Cleavage and activation of human factor IX by serine proteases
DL Enfield and AR Thompson
Human factor IX circulates as a single-chain glycoprotein. Upon activation
in vitro, it is cleaved into disulfide-linked light and heavy chains and an
activation peptide. After reduction of activated 125I-factor IX, the heavy
and light chains are readily identified by gel electrophoresis. A direct,
immunoradiometric assay for factor IXa was developed to assess activation
of factor IX for proteases that cleaved it. The assay utilized radiolabeled
antithrombin III with heparin to identify the active site and antibodies to
distinguish factor IX. After cleavage of factor IX by factor XIa, factor
VIIa- tissue thromboplastin complex, or the factor X-activating enzyme from
Russell's viper venom, antithrombin III bound readily to factor IXa.
Cleavage of 125I-factor IX by trypsin, chymotrypsin, and granulocyte
elastase in the presence of calcium yielded major polypeptide fragments of
the sizes of the factor XIa-generated light and heavy chains. Kallikrein
did not cleave the zymogen. Nonactivation cleavage was noted by thrombin,
but only in the absence of calcium. When the immunoradiometric assay was
used to assess trypsin-cleaved factor IX, the product bound antithrombin
III, but not maximally. After digesting with insolubilized trypsin,
clotting activity confirmed activation. In contrast, incubation of factor
IX with elastase (Takaki A et al, J Clin Invest 71:1706, 1983) or
chymotrypsin did not lead to generation of an antithrombin III-binding
site, despite their digestion of 125I-factor IX into heavy and light
chain-sized fragments. In evaluating activation of factor IX, physical
evidence of activation cleavages does not necessarily correlate with
generation of an active site.
Volume 64,
Issue 4,
pp. 821-831,
10/01/1984
Copyright © 1984 by The American Society of Hematology
