Neutrophil cathepsin G potentiates biased signaling through Protease Activated Receptor 4 Open Access

• Cathepsin G creates a novel PAR4 tethered ligand and biases PAR4 signaling through Gαq and β-arrestin pathways.

• PAR4 activation at Ser67 is not inhibited by BMS-986120

Platelets and neutrophils play a complex role in inflammation and thrombosis. Platelets are crucial for hemostasis, forming platelet plugs at the site of vascular injury. They are also drivers of venous thromboembolism (VTE), and they participate in cell immune responses by interacting with neutrophils. Neutrophil recruitment to platelet plugs is integral to resolve vascular injury, but excessive neutrophil infiltration can shift the response from hemostatic to thrombotic. The neutrophil protease cathepsin G (CatG) cleaves Protease Activated Receptor 4 (PAR4) at alternative sites than thrombin, but the signaling and physiological outcomes of this interaction are understudied. Considering the interaction between platelets and neutrophils, CatG may provide a mechanism for context specific signaling through PAR4. We used light transmission aggregometry and flow cytometry to measure platelet aggregation, integrin activation, and P-selectin surface expression on human platelets in response to CatG or the RALL 11-mer, which mimics PAR4 cleavage by CatG at Ser67. We observed calcium mobilization, RhoA activation, and Akt phosphorylation in platelets to investigate which PAR4 signaling pathways are activated by CatG. Here, we show that CatG cleaves PAR4 expressed on the surface of cells at a different site than thrombin. Both CatG and the RALL 11-mer increase platelet aggregation, integrin activation, and P-selectin surface expression. Additionally, CatG activation of PAR4 increases calcium mobilization and Akt phosphorylation in platelets. The RALL 11-mer increases Akt phosphorylation to activate platelets. These results indicate that CatG utilizes Gαq and β-arrestin pathways to facilitate platelet aggregation in the absence of Gα12/13 signaling.