光晕荧光纤维蛋白溶解试验：一种新的定量分析方法来评估已建立的血浆凝块的纤维蛋白溶解

IF 3.4 3区医学 Q2 HEMATOLOGY

Research and Practice in Thrombosis and Haemostasis Pub Date : 2025-05-01 DOI:10.1016/j.rpth.2025.102874

Zikou Liu , Orr Zaacks , Be’eri Niego , Robert L. Medcalf

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引用次数: 0

摘要

背景：纤溶对于溶解血凝块和维持止血至关重要。这个过程主要是由组织型纤溶酶原激活剂介导的，它将纤溶酶原转化为纤溶酶，从而分解纤维蛋白凝块。传统的纤溶试验通常测量纤溶酶的产生，而不直接评估纤维蛋白降解，而血栓弹性成像经常忽略凝块成熟，这显着影响纤维蛋白溶解抵抗。目的为了解决这些局限性，我们提出了一种新的定量分析凝块纤维蛋白溶解的方法，称为晕荧光纤维蛋白溶解（HoFF）试验。方法HoFF试验采用荧光基团偶联纤维蛋白原形成晕状血浆凝块。用组织型纤溶酶原激活剂或其变体tenecteplase诱导纤维蛋白溶解，并通过微孔板读取器实时荧光检测监测凝块分解。分析荧光信号，计算纤溶指数，表明纤溶能力。它的特异性纤溶超过纤溶酶的产生，与使用纤溶酶底物的传统纤溶测定相比得到了验证。结果荧光标记纤维蛋白原是一种可靠的纤维蛋白降解标志物。霍夫试验显示纤溶酶指数和纤溶酶原激活剂浓度之间有很强的线性相关性，具有很强的重复性。它还有效地评估了tenectepase诱导的纤维蛋白溶解，并证明了凝块类型的通用性，包括小鼠血浆和人类全血模型。此外，该试验区分了纤溶和纤溶酶的产生，证明了氨甲环酸抑制的不同效果。结论HoFF试验是一种灵敏、可靠、高通量的定量评价人、鼠血浆及全血凝块纤维蛋白溶解的方法。

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Halo fluorescence fibrinolysis test: a novel quantitative assay to evaluate fibrinolysis on established plasma clots

Background

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.

Objectives

To address these limitations, we present a novel quantitative assay for analyzing fibrinolysis on established clots, termed the halo fluorescence fibrinolysis (HoFF) test.

Methods

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.

Results

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.

Conclusion

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.

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