The binding of antifibrinolytic amino acids to kringle-4-containing fragments of plasminogen

A monoclonal antibody to human plasminogen, 10-F-1, was found to interact with the lysine-binding site (LBS) on the kringle 4 (K 4) region of the molecule. This observation has been employed to measure the binding of various antifibrinolytic amino acid analogs of epsilon-aminocaproic acid (epsilon ACA) to its site on K 4 in appropriate elastolytic-derived fragments of human plasminogen and to other species of plasminogen to which antibody 10-F-1 cross-reacts. By analysis of the concentration dependence of epsilon ACA displacement of [125I]10-F-1 from human Glu1Pg, a KD for epsilon ACA of 7.1 +/- 1.0 mM was calculated. Similar experiments with K 4-containing fragments of Glu1Pg, viz., Lys77Pg, K 4, Lys77H and Val354Pg, yielded KD values of 6.6 +/- 1.0, 7.5 +/- 1.0, 6.6 +/- 1.0, and 12.0 +/- 2.0 mM, respectively. When baboon, goat, monkey, rabbit, and sheep plasminogens were substituted for human plasminogen, the KD values calculated ranged from 2.1 to 7.1 mM. The KD values for several analogs of epsilon ACA, i.e., 4-aminobutyric acid, 5-aminopentanoic acid, 8-aminooctanoic acid, L-lysine, and trans-aminomethyl cyclohexane-1-carboxylic acid, were measured to the K 4 region of Lys77Pg. The values obtained were 11.3 +/- 1.5, 9.0 +/- 1.0, 71.0 +/- 10, 38.0 +/- 5.0, and 1.1 +/- 0.4 mM, respectively. Additionally, the KD of trans-aminomethylcyclohexane-1-carboxylic acid towards the K 4 region of Glu1Pg, Lys77Pg, and isolated K 4 was found to be 2.4 +/- 0.5, 1.1 +/- 0.3, and 2.0 +/- 0.6 mM, respectively. These studies show directly that the LBS on the K 4 domain of plasminogen represents one of its 4-5 weak binding sites and that this site can be specifically probed with the use of monoclonal antibody 10-F-1. Furthermore, it appears as though this site is conserved in several important proteolytic fragments of plasminogen, providing additional evidence that these fragments exist as independent domains in the native molecule. Finally, this weak LBS on the K 4 domain of human plasminogen is also present in other species of plasminogen.