Identification of a receptor‐induced binding site (RIBS) in plasminogen induced by its interaction with cells

Abstract

When plasminogen is bound to cells its activation is markedly enhanced compared to the reaction in solution, a key step in regulation of thrombolysis and cell migration. To study the mechanism of cell‐surface plasminogen activation we developed a monoclonal antibody (mAb 51) that reacts with cell‐associated plasminogen in blood in the presence of a high molar excess of soluble plasminogen. Thus, mAb 51 detects receptor‐induced binding sites (RIBS) induced in plasminogen upon its interaction with cells. The objective of our study was to define the RIBS epitope in plasminogen recognized by mAb 51. Plasminogen was denatured, reduced and alkylated and then digested with trypsin. Using western blotting and a specific Mab 51‐based ELISA for plasminogen, we established the concentration of trypsin that resulted in maximal digestion of plasminogen, while still retaining reactivity with mAb 51. The plasminogen digest was applied to a mAb 51 immunoaffinity column. MALDI analysis of the eluate yielded a single peak with a mass of 3,076 m/z. In LC‐MS‐MS, a peak with the same mass was obtained and assigned to a peptide corresponding to K177‐Y154, spanning a domain linking plasminogen kringles 1 and 2. Our data suggest that this domain becomes accessible when plasminogen interacts with cells. Thus, this conformational change detected by mAb 51 is likely to contribute to the ability of plasminogen to be activated on the cell surface.