CHARACTERIZATION OF RECEPTORS INTERACTING SPECIFICALLY WITH THE B-CHAIN OF TISSUE-PLASMINOGEN ACTIVATOR ON ENDOTHELIAL-CELLS

We have previously shown that the catalytic domain of tissue plasminog en activator (t-PA), the B-chain, binds specifically to hydrophobic co mponents on the endothelial cell (EC) surface. The binding is mediated by amino acids within the sequence, AKHRRSPGER (B-20-29). The complex imparts a strong fibrinolytic potential to the EC surface, which is n ot inhibited by plasminogen activator inhibitor 1 (PAI-1). Here we cha racterize membrane proteins from EC that interact with the binding pep tide and have hydrophobic properties. Specific labelling of cell surfa ce components with affinity for the t-PA B-chain was performed using B -chain derivatized with the heterobifunctional and cleavable cross-lin ker SASD labelled with I-125. Analysis of the cell extract by SDS/PAGE before and after reduction demonstrated the presence of a complex bet ween the B-chain and a membrane protein of about 56 kDa. Radiolabelled proteins from the surface of EC were affinity purified on B-19-30-Sep harose. The adsorbed proteins were eluted with lysine and the peptide B-20-29. The hydrophobic fraction from the peptide eluate contained on e main component with a molecular mass 56 kDa, which was absent in the hydrophilic fraction. In addition, four minor components were observe d. Only traces of radiolabelled proteins were found in the hydrophobic fraction of the lysine eluate. Complexes between I-125-t-PA and hydro phobic proteins from EC obtained by affinity chromatography on B-19-30 -Sepharose were crosslinked with disuccinylsuberate (DSS). Two radioac tive complexes were identified after electroblotting to a PVDF membran e, one major component with a molecular mass of about 120 kDa and one minor 200 kDa complex. The 120 kDa compound had a significant activato r activity before and after treatment with an excess of PAI-1. This is in contrast to free t-PA, which completely loses its activity in the presence of PAI-1. This indicates that t-PA (about 65 kDa) can form co mplexes with membrane components from EC with an approximate molecular mass of 55 kDa.