Development of new analytical method for antithrombin resistance

引言：我们报告了一个变异的凝血酶原（p.Arg596Leu）在遗传性血栓形成患者中传递抗凝血酶抵抗。为了检测血浆中的抗凝血酶抗性，我们设计了一种使用抗凝血酶分析凝血酶失活动力学的实验室测试。材料与方法：与凝血酶原激活剂组分（磷脂、CaCl 2和蛇毒）孵育后，在存在或不存在肝素的情况下，用过量抗凝血酶处理样品不同时间段。随后，加入H-D-Phe-Pip- Arg-p-硝基苯胺，并在405 nm处测量吸光度/min的变化（ΔA/min）。结果和结论：经抗凝血酶和肝素灭活5 min后，重组突变凝血酶原的相对残留凝血酶活性（40%）高于野生型（0.5%）。此外，在没有肝素的情况下30分钟后，重组突变体凝血酶原的相对残余凝血酶活性（99%）高于野生型的相对残余凝血酶活性（20%），表明该测定能够检测变体596 Leu凝血酶原的抗凝血酶抗性。对于重组凝血酶原的重建血浆，有或没有肝素的测定表明，相对残留凝血酶活性的突变体血浆高于野生型血浆。对于含有596 Leu凝血酶原的华法林化血浆，有或没有肝素的测定表明，与正常混合血浆相比，最终相对残留凝血酶活性较高，尽管其初始凝血酶活性较低。总之，我们设计了一个实验室测试检测抗凝血酶抵抗血浆中的凝血酶灭活动力学分析使用抗凝血酶。该测定可用于检测血浆中的抗凝血酶抗性，即使在华法林化的患者中。

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.