DETECTION OF THE 3 KUNITZ-TYPE SINGLE DOMAINS OF MEMBRANE-BOUND TISSUE FACTOR PATHWAY INHIBITOR (TFPI) BY FLOW-CYTOMETRY

Tissue factor pathway inhibitor, a natural anticoagulant in the extrin sic pathway of blood coagulation, is associated with the endothelial m embrane and presumed to be released by heparin. For flow cytometric de tection of membrane-bound tissue factor pathway inhibitor we synthesiz ed polyclonal monospecific antibodies directed against each of the thr ee Kunitz-type domains. Antisera were obtained by immunisation of rabb its with synthetic oligopeptides representing the reactive site of eac h domain. Kunitz-domain delta 1: (26)CAFKDDGPCKAIMKR(41), domain delta 2: (101)EDPGICRGYITR(112) and domain delta 3: (192)PADRGLCRANENR(204) . Different cell lines (chondrosarcoma, synovial sarcoma, synovial cel ls, leukaemic monocytes) and endothelial cells were investigated by fl ow cytometric analysis using these antibodies. The three tissue factor pathway inhibitor domains were detected on the surface of all cells b y the corresponding antisera. Similar results were obtained by immuno- histochemical staining. Since domain delta 3 was recognised by the app ropriate antibody, it would seem that this third domain is not the mem brane binding site. To investigate the cellular tissue factor pathway inhibitor release, endothelial cells were cultivated with heparin. Pro tein resynthesis and translocation were inhibited by puromycin and mon ensin, respectively. After heparin incubation an increased tissue fact or pathway inhibitor concentration was determined in the cell culture medium by a chromogenic substrate assay. However, the tissue factor pa thway inhibitor density on the cell surface was not influenced by hepa rin, as shown by flow cytometry using the three tissue factor pathway inhibitor antisera. Our results suggest that functionally active tissu e factor pathway inhibitor is not released from the cell surface. Ther efore, the effect of heparin appears to be mediated by secretion of ti ssue factor pathway inhibitor from intracellular stores.