Liu Zikou, Zaacks Orr, Niego Be'eri, Medcalf Robert L
Molecular Neurotrauma and Haemostasis Laboratory, Australian Centre of Blood Diseases, School of Translational Medicine, Monash University, Melbourne, Victoria, Australia.
NanoBiotechnology Laboratory, Australian Centre for Blood Diseases, School of Translational Medicine, Monash University, Melbourne, Victoria, Australia.
Res Pract Thromb Haemost. 2025 Apr 27;9(4):102874. doi: 10.1016/j.rpth.2025.102874. eCollection 2025 May.
Fibrinolysis is essential for dissolving blood clots and maintaining hemostasis. This process is primarily mediated by tissue-type plasminogen activator, which converts plasminogen into plasmin, thereby breaking down fibrin clots. Traditional amidolytic assays often measure plasmin generation without directly assessing fibrin degradation, while thromboelastography frequently overlooks clot maturation, which significantly influences fibrinolysis resistance.
To address these limitations, we present a novel quantitative assay for analyzing fibrinolysis on established clots, termed the halo fluorescence fibrinolysis (HoFF) test.
The HoFF test used fluorophore-conjugated fibrinogen to form halo-shaped plasma clots. Fibrinolysis was induced with tissue-type plasminogen activator or its variant tenecteplase, and clot breakdown was monitored via real-time fluorescence detection by a microplate reader. The fluorescence signal was analyzed to calculate a fibrinolysis index, indicating fibrinolytic capacity. Its specificity for fibrinolysis over plasmin generation was validated against traditional amidolytic assays using a plasmin substrate.
Fluorescence-labeled fibrinogen was confirmed as a reliable marker of fibrin degradation. The HoFF test exhibited strong linear correlations between the fibrinolysis index and plasminogen activator concentrations, with robust reproducibility. It also effectively evaluated tenecteplase-induced fibrinolysis and demonstrated versatility across clot types, including mouse plasma and human whole-blood models. Furthermore, the test distinguished fibrinolysis from plasmin generation, demonstrated by the differential effects of tranexamic acid inhibition.
The HoFF test offers a sensitive, reliable, and high-throughput tool for quantitatively evaluating fibrinolysis on established human and mouse plasma and whole blood clots.
纤维蛋白溶解对于溶解血栓和维持止血至关重要。这一过程主要由组织型纤溶酶原激活物介导，该激活物将纤溶酶原转化为纤溶酶，从而分解纤维蛋白凝块。传统的酰胺水解测定法通常测量纤溶酶的生成，而不直接评估纤维蛋白的降解，而血栓弹力图常常忽略凝块成熟，这对纤维蛋白溶解抵抗有显著影响。
为解决这些局限性，我们提出了一种用于分析已形成凝块上纤维蛋白溶解的新型定量测定法，称为光晕荧光纤维蛋白溶解（HoFF）试验。
HoFF试验使用荧光团偶联的纤维蛋白原形成光晕状血浆凝块。用组织型纤溶酶原激活物或其变体替奈普酶诱导纤维蛋白溶解，并通过酶标仪进行实时荧光检测来监测凝块分解。分析荧光信号以计算纤维蛋白溶解指数，该指数表明纤维蛋白溶解能力。使用纤溶酶底物，通过与传统酰胺水解测定法对比，验证了其对纤维蛋白溶解而非纤溶酶生成的特异性。
荧光标记的纤维蛋白原被确认为纤维蛋白降解可靠标记物。HoFF试验在纤维蛋白溶解指数与纤溶酶原激活物浓度之间表现出强线性相关性，且具有良好的可重复性。它还有效评估了替奈普酶诱导的纤维蛋白溶解，并在包括小鼠血浆和人全血模型在内的不同凝块类型中显示出通用性。此外，通过氨甲环酸抑制的不同作用证明，该试验能区分纤维蛋白溶解和纤溶酶生成。
HoFF试验为定量评估已形成的人及小鼠血浆和全血凝块上的纤维蛋白溶解提供了一种灵敏、可靠且高通量的工具。
