title: Thrombin-variable region 1 (VR1). Evidence for the dominant contribution of VR1 of serine proteases to their interaction with plasminogen activator inhibitor 1.
abstract: The importance of a specific variable region in different serine proteases for the interaction with plasminogen activator inhibitor 1 (PAI-1) is studied. To that end, we have constructed a thrombin substitution variant, thrombin-VR1, in which the entire variable region 1 (VR1) of the protease domain (Phe-34 to Leu-40) has been replaced by the corresponding sequence (Phe-294 to Phe-305) of tissue-type plasminogen activator. The substitution resulted in a 2000-fold increase of the second-order rate constant of inhibition by PAI-1 (k2 = 2.2 x 10(6) M-1 s-1) as compared to alpha-thrombin (k2 = 1.1 x 10(3) M-1 s-1). Inhibition of thrombin-VR1 by PAI-1 is mediated by the formation of SDS-stable, enzyme-inhibitor complexes. The substitution did not affect the rate constant of inhibition by antithrombin III, whereas clotting efficiency and the rate of inhibition by heparin cofactor II were decreased 3-fold. These results demonstrate the importance and specificity of the protease domain VR1 region for the interaction of PAI-1 with its target proteases.
authors.affiliation: Department of Molecular Biology, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam.
authors.forename: A J
authors.initials: AJ
authors.lastname: Horrevoets
authors.forename: G
authors.initials: G
authors.lastname: Tans
authors.forename: A E
authors.initials: AE
authors.lastname: Smilde
authors.forename: A J
authors.initials: AJ
authors.lastname: van Zonneveld
authors.forename: H
authors.initials: H
authors.lastname: Pannekoek
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D014644:Genetic Variation; D006801:Humans; D007668:Kidney; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010649:Phenylalanine; D017395:Plasminogen Activator Inhibitor 1 / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics* / Q000378:metabolism*; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D010959:Tissue Plasminogen Activator / Q000037:antagonists & inhibitors
chemical_list: D009838:Oligodeoxyribonucleotides; D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins; D010649:Phenylalanine; D012697:Serine Endopeptidases; D013917:Thrombin; D010959:Tissue Plasminogen Activator