B. de Laat, PhD, Synapse bv, Oxfordlaan 70, 6229 EV Maastricht, The Netherlands, Tel.: +31 43 3885895, Fax: +31 43 3884159, E-mail:
Thromb Haemost. 2014 Aug;112(2):271-5. doi: 10.1160/TH13-11-0910. Epub 2014 Apr 17.
The Calibrated Automated Thrombogram (CAT) assay that measures thrombin generation (TG) in platelet-poor and -rich plasma, is increasingly being recognised as a more sensitive tool to determine the overall function of the haemostatic system. We developed a method enabling the measurement of TG in a small aliquot of blood. The objective was to validate this assay in mouse blood and to examine the rate and extent of TG in a mouse model of premature aging. TG was assayed in blood from 20- to 28-week-old brain and muscle ARNT-like protein-1 (Bmal1)-deficient (knockout, KO) mice and wild-type (WT) littermates. Bmal1-KO mice are known to display symptoms of premature aging. TG was initiated by adding calcium, tissue factor and a thrombin specific substrate. After TG, the samples were prepared for scanning electron microscopy (SEM). The intra-assay variations (%) in mouse blood of the endogenous thrombin potential (ETP), peak height, lag time, time-to-peak and velocity index were 10% or less (n=24). We found that Bmal1-KO mice have a significantly (p<0.001) higher ETP (437 ± 7 nM.min; mean ± SD, n=7) when compared with WT mice (ETP=220 ± 45 nM.min; mean ± SD, n=5). The peak heights also differed significantly (p=0.027). By applying SEM we found that Bmal1 deficient mice display a denser fibrin network with smaller pores compared to WT mice. In conclusion, the whole blood TG assay in mice revealed to be reproducible. As a proof-of-principle we have shown that the whole blood TG assay is capable of detecting a prothrombotic phenotype in Bmal1-KO mice.
校准的自动化血栓图（CAT）检测血小板贫浆和富浆中的凝血酶生成（TG），被越来越多地认为是一种更敏感的工具，用于确定止血系统的整体功能。我们开发了一种在小体积血液中测量 TG 的方法。目的是验证该方法在小鼠血液中的适用性，并在早老症小鼠模型中检查 TG 的速率和程度。从 20 至 28 周龄的大脑和肌肉 ARNT 样蛋白-1（Bmal1）缺陷（敲除，KO）小鼠和野生型（WT）同窝仔鼠中测定 TG。已知 Bmal1-KO 小鼠表现出早老症的症状。通过添加钙、组织因子和凝血酶特异性底物来启动 TG。TG 后，将样品准备用于扫描电子显微镜（SEM）检查。内源性凝血酶潜能（ETP）、峰高、滞后时间、达峰时间和速度指数在小鼠血液中的批内变异（%）为 10%或更低（n=24）。我们发现 Bmal1-KO 小鼠的 ETP（437±7 nM.min；平均值±SD，n=7）明显高于 WT 小鼠（ETP=220±45 nM.min；平均值±SD，n=5）（p<0.001）。峰高也有显著差异（p=0.027）。通过 SEM 分析，我们发现 Bmal1 缺陷小鼠的纤维蛋白网络比 WT 小鼠更密集，小孔更小。总之，小鼠全血 TG 检测重复性好。作为原理验证，我们已经表明，全血 TG 检测能够检测到 Bmal1-KO 小鼠的血栓形成表型。
