Protac

Five μg of purified proteins in TBS were incubated with 0.1 unit of Protac (Sekisui Diagnostics, Stamford, CT), an activator of human [22 (link)] and canine [24 ] protein C and a substance derived from the venom of Agkistrodon c. contortrix, or same volume of TBS at 37°C for 15 min. All samples were then subjected to non-reducing 12% SDS-PAGE, followed by silver-staining or Western immunoblotting using a polyclonal rabbit anti-protein C (HPC4) tag antibody as described above.

Recombinant human APC (hAPC, drotrecogin alfa [activated]) was purchased from Eli Lilly (Indianapolis, IN, USA); plasma-derived hAPC and hPC from Enzyme Research Laboratories (South Bend, IN, USA); calf thymus histones from Sigma-Aldrich (St. Louis, MO, USA); TM and human coagulation FVa from Hematologic Technologies (Essex Junction, VT, USA); Protac, a rapid activator of PC from Sekisui Diagnostics (Lexington, MA, USA); hAPC substrate Spectrozyme PCa from American Diagnostica (Pfungstadt, Germany) and Sekisui Diagnostics (Lexington, MA, USA), and S-2366 from Diapharma (West Chester, OH, USA).