Enhanced plasmin inhibition by a reactive center lysine mutant of the Kunitz-type protease inhibitor domain of the amyloid β-protein precursor

Abstract

The Alzheimer's disease related protein, amyloid β-protein precursor (AβPP), contains a domain homologous to Kunitz-type serine protease inhibitors (KPI). The recombinant KPI domain of AβPP 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 AβPP (KPI-Lys17). An expression plasmid for the KPI-Lys17 domain of AβPP encoded amino acids 285-345 of the AβPP 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 AβPP. Both KPI domains equally inhibited trypsin, chymotrypsin, and coagulation factors IXa and Xa. However, the KPI-Lys17 domain was an ≃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 AβPP. On the other hand, the KPI- Lys17 domain was an ≃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 AβPP. 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 AβPP.