The autolysis loop of activated protein C interacts with factor Va and differentiates between the Arg506 and Arg306 cleavage sites.
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. (Blood. 2000;96:585-593)
Gale
A J
AJ
Departments of Molecular and Experimental Medicine and of Vascular Biology, The Scripps Research Institute, La Jolla, CA 92307, USA.
Heeb
M J
MJ
Griffin
J H
JH
D001120:Arginine; D001329:Autolysis; D001665:Binding Sites; D001777:Blood Coagulation / Q000187:drug effects; D015153:Blotting, Western; D015943:Factor Va / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010314:Partial Thromboplastin Time; D011486:Protein C / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship
D011486:Protein C; D011994:Recombinant Proteins; D015943:Factor Va; D001120:Arginine