title: Immunoradiometric assay for the calcium-stabilized conformation of human protein S.
abstract: Rabbit polyclonal anti-protein S serum was fractionated with immobilized human protein S to establish solid-phase immunoradiometric assays recognizing Ca(II)-dependent and NonCa(II)-dependent epitopes of human protein S. The two assays were specific for PS:Ca(II)Ag and PS:NonCa(II)Ag and highly sensitive with a lower limit of detection of about 2.5 ng/ml. PS:Ca(II)Ag and PS:NonCa(II)Ag levels were measured in immunopurified protein S, thrombin-modified protein S and chymotrypsin-cleaved protein S. Only in chymotrypsin-cleaved protein S an important discrepancy between the two antigen levels was observed. Ranges for the concentration of PS:Ca(II)Ag and PS:Non-Ca(II)Ag and their ratio were established in plasma of healthy individuals (0.92 +/- 0.13 U/ml, 0.98 +/- 0.21 U/ml, 0.96 +/- 0.17, respectively). In a group of patients using oral anticoagulant therapy the ratio PS:Ca(II)Ag/PS:NonCa(II)Ag decreased at increasing intensity of anticoagulation suggesting the presence of sub- and noncarboxylated protein S molecules. In plasma of patients with a hereditary type I protein S deficiency PS:Ca(II)Ag and PS:NonCa(II)Ag were reduced to the same extent: mean ratio 1.02 +/- 0.12 in the group not on oral anticoagulant treatment and 0.94 +/- 0.10 in the group on oral anticoagulant therapy. Analysis of patients with a history of unexplained thrombo-embolic disease did not reveal individual patients with a PS:Ca(II)Ag/PS:NonCa(II)Ag ration below the lower limit of the normal range (mean ratio 1.05 +/- 0.17), suggesting that the frequency of genetic protein S variants with defects in the Ca(II)-stabilized conformation is very low.
authors.affiliation: Department of Hematology, Leiden University Hospital, The Netherlands.
authors.forename: S R
authors.initials: SR
authors.lastname: Poort
authors.forename: P P
authors.initials: PP
authors.lastname: Deutz-Terlouw
authors.forename: A
authors.initials: A
authors.lastname: van Wijngaarden
authors.forename: R M
authors.initials: RM
authors.lastname: Bertina
mesh_terms: D000925:Anticoagulants / Q000494:pharmacology; D000941:Antigens / Q000032:analysis; D002118:Calcium / Q000097:blood; D002918:Chymotrypsin; D006023:Glycoproteins / Q000097:blood* / Q000172:deficiency / Q000276:immunology; D006801:Humans; D011487:Protein Conformation; D017293:Protein S; D011863:Radioimmunoassay / Q000379:methods*; D013917:Thrombin; D013923:Thromboembolism / Q000097:blood
chemical_list: D000925:Anticoagulants; D000941:Antigens; D006023:Glycoproteins; D017293:Protein S; D002918:Chymotrypsin; D013917:Thrombin; D002118:Calcium