title: Differences between binding of one-chain and two-chain tissue plasminogen activators to non-cross-linked and cross-linked fibrin clots.
abstract: Interaction of tissue plasminogen activator (t-PA) with fibrin plays a key role in regulation of plasminogen activation and clot dissolution. Previous investigations of t-PA-fibrin interaction, using incorporation of t-PA into polymerizing fibrin clots, have suggested that no significant differences exist in the binding of one-chain or two-chain t-PA to non-cross-linked or cross-linked fibrin. In the present study, binding of 125I-labeled and affinity-purified one-chain and two-chain forms of t-PA to preformed non-cross-linked or cross-linked, sonicated suspension of fibrin was investigated. Interaction of one-chain t-PA with cross-linked fibrin involved a single type of binding site with dissociation constant (kd) of 0.58 mumol/L and a stoichiometry (n) of 1.5. Interaction of one-chain t-PA with non-cross-linked fibrin, however, involved two classes of binding sites with dissociation constants of 0.32 and 1.5 mumol/L and corresponding number of binding sites equal to 0.57 and 2.0, respectively. In contrast to the binding of one-chain t-PA to cross-linked fibrin by a limited number of sites, two-chain t-PA appeared to involve a considerably greater number of sites (minimum six) whose dissociation constant was 3.2 mumol/L. Interaction of two-chain t-PA with non-cross-linked fibrin also showed the presence of many binding sites (minimum seven) with approximate dissociation constant of 6.4 mumol/L, as well as a few (n = 0.012) high-affinity sites with a kd of 0.011 mumol/L epsilon-Aminocaproic acid did not completely reverse the binding of either one-chain t-PA or two-chain t-PA to fibrin. The present findings suggest that the fibrin-binding properties of t-PA undergo considerable changes on proteolytic conversion from one-chain to two-chain t-PA, catalyzed under physiologic conditions by plasmin. The cleavage of one-chain t-PA to two-chain t-PA allows to bind to a large number of low-affinity binding sites on fibrin. Cross-linking of fibrin by factor XIIIa results in masking of high-affinity binding sites that are present in non-cross-linked fibrin. We propose that both plasmin and factor XIIIa play an important regulatory role in dissolution of blood clots by modulating t-PA-fibrin interaction.
authors.affiliation: Department of Biochemistry, Temple University School of Medicine, Philadelphia, PA 19140.
authors.forename: S S
authors.initials: SS
authors.lastname: Husain
authors.forename: A A
authors.initials: AA
authors.lastname: Hasan
authors.forename: A Z
authors.initials: AZ
authors.lastname: Budzynski
mesh_terms: D015119:Aminocaproic Acid / Q000494:pharmacology; D001667:Binding, Competitive; D001777:Blood Coagulation / Q000187:drug effects; D001780:Blood Coagulation Tests; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents; D005337:Fibrin / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes / Q000378:metabolism; D046911:Macromolecular Substances; D010959:Tissue Plasminogen Activator / Q000097:blood* / Q000302:isolation & purification
chemical_list: D003432:Cross-Linking Reagents; D007457:Iodine Radioisotopes; D046911:Macromolecular Substances; D005337:Fibrin; D010959:Tissue Plasminogen Activator; D015119:Aminocaproic Acid