Tilley Derek, Levit Irina, Samis John A
Applied Bioscience Program, University of Ontario Institute of Technology, Ontario.
J Vis Exp. 2012 Sep 9(67):3822. doi: 10.3791/3822.
In response to injury, blood coagulation is activated and results in generation of the clotting protease, thrombin. Thrombin cleaves fibrinogen to fibrin which forms an insoluble clot that stops hemorrhage. Factor V (FV) in its activated form, FVa, is a critical cofactor for the protease FXa and accelerator of thrombin generation during fibrin clot formation as part of prothrombinase (1, 2). Manual FV assays have been described (3, 4), but they are time consuming and subjective. Automated FV assays have been reported (5-7), but the analyzer and reagents are expensive and generally provide only the clot time, not the rate and extent of fibrin formation. The microplate platform is preferred for measuring enzyme-catalyzed events because of convenience, time, cost, small volume, continuous monitoring, and high-throughput (8, 9). Microplate assays have been reported for clot lysis (10), platelet aggregation (11), and coagulation Factors (12), but not for FV activity in human plasma. The goal of the method was to develop a microplate assay that measures FV activity during fibrin formation in human plasma. This novel microplate method outlines a simple, inexpensive, and rapid assay of FV activity in human plasma. The assay utilizes a kinetic microplate reader to monitor the absorbance change at 405 nm during fibrin formation in human plasma (Figure 1) (13). The assay accurately measures the time, initial rate, and extent of fibrin clot formation. It requires only μl quantities of plasma, is complete in 6 min, has high-throughput, is sensitive to 24-80 pM FV, and measures the amount of unintentionally activated (1-stage activity) and thrombin-activated FV (2-stage activity) to obtain a complete assessment of its total functional activity (2-stage activity - 1-stage activity). Disseminated intravascular coagulation (DIC) is an acquired coagulopathy that most often develops from pre-existing infections (14). DIC is associated with a poor prognosis and increases mortality above the pre-existing pathology (15). The assay was used to show that in 9 patients with DIC, the FV 1-stage, 2-stage, and total activities were decreased, on average, by 54%, 44%, and 42%, respectively, compared with normal pooled human reference plasma (NHP). The FV microplate assay is easily adaptable to measure the activity of any coagulation factor. This assay will increase our understanding of FV biochemistry through a more accurate and complete measurement of its activity in research and clinical settings. This information will positively impact healthcare environments through earlier diagnosis and development of more effective treatments for coagulation disorders, such as DIC.
作为对损伤的反应，血液凝固被激活，导致凝血蛋白酶即凝血酶的产生。凝血酶将纤维蛋白原裂解为纤维蛋白，后者形成不溶性凝块以阻止出血。活化形式的因子V（FV），即FVa，是蛋白酶FXa的关键辅因子，也是纤维蛋白凝块形成过程中凝血酶生成的促进因子，是凝血酶原酶的一部分（1,2）。已有手动FV检测方法的描述（3,4），但它们耗时且主观。也有自动FV检测方法的报道（5 - 7），但分析仪和试剂昂贵，且通常仅提供凝血时间，而非纤维蛋白形成的速率和程度。由于方便、省时、成本低、体积小、可连续监测以及高通量，微孔板平台更适合用于测量酶催化反应（8,9）。已有关于微孔板检测凝块溶解（10）、血小板聚集（11）和凝血因子（12）的报道，但尚未见用于检测人血浆中FV活性的报道。该方法的目标是开发一种微孔板检测方法，用于测量人血浆中纤维蛋白形成过程中的FV活性。这种新颖的微孔板方法概述了一种简单、廉价且快速的人血浆FV活性检测方法。该检测利用动力学微孔板读数器监测人血浆中纤维蛋白形成过程中405 nm处的吸光度变化（图1）（13）。该检测可准确测量纤维蛋白凝块形成的时间、初始速率和程度。它仅需微量血浆，6分钟内即可完成，具有高通量，对24 - 80 pM的FV敏感，可测量无意激活的（1期活性）和凝血酶激活的FV（2期活性），以全面评估其总功能活性（2期活性 - 1期活性）。弥散性血管内凝血（DIC）是一种获得性凝血病，最常由先前存在的感染发展而来（14）。DIC与预后不良相关，且会增加原有疾病的死亡率（15）。该检测用于表明，与正常混合人参考血浆（NHP）相比，9例DIC患者的FV 1期、2期和总活性平均分别降低了54%、44%和42%。FV微孔板检测方法易于调整以测量任何凝血因子的活性。通过在研究和临床环境中更准确、完整地测量FV活性，该检测将增进我们对FV生物化学的理解。这些信息将通过更早的诊断和开发针对凝血障碍（如DIC）的更有效治疗方法，对医疗环境产生积极影响。
