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Blood, Vol. 96 No. 2 (July 15), 2000:
pp. 585-593
The autolysis loop of activated protein C interacts with factor Va
and differentiates between the Arg506 and Arg306 cleavage
sites
Andrew J. Gale,
Mary J. Heeb, and
John H. Griffin
From the Departments of Molecular and Experimental Medicine and of
Vascular Biology, The Scripps Research Institute, La Jolla, CA.
The anticoagulant human plasma serine protease, activated protein C
(APC), inactivates blood coagulation factors Va (FVa) and VIIIa. The
so-called autolysis loop of APC (residues 301-316, equivalent to
chymotrypsin [CHT] residues 142-153) has been hypothesized to bind
FVa. In this study, site-directed mutagenesis was used to probe the
role of the charged residues in this loop in interactions between APC
and FVa. Residues Arg306 (147 CHT), Glu307, Lys308, Glu309, Lys311,
Arg312, and Arg314 were each individually, or in selected combinations,
mutated to Ala. The purified recombinant protein C mutants were
characterized using activated partial thromboplastin time (APTT)
clotting assays and FVa inactivation assays. Mutants 306A, 308A, 311A,
312A, and 314A had mildly reduced anticoagulant activity. Based on FVa
inactivation assays and APTT assays using purified Gln506-FVa and
plasma containing Gln506-FV, it appeared that these mutants were
primarily impaired for cleavage of FVa at Arg506. Studies of the
quadruple APC mutant (306A, 311A, 312A, and 314A) suggested that the
autolysis loop provides for up to 15-fold discrimination of the Arg506
cleavage site relative to the Arg306 cleavage site. This study shows
that the loop on APC of residues 306 to 314 defines an FVa binding site
and accounts for much of the difference in cleavage rates at the 2 major cleavage sites in FVa.
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