Characterization of functionally important domains in human vitamin K-dependent protein S using monoclonal antibodies.
Vitamin K-dependent protein S is an anticoagulant plasma protein functioning as a cofactor to activated protein C in the degradation of coagulation factors Va and VIIIa. To determine which regions in protein S are important for its cofactor activity, we have raised and characterized a large panel of monoclonal antibodies against human protein S. Several of the antibodies were directed against Ca2(+)-dependent epitopes, and they were found to be located either in the domain containing gamma-carboxyglutamic acid (Gla), the thrombin-sensitive region, or in the first epidermal growth factor (EGF)-like domain. The first two types of epitopes were exposed at approximately 1 mM Ca2+, whereas the epitope(s) in the EGF-like domains required less than 1 microM Ca2+, suggesting the presence of one or more high affinity Ca2(+)-binding site(s). The antibodies, as well as their Fab' fragments, against all three types of Ca2(+)-dependent epitopes efficiently inhibited the activated protein C cofactor function of protein S, but through different mechanisms. The antibodies against the Gla domain exerted their effects through inhibition of protein S binding to negatively charged phospholipid. Fab'-fragments of antibodies against the thrombin-sensitive region and the first EGF-like domain were the most potent inhibitors of the activated protein C cofactor function but did not inhibit phospholipid binding of protein S. In conclusion, we have identified the domains in protein S that are important for the activated protein C cofactor activity. The Gla domain is instrumental in the binding of protein S to phospholipid, whereas the thrombin-sensitive region and the first EGF-like domain may be directly involved in protein-protein interactions on the phospholipid surface.
Dahlbäck
B
B
Department of Clinical Chemistry, University of Lund, Malmö General Hospital, Sweden.
Hildebrand
B
B
Malm
J
J
D015055:1-Carboxyglutamic Acid; D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001665:Binding Sites; D001667:Binding, Competitive; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D002417:Cattle; D002846:Chromatography, Affinity; D002918:Chymotrypsin / Q000378:metabolism; D003169:Complement Inactivator Proteins; D004815:Epidermal Growth Factor; D000939:Epitopes / Q000276:immunology; D006023:Glycoproteins / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D010446:Peptide Fragments / Q000276:immunology / Q000302:isolation & purification; D010743:Phospholipids / Q000378:metabolism; D011486:Protein C / Q000378:metabolism; D017293:Protein S; D012697:Serine Endopeptidases / Q000378:metabolism; D013329:Structure-Activity Relationship; D013381:Subtilisins / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D003169:Complement Inactivator Proteins; D000939:Epitopes; D006023:Glycoproteins; D010446:Peptide Fragments; D010743:Phospholipids; D011486:Protein C; D017293:Protein S; D015055:1-Carboxyglutamic Acid; D004815:Epidermal Growth Factor; D012697:Serine Endopeptidases; D013381:Subtilisins; D002918:Chymotrypsin; D013917:Thrombin; C036813:lysyl endopeptidase; D002118:Calcium