Antifibrinolytic Activities of or-iV-Acetyl-L-Lysine Methyl Ester, e-Aminocaproic Acid, and Tranexamic Acid Importance of Kringle Interactions and Active Site Inhibition

cr-jV-acetyl-L-lysine methyl ester (NALME) is a lysine analogue that reportedly binds to low-affinity lysine binding sites in plasmin(ogen) and miniplasmin(ogen). In the studies presented here, we show that NALME has antifibrinolytic activity; however, unlike the therapeutic agents e-amino-n-caproic acid (eACA) and tranexamic acid (TEA), the activity of NALME is based on inhibition of the plasmin active site. NALME (0.1-10 mM) significantly inhibited the amidase activity of plasmin, miniplasmin, and streptokinase-plasmin complex without affecting er-thrombin or tissue plasminogen activator. eACA and TEA (0.1-10 mM) did not affect the amidase activity of plasmin or miniplasmin. A kinetic analysis showed that NALME is a competitive inhibitor of D-Val-L-Leu-L-Lys-p-nitroanilide HC1 (S-2251) hydrolysis by plasmin; NALME binding to plasmin completely prevented S-2251 binding. The K, for the plasminNALME interaction was 0.4 mM. eACA and TEA inhibited fibrin monomer digestion by plasmin and miniplasmin without binding to the active site of either enzyme. This result suggests that eACA and TEA function as antifibrinolytics by disrupting the noncovalent association of fibrin monomer with a domain common to both plasmin and miniplasmin (probably kringle 5). NALME inhibited fibrin monomer digestion principally by decreasing amidase activity. NALME was the only lysine analogue that prevented fragment X formation; TEA and eACA primarily inhibited the formation of fragments Y and D. When plasmin was incubated simultaneously with o^-antiplasmin and a2-macroglobulin, eACA increased the fraction of plasmin reacting with o^-macroglobulin; NALME had no effect on the plasmin distribution. eACA, TEA, and NALME increased the euglobulin clot lysis time of normal plasma. NALME did not prolong the prothrombin time or activated partial thromboplastin time. These studies demonstrate that the antifibrinolytic activity of NALME is based on inhibition of the plasmin active site, whereas eACA and TEA are active due to kringle domain interactions. (Arteriosclerosis and Thrombosis 1992;12:708-716)