Immunologic evidence for insertion of the reactive-bond loop of antithrombin into the A beta-sheet of the inhibitor during trapping of target proteinases.
Identical or highly similar antigenic determinants, not present in the intact inhibitor, were induced in antithrombin on cleavage of the reactive bond, on formation of a complex between antithrombin and a synthetic reactive-loop tetradecapeptide, and on partial denaturation of antithrombin at low concentrations of guanidinium chloride. Previous studies indicate that the common structural feature of these three modified forms of antithrombin is that the region of the reactive-bond loop on the amino-terminal side of the reactive bond, or the corresponding synthetic peptide, is inserted as a middle strand in the main beta-sheet of the inhibitor, the A sheet. The new epitopes in the three modified antithrombin forms therefore most likely are exposed as a result of this insertion. Identical or highly similar epitopes were exposed also in complexes between antithrombin and thrombin or factor Xa, strongly suggesting that a substantial segment of the reactive-bond loop is inserted into the A sheet also in these complexes. In contrast, the new epitopes were not exposed in antithrombin on binding of heparin, implying that the conformational change induced by heparin does not involve such loop insertion. These results provide the first experimental verification of recent hypotheses that insertion of the reactive-bond loop of serpins into the A beta-sheet is involved in the binding of target proteinases.
Björk
I
I
Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Uppsala.
Nordling
K
K
Olson
S T
ST
D000595:Amino Acid Sequence; D000818:Animals; D000991:Antithrombins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D001665:Binding Sites; D002417:Cattle; D000939:Epitopes / Q000032:analysis; D006801:Humans; D005779:Immunodiffusion; D008961:Models, Structural; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D013917:Thrombin / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*
D000991:Antithrombins; D000939:Epitopes; D013917:Thrombin