title: Enhanced plasmin inhibition by a reactive center lysine mutant of the Kunitz-type protease inhibitor domain of the amyloid beta-protein precursor.
abstract: The Alzheimer's disease related protein, amyloid beta-protein precursor (A beta PP), contains a domain homologous to Kunitz-type serine protease inhibitors (KPI). The recombinant KPI domain of A beta PP is a potent inhibitor of coagulation factors XIa and IXa and functions as an anticoagulant in vitro. Here we report the expression, purification, and characterization of a reactive center lysine mutant of the KPI domain of A beta PP (KPI-Lys17). An expression plasmid for the KPI-Lys17 domain of A beta PP encoded amino acids 285-345 of the A beta PP cDNA containing a lysine substitution at arginine 17 in the KPI domain. The secreted 61-amino acid product was purified to homogeneity and functionally characterized. The protease inhibitory properties of the KPI-Lys17 domain were compared to those of the native KPI domain of A beta PP. Both KPI domains equally inhibited trypsin, chymotrypsin, and coagulation factors IXa and Xa. However, the KPI-Lys17 domain was an approximately 25-fold less effective inhibitor of coagulation factor XIa resulting in markedly less prolongation of the activated partial thromboplastin time compared to the native KPI domain of A beta PP. On the other hand, the KPI-Lys17 domain was an approximately 10- and 5-fold better inhibitor of plasmin in a chromogenic substrate assay and in a fibrinolytic assay, respectively, than the native KPI domain of A beta PP. Together, these studies suggest that the KPI-Lys17 domain has enhanced anti-fibrinolytic and diminished factor XIa inhibitory properties compared to the native KPI domain of A beta PP.
authors.affiliation: Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine 92717-4025, USA.
authors.forename: W E
authors.initials: WE
authors.lastname: Van Nostrand
authors.forename: A H
authors.initials: AH
authors.lastname: Schmaier
authors.forename: R S
authors.initials: RS
authors.lastname: Siegel
authors.forename: S L
authors.initials: SL
authors.lastname: Wagner
authors.forename: W C
authors.initials: WC
authors.lastname: Raschke
mesh_terms: D000595:Amino Acid Sequence; D016564:Amyloid beta-Protein Precursor / Q000737:chemistry* / Q000494:pharmacology*; D007611:Aprotinin / Q000737:chemistry*; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D017931:DNA Primers; D005341:Fibrinolysin / Q000037:antagonists & inhibitors*; D006801:Humans; D007700:Kinetics; D008239:Lysine; D008969:Molecular Sequence Data; D010314:Partial Thromboplastin Time; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D013329:Structure-Activity Relationship
chemical_list: D016564:Amyloid beta-Protein Precursor; D017931:DNA Primers; D010446:Peptide Fragments; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D007611:Aprotinin; D005341:Fibrinolysin; D008239:Lysine