ENHANCED PLASMIN INHIBITION BY A REACTIVE CENTER LYSINE MUTANT OF THEKUNITZ-TYPE PROTEASE INHIBITOR DOMAIN OF THE AMYLOID BETA-PROTEIN PRECURSOR

The Alzheimer's disease related protein, amyloid beta-protein precurso r (A beta PP), contains a domain homologous to Kunitz-type serine prot ease inhibitors (KPI). The recombinant KPI domain of A beta PP is a po tent inhibitor of coagulation factors XIa and IXa and functions as an anticoagulant in vitro. Here we report the expression, purification, a nd characterization of a reactive center lysine mutant of the KPI doma in of A beta PP (KPI-Lys(17)). An expression plasmid for the KPI-Lys(1 7) domain of A beta PP encoded amino acids 285-345 of the A beta PP cD NA containing a lysine substitution at arginine 17 in the KPI domain. The secreted 61-amino acid product was purified to homogeneity and fun ctionally characterized. The protease inhibitory properties of the KPI -Lys(17) domain were compared to those of the native KPI domain of A b eta PP. Both KPI domains equally inhibited trypsin, chymotrypsin, and coagulation factors IXa and Xa. However, the KPI-Lys(17) domain was an approximate to 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-Lys(17) domain was an approximate to 10- and 5-fold better inhibitor of plasmin in a chromogenic substrate assay an d in a fibrinolytic assay, respectively, than the native KPI domain of A beta PP. Together, these studies suggest that the KPI-Lys(17) domai n has enhanced anti-fibrinolytic and diminished factor XIa inhibitory properties compared to the native KPI domain of A beta PP.