Detection of the three Kunitz-type single domains of membrane-bound tissue factor pathway inhibitor (TFPI) by flow cytometry.

ABSTRACT

Tissue factor pathway inhibitor, a natural anticoagulant in the extrinsic pathway of blood coagulation, is associated with the endothelial membrane and presumed to be released by heparin. For flow cytometric detection of membrane-bound tissue factor pathway inhibitor we synthesized polyclonal monospecific antibodies directed against each of the three Kunitz-type domains. Antisera were obtained by immunisation of rabbits with synthetic oligopeptides representing the reactive site of each domain. Kunitz-domain delta 1 26CAFKDDGPCKAIMKR41, domain delta 2 101EDPGICRGYITR112 and domain delta 3 192PADRGLCRANENR204. Different cell lines (chondrosarcoma, synovial sarcoma, synovial cells, leukaemic monocytes) and endothelial cells were investigated by flow cytometric analysis using these antibodies. The three tissue factor pathway inhibitor domains were detected on the surface of all cells by the corresponding antisera. Similar results were obtained by immuno-histochemical staining. Since domain delta 3 was recognised by the appropriate antibody, it would seem that this third domain is not the membrane binding site. To investigate the cellular tissue factor pathway inhibitor release, endothelial cells were cultivated with heparin. Protein resynthesis and translocation were inhibited by puromycin and monensin, respectively. After heparin incubation an increased tissue factor pathway inhibitor concentration was determined in the cell culture medium by a chromogenic substrate assay. However, the tissue factor pathway inhibitor density on the cell surface was not influenced by heparin, as shown by flow cytometry using the three tissue factor pathway inhibitor antisera. Our results suggest that functionally active tissue factor pathway inhibitor is not released from the cell surface. Therefore, the effect of heparin appears to be mediated by secretion of tissue factor pathway inhibitor from intracellular stores.