Missense mutations in the gene encoding prothrombin corresponding to Arg596 cause antithrombin resistance and thrombomodulin resistance
Missense mutations in the gene encoding prothrombin corresponding to Arg596 cause antithrombin resistance and thrombomodulin resistance
复制标题
DOI：
10.1160/th16-03-0223
复制
发表时间：
2016-09
影响因子：
6.7
通讯作者：
Y. Takagi;M. Murata;Toshihiro Kozuka;Y. Nakata;Ryo Hasebe;S. Tamura;A. Takagi;T. Matsushita;H. Saito;T. Kojima
中科院分区：
文献类型：
--
作者：
Y. Takagi;M. Murata;Toshihiro Kozuka;Y. Nakata;Ryo Hasebe;S. Tamura;A. Takagi;T. Matsushita;H. Saito;T. Kojima
抗凝血酶（AT）和血栓调节蛋白（TM）在体内天然抗凝过程中起重要作用。最近，我们报道凝血酶原Yukuhashi突变（p.Arg596Leu）与AT和TM耐药相关的血栓形成有关。为了评估与Arg 596密码子中的单碱基取代的其他错义突变相关的AT和TM抗性，我们产生了重组变体（596 Gln、596 Trp、596 Gly和596 Pro），并研究了对AT和TM抗凝功能的影响。除596 Pro外的所有变体的分泌量与野生型相当，但表现出可变的促凝血活性。经AT灭活30 min后，野生型凝血酶的相对残余活性降至15 ± 4.0%，而所有突变体的相对残余活性均保持在80%以上。凝血酶-AT复合物的形成，通过酶联免疫吸附试验测定，减少与所有测试的变体在肝素的存在和不存在。在可溶性TM（sTM）的存在下，野生型凝血酶的相对纤维蛋白原凝血活性下降至16 ± 0.12%，而测试变体的相对纤维蛋白原凝血活性为37%-56%。在表面等离子体共振测定中，错义Arg 596突变降低凝血酶-TM亲和力的程度类似于降低纤维蛋白原凝血抑制。在存在sTM或培养的内皮样细胞的情况下，变体凝血酶以凝血酶-TM亲和力依赖性方式不同地增强APC生成。这些数据表明凝血酶原Arg 596错义突变导致变体凝血酶中的AT和TM抗性，并表明凝血酶原Arg 596对于AT和TM介导的抗凝作用是重要的。
Summary Antithrombin (AT) and thrombomodulin (TM) play important roles in the process of natural anticoagulation in vivo. Recently, we reported that the prothrombin Yukuhashi mutation (p.Arg596Leu) was associated with AT and TM resistance-related thrombophilia. To assess the AT and TM resistances associated with other missense mutations by single base substitution in the Arg596 codon, we generated recombinant variants (596Gln, 596Trp, 596Gly, and 596Pro) and investigated the effects on AT and TM anticoagulant functions. All variants except 596Pro were secreted in amounts comparable to that of the wild-type but exhibited variable procoagulant activities. After a 30-minute inactivation by AT, the relative residual activity of wild-type thrombin decreased to 15 ± 4.0%, in contrast to values of all variants were maintained at above 80%. The thrombin–AT complex formation, as determined by enzyme-linked immunosorbent assay, was reduced with all tested variants in the presence and absence of heparin. In the presence of soluble TM (sTM), the relative fibrinogen clotting activity of wild-type thrombin decreased to 16 ± 0.12%, whereas that of tested variants was 37%–56%. In a surface plasmon resonance assay, missense Arg596 mutations reduced thrombin–TM affinity to an extent similar to the reduction of fibrinogen clotting inhibition. In the presence of sTM or cultured endothelial-like cells, APC generation was enhanced differently by variant thrombins in a thrombin–TM affinity- dependent manner. These data indicate that prothrombin Arg596 missense mutations lead to AT and TM resistance in the variant thrombins and suggest that prothrombin Arg596 is important for AT- and TM- mediated anticoagulation.