Development of new analytical method for antithrombin resistance

抗凝血酶抵抗新分析方法的开发

• 批准号: 22659114
• 负责人: TAKAGI Akira
• 金额: $ 1.98万
• 依托单位: Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Challenging Exploratory Research
• 财政年份: 2010
• 资助国家: 日本
• 起止时间: 2010 至 2012
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-22659114/
• 关键词: 臨床血液学; プロトロンビン変異; アンチトロンビン抵抗性; 病態解析; 変異プロトロンビン; 制御因子抵抗性; 血栓症リスク; 検査法開発; プロトロンビン; 遺伝子変異

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.

前言：我们报道了一种变异型凝血酶原(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抵抗，我们设计了一种实验室测试，分析了使用抗凝血酶灭活凝血酶的动力学。材料和方法：标本与凝血酶原激活剂成分(磷脂、CaCl2和蛇毒)孵育后，在有或没有肝素的情况下进行不同时间的过量抗凝血酶处理。随后，加入H-D-Phe-Pip-Arg-p-Nitoranilde，在405nm处测量吸光度/分钟(ΔA/分钟)的变化。结果与结论：经抗凝血酶和肝素灭活5min后，重组突变型凝血酶原的相对残余凝血酶活性(40%)高于野生型(0.5%)。此外，在无肝素作用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(20%)，表明本方法能够检测596Leu变异型凝血酶原的抗凝血酶抗性。对于含有重组凝血酶原的重组血浆，无论是否加入肝素，突变血浆的相对残存凝血酶活性均高于野生型血浆。对于含有596Leu凝血酶原的华法化血浆，无论有无肝素，其最终的相对残余凝血酶活性都高于正常混合血浆，尽管其初始凝血酶活性较低。总之，我们设计了一种通过分析使用抗凝血酶灭活凝血酶的动力学来检测血浆中抗凝血酶抵抗的实验室测试。这种检测方法可能有助于检测血浆中的抗凝血酶抵抗，即使在华法林患者中也是如此。

项目成果

アンチトロンビン抵抗性を検出するトロンビン不活化動態解析法の開発

开发检测抗凝血酶抵抗的凝血酶失活动力学分析方法

• 发表时间: 2011
• 作者: 高木 明; 宮脇由理; 鈴木敦夫;藤田絢子;牧明日加;奥山恵理子;村田 萌;村手 隆;松下 正; 小嶋哲人;高木明;高木明;Yuhri Miyawaki;高木明
• 通讯作者: 高木明

静脈血栓塞栓症リスク・アンチトロンビン抵抗性とそのスクリーニング検査法

静脉血栓栓塞风险/抗凝血酶抵抗及其筛查试验方法

• 发表时间: 2012
• 作者: Miyawaki Y;Suzuki A;Fujita J;Maki A;Okuyama E;Murata M;Takagi A;Murate T;Kunishima S;Sakai M;Okamoto K;Matsushita T;Naoe T;Saito H;Kojima T;高木明
• 通讯作者: 高木明

A NOVEL PROTHROMBIN GENE MUTATION LEADS TO AN AT-RESISTANT THROMBIN IN A FAMILY WITH INHERITED THROMBOPHILIA

一种新的凝血酶原基因突变导致遗传性血栓形成倾向家族出现凝血酶抗性

• 发表时间: 2011
• 作者: Miyawaki Y;Suzuki A;Fujimori Y;Fujita J;Maki A;Takagi A;Murate T;Sakai M;Okamoto K;Matsushita T;Kojima T
• 通讯作者: Kojima T

Thrombosis from a Prothrombin Mutastion Conveying Antithrombin Resistance

凝血酶原突变导致的血栓形成，导致抗凝血酶抵抗

• 发表时间: 2012
• 期刊: The NEW ENGLAND JOURNAL of MEDICINE
• 作者: Yuhri Miyawaki;Atsuo Sizuki;Junko Fujita;Asuka Maki;Eriko Okuyama;Moe Murata;Akira Takagi;Takashi Murate;Shinji Kunishima;Michio Sakata;Kohji Okamoto;Tadashi Matsushita;Tomoki Naoe;Hidehiko Saito;Tetsuhito Kojima
• 通讯作者: Tetsuhito Kojima

静脈血栓塞栓症リスク・アンチトロンビン抵抗性とそのスクリーニング検査

静脉血栓栓塞风险/抗凝血酶抵抗及其筛查试验

• 发表时间: 2012
• 作者: 高木 明; 宮脇由理; 鈴木敦夫;藤田絢子;牧明日加;奥山恵理子;村田 萌;村手 隆;松下 正; 小嶋哲人
• 通讯作者: 小嶋哲人