Characterization of receptors interacting specifically with the B-chain of tissue plasminogen activator on endothelial cells

Abstract

Summary We have previously shown that the catalytic domain of tissue plasminogen activator (t-PA), the B-chain, binds specifically to hydrophobic components 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 not inhibited by plasminogen activator inhibitor 1 (PAI-1). Here we characterize membrane proteins from EC that interact with the binding peptide and have hydrophobic properties. Specific labelling of cell surface components with affinity for the t-PA B-chain was performed using B-chain derivatized with the heterobifunctional and cleavable cross-linked SASD labelled with 125 I. Analysis of the cell extract by SDS/PAGE before and after reduction demonstrated the presence of a complex between 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 -Sepharose. The adsorbed proteins were eluted with lysine and the peptide B 20–29 . The hydrophobic fraction from the peptide eluate contained one main component with a molecular mass 56 kDa, which was absent in the hydrophilic fraction. In addition, four minor components were observed. Only traces of radiolabelled proteins were found in the hydrophobic fraction of the lysine eluate. Complexes between 125 I-t-PA and hydrophobic proteins from EC obtained by affinity chromatography on B 19–30 -Sepharose were crosslinked with disuccinylsuberate (DSS). Two radioactive complexes were identified after electroblotting to a PVDF membrane, one major component with a molecular mass of about 120 kDa and one minor 200 kDa complex. The 120 kDa compound had a significant activator 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 complexes with membrane components from EC with an approximate molecular mass of 55 kDa.