REACTIVITIES OF THE S2 AND S3 SUBSITE RESIDUES OF THROMBIN WITH THE NATIVE AND HEPARIN-INDUCED CONFORMERS OF ANTITHROMBIN

A pentasaccharide (PS) fragment of heparin capable of activating antit hrombin (AT) markedly accelerates the inhibition of factor Xa by AT, b ut has insignificant effect on inhibition of thrombin. For inhibition of thrombin, the bridging function of a longer polysaccharide chain is required to accelerate the reaction. To study the basis for the simil ar reactivity of thrombin with the native or heparin-activated conform ers of AT, several residues surrounding the active site pocket of thro mbin were targeted for mutagenesis study. Leu99 and Glu192, the varian t residues influencing the S2 and S3 subsite specificity of thrombin w ere replaced with Tyr and Gin. The Tyr60a, Pro60b, Pro60c, and Trp60d residues forming part of the S2 specificity pocket were deleted from t he B-insertion loop of the wild-type and Leu99/Glu192 --> Tyr/Gln thro mbins. Kinetic studies indicated that the reactivities of all mutants with AT were moderately or severely impaired. Although heparin largely corrected the defect in reactivities, it also markedly elevated the s toichiometries of inhibition with the mutants. Interestingly, PS also accelerated AT inhibition of the mutants 5-68-fold, suggesting that th e mutants are able to discriminate between the native and activated co nformers of AT. Based on these results and the recent crystal structur e determination of AT in complex with FS, a model for thrombin-AT inte raction is proposed in which the S2 and S3 subsite residues of thrombi n are critical for recognition of the P2 and P3 residues of AT in the native conformation. In the activated conformation, other residues are made accessible for interaction with the protease, and the similar re activity of thrombin with the native and heparin-activated conformers of AT may be coincidental. The results further suggest that the S2 and S3 subsite residues are crucial in controlling the partitioning of th e thrombin-AT intermediate into the alternative inhibitory or substrat e pathways of the reaction.