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 ?-aminocaproic acid (?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 ?ACA displacement of [¹²⁵I]10-F-1 from human Glu₁Pg, a K_D for ?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 K_D 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 K_D values calculated ranged from 2.1 to 7.1 mm. The K_D values for several analogs of ?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 K_D 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.