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Inhibition of Intrinsic Thrombin Generation

机译：内在凝血酶生成的抑制

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Background: The contact phase of coagulation is of physiologic/pathophysiologic importance, whenever unphysiologic polynegative substances such as cell fragments (microparticles) get in contact with blood. There are several clinically used inhibitors of intrinsic thrombin generation. Here the inhibitory concentrations 50% (IC50) of these anticoagulants are measured by the highly specifi c thrombin generation assay INCA.Methods: Unfrozen pooled normal citrated plasma in polystyrole tubes was supplemented at 23°C in duplicate with 0–2 IU/ml low molecular weight heparin (dalteparin), 0–2 IU/ml unfractionated heparin, 0–500 KIU/ml aprotinin, or 0–40 mM arginine. 50 μl plasma or 1 IU/ml thrombin standard were pipetted into a polystyrole microtiter plate with fl at bottom. 5 μl SiO2/ CaCl2 - reagent (INCA activator) were added and after 0–30 min incubation at 37°C 100 μl 2.5 M arginine, pH 8.6, were added; arginine inhibits hemostasis activation and depolymerizes generated fi brin within 20 min at 23°C. The in the physiologic 37°C incubation phase generated thrombin was then chromogenically detected. The intra-assay CV values were < 5%.Results and Discussion: The approximate IC50 were 0.01 IU/ml dalteparin, 0.02 IU/ml heparin, 25 KIU/ml aprotinin, and 12 mM arginine. The effi ciency of any anticoagulant on intrinsic thrombin generation should be measured for each individual patient.Abbreviations: IIa, thrombin; ΔA, increase in absorbance; APTT, activated partial thromboplastin time; CRT, coagulation reaction time (at 37°C in water-bath); F-wells, polystyrole microtiter plates with fl at bottom; IC50, inhibitory concentration 50%; INCA, intrinsic coagulation activity assay; IU, international units; KIU, kallikrein inhibiting unis; LMWH, low molecular weight heparin; mA, milli-absorbance units; PSL, pathromtin SL?; RT, room temperature (23°C); U-wells, polystyrole microtiter plates with round bottom.
机译：背景：每当非生理性多阴性物质（例如细胞碎片（微粒））与血液接触时，凝血的接触阶段就具有生理/病理生理重要性。有几种临床上使用的内在凝血酶生成抑制剂。在这里，这些抗凝剂的抑制浓度50％（IC50）通过高特异性凝血酶生成测定INCA进行测量。方法：在23°C下以0–2 IU / ml的浓度在多苯乙烯管中补充未冷冻的合并的正常柠檬酸血浆。低分子量肝素（达肝素），0–2 IU / ml普通肝素，0–500 KIU / ml抑肽酶或0–40 mM精氨酸。将50μl血浆或1 IU / ml凝血酶标准液吸移到底部装有fl的多苯乙烯微量滴定板中。加入5μlSiO2 / CaCl2-试剂（INCA活化剂），并在37°C下孵育0–30分钟后，加入100μl2.5 M精氨酸，pH 8.6;精氨酸可抑制止血活化，并在23°C下20分钟内解聚生成的纤维蛋白。然后在37°C的生理温育阶段，对产生的凝血酶进行显色检测。试验内CV值<5％。结果与讨论：IC50约为0.01 IU / ml达肝素，0.02 IU / ml肝素，25 KIU / ml抑肽酶和12 mM精氨酸。应针对每位患者测量任何抗凝剂对内在凝血酶生成的效率。 ΔA，吸光度增加; APTT，激活部分凝血活酶时间; CRT，凝固反应时间（在37°C水浴中）; F孔，底部有fl的多苯乙烯微量滴定板; IC50，抑制浓度50％; INCA，固有凝血活性测定; IU，国际单位; KIU，激肽释放酶抑制unis; LMWH，低分子量肝素; mA，毫吸光度单位; PSL，pathromtin SL？室温，室温（23℃）; U型孔，带圆底的多苯乙烯微量滴定板。

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