Binding site of alpha 2-plasmin inhibitor to plasminogen

Peptide T-11, a carboxyl terminal tryptic fragment of alpha 2-plasmin inhibitor, inhibits the reversible first step of the reaction between plasmin and alpha 2-plasmin inhibitor. To elucidate which amino-acid residues played a important role in the inhibitory activity of peptide T-11, we prepared the various synthetic derivatives of peptide T-11 and determined the peptide concentration that inhibited the apparent rate constant of the reaction between plasmin and alpha 2-plasmin inhibitor by 50% (IC50). Peptide III, which lacked the residues Gly-1 to Pro-7 of peptide I (peptide T-11), had a strong inhibitory activity, like peptide I (IC50: peptide I, 7 microM; peptide III, 13 microM). The peptides that lacked the Leu-9 and Lys-10 or Lys-26 of peptide III showed much weaker activity, and the loss or amidation of the C-terminal lysine of peptide III also markedly reduced the inhibitory activity. Peptide III competitively inhibited the binding of [14C]tranexamic acid to kringle 1 + 2 + 3 (K1-3) and kringle 4 (K4) in a binding assay performed by the gel-diffusion method. The respective dissociation constants (Kd) of peptide III for K1-3 and K4 were 0.85 microM and 35.2 microM. These data suggest that the amino residue of Lys-10 and the carboxylic acid of Lys-26 in peptide T-11 play crucial roles in the ionic binding of alpha 2-plasmin inhibitor to the tranexamic acid-binding site (lysine-binding site) of plasminogen. Peptide T-11: H-G-D-K-L-F-G-P-D-L-K-L-V-P-P-M-E-E-D-Y-P-Q-F-G-S-P-K-OH.