Myles T, Church F C, Whinna H C, Monard D, Stone S R
Department of Haematology, University of Cambridge, MRC Centre, Hills Road, Cambridge, CB2-2QH, United Kingdom.
J Biol Chem. 1998 Nov 20;273(47):31203-8. doi: 10.1074/jbc.273.47.31203.
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 (kon) 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 kon 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 kon 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 kon values were decreased 2-4-fold, compared with rIIa. For HCII/heparin, kon 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 kon 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 kon 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.
在有无糖胺聚糖存在的情况下，采用定点诱变技术研究了凝血酶阴离子结合外位点-I中碱性残基在凝血酶-抗凝血酶III（ATIII）、凝血酶-蛋白酶连接蛋白1（PN1）和凝血酶-肝素辅因子II（HCII）抑制复合物形成过程中的作用。在无糖胺聚糖存在时，ATIII和PN1对突变型凝血酶（R35Q、K36Q、R67Q、R73Q、R75Q、R77(a)Q、K81Q、K109Q、K110Q和K149(e)Q）的抑制作用的结合速率常数（kon）值与野生型重组凝血酶（rIIa）相似，而HCII对大多数外位点-I突变体的kon值降低了2 - 3倍。外位点-I突变体对ATIII的肝素加速抑制作用没有显著影响，最大kon值与rIIa相似。对于PN1/肝素对R35Q、R67Q、R73Q、R75Q和R77(a)Q外位点-I突变体的抑制作用，观察到较小的影响，与rIIa相比，kon值降低了2 - 4倍。对于HCII/肝素，外位点-I突变体（R67Q、R73Q和K149(e)Q除外）的抑制作用的kon值比rIIa低2 - 3倍。对于R67Q和R73Q凝血酶，HCII/肝素的kon值分别降低了441倍和14倍，而K149(e)Q不变。对于HCII/硫酸皮肤素，R67Q和R73Q的kon值分别降低了720倍和48倍，而其余突变体相对于rIIa降低了3 - 7倍。结果表明，无论有无肝素，ATIII与凝血酶的外位点-I均无主要相互作用。与肝素结合的PN1在一定程度上利用外位点-I，可能是通过利用外位点-I的正静电场来增强定向和凝血酶复合物的形成。凝血酶外位点-I突变体对HCII与肝素和硫酸皮肤素的抑制作用有较大影响，表明其需要外位点-I，推测是通过HCII的“水蛭素样”结构域与凝血酶的外位点-I接触实现的。
