Characterization of alpha 2-macroglobulin-plasmin complexes: complete subunit cleavage alters receptor recognition in vivo and in vitro.
When human alpha 2-macroglobulin (alpha 2M) binds proteinases, it undergoes subunit cleavage. Binding of small proteinases such as trypsin results in proteolysis of each of the four subunits of the inhibitor. By contrast, previous studies suggest that reaction of plasmin with alpha 2M results in cleavage of only two or three of the inhibitor subunits. In this paper, we demonstrate that the extent of subunit cleavage of alpha 2M is a function of plasmin concentration. When alpha 2M was incubated with a 2.5-fold excess of plasmin, half of the subunits were cleaved; however, at a 20-fold enzyme to inhibitor ratio, greater than 90% of the subunits were cleaved with no additional plasmin binding. This increased cleavage was catalyzed by free rather than bound plasmin. It is concluded that this "nonproductive" subunit cleavage is dependent upon the molar ratio of proteinase to inhibitor. The consequence of complete subunit cleavage on receptor recognition of alpha 2M-plasmin (alpha 2M-Pm) complexes was studied. Preparations of alpha 2M-Pm with only two cleaved subunits bound to the murine macrophage receptor with a Kd of 0.4 nM and 60 fmol of bound complex/mg of cell protein. When preparations of alpha 2-M-Pm with four cleaved subunits were studied, the Kd was unaltered but ligand binding increased to 140 fmol/mg of cell protein. The receptor binding behavior of the latter preparation is equivalent to that observed when alpha 2M is treated with small proteinases such as trypsin. This study suggests that receptor recognition site exposure is not complete in the alpha 2M-Pm complex with half of the subunits cleaved. Proteolytic cleavage of the remaining subunits of the inhibitor results in a further conformational change exposing the remaining receptor recognition sites.
D000818:Animals; D005341:Fibrinolysin / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D046911:Macromolecular Substances; D008264:Macrophages / Q000378:metabolism*; D008657:Metabolic Clearance Rate; D051379:Mice; D011971:Receptors, Immunologic / Q000378:metabolism*; D000511:alpha-Macroglobulins / Q000378:metabolism*
D026503:Low Density Lipoprotein Receptor-Related Protein-1; D046911:Macromolecular Substances; D011971:Receptors, Immunologic; D000511:alpha-Macroglobulins; D005341:Fibrinolysin