Kinetic characterization of the substrate reaction between a complex of antithrombin with a synthetic reactive-bond loop tetradecapeptide and four target proteinases of the inhibitor.

A tetradecapeptide corresponding to the P1 to P14 region of the reactive-bond loop of antithrombin (AT) binds to the inhibitor, presumably as a middle strand of the A beta-sheet, thereby converting AT from an inhibitor to a substrate of thrombin (Björk, I., Ylinenjärvi, K., Olson, S.T., and Bock, P. E. (1992) J. Biol. Chem. 267, 1976-1982). The kinetics of cleavage of the AT reactive bond in the AT-peptide complex by four target proteinases were quantified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometry. The kcat/Km values for thrombin and factor IXa were indistinguishable from the second-order rate constants for AT inhibition of these enzymes, whereas the values for factor Xa and plasmin were 10-17-fold higher than the inhibition rate constants. Heparin with high affinity for AT accelerated the substrate reaction with thrombin to an extent consistent with the reduced heparin affinity of the AT-peptide complex. These data show that blocking by the peptide of the putative intramolecular association of the P1 to P14 region of the AT reactive-bond loop with the A beta-sheet leads to AT functioning as a substrate of its target enzymes with an efficiency that equals or exceeds the action of uncomplexed AT as an inhibitor and with the expected heparin activation. The results thus suggest that a substrate-like attack of the proteinase on the inhibitor reactive bond in an exposed loop initiates the inhibition reaction. This attack presumably induces the subsequent trapping of the enzyme by the insertion of the reactive-bond loop into the A beta-sheet.