Inhibition of human vitamin-K-dependent protein-S-cofactor activity by a monoclonal antibody specific for a Ca2+-dependent epitope.
Protein S is an anticoagulant vitamin-K-dependent plasma protein functioning as a cofactor to activated protein C in the degradation of factors Va and VIIIa. A murine monoclonal antibody, HPS 7, specific for a calcium-stabilized epitope in human protein S, is described. The epitope was available in intact protein S, both in its free form and when protein S was bound to C4b-binding protein. It disappeared upon reduction of disulfide bridges and also after thrombin of chymotrypsin cleavage of protein S. Thrombin cleaves protein S close to the calcium-binding region containing gamma-carboxyglutamic acid (Gla). The cleaved protein still contains the Gla region, linked by a disulfide bridge, but it has a lower affinity for calcium and no protein C cofactor activity. The thrombin-mediated cleavage of protein S could be inhibited by HPS 7. The Ka for the interaction between protein S and the monoclonal was estimated to be approximately 0.7 X 10(8) M-1. Half-maximal binding between HPS 7 and protein S was observed at a calcium concentration of 0.50 mM, indicating that saturation of the Gla region with calcium was required for the interaction. The recently reported Gla-independent high-affinity calcium binding did not induce the epitope. The calcium-dependent binding of protein S to phospholipid vesicles as well as the protein C cofactor activity was inhibited by HPS 7. The data suggests that the epitope for HPS 7 is located in the Gla region of protein S or in the closely positioned thrombin-sensitive region.
D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D002118:Calcium / Q000378:metabolism*; D000939:Epitopes / Q000032:analysis*; D006023:Glycoproteins / Q000037:antagonists & inhibitors* / Q000276:immunology; D006801:Humans; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D017293:Protein S; D013917:Thrombin
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000939:Epitopes; D006023:Glycoproteins; D017293:Protein S; D013917:Thrombin; D002118:Calcium