Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, Missouri, USA.
Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, Missouri, USA.
J Thromb Haemost. 2023 Jul;21(7):1769-1778. doi: 10.1016/j.jtha.2023.03.008. Epub 2023 Mar 15.
Current assays that monitor thrombin generation in plasma rely on fluorogenic substrates to follow the kinetics of zymogen activation, which may be complicated by substrate cleavage from other proteases. In addition, these assays depend on activation following cleavage at the prothrombin R320 site and fail to report the cleavage at the alternative R271 site, leading to the shedding of the auxiliary Gla and kringle domains of prothrombin.
To develop a plasma assay that directly monitors prothrombin activation independent of fluorogenic substrate hydrolysis.
Cleavage at the R271 site of prothrombin is monitored through loss of Förster resonance energy transfer in plasma coagulated along the extrinsic or intrinsic pathway.
The availability of factor (F)V in plasma strongly influences the rate of prothrombin activation. The rate of thrombin formation is equally perturbed in FV or prothrombin-depleted plasma, implicating that the thrombin-catalyzed feedback reactions that amplify the coagulation response play an important role in generating sufficient amounts of FVa required for the assembly of prothrombinase. Congenital deficiencies in FVIII and FIX significantly slow down cleavage at R271 in plasma coagulated along the extrinsic and intrinsic pathways. Prothrombin activation in FXI-deficient plasma is only perturbed when coagulation is triggered along the intrinsic pathway.
The Förster resonance energy transfer assay enables direct monitoring of prothrombin activation through cleavage at R271 without the need for fluorogenic substrates. The assay is sensitive enough to assess how deficiencies in coagulation factors affect thrombin formation.
目前监测血浆中凝血酶生成的检测方法依赖于荧光底物来跟踪酶原激活的动力学，这可能会因其他蛋白酶对底物的裂解而变得复杂。此外，这些检测方法依赖于在凝血酶原 R320 位点切割后的激活，并且无法报告在替代 R271 位点的切割，导致凝血酶原辅助 Gla 和 Kringle 结构域的脱落。
开发一种不依赖于荧光底物水解即可直接监测凝血酶原激活的血浆检测方法。
通过沿外源性或内源性途径凝固的血浆中 Förster 共振能量转移的损失来监测凝血酶原在 R271 位点的切割。
血浆中 FV 因子的可用性强烈影响凝血酶原的激活速率。FV 或缺乏凝血酶原的血浆中血栓形成酶的形成速率同样受到干扰，这表明放大凝血反应的血栓形成酶催化的反馈反应在产生组装凝血酶原酶所需的足够量的 FVa 方面起着重要作用。FVIII 和 FIX 的先天性缺乏显著减缓了沿外源性和内源性途径凝固的血浆中 R271 的切割。只有当沿内源性途径触发凝血时，FXI 缺乏的血浆中凝血酶原的激活才会受到干扰。
Förster 共振能量转移检测法能够通过 R271 的切割直接监测凝血酶原的激活，而无需使用荧光底物。该检测方法足够灵敏，可以评估凝血因子缺乏如何影响血栓形成酶的形成。
