Wu Chengliang, Stafford Alan R, Fredenburgh James C, Weitz Jeffrey I, Gils Ann, Declerck Paul J, Kim Paul Y
Paul Y. Kim, Thrombosis and Atherosclerosis Research Institute, 237 Barton St. East, Hamilton, Ontario, L8L 2X2, Canada, Tel.: +1 905 521 2100, Fax: +1 905 575 2646, E-mail:
Thromb Haemost. 2017 Jul 26;117(8):1509-1517. doi: 10.1160/TH17-01-0054. Epub 2017 Jun 22.
The thrombin-thrombomodulin (TM) complex activates thrombin-activable fibrinolysis inhibitor (TAFI) more efficiently than thrombin alone. The exosite on TAFI required for its TM-dependent activation by thrombin has not been identified. Based on previous work by us and others, we generated TAFI variants with one or more of residues Lys 42, Lys 43, Lys 44 and Arg 12 within the activation peptide mutated to alanine. Mutation of one, two, or three Lys residues or the Arg residue alone decreased the catalytic efficiency of TAFI activation by thrombin-TM by 2.4-, 3.2-, 4.7-, and 15.0-fold, respectively, and increased the TAFI concentrations required for half-maximal prolongation of clot lysis times (K) by 3-, 4,- 15-, and 24-fold, respectively. Mutation of all four residues decreased the catalytic efficiency of TAFI activation by 45.0-fold, increased the K by 130-fold, and abolished antifibrinolytic activity in a clot lysis assay at physiologic levels of TAFI. Similar trends in the antifibrinolytic activity of the TAFI variants were observed when plasma clots were formed using HUVECs as the source of TM. When thrombin was used as the activator, mutation of all four residues reduced the rate of activation by 1.1-fold compared with wild-type TAFI, suggesting that these mutations only impacted activation kinetics in the presence of TM. Surface plasmon resonance data suggest that mutation of the four residues abrogates TM binding with or without thrombin. Therefore, Lys 42, Lys 43, Lys 44 and Arg 12 are critical for the interaction of TAFI with the thrombin-TM complex, which modulates its antifibrinolytic potential.
凝血酶 - 血栓调节蛋白（TM）复合物比单独的凝血酶更有效地激活凝血酶激活的纤维蛋白溶解抑制剂（TAFI）。尚未确定TAFI上其依赖TM的凝血酶激活所需的外部位点。基于我们和其他人之前的工作，我们生成了激活肽内一个或多个残基赖氨酸42、赖氨酸43、赖氨酸44和精氨酸12突变为丙氨酸的TAFI变体。单个赖氨酸残基、两个赖氨酸残基、三个赖氨酸残基或单独的精氨酸残基发生突变，分别使凝血酶 - TM激活TAFI的催化效率降低2.4倍、3.2倍、4.7倍和15.0倍，并使凝块溶解时间延长至最大值一半所需的TAFI浓度（K）分别增加3倍、4倍、15倍和24倍。所有四个残基发生突变使TAFI激活的催化效率降低45.0倍，使K增加130倍，并在生理水平的TAFI的凝块溶解试验中消除抗纤维蛋白溶解活性。当使用人脐静脉内皮细胞（HUVECs）作为TM来源形成血浆凝块时，观察到TAFI变体抗纤维蛋白溶解活性的类似趋势。当使用凝血酶作为激活剂时，与野生型TAFI相比，所有四个残基发生突变使激活速率降低1.倍，这表明这些突变仅在存在TM的情况下影响激活动力学。表面等离子体共振数据表明，无论有无凝血酶，这四个残基的突变都会消除TM结合。因此，赖氨酸42、赖氨酸43、赖氨酸44和精氨酸12对于TAFI与凝血酶 - TM复合物的相互作用至关重要，而这种相互作用调节其抗纤维蛋白溶解潜力。
