Argatroban monohydrate

PEG-T1Q (1 μM by peptide) was mixed with recombinant human thrombin (~10 nM working concentration, Haematologic Technologies) in a 384-well plate at 37°C in PBS BSA (1% wt/vol). Similar conditions were used for Factor IX. Fluorescence was monitored using a microplate reader (either SpectroMax Gemini EM or Tecan Infinite). For plasma studies, PEG-T1Q was mixed with 50 μL of control human plasma (Thermo Scientific) and 50 μL of 80 mM CaCl₂ (Sigma) to initiate the clotting cascade with or without the thrombin inhibitor (Argatroban Monohydrate, Sigma) or PBS. For serum stability, PEG-T1Q was added to control human serum and placed at 37°C overnight and then tested for responsiveness to thrombin compared to fresh PEG-T1Q.
PEG-M1Q (1 μM by peptide) was mixed with recombinant human MMP9 or MMP19 (~100 nM working concentration, Enzo Life Sciences) in activity buffer (50 mM Tris, 150 mM NaCl, 5 mM CaCl₂, 1 μM ZnCl₂) containing 1% BSA and fluorescence dequenching was monitored. Marimastat (Tocris Bioscience) was added at a final concentration of 5 μM. A similar approach was used for storage stability experiments.

Human fibrinogen and thrombin were obtained from Enzyme Research Laboratories (South Bend, IN). Monoclonal antibody (mAb) 59D8 which recognizes the N-terminal end of the β-chain of human fibrin [31 (link),32 (link)] was obtained from Dr. H. Weiler, Blood Center of Wisconsin. MAb FPA directed against the fibrinopeptide A of Human fibrinogen was a gift from Dr. J. Shainoff, the Cleveland Clinic. Polyclonal antibody against β₃ integrin subunit was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). The following secondary antibodies were obtained from Invitrogen (Life Technologies, Grand Island, NY): goat anti-mouse antibodies conjugated to Alexa 488, 633 or 647, and donkey anti-rabbit antibodies conjugated to Alexa 568. Alexa 488-labeled fibrinogen was from Invitrogen (Life Technologies, Grand Island, NY). MAb 7E3 was a gift from Dr. B. Coller. thrombin inhibitors argatroban monohydrate and hirudin were from Sigma (St. Louis, MO) and D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK) was from Haematologic Technologies Inc. (Essex Junction, VT). The chromogenic thrombin substrate S-2238 was purchased from Sigma.