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2 protocols using trypsin substrate s 2222

1

Coagulation Enzyme Assay Protocols

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Pentamidine isethionate was purchased from Millipore-Sigma (St. Louis, MO, USA). Reagents for clotting assays including thromboplastin-D, APTT reagent, and thrombin time reagent were all from Fisher Scientific (Pittsburgh, PA, USA). Chemicals used to prepare enzyme assay buffers were from Millipore-Sigma or Fisher Scientific. N,N–dimethylcasein, dansylcadaverine, and dithiothreitol were also from Millipore-Sigma. All types of plasmas were purchased from George King Bio-Medical, Inc. (Overland Park, KS, USA). Coagulation enzymes including thrombin, factor Xa (FXa), factor XIa (FXIa), and factor XIIIa (FXIIIa) were from Haematologic Technologies, Inc. (Essex Junction, VT, USA). Digestive enzymes including trypsin and chymotrypsin were from Millipore-Sigma. Neutrophil elastase was from Elastin Products Company (Owensville, MO, USA). Chromogenic substrates: Spectrozyme TH, Spectrozyme FXa, and Spectrozyme PL, were obtained from Biomedica-Diagnostics (Windsor, NS, Canada). FXIa chromogenic substrate (S-2366) and trypsin substrate (S-2222) were obtained from Diapharma (West Chester, OH, USA). Chromogenic substrate for chymotrypsin (N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide) and that for neutrophil elastase (S-1384) were from Millipore-Sigma.
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2

Assay of Serine Proteases

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Human plasma serine proteases, including thrombin, FXa, FXIa, FIXa, FVIIa/tissue factor, and plasmin were obtained from Haematologic Technologies (Essex Junction, VT). FXIIa was purchased from Enzyme Research Laboratories (South Bend, IN). Bovine α-chymotrypsin, bovine trypsin, and human plasma kallikrein were obtained from Sigma-Aldrich (St. Louis, MO). The substrates Spectrozyme TH, Spectrozyme FXa, Spectrozyme FIXa, and Spectrozyme PL. Spectrozyme VIIa, Spectrozyme FXIIa, and Spectrozyme PK were obtained from Biomedica Diagnostics (Windsor, NS Canada). Factor XIa substrate (S-2366) and trypsin substrate (S-2222) were obtained from Diapharma (West Chester, OH). N-succinyl Ala-Ala-Pro-Phe-p-nitroanilide substrate for chymotrypsin is from Sigma-Aldrich. All enzymes and substrates were prepared in 20-50 mM TrisHCl buffer, pH 7.4, containing 100-150 mM NaCl, 2.5 mM CaCl2, 0.1% PEG8000, and 0.02% Tween80. In the case of FIXa, 33% v/v ethyleneglycol was also added to the buffer.
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