Figure 1.
Neutrophil cathepsin G cleaves PAR4 at Ser67-Arg68 to induce platelet aggregation. (A) Washed platelets were treated with varying concentrations of CatG in the presence or absence of the PAR4 inhibitor, BMS-986120 (400 nM BMS; n = 5), and maximum(max)platelet aggregation (percent ± SEM) recorded. The PAR1 activation peptide (SFLLRN; 10 μM) served as a negative control (n = 5) for PAR4 inhibition. (B) Washed platelets were stimulated with increasing concentrations of CatG, and platelet activation was measured by PAC-1 binding (Ala: n = 7; Thr: n = 7) and displayed as MFI (mean ± SEM). (C) PAR4 amino acid sequence targeted by RC3 monoclonal antibody shown above. The underline represents the tethered ligand generated by thrombin. The arrow represents the location of the canonical thrombin cleavage site. All aggregation studies were performed with PAR1 blockade using 100 nM vorapaxar. Representative tracing of washed platelets treated with 0.25 U/mL thrombin or 200 nM CatG in the presence or absence of RC3. (D) Quantification of maximum (max) platelet aggregation (mean percent ± SEM) (n = 3 different subjects). (E) PAR4-B and PAR4-C peptide sequences used in CatG proteolysis analysis by LC-MS/MS. Red sequence indicates novel tethered ligand generated by CatG. (F) LC-MS/MS performed on PAR4-C in the absence of CatG. Time of flight analysis showed an experimental peak with the correct mass (m) over charge (z) ratio. (G) LC-MS/MS analysis performed on PAR4-C after incubation with 400 nM CatG at 37°C for 15 minutes. Time of flight analysis observed a peak with the correct m/z ratio of a fragment containing the amino acids DSDTLELPSS (the last residue is Ser67), indicating CatG cleaved PAR4-C between Ser67 and Arg68. For reference, the expected m/z ratio of DSDTLELPSS is shown below. (H) Calcium mobilization of WT, mutated PAR4, or empty vector (mock) expressed in HEK293T/17 cells treated with or without CatG (2.5 µM) in the presence of PAR1 blockade with 100 nM vorapaxar. (I) WT, mutated PAR4, or empty vector (mock) were expressed in HEK293T/17 cells and treated with thrombin (1.5 U/mL) in the presence of 100 nM vorapaxar. Solid thick lines and thin vertical lines are means and SEMs, respectively. n = 4 independent experiments performed in duplicate in panels H-I.

Neutrophil cathepsin G cleaves PAR4 at Ser67-Arg68 to induce platelet aggregation. (A) Washed platelets were treated with varying concentrations of CatG in the presence or absence of the PAR4 inhibitor, BMS-986120 (400 nM BMS; n = 5), and maximum(max)platelet aggregation (percent ± SEM) recorded. The PAR1 activation peptide (SFLLRN; 10 μM) served as a negative control (n = 5) for PAR4 inhibition. (B) Washed platelets were stimulated with increasing concentrations of CatG, and platelet activation was measured by PAC-1 binding (Ala: n = 7; Thr: n = 7) and displayed as MFI (mean ± SEM). (C) PAR4 amino acid sequence targeted by RC3 monoclonal antibody shown above. The underline represents the tethered ligand generated by thrombin. The arrow represents the location of the canonical thrombin cleavage site. All aggregation studies were performed with PAR1 blockade using 100 nM vorapaxar. Representative tracing of washed platelets treated with 0.25 U/mL thrombin or 200 nM CatG in the presence or absence of RC3. (D) Quantification of maximum (max) platelet aggregation (mean percent ± SEM) (n = 3 different subjects). (E) PAR4-B and PAR4-C peptide sequences used in CatG proteolysis analysis by LC-MS/MS. Red sequence indicates novel tethered ligand generated by CatG. (F) LC-MS/MS performed on PAR4-C in the absence of CatG. Time of flight analysis showed an experimental peak with the correct mass (m) over charge (z) ratio. (G) LC-MS/MS analysis performed on PAR4-C after incubation with 400 nM CatG at 37°C for 15 minutes. Time of flight analysis observed a peak with the correct m/z ratio of a fragment containing the amino acids DSDTLELPSS (the last residue is Ser67), indicating CatG cleaved PAR4-C between Ser67 and Arg68. For reference, the expected m/z ratio of DSDTLELPSS is shown below. (H) Calcium mobilization of WT, mutated PAR4, or empty vector (mock) expressed in HEK293T/17 cells treated with or without CatG (2.5 µM) in the presence of PAR1 blockade with 100 nM vorapaxar. (I) WT, mutated PAR4, or empty vector (mock) were expressed in HEK293T/17 cells and treated with thrombin (1.5 U/mL) in the presence of 100 nM vorapaxar. Solid thick lines and thin vertical lines are means and SEMs, respectively. n = 4 independent experiments performed in duplicate in panels H-I.

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