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Antithrombin-III-Hamilton, Ala 382 to Thr: an antithrombin-III variant that
acts as a substrate but not an inhibitor of alpha-thrombin and factor Xa
RC Austin, RA Rachubinski, FA Ofosu and MA Blajchman
Canadian Red Cross Blood Transfusion Service, Hamilton, Ontario.
Antithrombin-III-Hamilton has been shown to be a structural variant of
antithrombin-III (AT-III) with normal heparin affinity but impaired
protease inhibitory activity. The molecular defect of AT-III-Hamilton is
the substitution of Thr for Ala at amino acid residue 382. The plasma of
affected individuals contains approximately equal quantities of normal
AT-III and AT-III-Hamilton. When AT-III was isolated from the plasma of the
propositus by heparin-Sepharose chromatography, it had identical mobility
on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to
normal plasma-derived AT-III, under both reducing and nonreducing
conditions. However, the AT-III-Hamilton species, separated from the
propositus' normal AT-III by a combination of heparin-Sepharose and
thrombin-Sepharose chromatography, had increased mobility on reductive
SDS-PAGE compared with AT-III from the propositus isolated by
heparin-Sepharose chromatography alone. Under nonreducing conditions this
AT-III-Hamilton species had decreased mobility compared with AT-III from
the propositus (or normal AT-III) isolated only by heparin-Sepharose
chromatography. When incubated with either human alpha-thrombin or human
factor Xa, this AT-III-Hamilton species was unreactive. Approximately 50%
of the AT-III from the propositus isolated by heparin-Sepharose
chromatography, when incubated with either human alpha-thrombin or factor
Xa, did not form complex but was cleaved, presumably at the reactive center
Arg393-Ser394. To further substantiate the biological behavior of this
variant, AT-III- Hamilton polypeptides were synthesized in a cell-free
system. This recombinantly produced AT-III-Hamilton, when incubated with
either human alpha-thrombin or factor Xa, was cleaved by both these
proteases, but did not show any complex formation. The results indicate
that AT- III-Hamilton does not form a stable covalent inhibitory complex
with these serine proteases but can be cleaved at the reactive center.
Thus, the inhibition of serine proteases by their natural inhibitors (the
serpins) involves at least two separate, but interrelated events;
hydrolysis at the reactive center followed by complex formation. AT-III-
Hamilton is capable of only the first of these events.
Volume 77,
Issue 10,
pp. 2185-2189,
05/15/1991
Copyright © 1991 by The American Society of Hematology
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