Antifibrinolytic activities of alpha-N-acetyl-L-lysine methyl ester, epsilon-aminocaproic acid, and tranexamic acid. Importance of kringle interactions and active site inhibition.

Abstract

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 plasmin-NALME 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 a 2 -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. ( I n the circulation, fibrinolysis and fibrinogenolysis are mediated primarily by plasmin, the active serine proteinase counterpart of the zymogen plasminogen. 1 ' 2 The intact structure of plasminogen includes 791 amino acids with an Af-terminal glutamic acid 2 " 5 and either one or two oligosaccharide chains. 6 -8 Plasminogen activation results from the cleavage of the Arg^-Val 56 ' peptide bond. 9 Low concentrations of plasmin cleave [Glu'J-plasminogen to form [Lys 78 ]-plasminogen, a second more readily activatable form of the zymogen. 10 Plasminogen activation and the subsequent function of plasmin are modulated by noncovalent binding inter-

Inhibitory effects of lysine analogues on t-PA induced whole blood clot lysis

“…At these concentrations, no inhibitory effect towards the amidolytic effect of plasmin was shown by these analogues. The ability of lysine analogues to inhibit the proteolytic activity of plasmin without affecting the amidolytic activity has been previously reported (24).…”

Plasmin-Mediated Fibroblast Growth Factor-2 Mobilisation Supports Smooth Muscle Cell Proliferation in Human Saphenous Vein

“…The K i for NALME is 1–2 mmol/l [27]. We therefore chose a concentration of 10 mmol/l, approximately 10-fold greater than the IC 50 , in order to produce a profound inhibition of plasmin.…”

“…We therefore chose a concentration of 10 mmol/l, approximately 10-fold greater than the IC 50 , in order to produce a profound inhibition of plasmin. This concentration does not inhibit tPA or uPA, or other serine proteases [27]. NALME had no cytotoxic effect as measured by lactate dehydrogenase release into the culture medium (table 3), and it had no significant effect on cell density in the media or intima (data not shown).…”

Fibrin Degradation Enhances Vascular Smooth Muscle Cell Proliferation and Matrix Deposition in Fibrin-Based Tissue Constructs Fabricated In Vitro

“…18 Fibrin degradation can be modulated by the addition of plasmin inhibitors, such as aprotinin or e-aminocaproic acid (ACA). ACA inhibits uPA, 19 tPA, 20 and plasmin, 21,22 and is widely used in tissue engineering to slow fibrin degradation. 6,[23][24][25][26] Controlling the rate of fibrinolysis is of great importance when using fibrin gel as a tissue scaffold.…”