IMMUNOLOGICAL 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 rea ctive bond, on formation of a complex between antithrombin and a synth etic reactive-loop tetradecapeptide, and on partial denaturation of an tithrombin at low concentrations of guanidinium chloride. Previous stu dies indicate that the common structural feature of these three modifi ed 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-she et of the inhibitor, the A sheet. The new epitopes in the three modifi ed antithrombin forms therefore most likely are exposed as a result of this insertion. Identical or highly similar epitopes were exposed als o in complexes between antithrombin and thrombin or factor Xa, strongl y suggesting that a substantial segment of the reactive-bond loop is i nserted into the A sheet also in these complexes. In contrast, the new epitopes were not exposed in antithrombin on binding of heparin, impl ying that the conformational change induced by heparin does not involv e such loop insertion. These results provide the first experimental ve rification of recent hypotheses that insertion of the reactive-bond lo op of serpins into the A beta-sheet is involved in the binding of targ et proteinases.