凝血酶的诱变选择性地调节丝氨酸蛋白酶抑制剂肝素辅因子II和抗凝血酶III的抑制作用。与阴离子结合外位点的相互作用决定了肝素辅因子II的特异性。

Mutagenesis of thrombin selectively modulates inhibition by serpins heparin cofactor II and antithrombin III. Interaction with the anion-binding exosite determines heparin cofactor II specificity.

作者信息

Sheehan J P, Wu Q, Tollefsen D M, Sadler J E

机构信息

Howard Hughes Medical Institute, Department of Medicine, Biochemistry & Molecular Biophysics, Jewish Hospital of St. Louis, Washington University School of Medicine, Missouri 63110.

出版信息

J Biol Chem. 1993 Feb 15;268(5):3639-45.

PMID:8429040

Abstract

Thrombin is a multifunctional serine protease that plays a critical role in hemostasis. Thrombin is inhibited by the serpins antithrombin III and heparin cofactor II in a reaction that is dramatically accelerated by glycosaminoglycans. The structural basis of the interaction with these inhibitors was investigated by introducing single amino acid substitutions into the anion-binding exosite (R68E, R70E) and unique insertion loops (K52E, K154A) of thrombin. The rate of inhibition of these recombinant thrombins by antithrombin III and heparin cofactor II was determined in the absence and presence of glycosaminoglycan. The second order rate constant (k2) for inhibition by antithrombin III without heparin was 3.7 x 10(5) M-1 min-1 for wild-type thrombin; rates for the mutant thrombins varied less than 2-fold. For inhibition by antithrombin III with heparin, the rate constant was 4.5 x 10(8) M-1 min-1 for wild-type thrombin with no significant differences between any of the recombinant thrombins. In contrast, the rate constant for inhibition by heparin cofactor II without glycosaminoglycan was 4.3 x 10(4) M-1 min-1 for wild-type thrombin; rates were 10-fold slower for thrombin K52E and 2- to 3-fold slower for thrombins R68E and R70E. The rate constants for inhibition of wild-type thrombin by HCII in the presence of heparin or dermatan sulfate were 9.2 x 10(8) M-1 min-1 and 9.0 x 10(8) M-1 min-1, respectively. Compared to wild-type thrombin, the rate of inhibition by HCII with glycosaminoglycan was 5- to 15-fold slower for thrombins K52E and R70E and 50- to over 100-fold slower for thrombin R68E. Thrombin K154A was inhibited by heparin cofactor II with rates similar to wild-type thrombin in all assays. These results suggest that heparin cofactor II interacts with residue Lys-52 in the proposed S1' subsite and with residues Arg-68 and Arg-70 in the anion-binding exosite of thrombin, and that these interactions contribute to the molecular basis of heparin cofactor II specificity for thrombin.

摘要

凝血酶是一种多功能丝氨酸蛋白酶，在止血过程中起关键作用。凝血酶在一种反应中受到丝氨酸蛋白酶抑制剂抗凝血酶III和肝素辅因子II的抑制，糖胺聚糖可显著加速该反应。通过将单个氨基酸取代引入凝血酶的阴离子结合外位点（R68E、R70E）和独特插入环（K52E、K154A）来研究与这些抑制剂相互作用的结构基础。在不存在和存在糖胺聚糖的情况下，测定了抗凝血酶III和肝素辅因子II对这些重组凝血酶的抑制率。对于野生型凝血酶，无肝素时抗凝血酶III抑制的二级速率常数（k2）为3.7×10⁵ M⁻¹ min⁻¹；突变型凝血酶的速率变化小于2倍。对于有肝素时抗凝血酶III的抑制，野生型凝血酶的速率常数为4.5×10⁸ M⁻¹ min⁻¹，任何重组凝血酶之间均无显著差异。相比之下，无糖胺聚糖时肝素辅因子II抑制的速率常数，野生型凝血酶为4.3×10⁴ M⁻¹ min⁻¹；凝血酶K52E的速率慢10倍，凝血酶R68E和R70E的速率慢2至3倍。在存在肝素或硫酸皮肤素的情况下，肝素辅因子II抑制野生型凝血酶的速率常数分别为9.2×10⁸ M⁻¹ min⁻¹和9.0×10⁸ M⁻¹ min⁻¹。与野生型凝血酶相比，对于凝血酶K52E和R70E，有糖胺聚糖时肝素辅因子II的抑制速率慢5至15倍，对于凝血酶R68E则慢50至100倍以上。在所有测定中，凝血酶K154A被肝素辅因子II抑制的速率与野生型凝血酶相似。这些结果表明，肝素辅因子II与凝血酶拟议的S1'亚位点中的赖氨酸-52残基以及阴离子结合外位点中的精氨酸-68和精氨酸-70残基相互作用，并且这些相互作用构成了肝素辅因子II对凝血酶特异性的分子基础。