CHARACTERIZATION OF CELL-SURFACE PROCOAGULANT ACTIVITIES USING A MICROCARRIER MODEL

A novel model is described for characterisation of cell-surface procoa gulant activities and their inhibitors. Microcarrier beads were used t o present living cells to recalcified blood plasma in the stirred meas uring wells of an electromagnetic coagulometer. By this means the proc oagulant activity on the surface of the cells could be automatically d etermined as clotting time. Procoagulant activity was investigated on normal and transformed cells, and representing hemopoietic, endothelia l, muscle and connective tissue phenotypes. The procoagulant activity on each cell type was characterised by the use of specifically immunod epleted plasmas and specific inhibitors, including monoclonal antibodi es. The predominant cell surface trigger of coagulation found in this series was tissue factor, and only blood monocytes provided some evide nce for direct activation of factor X independent of FVII. Human ECV30 4 transformed endothelial cells were more closely studied as represent ative of a cell type constitutively expressing procoagulant. Coagulati on mediated by ECV304 cells was found to be strictly dependent on tiss ue factor, as shown by an inhibitory monoclonal antibody, and on coagu lation factors V, VII and X. ECV304 procoagulant activity was strongly inhibited by active-site-inactivated FVIla, a synthetic peptide inhib itor of FXa (Tenstop) and the thrombin inhibitor, hirudin. While not a ppropriate for routine clinical assessment of coagulation factor funct ion, we have found this model to be valuable in characterising the pro coagulant activity on different cell types and particularly useful as a drug discovery tool in the search for new anticoagulants. Copyright (C) 1996 Elsevier Science Ltd