Neutrophil cathepsin G proteolysis of protease-activated receptor 4 generates a novel, functional tethered ligand.

Michelle L Stoller, Indranil Basak, Frederik Denorme, Jesse W Rowley, James Alsobrooks, Krishna Parsawar, Marvin T Nieman, Christian Con Yost, Justin R Hamilton, Paul F Bray, Robert A Campbell

Blood advances 2022 Apr 12

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Platelet-neutrophil interactions regulate ischemic vascular injury. Platelets are activated by serine proteases that cleave protease-activated receptor (PAR) amino termini, resulting in an activating tethered ligand. Neutrophils release cathepsin G (CatG) at sites of injury and inflammation, which activates PAR4 but not PAR1, although the molecular mechanism of CatG-induced PAR4 activation is unknown. We show that blockade of the canonical PAR4 thrombin cleavage site did not alter CatG-induced platelet aggregation, suggesting CatG cleaves a different site than thrombin. Mass spectrometry analysis using PAR4 N-terminus peptides revealed CatG cleavage at Ser67-Arg68. A synthetic peptide, RALLLGWVPTR, representing the tethered ligand resulting from CatG proteolyzed PAR4, induced PAR4-dependent calcium flux and greater platelet aggregation than the thrombin-generated GYPGQV peptide. Mutating PAR4 Ser67or Arg68 reduced CatG-induced calcium flux without affecting thrombin-induced calcium flux. Dog platelets, which contain a conserved CatG PAR4 Ser-Arg cleavage site, aggregated in response to human CatG and RALLLGWVPTR, while mouse (Ser-Gln) and rat (Ser-Glu) platelets were unresponsive. Thus, CatG amputates the PAR4 thrombin cleavage site by cleavage at Ser67-Arg68 and activates PAR4 by generating a new functional tethered ligand. These findings support PAR4 as an important CatG signaling receptor and suggest a novel therapeutic approach for blocking platelet-neutrophil-mediated pathophysiologies. © 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.

Citation

Michelle L Stoller, Indranil Basak, Frederik Denorme, Jesse W Rowley, James Alsobrooks, Krishna Parsawar, Marvin T Nieman, Christian Con Yost, Justin R Hamilton, Paul F Bray, Robert A Campbell. Neutrophil cathepsin G proteolysis of protease-activated receptor 4 generates a novel, functional tethered ligand. Blood advances. 2022 Apr 12;6(7):2303-2308