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Role of individual gamma-carboxyglutamic acid residues of activated human
protein C in defining its in vitro anticoagulant activity
L Zhang, A Jhingan and FJ Castellino
Department of Chemistry and Biochemistry, University of Notre Dame, IN
46556.
To evaluate the contributions of individual gamma-carboxyglutamic acid
(gla) residues to the overall Ca(2+)-dependent anticoagulant activity of
activated human protein C (APC), we used recombinant (r) DNA technology to
generate protein C (PC) variants in which each of the gla precursor
glutamic acid (E) residues (positions 6, 7, 14, 16, 19, 20, 25, 26, and 29)
was separately altered to aspartic acid (D). In one case, a gla26V mutation
([gla26V]r-PC) was constructed because a patient with this particular
substitution in coagulation factor IX had been previously identified. Two
additional r-PC mutants were generated, viz, an r-PC variant containing a
substitution at arginine (R) 15 ([R15]r-PC), because this particular R
residue is conserved in all gla- containing blood coagulation proteins, as
well as a variant r-PC with substitution of an E at position 32 ([F31L,
Q32E]r-PC), because gla residues are found in other proteins at this
sequence location. This latter protein did undergo gamma-carboxylation at
the newly inserted E32 position. For each of the 11 recombinant variants, a
subpopulation of PC molecules that were gamma-carboxylated at all
nonmutated gla- precursor E residues has been purified by anion exchange
chromatography and, where necessary, affinity chromatography on an
antihuman PC column. The r-PC muteins were converted to their respective
r-APC forms and assayed for their amidolytic activities and
Ca(2+)-dependent anticoagulant properties. While no significant differences
were found between wild-type (wt) r-APC and r-APC mutants in the amidolytic
assays, lack of a single gla residue at any of the following locations,
viz, 7, 16, 20, or 26, led to virtual complete disappearance of the
Ca(2+)-dependent anticoagulant activity of the relevant r-APC mutant, as
compared with its wt counterpart. On the other hand, single eliminations of
any of the gla residues located at positions 6, 14, or 19 of r-APC resulted
in variant recombinant molecules with substantial anticoagulant activity
(80% to 92%), relative to wtr-APC. Mutation of gla residues at positions 25
and 29 resulted in r-APC variants with significant but low (24% and 9% of
wtr-APC, respectively) levels of anticoagulant activity. The variant,
[R15L]r-APC, possessed only 19% of the anticoagulant activity of wrt-APC,
while inclusion of gla at position 32 in the variant, [F31L, Q32gla]r-APC,
resulted in a recombinant enzyme with an anticoagulant activity equivalent
to that of wtr-APC.
Volume 80,
Issue 4,
pp. 942-952,
08/15/1992
Copyright © 1992 by The American Society of Hematology
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