Localization of Individual Lysine‐Binding Regions in Human Plasminogen and Investigations on Their Complex‐Forming Properties

Abstract

After partial digestion of human plasminogen with elastase, followed by chymotryptic cleavage of one of the fragments produced, two polypeptides with molecular weights of approximately 10000 and with lysine‐binding sites still intact were isolated by means of affinity chromatography and gel filtration. One fragment, which was completely sequenced (88 residues), was identified as the fourth kringle, whereas the other, according to partial sequence analysis represented the first kringle. Equilibrium, dialysis against 6‐aminohexanoic acid yielded for the first kringle one high‐affinity binding site (Ka= 60 mM−1) and for the fourth kringle one single low‐affinity binding site (Ka= 28 mM−1). Moreover, interactions were detected between the first kringle and the N‐terminal CNBr fragment of plasminogen and also fibrin. In these cases an additional lysine‐ binding site, though of low affinity, appears to be involved. Thus, the first kringle seems to play important roles, structurally by contributing to the maintenance of a compact structure of plasminogen through an intramolecular interaction with its N‐terminal polypeptide region, and functionally by increasing the fibrin affinity of Lys‐plasminogen (plasminogen lacking the first 76 residues) and plasmin.

A Fast-acting, Modular-structured Staphylokinase Fusion with Kringle-1 from Human Plasminogen as the Fibrin-targeting Domain Offers Improved Clot Lysis Efficacy

“…Glycosylation Based on the average of duplicate experiments, K d for the interaction between SAKM3-L-K1 and EACA was found to be 15 M. As a reference, the binding affinity of Kringle-1 domain and its derivative carrying the N-terminal proactivation peptide of human plasminogen to EACA, expressed in terms of the dissociation constant, has been determined to be 17 M by equilibrium dialysis (24), 12 M by isothermal titration calorimetry (25), and 13.7 M by fluorescence study (42). Thus, the K d obtained with SAKM3-L-K1 is within the range of the reported values.…”

Rapid-reaction Kinetic Characterization of the Pathway of Streptokinase-Plasmin Catalytic Complex Formation

“…Removal of the NH 2 -terminal peptide by Pm cleavage results in formation of [Lys]Pg, which assumes a partially extended ␤-conformation that is more rapidly activated by tissue-type plasminogen activator and urokinase and by the SK⅐Pg* and SK⅐Pm catalytic complexes (2, 29 -32). The lysine analog 6-aminohexanoic acid (6-AHA) binds with high affinity to K1 (K D 11-13 M) and to lower affinity sites on K4 (K D 20 -60 M) and K5 (K D 95-140 M) based on studies of the isolated kringle domains (17,18,33,34). Benzamidine binds isolated K5 with highest affinity (K D 290 M), interacts weaker with K1 (K D 12.5 mM) and K2 (K D 33 mM), and displays insignificant affinity for K3 and K4 (18,35,36).…”

Mutational Analysis of Affinity and Selectivity of Kringle-Tetranectin Interaction

“…Purification of Plasminogen, Mini-Plasminogen, and Plasminogen Kringle 1-3-Plg was purified from pooled outdated blood bank plasma and fragmented by digestion with elastase (35). Affinity chromatography on lysine-Sepharose yielded mini-Plg in the flow-trough, while fragments corresponding to kringle 4, kringle 1-3, and Lys-and Glu-Plg were eluted with 6-aminohexanoic acid (6-AHA).…”