CatG-generated PAR4 tethered ligand RALLLGWVPTR induces platelet activation and aggregation. (A) WT human PAR4 (hPAR4, black) or empty vector (mock, blue) was expressed in HEK293T/17 cells and treated with 1.5 mM RALLLGWVPTR (RA-11mer, solid line) or tyrodes (dash lines). Thick lines and thin vertical lines are means and SEMs, respectively. n = 3 independent experiments performed in duplicate. (B-C) Washed platelets (n = 6) were treated with buffer or 1 mM RA-11mer, and platelet activation was measured by (B) PAC-1 binding (mean percent ± SEM) and (C) P-selectin expression (mean percent ± SEM). (D) Representative aggregation tracing of washed platelets treated with 1 mM GYPGQV (GYP), ALLLGWVPTR (AL-10mer), or RA-11mer. (E) Maximum (max) aggregation of platelets treated with buffer or 1 mM of each indicated peptide (mean percent ± SEM; n = 5). (F) Representative tracing of platelet calcium flux induced by RA-11mer (2 mM, black line). Tyrodes buffer (red) served as a negative control. n = 4 independent experiments performed in duplicate. (G) Representative tracing of washed platelets treated with 1 mM or 10 mM RA-6mer or 1 mM RA-11mer. (H) Quantification of maximum (max) aggregation (percent ± SEM) elicited by treatment of same subjects’ platelets with RA-11mer or RA-6mer (1 mM, n = 3). (I) Dog, human, mouse, and rat PAR4 sequence alignment of the 12 amino acids adjacent to the plasma membrane of the first (N-terminal) PAR4 extracellular domain. Arrow indicates Arg⁶⁸ in humans where CatG cleaves PAR4. (J-L) Representative aggregation tracing of dog (blue), human (red), mouse (green), and rat (purple) washed platelets treated with 1 U/mL human thrombin (J), 1 µM human CatG (K), or 1 mM RA-11mer (L). n > 3 for human and mouse (J-L); n = 2 for dog and rat (J-K); n = 2 for rat (L); n = 1 for dog (L).