title: A simplified and high-throughput chromogenic assay for testing tissue factor-dependent procoagulant activity.
abstract: Tissue factor (TF), the primary initiator of the coagulation cascade, plays a critical role in hemostasis and thrombosis, and inhibition of TF activity appears to be an attractive target for the treatment of cardiovascular diseases. However, few selective small-molecule inhibitors of TF are available, and the present assays for measuring TF activity are relatively expensive and complex. The authors present a simple and high-throughput chromogenic assay for screening TF inhibitors based on using commercial human prothrombin complex instead of purified coagulation factors, reducing the dosage, and performing with a one-stage procedure. In the optimized assay, <45 µL cell lysates was incubated with Tris-CaCl(2) buffer (pH 7.3) containing human prothrombin complex at 37°C for 15 min in 96-well or 384-well plates. Tris-EDTA buffer (pH 8.4) containing chromogenic substrate Xa was then added and the absorbance measured at 405 nm. This simplified assay was more sensitive or precise than some reported methods for TF procoagulant activities. Two known active compounds (curcumin and simvastatin) inhibiting TF activity were tested by the simplified assay to validate the screening method. Furthermore, berberine and cryptotanshinone suppressed TF activity induced by lipopolysaccharides in human monocytes by this assay and might be promising new TF inhibitors.
authors.affiliation: Department of Complex Prescription of TCM, School of Chinese Material Medicine, China Pharmaceutical University, Nanjing, China.
authors.forename: Wenwen
authors.initials: W
authors.lastname: Jiang
authors.forename: Junping
authors.initials: J
authors.lastname: Kou
authors.forename: Shengtao
authors.initials: S
authors.lastname: Yuan
authors.forename: Li
authors.initials: L
authors.lastname: Sun
authors.forename: Boyang
authors.initials: B
authors.lastname: Yu
mesh_terms: D001599:Berberine / Q000378:metabolism / Q000494:pharmacology; D001779:Blood Coagulation Factors / Q000378:metabolism; D002478:Cells, Cultured; D002863:Chromogenic Compounds / Q000378:metabolism; D003474:Curcumin / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D057166:High-Throughput Screening Assays; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism / Q000494:pharmacology; D008074:Lipoproteins / Q000378:metabolism; D019821:Simvastatin / Q000494:pharmacology; D013925:Thromboplastin / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D001779:Blood Coagulation Factors; D002863:Chromogenic Compounds; D004791:Enzyme Inhibitors; D008070:Lipopolysaccharides; D008074:Lipoproteins; D014409:Tumor Necrosis Factor-alpha; C045071:leukocyte procoagulant activity; C051928:lipoprotein-associated coagulation inhibitor; D001599:Berberine; D013925:Thromboplastin; D019821:Simvastatin; D003474:Curcumin