Cathepsin G Cleavage of PAR4 Generates a Novel Tethered Ligand That Induces Platelet Activation

Atherosclerotic vessel injury induces recruitment of both platelets and neutrophils where multiple proteases induce platelet activation and aggregation. Platelets contain two protease activated receptors, PAR1 and PAR4, the cleavage of which results in exposure of a new amino terminus to serve as a tethered ligand. Released neutrophil cathepsin G (CatG) has been shown to be a physiologic modulator of platelet thrombus formation in mice. CatG activates PAR4 and not PAR1, presumably because CatG cleaves PAR1 by removing its tethered ligand. However, neither the CatG biochemical cleavage of PAR4 nor the resulting tethered ligands have been reported. The goals of the current study are to (1) identify the CatG-PAR4 cleavage sites and resulting tethered ligands and (2) determine how CatG-stimulated PAR4 signaling is altered by the PAR4 Ala120Thr variant. We synthesized two portions of the PAR4 extracellular N-terminus: amino acids Asp38-Ser58 (PAR4-B) and Asp57-Arg78 (PAR4-C) and exposed each peptide to purified CatG. Mass spectrometry identified two major cleavage sites for PAR4-B: the previously documented CatG and thrombin site Arg47-Gly48 and a novel Cys54-Ala55. Analysis of PAR4-C digestion yielded an additional three novel CatG cleavage sites, two major: Arg68-Ala69 and Leu71-Leu72, and one minor: Leu70-Leu71. Neither concentration or time of exposure appeared to alter the CatG cleavage sites. To assess functionality, we generated peptides based on the novel cleavage sites produced by CatG cleavage. Human washed platelets were treated with each peptide, and platelet activation was assessed by PAC-1 binding. As expected, the known tethered ligand sequence GYPGQV showed a statistically significant increase in PAC-1 binding (p=.02) compared to resting platelets. Three of the remaining four novel peptides generated no significant change in PAC-1 binding compared to baseline. However, peptide 3, representing novel tethered ligand ALLLGW, induced a substantial increase (462%) in PAC-1 binding compared to resting platelets. To assess the effect of the PAR4Ala120Thr variant on CatG-stimulated platelet reactivity, human washed platelets were collected from donors homozygous for Ala120 or Thr120 and stimulated with CatG. Platelets expressing the Thr120 variant displayed a significant increase in PAC-1 compared to Ala120 platelets (54%; p=.004). Addition of a CatG inhibitor caused a significant decrease in platelet activation triggered by CatG for both groups (Thr: -81%, p=.000001; Ala: -78%, p=.00017), and abrogated the significant increase in platelet activation displayed by Thr120 platelets (p=.99). To further examine the relationship between the PAR4 Ala120Thr variant and CatG, platelets from each variant were subjected to increasing amounts of CatG. Platelet activation was measured by PAC-1 binding and P-selectin expression. Both PAC-1 binding and P-selectin expression were significantly increased in platelets from Thr120 donors compared to Ala120 platelets (PAC-1, p=.026; P-selectin, p=.025). Overall, our study identified a previously unidentified CatG cleavage site in PAR4, which produced a novel tethered ligand capable of activating platelets. Because cleavage resulting in the ALLLGW ligand is downstream of the thrombin cleavage site, CatG may modulate in vivo thrombin-induced signaling in platelets or other cell types. Our findings also indicate the hyperactive response of 120Thr platelets is not dependent on a specific PAR4 protease. These new insights into PAR4 biology may provide targets for future antithrombotic therapies.