Identification of the domains of tissue-type plasminogen activator involved in the augmented binding to fibrin after limited digestion with plasmin.
The binding of recombinant tissue-type plasminogen activator (rt-PA) to fibrin increases upon digestion of fibrin with plasmin. Optimal binding is observed following a limited plasmin digestion of fibrin, coinciding with the generation of fibrin fragment X polymers. We studied the involvement of the separate domains of the amino-terminal "heavy" (H) chain of rt-PA in this augmentation of fibrin binding. The fibrin-binding characteristics of a set of rt-PA deletion mutants, lacking either one or more of the structural domains of the H chain, were determined on intact fibrin matrices and on fibrin matrices that were subjected to limited digestion with plasmin. The augmented fibrin binding of rt-PA is partially abolished when the plasmin-degraded fibrin matrices are subsequently treated with carboxypeptidase B, demonstrating that this increased binding is dependent on the generation of carboxyl-terminal lysine residues in the fibrin matrix. Evidence is provided that this increase of fibrin binding is mediated by the kringle 2 (K2) domain that contains a lysine-binding site. Further increase of the fibrin binding of rt-PA is independent of the presence of carboxyl-terminal lysines. It is shown that the latter increase is not mediated by the K2 domain. Based on our data, we propose that the increase in fibrin binding, unrelated to the presence of carboxyl-terminal lysine residues, is mediated by the finger (F) domain, provided that this domain is correctly exposed in the remainder of the protein.
de Vries
C
C
Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Department of Molecular Biology, Amsterdam.
Veerman
H
H
Pannekoek
H
H
D000818:Animals; D001483:Base Sequence; D002872:Chromosome Deletion; D005337:Fibrin / Q000378:metabolism*; D005341:Fibrinolysin; D007700:Kinetics; D007739:L Cells / Q000201:enzymology; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D010446:Peptide Fragments; D011994:Recombinant Proteins; D005337:Fibrin; D010959:Tissue Plasminogen Activator; D005341:Fibrinolysin