Kinetics of inhibition of human plasma kallikrein by a site-specific
modified inhibitor Arg15-aprotinin: evaluation using a microplate system
and comparison with other proteases
CF Scott, HR Wenzel, HR Tschesche and RW Colman
Human plasma kallikrein, a product of contact-activated plasma proteolysis,
is moderately inhibited by aprotinin, a small polypeptide from bovine lung
that has been used as an experimental drug in human disease states.
Aprotinin has a Lys residue in the P1 (reactive center) position occupying
residue 15. Since kallikrein is an arginine-directed serine protease, we
hypothesized that an altered form of aprotinin, Arg15-aprotinin, might be a
better inhibitor. Kinetic evaluations were performed in 96-well
microplates. We found that the KL (loose or Michaelis-Menten complex) was
unchanged by the modification. However, the association rate constant was
increased from 1.14 X 10(4) (mol/L)- 1s-1 to 1.5 X 10(5) (mol/L)-1s1, thus
indicating that the inhibition rate was increased 14-fold for the modified
protein. The Ki (at equilibrium) was decreased from 3.2 X 10(-7) mol/L to
1.5 X 10(-8) mol/L after substituting Arg for Lys in the P1 position.
Therefore, the modified inhibitor binds to plasma kallikrein more tightly
than the natural protein. We also investigated the effect of
Arg15-aprotinin on tissue kallikrein, plasmin, factor XIIa, factor XIa, and
thrombin and found that the Ki slightly decreased from 5.1 X 10(-7) mol/L
to 1.2 X 10(-7) mol/L for tissue kallikrein and slightly decreased from 2 X
10(- 8) mol/L to 1 X 10(-8) mol/L for plasmin. Arg15-aprotinin did not
inhibit thrombin or factor XIIa, even though both enzymes are arginine-
directed serine proteases. However, factor XIa, although it was not
inhibited by aprotinin, had a Ki of 3.4 X 10(-8) mol/L for Arg15-
aprotinin. Therefore, Arg15-aprotinin is a more effective inhibitor of
plasma kallikrein as well as factor XIa but shows minimal preference for
plasmin and tissue kallikrein. This study also indicates that it is
possible and practical to perform kinetic analyses directly in microplates.
Volume 69,
Issue 5,
pp. 1431-1436,
05/01/1987
Copyright © 1987 by The American Society of Hematology
