APC cleaves a synthetic PAR1 N-terminal peptide at Arg41 and Arg46. Cleavage of a PAR1 N-terminal tail peptide (TR33-62) by APC (500nM) or thrombin (10nM) in the presence of CaCl2 (4mM) and MgCl2 (0.6mM) and resolution of cleavage fragments by HPLC using C18 reverse phase chromatography. (A) Chromatograms of APC-mediated TR33-62 cleavage over time. (B) Time course of TR33-62 cleavage by APC (□) or thrombin (■). (C) Generation of the N-terminal cleavage fragment derived from cleavage at Arg41 (TR33-41) by APC (▿) or thrombin (▾). (D) Generation of the C-terminal cleavage fragment derived from cleavage at Arg41 (TR42-62) by APC (Δ) or thrombin (▴). (E) Generation of the N-terminal TR42-46 (♢, ♦) and C-terminal TR47-62 fragments (○, ●) derived from cleavage at Arg46 by APC (open symbols) or by thrombin (closed symbols). (F) Time courses for cleavage of peptide TR42-66 (□, ■) and generation of TR42-46 (♢, ♦) and TR47-66 (○, ●) cleavage fragments by APC (open symbols) or thrombin (closed symbols). Representative chromatograms (A) and (B-F) data points represent the mean ± SD (N ≥ 3).
