Thrombin interaction with fibrin polymerization sites.
Thrombin is central to hemostasis, and postclotting fibrinolysis and wound healing. During clotting, thrombin transforms plasma fibrinogen into polymerizing fibrin, which selectively adsorbs the enzyme into the clot. This protects thrombin from heparin-antithrombin inactivation, thus preserving the enzyme for postclotting events. To determine how the fibrin N-terminal polymerization sites of A alpha 17-23 (GPRVVER) and B beta 15-25 (GHRPLDKKREE) and their analogs may interact with thrombin, amidolysis vs. plasma- and fibrinogen-clotting assays were used to differentiate blockade of catalytic site vs. other thrombin domains. Amidolysis studies suggest GPRVVER inhibition of thrombin catalytic site through hydrophobic interaction, and GPRVVER inhibited clotting. Neither GPRP nor VVER nor the B beta 15-25 homologs inhibited amidolysis. Contrary to heparin, acyl-DKKREE promoted plasma-clotting, but inhibited fibrinogen-clotting. In addition, acyl-DKKREE reversed the anticoagulant effect of heparin (0.1 U/ml) in plasma. The results suggest fibrin B beta 15-25 interaction with thrombin, possibly by blocking the heparin-binding site. Together with the reported fibrin A alpha 27-50 binding to thrombin, polymerizing fibrin appears to initially bind to thrombin catalytic site and exosite-1 through A alpha 17-50, and to another thrombin site through B beta 15-25. As these fibrin sites are also involved in polymerization, competition of the polymerization process with thrombin-binding could subsequently dislodge thrombin from fibrin alpha-chain. This may re-expose the catalytic site and exosite-1, thus explaining the thrombogenicity of clot-bound thrombin. The implications of these findings in polymerization mechanism and anticoagulant design are discussed.
Hsieh
K
K
Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology Washington State University, Pullman 99164-6520, USA.
D000595:Amino Acid Sequence; D001665:Binding Sites; D001777:Blood Coagulation / Q000187:drug effects; D002384:Catalysis; D005337:Fibrin / Q000378:metabolism* / Q000502:physiology; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D011108:Polymers / Q000378:metabolism*; D011485:Protein Binding; D017421:Sequence Analysis; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000378:metabolism*
D010446:Peptide Fragments; D011108:Polymers; D005337:Fibrin; D006493:Heparin; D013917:Thrombin