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Role of Thrombin Anion-binding Exosite-I in the Formation of Thrombin-Serpin Complexes

1998; Elsevier BV; Volume: 273; Issue: 47 Linguagem: Inglês

10.1074/jbc.273.47.31203

1083-351X

Timothy Myles, Frank Church, Herbert C. Whinna, Denis Monard, Stuart R. Stone,

Coagulation, Bradykinin, Polyphosphates, and Angioedema

Site-directed mutagenesis was used to investigate the role of basic residues in the thrombin anion-binding exosite-I during formation of thrombin-antithrombin III (ATIII), thrombin-protease nexin 1 (PN1), and thrombin-heparin cofactor II (HCII) inhibitor complexes, in the absence and presence of glycosaminoglycans. In the absence of glycosaminoglycan, association rate constant ( k on ) values for the inhibition of the mutant thrombins (R35Q, K36Q, R67Q, R73Q, R75Q, R77 a Q, K81Q, K109Q, K110Q, and K149 e Q) by ATIII and PN1 were similar to wild-type recombinant thrombin (rIIa), whereas k on values were decreased 2–3-fold for HCII against the majority of the exosite-I mutants. The exosite-I mutants did not have a significant effect on heparin-accelerated inhibition by ATIII with maximal k on values similar to rIIa. A small effect was seen for PN1/heparin inhibition of the exosite-I mutants R35Q, R67Q, R73Q, R75Q, and R77 a Q, where k on values were decreased 2–4-fold, compared with rIIa. For HCII/heparin, k on values for inhibition of the exosite-I mutants (except R67Q, R73Q, and K149 e Q) were 2–3-fold lower than rIIa. Larger decreases in k on values for HCII/heparin were found for R67Q and R73Q thrombins with 441- and 14-fold decreases, respectively, whereas K149 e Q was unchanged. For HCII/dermatan sulfate, R67Q and R73Q had k on values reduced 720- and 48-fold, respectively, whereas the remaining mutants were decreased 3–7-fold relative to rIIa. The results suggest that ATIII has no major interaction with exosite-I of thrombin with or without heparin. PN1 bound to heparin uses exosite-I to some extent, possibly by utilizing the positive electrostatic field of exosite-I to enhance orientation and thrombin complex formation. The larger effects of the thrombin exosite-I mutants for HCII inhibition with heparin and dermatan sulfate indicate its need for exosite-I, presumably through contact of the "hirudin-like" domain of HCII with exosite-I of thrombin.