Cathepsin G Potentiates Biased Signaling through Protease Activated Receptor 4
Authors: Kendrick NaShea , Nieman Marvin T. 
Published: December 2024

Venous thromboembolism (VTE) is a condition in which the blood clots inappropriately. Platelets are essential drivers of VTE, and they are active participants in cell immune responses by interacting with neutrophils. Protease activated receptors (PARs) mediate intracellular signaling initiated by proteases on platelets. Multiple proteases can cleave the N-terminus of PARs to generate unique tethered ligands that potentiate biased signaling. Recently, a role for PAR4 in VTE has been established.

Thrombin cleavage of platelet PAR4 at arginine 47 (R47) leads to sustained signaling through the Gαq and Gα12/13 pathways which help stabilize a growing thrombus through platelet aggregation and adhesion. PAR4 is also activated by the neutrophil protease cathepsin G (CatG), but the signaling and physiological outcomes of this activation in platelets is unknown. Our collaborators demonstrated that CatG cleaves PAR4 at a novel site, serine 67 (S67), in addition to the canonical R47 site. Given the interplay between platelets and neutrophils, CatG may provide a mechanism for context-specific signaling via PAR4 in the progression of VTE. Here, we show that alternative cleavage of platelet PAR4 by CatG potentiates biased signaling.

Previously, the CatG cleavage sites on PAR4 were determined using mass spectrometry of PAR4 N-terminus peptides after incubation with purified human CatG. However, CatG cleavage sites have not been confirmed on receptors. Therefore, we sought to confirm that CatG cleaves PAR4 at an alternative site(s) than thrombin on cells. We used flow cytometry to measure cleavage rates of PAR4-WT, PAR4-R47Q, PAR4-S67A, or a double mutant (DM) expressed on HEK293 cells by CatG. CatG cleaves PAR4-WT, PAR4-R47Q, PAR4-S67A, and PAR4-DM, indicating that CatG can create a unique PAR4 ligand, but S67 and R47 are not the only cleavage sites. We next observed platelet aggregation in response to CatG or an activation peptide derived from the S67 cleavage site, RALLLGWVPTR (RALL-11mer). We used PAR-specific inhibitors, BMS-986120 (PAR4) and vorapaxar (PAR1) to show specificity of signaling. At 0.4-0.8µM of CatG, platelet aggregation was ablated by BMS-986120 but not vorapaxar. At 2µM CatG, aggregation was only blocked 50% of the time by BMS-986120. Conversely, RALL-11mer-induced aggregation was unaffected by PAR1 or PAR4 inhibition. This suggests CatG-dependent platelet aggregation is mediated by PAR4, and activation of PAR4 at S67 bypasses BMS-986120 inhibition which infers a novel ligand binding site. Platelet aggregation is a result of degranulation and integrin activation. We quantified integrin αIIbβ3 activation and P-selectin surface expression response to CatG (0.4-2µM) or RALL-11mer (125-500µM) using flow cytometry. CatG increased αIIbβ3 activation and P-selectin exposure in a dose-dependent manner. Similar to aggregation, the response is inhibited by BMS-986120 only at low concentrations. In contrast, the RALL-11mer did not induce αIIbβ3 activation or P-selectin exposure.

To determine initial signaling consequences of PAR4 cleavage by CatG at R47 versus S67, we measured calcium mobilization in platelets in response to CatG, RALL-11mer, or AYPGKF, which mimics the tethered ligand created by PAR4 cleavage at R47. In comparison to AYPGKF, CatG modestly increases calcium flux while RALL-11mer fails to elicit a calcium response. Since calcium mobilization is a critical step in both degranulation and integrin activation, this provides a potential mechanism for how CatG potentiates biased signaling through PAR4.