On the Rolw of the Carbohydrate Side Chains of Human Plasminogen in Its Interaction with α 2 ‐Antiplasmin and Fibrin

The Dissociation constants ( K d ) for the interaction between different plasminogen derivatives and α 2 ‐antiplasmin were determined as the concentrations of ligand that decrease the rate of the reaction between plamin and α 2 ‐antiplasminby 50%. The K d of Lys‐plasminogen (residues 77–790) type I (with a glucosamine‐based oligosaccharide chain attached to Asn‐288) and type II (with an unsubstituted Asn‐288) was found to be 1.9 μM and 0.2 μM respectively. The isolated plasminogen type I fragments containg the high‐affinity lysine‐binding site (representing residues 79–337 with glucosamine‐based carbohydrate on Asn‐288 or residues 79–353 with glucosamine‐based carbohydrate on Asn‐288 and galactosamine‐based carbohydrate on Thr‐345) both had a dissociation constant of 1.3 μM. The isolated plasminogen type II fragments containg the high‐affinity lysine‐binding site (residues 79–337 without carbohydrate or residues 79–353 with galactosamine‐based carbohydrate on Thr‐345) both had a dissociation constant of 0.24μM. These findings indicate that the difference in K d is related to the presence of an oligosaccharide side chain on Asn‐288 but is unrelated to the presence of carbohydrate on Thr‐345. The dissociation constants of type I and type II Glu‐plasminogen (residues 1–790) were 4.9 μM and 2.9 μM. These higher values and smaller differences between type I and type II can probably be explained by the presence of the NH 2 ‐terminal preactivation peptide (residues 1–76) which interacts with the high‐affinity lysine‐binding site in Glu‐plasminogen. Using 125 I‐labelled plasminogen derivatives, direct binding studies to fibrin were performed. This revealed that the binding of the purified fragments obtained from type II plasminogen was 7‐times higher than that of fragments obtained from type I plasminogen. For both Glu‐plasminogen and Lys‐plasminogen no difference in extent of binding was observed for type I and type II. These finding indicate that the presence of a carbohydrate chain on Asn‐288 to both α 2 ‐antiplasmin and fibrin. This decrease was still observed for the interaction between Lys‐plasminogen and α 2 ‐antiplasmin, but not for the extent of binding to fibrin, suggesting that other interactions than via the high‐affinity lysine‐binding site might contribute in the binding of plasminogen to fibrin.