title: Inhibition of thrombin activity by prothrombin activation fragment 1.2.
abstract: Prothrombin is the precursor of thrombin, the central enzyme in coagulation. Prothrombin is activated in vivo by the prothrombinase complex to form fragment 1.2 and thrombin. Fragment 1.2 has an amino-terminal gla domain and two kringle domains. The second kringle domain (kringle 2) binds to the exosite II on thrombin. Nascent thrombin generated on platelet surface remains non-covalently bound to fragment 1.2 by kringle 2-exosite II interaction. To determine whether this interaction can modulate coagulant activity of thrombin, we labeled thrombin at the active site with fluorescein-Phe-Pro-Arg chloromethylketone and monitored the fluorescence changes upon ligand binding. Anionic phospholipid-bound fragment 1.2 and fragment 2 bound to FPR-thrombin and induced changes in the active site with half maximal effects at 7.2 microM and 8.8 microM, respectively. We also tested the effect of anionic phospholipid-bound fragment 1.2 (0-10 microM) on thrombin clotting activity. Phospholipid-bound fragment 1.2 inhibited fibrinogen clotting in a concentration-dependent manner but had no significant effect on amidolytic activity towards S2238, suggesting a competitive inhibition of the fibrinogen binding site. Furthermore, fragment 1.2 inhibited FPR-thrombin binding to platelet. Consistent with these findings fragment 1.2 inhibited thrombin-induced aggregation of gel filtered platelets in a concentration-dependant manner. These results suggest that the membrane-bound prothrombin fragment 1.2 may play a role in hemostasis by down regulating the procoagulant activity of newly formed thrombin.
authors.affiliation: Department of Pathology and Medicine (Thrombosis Research), Baylor College of Medicine, Michael E. DeBakey VA Medical Center, Houston, TX 77030, USA. swapand@bcm.tmc.edu
authors.forename: Swapan Kumar
authors.initials: SK
authors.lastname: Dasgupta
authors.forename: Perumal
authors.initials: P
authors.lastname: Thiagarajan
mesh_terms: D001665:Binding Sites; D001667:Binding, Competitive; D001777:Blood Coagulation / Q000187:drug effects; D001792:Blood Platelets / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D005340:Fibrinogen / Q000378:metabolism; D006487:Hemostasis / Q000187:drug effects*; D006801:Humans; D010446:Peptide Fragments / Q000494:pharmacology*; D010974:Platelet Aggregation / Q000187:drug effects; D011516:Prothrombin / Q000378:metabolism / Q000494:pharmacology*; D013917:Thrombin / Q000037:antagonists & inhibitors*
chemical_list: D010446:Peptide Fragments; C045879:prothrombin fragment 1.2; D011516:Prothrombin; D005340:Fibrinogen; D013917:Thrombin