ROLE OF THROMBIN ANION-BINDING EXOSITE-I IN THE FORMATION OF THROMBIN-SERPIN COMPLEXES

Site-directed mutagenesis was used to investigate the role of basic re sidues in the thrombin anion-binding exosite-I during formation of thr ombin-antithrombin III (ATIII), thrombin-protease nexin 1 (PN1), and t hrombin-heparin cofactor II (HCII) inhibitor complexes, in the absence and presence of glycosaminoglycans, In the absence of glycosaminoglyc an, association rate constant (k(on)) values for the inhibition of the mutant thrombins (R35Q, H36Q, R67Q, R73Q, R75Q, R77(a)Q, H81Q, K109Q, K110Q, and K149(e)Q) by ATIII and PN1 were similar to wild-type recom binant thrombin (rIIa), whereas k(on) values were decreased 2-3-fold f or HCII against the majority of the exosite-I mutants. The exosite-I m utants did not have a significant effect on heparin-accelerated inhibi tion by ATIII with maximal k(on) values similar to rIIa, A small effec t was seen for PN1/heparin inhibition of the exosite-I mutants R35Q, R 67Q, R73Q, R75Q, and R77(a)Q, where k(on) values were decreased 2-4-fo ld, compared with rIIa, For HCII/heparin, k(on) values far inhibition of the exosite-I mutants (except R67Q, R73Q, and K149(e)Q) were 2-3-fo ld lower than rIIa. Larger decreases in It,, values for HCII/heparin w ere found for R67Q and R73Q thrombins with 441- and 14-fold decreases, respectively, whereas K149(e)Q was unchanged. For HCII/dermatan sulfa te, R67Q and R73Q had k(on) values reduced 720- and 48-foId, respectiv ely, whereas the remaining mutants were decreased 3-7-fold relative to rIIa, The results suggest that ATIII has no major interaction with ex osite-I of thrombin with or without heparin, PN1 bound to heparin uses exosite-I to some extent, possibly by utilizing the positive electros tatic field of exosite-I to enhance orientation and thrombin complex f ormation. The larger effects of the thrombin exosite-I mutants for HCI I inhibition with heparin and dermatan sulfate indicate its need for e xosite I, presumably through contact of the ''hirudin-like'' domain of HCII with exosite-I of thrombin.