Cooper Scott T, Rezaie Alireza R, Esmon Charles T, Church Frank C
Department of Pathology, The University of North Carolina at Chapel Hill, School of Medicine, Chapel Hill, NC 27599, USA.
Thromb Res. 2002 Jul 15;107(1-2):67-73. doi: 10.1016/s0049-3848(02)00180-9.
Antithrombin (ATIII), heparin cofactor II (HCII) and protein C inhibitor (PCI; also named plasminogen activator inhibitor-3) are serine protease inhibitors (serpins) whose thrombin inhibition activity is accelerated in the presence of glycosaminoglycans. We compared the inhibition properties of PCI and HCII to ATIII using R93A/R97A/R101A thrombin, an anion-binding exosite-2 (exosite-2) mutant that has greatly reduced heparin-binding properties. Heparin-enhanced PCI inhibition of R93A/R97A/R101A thrombin was only approximately 2-fold compared to 40-fold enhancement with wild-type recombinant thrombin. Thrombomodulin (TM) (with or without the chondroitin sulfate moiety) accelerated PCI inhibition of both wild-type and R93A/R97A/R101A thrombins. HCII achieved the same maximum activity in the presence of heparin with both wild-type and R93A/R97A/R101A thrombins; however, the optimum heparin concentration was 20 times greater than the reaction with wild-type thrombin, indicative of a decrease in heparin affinity. Dermatan sulfate (DSO4)-catalyzed HCII thrombin inhibition was unchanged in R93A/R97A/R101A thrombin compared to wild-type recombinant thrombin. These results suggest that PCI is similar to ATIII and depends upon ternary complex formation with heparin and these specific thrombin exosite-2 residues to accelerate thrombin inhibition. In contrast, HCII does not require Arg(93), Arg(97) and Arg(101) of thrombin exosite-2 and further supports the hypothesis that HCII uses an allosteric process following glycosaminoglycan binding to inhibit thrombin.
抗凝血酶（ATIII）、肝素辅因子II（HCII）和蛋白C抑制剂（PCI；也称为纤溶酶原激活物抑制剂-3）是丝氨酸蛋白酶抑制剂（丝氨酸蛋白酶抑制因子），其凝血酶抑制活性在存在糖胺聚糖的情况下会加速。我们使用R93A/R97A/R101A凝血酶比较了PCI和HCII对ATIII的抑制特性，R93A/R97A/R101A凝血酶是一种阴离子结合外位点2（外位点2）突变体，其肝素结合特性大大降低。与野生型重组凝血酶增强40倍相比，肝素增强的PCI对R93A/R97A/R101A凝血酶的抑制作用仅约为2倍。血栓调节蛋白（TM）（有或没有硫酸软骨素部分）加速了PCI对野生型和R93A/R97A/R101A凝血酶的抑制作用。在存在肝素的情况下，HCII对野生型和R93A/R97A/R101A凝血酶均达到相同的最大活性；然而，最佳肝素浓度比与野生型凝血酶反应时高20倍，表明肝素亲和力降低。与野生型重组凝血酶相比，硫酸皮肤素（DSO4）催化的HCII对R93A/R97A/R101A凝血酶的抑制作用没有变化。这些结果表明，PCI与ATIII相似，依赖于与肝素和这些特定的凝血酶外位点2残基形成三元复合物来加速凝血酶抑制。相比之下，HCII不需要凝血酶外位点2的Arg(93)、Arg(97)和Arg(101)，进一步支持了HCII在糖胺聚糖结合后使用变构过程来抑制凝血酶的假设。
