Global measurement of coagulation in plasma from normal and haemophilia dogs using a novel modified thrombin generation test – Demonstrated <i>in vitro</i> and <i>ex vivo</i>

Introduction Canine models of severe haemophilia resemble their human equivalents both regarding clinical bleeding phenotype and response to treatment. Therefore pre-clinical studies in haemophilia dogs have allowed researchers to make valuable translational predictions regarding the potency and efficacy of new anti-haemophilia drugs (AHDs) in humans. To refine in vivo experiments and reduce number of animals, such translational studies are ideally preceded by in vitro prediction of compound efficacy using a plasma based global coagulation method. One such widely used method is the thrombin generation test (TGT). Unfortunately, commercially available TGTs are incapable of distinguishing between normal and haemophilia canine plasma, and therefore in vitro prediction using TGT has so far not been possible in canine plasma material. Aim Establish a modified TGT capable of: 1) distinguishing between normal and haemophilia canine plasma, 2) monitoring correlation between canine plasma levels of coagulation factor VIII (FVIII) and IX (FIX) and thrombin generation, 3) assessing for agreement between compound activity and thrombin generation in ex vivo samples. Methods A modified TGT assay was established where coagulation was triggered using a commercially available activated partial thromboplastin time reagent. Results With the modified TGT a significant difference was observed in thrombin generation between normal and haemophilia canine plasma. A dose dependent thrombin generation was observed when assessing haemophilia A and B plasma spiked with dilution series of FVIII and FIX, respectively. Correlation between FVIII activity and thrombin generation was observed when analyzing samples from haemophilia A dogs dosed with canine FVIII. Limit of detection was 0.1% (v/v) FVIII or FIX. Conclusion A novel modified TGT suitable for monitoring and prediction of replacement therapy efficacy in plasma from haemophilia A and B dogs was established.## 引言 重度血友病犬模型在临床出血表型与治疗应答方面均与人类血友病患者高度相似。因此，针对血友病犬的临床前研究，可为研究者评估新型抗血友病药物（anti-haemophilia drugs, AHDs）在人类中的药效与疗效提供极具价值的转化预测依据。为优化体内（in vivo）实验并减少实验动物使用量，此类转化研究理想情况下应先通过基于血浆的整体凝血法开展化合物疗效的体外（in vitro）预测。其中应用最为广泛的方法之一即为凝血酶生成试验（thrombin generation test, TGT）。遗憾的是，市售凝血酶生成试验无法区分正常犬血浆与血友病犬血浆，因此截至目前，暂无法利用犬血浆通过TGT开展体外预测研究。 ## 研究目的 本研究旨在构建一种改良版凝血酶生成试验，使其能够实现以下目标：1）区分正常犬血浆与血友病犬血浆；2）监测犬血浆中凝血因子VIII（coagulation factor VIII, FVIII）与IX（coagulation factor IX, FIX）水平与凝血酶生成之间的相关性；3）评估离体（ex vivo）样本中化合物活性与凝血酶生成的一致性。 ## 研究方法 本研究构建了一种改良版TGT检测体系，采用市售活化部分凝血活酶时间试剂触发凝血过程。 ## 研究结果 利用该改良版TGT，可在正常犬血浆与血友病犬血浆的凝血酶生成水平间检测到显著差异。分别向A型与B型血友病犬血浆中掺入梯度稀释的FVIII与FIX后，可观察到凝血酶生成呈剂量依赖性变化。对给予犬源FVIII的A型血友病犬样本进行分析时，可观察到FVIII活性与凝血酶生成之间存在相关性。该检测体系的检出限为0.1%（v/v）的FVIII或FIX。 ## 结论 本研究成功构建了一款新型改良版TGT，可用于监测与预测A型及B型血友病犬血浆替代疗法的疗效。