (18).…”

Protein targets of inflammatory serine proteases and cardiovascular disease

“…PAR 1 and PAR 4 signaling show considerably different kinetics and indeed appear to have distinct functions in platelet aggregation [14]. PAR 1 is a high-affinity receptor for thrombin by virtue of a hirudin-like sequence that resides in its N-terminal extracellular domain [18,19]. Signal transduction via PAR 1 is fast and transient, and is followed by a prolonged signaling arising from PAR 4 , a receptor normally more slowly activated by thrombin.…”