Evaluation and clinical application of a new method for measuring activity of von Willebrand factor-cleaving metalloprotease (ADAMTS13)
Evaluation and clinical application of a new method for measuring activity of von Willebrand factor-cleaving metalloprotease (ADAMTS13)
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DOI：
10.1007/s00277-002-0502-3
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发表时间：
2002-08-01
影响因子：
3.5
通讯作者：
Scharrer, I
中科院分区：
文献类型：
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作者：
Böhm, M;Vigh, T;Scharrer, I
血栓性血小板减少性紫癜（TTP）与获得性或先天性血浆血管性血友病因子切割蛋白酶（VWFcp）缺乏有关。基于对先天性TTP家系的部分氨基酸序列和全基因组连锁分析，VWFcp最近被确定为ADAMTS家族的新成员，并命名为ADAMTS13。基于VWF多聚体大小与瑞斯托霉素辅因子活性（VWF:RCo）呈正相关关系，建立了一种新的、快速、简便的测定VWFcp活性的方法。在用低离子Tris缓冲液稀释血浆和用氯化钡激活蛋白酶后，在尿素存在下消化VWF浓缩物。随后，评估样品的残余VWF:RCo，并用于计算样品的VWFcp活性。通过使用基于rco的测定法和原始免疫印迹测定法对282份血浆样品进行VWFcp活性评估，验证了新技术的准确性。该方法重复性好，正常样品的内、间变异系数较低（分别为2.8%和7.5%，异常样品的内、间变异系数分别为8.7%和12.9%）。此外，通过测量14例22次急性TTP发作以及其他血栓性、血小板减少性或溶血性疾病患者的VWFcp，说明了新方法的临床应用。严重VWFcp缺乏仅限于急性、经典TTP患者。大多数低效价抑制剂患者对血浆交换治疗的反应是VWFcp活性增加，而一些高效价抑制剂患者尽管临床缓解，但VWFcp缺乏仍然存在。
Thrombotic thrombocytopenic purpura (TTP) is associated with acquired or congenital deficiency of a plasma von Willebrand factor-cleaving protease (VWFcp). Based on partial amino acid sequence and genomewide linkage analysis of pedigrees with congenital TTP, VWFcp was recently identified as a new member of the ADAMTS family and designated ADAMTS13. We developed a new, rapid, and simple method for measuring VWFcp activity based on the positive correlation between VWF multimeric size and Ristocetin cofactor activity (VWF:RCo). After dilution of plasma with low ionic Tris buffer and activation of the protease with barium chloride, a VWF concentrate is digested in the presence of urea. Subsequently, the residual VWF:RCo of the samples is assessed and used to calculate the VWFcp activity of the samples. The accuracy of the new technique is verified by estimating VWFcp activity for 282 plasma samples with the RCo-based assay and the original immunoblotting assay. The method is reproducible as shown by low intra-and interassay coefficients of variation (2.8% and 7.5% for normal samples, respectively, and 8.7% and 12.9% for abnormal samples, respectively). Furthermore, the clinical application of the new method is illustrated by measuring VWFcp of 14 patients with 22 episodes of acute TTP as well as other thrombotic, thrombocytopenic, or hemolytic disorders. Severe VWFcp deficiency was restricted to patients with acute, classic TTP. The majority of patients with low titer inhibitor respond to plasma exchange treatment with increase of VWFcp activity, whereas VWFcp deficiency persists in some patients with high titer inhibitor despite clinical remission.