Spannagl M, Bichler H, Lill H, Schramm W
Department of Medicine, Ludwig-Maximilians University, Munich, FRG.
Haemostasis. 1991;21 Suppl 1:36-40. doi: 10.1159/000216261.
Recombinant hirudin, a potent and specific thrombin inhibitor, is considered for anticoagulant therapy. Therefore we developed a fast and sensitive chromogenic assay for its determination in plasma. Samples can be assayed directly if aprotinin, Polybrene and urea are added to the reagent mixture. The influence of progressive inhibitors is excluded by a short incubation time. The samples (20 microliters) are diluted with 1 ml reagent mixture (0.2 M Tris, 0.025 M NaCl, pH 8.1, containing 0.833 M urea, 0.7 trypsin inhibitory units/ml aprotinin, 100 ng/ml Polybrene and 0.31 NIH units/ml bovine thrombin). After an incubation time of 1 min, 100 microliters Chromoyzm TH (Tos-Gly-Pro-Arg-pNA, 1.9 mM) is added. delta absorbance/min is linear at least up to 3 min. The calibration curve is linear up to at least 800 ng hirudin/ml plasma. The inter- and intraassay coefficient of variation in hirudin spiked normal plasma is below 5%. The detection limit is at 25 ng hirudin/ml. In plasma samples obtained from healthy subjects under hirudin therapy, a good correlation was found to the activated partial thromboplastin time (r = 0.89). In conclusion, we describe a fast and simple chromogenic substrate test to assay hirudin in plasma. Under assay conditions, the influence of endogenous thrombin inhibitors can be neglected.
重组水蛭素是一种强效且特异的凝血酶抑制剂，正被考虑用于抗凝治疗。因此，我们开发了一种快速灵敏的显色测定法来测定血浆中的重组水蛭素。如果在试剂混合物中加入抑肽酶、聚凝胺和尿素，样品可直接进行测定。通过短时间孵育可排除渐进性抑制剂的影响。将样品（20微升）用1毫升试剂混合物（0.2M Tris、0.025M NaCl，pH 8.1，含有0.833M尿素、0.7胰蛋白酶抑制单位/毫升抑肽酶、100纳克/毫升聚凝胺和0.31 NIH单位/毫升牛凝血酶）稀释。孵育1分钟后，加入100微升Chromoyzm TH（Tos-Gly-Pro-Arg-pNA，1.9 mM）。至少在3分钟内，吸光度变化率/分钟呈线性。校准曲线至少在800纳克水蛭素/毫升血浆范围内呈线性。在添加了水蛭素的正常血浆中，批间和批内变异系数均低于5%。检测限为25纳克水蛭素/毫升。在接受水蛭素治疗的健康受试者的血浆样本中，发现其与活化部分凝血活酶时间有良好的相关性（r = 0.89）。总之，我们描述了一种快速简便的显色底物试验来测定血浆中的水蛭素。在测定条件下，内源性凝血酶抑制剂的影响可忽略不计。
