Evaluation of tissue factor procoagulant activity on the surface of feline leukocytes in response to treatment with lipopolysaccharide and heat-inactivated fetal bovine serum

Abstract

To use a chromogenic assay to measure tissue factor (TF) activity on the cell surface and in whole cell lysates of feline monocytes in response to treatment with lipopolysaccharide (LPS) and fetal bovine serum (FBS). 14 healthy cats. Peripheral blood monocytes were isolated via density gradient centrifugation followed by adhesion to plastic. Tissue factor procoagulant activity was measured by use of an assay that detects TF-activated factor X, on the basis of cleavage of a chromogenic TF-activated factor X-dependent substrate. Activity was quantified by comparison with a serially diluted human recombinant TF-activated factor x curve. The TF procoagulant activity assay was sensitive and specific for TF. Treatment with LPS stimulated TF procoagulant activity on the surface and in whole cell lysates of isolated feline leukocytes. The LPS response in intact cells was dose dependent and cell number dependent and was inhibited by FBS. Monocyte isolation was inefficient, with monocytes comprising a mean of 22% of the isolated cells. A TF-activated factor X-dependent chromogenic assay that uses human reagents successfully measured surface-expressed and intracellular TF activity of feline monocytes. Treatment with LPS induced TF expression on feline monocytes, but this response was inhibited by FBS. The chromogenic assay was a useful method for measuring TF procoagulant activity in feline cells in vitro and can be used as a research tool to investigate the role of cell-associated TF in thrombotic disorders in cats.