Binding and endocytosis of .alpha.2-macroglobulin-plasmin complexes

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Abstract
The clearance of 125I-labeled .alpha.2-macroglobulin-plasmin complexes (125I-.alpha.2M-PM) from mouse circulation is slower than that of 125I-labeled .alpha.2M-methylamine complexes (125I-.alpha.2M-CH3 NH2). In addition, clearance of 125I-.alpha.2M-PM is biphasic, but that of 125I-.alpha.2M-CH3NH2 follows simple first-order kinetics. Treatment of .alpha.2M-PM with trypsin yields a complex that clears like .alpha.2M-CH3NH2. Complexes of .alpha.2M with Val442-plasmin (.alpha.2M-Val442-PM) were prepared; .alpha.2M-Val442-PM has a stoichiometry of 2 mol of Val442-PM to 1 mol of .alpha.2M and also clears like .alpha.2M-CH3NH2. In vitro 4.degree. C binding inhibition studies with mouse peritoneal macrophages show that .alpha.2M-CH3NH2, .alpha.2M-PM, trypsin-treated .alpha.2M-PM, and .alpha.2M-Val442-PM bind with the same affinity, apparent Kd = 0.4 nM. The binding isotherms at 4.degree. C are the same for 125I-.alpha.2M-CH3NH2, 125I-.alpha.2M-PM, and 125I-trypsin-treated .alpha.M-PM in both mouse peritoneal macrophages and 3T3-L1 fibroblasts. The Scatchard plots for the binding isotherms in macrophages were curved; those in 3T3-L1 fibroblasts were linear with an apparent Kd of 0.48 nM and a receptor activity of 140 fmol/mg of cell protein for .alpha.2M-CH3NH2, an apparent Kd of 0.29 nM and a receptor activity of 110 fmol/mg of cell protein for .alpha.2M-PM, and an apparent Kd of 0.35 nM and a receptor activity of 210 fmol/mg of cell protein for trypsin-treated .alpha.2M-PM. The time course of uptake and degradation and the concentration dependence of uptake at 37.degree. C are indistinguishable for 125I-.alpha.2M-PM and 125I-.alpha.2M-CH3NH2 in mouse peritoneal macrophages. The uptake of both complexes reaches a steady state after 1-2 h. Maximum cell-associated ligand at steady state is 2.0 pmol/mg of cell protein, and the concentration of half-maximal uptake is 50 nM for both complexes. These results suggest that the slower clearance of .alpha.2M-PM from mouse circulation is not due to altered receptor recognition of the .alpha.2M-PM complex but may be due to .alpha.2M-PM interaction with some factor in mouse blood.