Cabbage seed protease inhibitor: a slow, tight-binding inhibitor of trypsin
with activity toward thrombin, activated Stuart factor (factor Xa),
activated Hageman factor (factor XIIa), and plasmin
TH Carter, BA Everson and OD Ratnoff
Department of Medicine, Case Western Reserve University, Cleveland, OH.
An inhibitor of procoagulant and fibrinolytic enzymes was derived from
cabbage seeds by a procedure using acetone precipitation, ion-exchange
chromatography, and gel filtration. The cabbage seed inhibitor was a 10- Kd
monomeric protein with intrachain disulfide bonds. This preparation
prevented clot formation in whole blood and blocked the ability of thrombin
to induce clot formation in plasma and to induce platelet aggregation. A
number of proteases were inhibited, as demonstrated by using purified
enzymes in amidolytic assays. Tight-binding inhibition was observed for
activated Stuart factor (factor Xa) and plasmin. Inhibition of thrombin and
activated Hageman factor (factor XIIa) was observed with a molar excess of
inhibitor. No inhibition was detected for activated plasma thromboplastin
antecedent (factor XIa), plasma kallikrein, or C1 esterase. Reaction
progress curves for trypsin indicated slow, tight-binding inhibition, with
an apparent inhibition constant in the nanomolar range or less. The
electrophoretic mobility of trypsin was altered by the inhibitor in
nondenaturing polyacrylamide gel electrophoresis (PAGE) but not in sodium
dodecyl sulfate (SDS)- PAGE, indicating noncovalent bonding. Only partial
reversal of trypsin inhibition could be demonstrated by washing the
inhibitor from enzyme immobilized on solid beads. A dot-blot technique with
cabbage seed inhibitor was capable of detecting 10 ng nitrocellulose-bound
trypsin. The dot-blot technique also appeared capable of detecting plasmin.
These findings demonstrated the potential utility of this inhibitor as a
probe for detection of tightly bound proteases. In summary, cabbage seed
extracts contain an inhibitor with activity toward a broad range of
proteases important to hemostasis. To our knowledge, this agent represents
the first inhibitor isolated from a plant source that inhibits thrombin.
Volume 75,
Issue 1,
pp. 108-115,
01/01/1990
Copyright © 1990 by The American Society of Hematology
