Gerritsen H E, Turecek P L, Schwarz H P, Lämmle B, Furlan M
Central Hematology Laboratory, Inselspital, University Hospital, Bern, Switzerland.
Thromb Haemost. 1999 Nov;82(5):1386-9.
Patients with thrombotic thrombocytopenic purpura (TTP) have a deficiency of von Willebrand factor (vWF)-cleaving protease, whereas patients with hemolytic-uremic syndrome (HUS) show normal activity of this protease. Present methods for assaying vWF-cleaving protease by immunoblotting are time-intensive and cumbersome. We therefore developed a new functional assay based on the preferential binding of high-molecular-weight forms of vWF to collagen. In this assay, the diluted plasma sample to be tested is added to normal human plasma in which protease activity had been abolished. The vWF present in the protease-depleted plasma is digested by the vWF-cleaving protease in the test plasma. The proteolytic degradation leads to low-molecular-weight forms of vWF, which show impaired binding to microtiter plates coated with human collagen type III. The collagen-bound vWF is quantified using a peroxidase-conjugated rabbit antibody against human vWF. The values of vWF-cleaving protease activity in tested plasma samples are read from a calibration curve achieved by incubating the vWF-substrate with dilutions of a normal human plasma pool (NHP). Testing of plasma from patients with TTP and HUS showed that the assay can be used to distinguish between these two syndromes. The presence of an inhibitor can be detected by carrying out the test after incubation of NHP with the patient plasma sample, thus enabling differentiation of patients with familial TTP from those with nonfamilial TTP.
血栓性血小板减少性紫癜(TTP)患者缺乏血管性血友病因子(vWF)裂解蛋白酶,而溶血尿毒综合征(HUS)患者该蛋白酶活性正常。目前通过免疫印迹法检测vWF裂解蛋白酶的方法耗时且繁琐。因此,我们基于高分子量形式的vWF与胶原蛋白的优先结合开发了一种新的功能检测方法。在该检测中,将待检测的稀释血浆样本加入蛋白酶活性已被消除的正常人血浆中。蛋白酶缺陷血浆中存在的vWF被检测血浆中的vWF裂解蛋白酶消化。蛋白水解降解导致低分子量形式的vWF,其与包被有人类III型胶原蛋白的微量滴定板的结合受损。使用抗人vWF的过氧化物酶偶联兔抗体对结合在胶原蛋白上的vWF进行定量。通过将vWF底物与正常人血浆库(NHP)的稀释液孵育得到校准曲线,从该曲线上读取检测血浆样本中vWF裂解蛋白酶活性的值。对TTP和HUS患者的血浆检测表明,该检测方法可用于区分这两种综合征。通过在NHP与患者血浆样本孵育后进行检测,可以检测到抑制剂的存在,从而能够区分家族性TTP患者和非家族性TTP患者。