Development of a continuous assay for the measurement of tissue factor procoagulant activity on intact cells.
Tissue factor (TF) is the major physiological initiator of the coagulation cascade and has an important function in the morbidity and mortality associated with many disease states, including cancer-associated thrombosis and atherosclerosis. TF normally exists in a partially encrypted state and its de-encryption on circulating monocytes, platelets or endothelial cells by inflammatory mediators can lead to thrombosis. Furthermore, many cancer cells express large amounts of TF and these cells communicate readily with the circulation through the fenestrated tumor endothelium. To assess agents or conditions that modulate the encryption state of TF, we developed a continuous assay for the determination of TF procoagulant activity (PCA) in a cell-based system. We have shown the use of this assay at detecting agents that de-encrypt TF thereby leading to an increase in TF PCA in three cancer cell lines, namely, T24/83 bladder carcinoma cells and PC-3 and DU145 prostate cancer cells. Further, through use of this assay, we have shown that the endoplasmic reticulum calcium pump inhibitor, thapsigargin, stimulates the de-encryption of TF. The continuous assay for the determination of TF PCA proved to have inherently less intra- and inter-assay variability than the widely used discontinuous assay and is considerably less labor intensive. Further, the continuous assay produced progress curves that were compatible with curve fitting to allow for the determination of the nature of reaction as well as rate constants for the underlying enzymes, TF/FVIIa and FXa. The continuous assay for the assessment of TF PCA on intact cells is applicable for high-throughput screening to allow for the determination of compounds that modulate TF PCA.
Caldwell
Jennifer A
JA
Division of Nephrology, Department of Medicine, St Joseph's Healthcare Hamilton and Hamilton Centre for Kidney Research, McMaster University, Hamilton, ON, Canada.
Dickhout
Jeffrey G
JG
Al-Hashimi
Ali A
AA
Austin
Richard C
RC
D014408:Biomarkers, Tumor / Q000032:analysis; D002277:Carcinoma / Q000473:pathology; D045744:Cell Line, Tumor; D002468:Cell Physiological Phenomena; D002470:Cell Survival / Q000187:drug effects; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D009363:Neoplasm Proteins / Q000032:analysis / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology; D013925:Thromboplastin / Q000032:analysis / Q000378:metabolism*; D001749:Urinary Bladder Neoplasms / Q000473:pathology
D014408:Biomarkers, Tumor; D004791:Enzyme Inhibitors; D009363:Neoplasm Proteins; D019284:Thapsigargin; D013925:Thromboplastin