Dimeric αs2-casein, a novel matrix for tissue-type plasminogen activator catalyzed plasminogen activation

Abstract

Abstract In previous studies, we demonstrated a differential distribution of plasminogen, tissue-type plasminogen activator (t-PA), and urokinase-type plasminogen activator (u-PA) between various milk fractions. Plasminogen and t-PA were mainly confined to the casein micelle fraction, whereas u-PA was restricted to the cell fraction. These studies also showed that the differential distribution of these components was caused by specific binding components in milk. The u-PA binding component in the cell fraction was identified as the u-PA receptor. The t-PA binding components of bovine casein micelles were identified as dimeric αs2-casein and multimeric forms of κ-casein. In order to further explore the action of the plasminogen activation system that influences endogenous degradation of proteins in bovine milk, we evaluated the effect of individual bovine caseins on the rate of plasminogen activation by t-PA. Using a coupled peptidyl anilide plasminogen activator assay, we found that dimeric αs2-casein induced a concentration-dependent enhancement of the t-PA-catalyzed plasminogen activation. On a weight basis, the stimulating effect of dimeric αs2-casein was comparable to that of cyanogen bromide cleaved fibrinogen fragments. In a direct catalytic assay with a peptidyl anilide substrate, dimeric αs2-casein had no effect on t-PA activity. Ligand blotting experiments showed binding of both plasminogen and t-PA to the dimeric αs2-casein. The combined results from the binding experiments and the enzymatic assays suggest that the observed enhancement in plasminogen activation is mediated via formation of a ternary complex between t-PA, plasminogen, and dimeric αs2-casein. We further investigated the binding specificity of t-PA towards dimeric αs2-casein using t-PA deletion mutants and monoclonal antibodies. The results were compatible with the binding being mediated by kringle 2 and the finger domain of t-PA. These findings suggest that casein, analogous to fibrin clots in the intravascular space, serve as a molecular matrix for t-PA-catalyzed plasminogen activation.