A novel exosite in the light chain of human activated protein C essential for interaction with blood coagulation factor Va.
Activated protein C (APC) exerts its physiologic anticoagulant role by proteolytic inactivation of the blood coagulation cofactors Va and VIIIa. To identify regions on the surface of the light chain of APC that mediate anticoagulant activity, 10 synthetic peptides were prepared and tested for their ability to inhibit APC anticoagulant activity. The synthetic peptide-(142-155) inhibited APC anticoagulant activity in Xa-1-stage coagulation assays in normal and protein S-depleted plasma with 50% inhibition at 5-25 microM peptide. In a system using purified clotting factors, peptide-(142-155) inhibited APC catalyzed inactivation of factor Va in the presence or absence of phospholipids with 50% inhibition at 50 microM peptide. However, peptide-(142-155) had no effect on APC amidolytic activity or on the reaction of APC with the serpin, recombinant [Arg358]-alpha 1-antitrypsin. Moreover, peptide-(142-155) inhibited factor Xa clotting activity in normal plasma as well as in a prothrombinase assay in the presence of factor Va with 50% inhibition at 5 microM and 50 microM peptide, respectively, under the assay conditions. The peptide had no significant effect on factor Xa or thrombin amidolytic activity and no effect on the clotting of purified fibrinogen by thrombin, suggesting that it does not directly inhibit these enzymes' active sites. Peptide-(142-155) was shown to bind directly to immobilized factor Va.(ABSTRACT TRUNCATED AT 250 WORDS)
Mesters
R M
RM
Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, California 92037.
Heeb
M J
MJ
Griffin
J H
JH
D000577:Amides / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D001777:Blood Coagulation / Q000187:drug effects; D015943:Factor Va / Q000378:metabolism*; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology; D011486:Protein C / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism; D013925:Thromboplastin / Q000032:analysis
D000577:Amides; D010446:Peptide Fragments; D011486:Protein C; D015943:Factor Va; D005340:Fibrinogen; D013925:Thromboplastin; D013917:Thrombin