Fortenberry Y M, Whinna H C, Cooper S T, Myles T, Leung L L K, Church F C
Departments of Pathology and Laboratory Medicine, and Pharmacology and Medicine, University of North Carolina at Chapel Hill, School of Medicine, Chapel Hill, NC, USA.
J Thromb Haemost. 2007 Jul;5(7):1486-92. doi: 10.1111/j.1538-7836.2007.02574.x.
Protein C inhibitor (PCI) and antithrombin (AT) are serine protease inhibitors (serpins) that inhibit a wide array of blood coagulation serine proteases including thrombin.
Fifty-five Ala-scanned recombinant thrombin mutants were used to determine thrombin residues important for inhibition by PCI with and without the cofactors heparin and thrombomodulin (TM) and compared with the prototypical serpin, AT.
Residues around the active site (Tyr50 and Glu202) and the sodium-binding site (Glu229 and Arg233) were required for thrombin inhibition by PCI with and without cofactors. Exosite-2 residues (Arg89, Arg93, Glu94, Arg98, Arg245, Arg248, and Gln251) were critical for heparin-accelerated inhibition of thrombin by PCI. Exosite-1 residues (especially Lys65 and Tyr71) were required for enhanced PCI inhibition of thrombin-TM. Interestingly, we also found that the TM chondroitin sulfate moiety is not required for the approximately 150-fold enhanced rate of thrombin inhibition by PCI. Using the aforementioned thrombin exosite-2 mutants that were essential for heparin-catalyzed PCI-thrombin inhibition reactions we found no change in PCI inhibition rates for thrombin-TM.
Collectively, these results show that (i) similar thrombin exosite-2 residues are critical for the heparin-catalyzed inhibition by PCI and AT, (ii) PCI and AT are different in their thrombin-TM inhibition properties, and (iii) PCI has a distinct advantage over AT in the regulation of the activity of thrombin-TM.
蛋白C抑制剂（PCI）和抗凝血酶（AT）是丝氨酸蛋白酶抑制剂（丝氨酸蛋白酶抑制因子），可抑制多种血液凝固丝氨酸蛋白酶，包括凝血酶。
使用55个丙氨酸扫描重组凝血酶突变体来确定对于PCI在有或没有辅因子肝素和血栓调节蛋白（TM）情况下抑制作用重要的凝血酶残基，并与典型的丝氨酸蛋白酶抑制因子AT进行比较。
无论有无辅因子，PCI抑制凝血酶都需要活性位点（Tyr50和Glu202）以及钠结合位点（Glu229和Arg233）周围的残基。外位点2残基（Arg89、Arg93、Glu94、Arg98、Arg245、Arg248和Gln251）对于肝素加速PCI抑制凝血酶至关重要。外位点1残基（尤其是Lys65和Tyr71）是增强PCI对凝血酶-TM抑制作用所必需的。有趣的是，我们还发现PCI使凝血酶抑制率提高约150倍并不需要TM的硫酸软骨素部分。使用上述对肝素催化的PCI-凝血酶抑制反应至关重要的凝血酶外位点2突变体，我们发现PCI对凝血酶-TM的抑制率没有变化。
总体而言，这些结果表明：（i）类似的凝血酶外位点2残基对于PCI和AT的肝素催化抑制作用至关重要；（ii）PCI和AT在凝血酶-TM抑制特性方面存在差异；（iii）在调节凝血酶-TM活性方面，PCI相对于AT具有明显优势。
