On the specific interaction between the lysine-binding sites in plasmin and complementary sites in α2-antiplasmin and in fibrinogen

Abstract

Plasminogen and plasminogen derivatives which contain lysine-binding sites were found to decrease the reaction rate between plasmin and α 2-antiplasmin by competing with plasmin for the complementary site(s) in α 2-antiplasmin. The dissociation constant K d for the interaction between intact plasminogen (Glu-plasminogen) and α 2-antiplasmin is 4.0 μM but those for Lys-plasminogen or TLCK-plasmin are about 10-fold lower indicating a stronger interaction. The lysine-binding site(s) which is situated in triple-loops 1–3 in the plasmin A-chain is mainly responsible for the interaction with α 2-antiplasmin. The interaction between Glu-plasminogen and α 2-antiplasmin furthermore enhances the activation of Glu-plasminogen by urokinase to a comparable extent as 6-aminohexanoic acid, suggesting that similar conformational changes occur in the proenzyme after complex formation. Fibrinogen, fibrinogen digested with plasmin, purified fragment E and purified fragment D interfere with the reaction between plasmin and α 2-antiplasmin by competing with α 2-antiplasmin for the lysine-binding site(s) in the plasmin A-chain. The K d obtained for these interactions varied between 0.2 μM and 1.4 μM; fragment E being the most effective. Thus the fibrinogen molecule contains several complementary sites to the lysine-binding sites located both in its NH 2-terminal and COOH-terminal regions; these sites are to a large extent preserved after plasmic degradation. Fibrinogen also competes with the plasmin catalyzed cleavage of d-Val-Leu-Lys-Nan with a K i value (obtained from a Dixon plot) of 1.9 μM which was increased 4-fold in the presence of 6-amino-hexanoic acid. The lysine-binding sites therefore are of importance for the enzyme-substrate binding.