Development of new analytical method for antithrombin resistance

我们报道了一种变异凝血酶原（p.a g596leu）在遗传性血栓患者中传递抗凝血酶耐药性。为了检测血浆中抗凝血酶的耐药性，我们设计了一个实验室测试来分析使用抗凝血酶的凝血酶失活动力学。材料和方法：用凝血酶原激活剂成分（磷脂、CaCl2和蛇毒）孵育后，在肝素存在或不存在的情况下，用过量的抗凝血酶处理不同时间段的样品。随后，加入h - d - phep - pip - Arg-p-nitoranilide，在405 nm处测量吸光度/min （ΔA/min）的变化。结果与结论：抗凝血酶和肝素灭活5 min后，重组突变型凝血酶原的相对残留凝血酶活性（40%）高于野生型（0.5%）。此外，在不使用肝素30min后，重组突变型凝血酶原的相对残留凝血酶活性（99%）高于野生型（20%），表明该方法能够检测变异596Leu凝血酶原的抗凝血酶耐药性。对于重组凝血酶原的重组血浆，加肝素或不加肝素的检测表明，突变型血浆的相对残留凝血酶活性高于野生型血浆。对于含有596Leu凝血酶原的华法化血浆，加肝素或不加肝素的测定表明，尽管初始凝血酶活性较低，但与正常血浆相比，最终的相对残留凝血酶活性较高。总之，我们设计了一种通过分析凝血酶失活动力学来检测血浆中抗凝血酶耐药性的实验室试验。该试验可用于检测血浆中抗凝血酶耐药性，甚至在华法林化患者中也是如此。

Introduction: We have reported a variant prothrombin (p.Arg596Leu) conveying antithrombin resistance in a patient with hereditary thrombosis. To detect antithrombin resistance in plasma, we devised a laboratory test analyzing kinetics of thrombin inactivation using antithrombin. Materials and Methods: After incubation with prothrombin activator components (phospholipids, CaCl2, and snake venom), samples were treated with excess antithrombin in the presence or absence of heparin for various time periods. Subsequently, H-D-Phe-Pip- Arg-p-nitoranilide was added and changes in absorbance/min (ΔA/min) were measured at 405 nm. Results and Conclusions: After inactivation for 5 min using antithrombin and heparin, relative residual thrombin activity of recombinant mutant prothrombin (40%) was higher than that of wild type (0.5%). In addition, after 30 min without heparin, relative residual thrombin activity of recombinant mutant prothrombin (99%) was higher than that of wild-type (20%), indicating that this assay was able to detect antithrombin resistance of the variant 596Leu prothrombin. For reconstituted plasmas with recombinant prothrombins, the assay with or without heparin showed that relative residual thrombin activity of mutant plasma was higher than that of wild-type plasma. For the warfarinized plasma containing 596Leu prothrombin, the assay with or without heparin demonstrated that final relative residual thrombin activity was high compared with that for normal pooled plasma, although its initial thrombin activity was low. In conclusion, we devised a laboratory test detecting antithrombin resistance in plasma by analyzing kinetics of thrombin inactivation using antithrombin. This assay may be useful to detect antithrombin resistance in plasma, even in warfarinized patients.