Monoclonal antibodies against the C-terminal peptide of human tissue factor for studies of the cytoplasmic domain

Abstract

The carboxyl-terminal cytoplasmic domain of human tissue factor (the essential cofactor for coagulation factor VII) is a prospective regulatory domain through which viable cells may control the expression of tissue factor activity. Furthermore, this domain is subject to post-translational modifications of as yet unknown functional significance. Hybridomas producing antibodies against the C-terminal domain of tissue factor were obtained using splenocytes harvested from mice immunized with a synthetic peptide corresponding to the nine terminal residues of the protein sequence. These antibodies, C28 1.1 and C28 2.1, react with purified placental tissue factor, but not with a recombinant soluble tissue factor lacking the cytoplasmic and membrane-spanning domains. This confirms that tissue factor from biological membranes contains the entire cytoplasmic domain predicted from the cDNA sequence, and provides a mechanism to determine whether tissue factor used experimentally retains this proteolytically sensitive epitope. The antibodies have been employed to demonstrate selective proteolytic removal of the carboxyl-terminal peptide from tissue factor. Limited experiments indicate that they will be useful for characterizing the distribution of vesicles with regard to the possible orientations of tissue factor on their surface.