Kokame Koichi, Miyata Toshiyuki
National Cardiovascular Center Research Institute, Suita 565-8565.
Rinsho Byori. 2005 Jul;53(7):639-45.
A plasma glycoprotein, von Willebrand factor (VWF), is essential for normal platelet aggregation. In healthy individuals, the homo-multimeric forms (VWF multimers) are partially cleaved by a plasma metalloprotease, ADAMTS13. Congenital or acquired deficiency of ADAMTS13 activity leads to the accumulation of hyperactive large VWF multimers, inducing a life-threatening disease, thrombotic thrombocytopenic purpura (TTP). As measuring ADAMTS13 activity is important in TTP diagnosis, a number of assay methods have been developed in the past few years. However, the time and skill required for these methods prohibited the progress of clinical usage. Recently, we have developed a fluorescence resonance energy transfer (FRET) assay for ADAMTS13 activity. A synthetic 73-amino-acid peptide, FRETS-VWF73, which is now commercially available, is used as a substrate. Cleavage of this peptide between two modified residues relieves the fluorescence quenching in the intact form. Incubation of FRETS-VWF73 with normal plasma quantitatively increased fluorescence over time, while TTP-patient plasma had little or no effect. The measurement can be achieved within a one-hour period using a 96-well format in commercial plate readers with common filters. The FRET assay will be useful not only for TTP diagnosis but also characterization of thrombotic microangiopathies.
血浆糖蛋白血管性血友病因子（VWF）对于正常血小板聚集至关重要。在健康个体中，同多聚体形式（VWF多聚体）会被血浆金属蛋白酶ADAMTS13部分切割。ADAMTS13活性的先天性或获得性缺乏会导致高活性大VWF多聚体的积累，引发一种危及生命的疾病——血栓性血小板减少性紫癜（TTP）。由于检测ADAMTS13活性在TTP诊断中很重要，在过去几年里已经开发了许多检测方法。然而，这些方法所需的时间和技能阻碍了其临床应用的进展。最近，我们开发了一种用于检测ADAMTS13活性的荧光共振能量转移（FRET）检测法。一种合成的73个氨基酸的肽段FRETS-VWF73，现已商业化，用作底物。该肽段在两个修饰残基之间的切割消除了完整形式中的荧光猝灭。将FRETS-VWF73与正常血浆一起孵育，荧光会随时间定量增加，而TTP患者血浆几乎没有影响或没有影响。使用配备普通滤光片的商用酶标仪，采用96孔板形式，在一小时内即可完成检测。FRET检测法不仅对TTP诊断有用，而且对血栓性微血管病的特征描述也有用。
