Hoekema L, Rosing J, Tans G
Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, The Netherlands.
Thromb Haemost. 2000 Dec;84(6):1066-71.
Blood coagulation factor V (FV) circulates in the blood in two forms designated FV1 and FV2. In model systems containing purified proteins FV1 appears to be more thrombogenic than FV2. Recently, we reported that in plasma from carriers of the R2 haplotype, a polymorphism which encodes several amino acid changes in FV and which is associated with an increased risk of thrombosis, the FV1/FV2 ratio is shifted in favor of the more thrombogenic form FV1. Here we describe in detail the assay that enables quantification of the plasma levels of FV1 and FV2. FV present in highly diluted plasma samples was activated with thrombin and the FVa generated was subsequently quantified in two prothrombinase-based assay systems. In the first assay, which is performed at saturating amounts of FXa and phospholipid vesicles with a high mole fraction phosphatidylserine, FVa1 and FVa2 express the same cofactor activity in prothrombin activation. Hence, this assay quantifies the total FV level (FV1 + FV2) present in plasma. In the second assay, which is performed at suboptimal amounts of FXa and phospholipid vesicles with a low mole fraction phosphatidylserine, FVa2 has approximately an 8-fold higher cofactor activity than FVa1. Therefore, the response in this assay depends on the relative amounts of FV1 and FV2 in the plasma sample. Calibration curves made with samples containing known concentrations of purified FVa1 and FVa2 subsequently allowed calculation of the amounts of FV1 and FV2 present in plasma.
血液凝固因子V（FV）以两种形式存在于血液中，分别称为FV1和FV2。在含有纯化蛋白质的模型系统中，FV1似乎比FV2更具血栓形成性。最近，我们报道在R2单倍型携带者的血浆中，一种编码FV中多个氨基酸变化且与血栓形成风险增加相关的多态性，FV1/FV2比值向更具血栓形成性的FV1形式偏移。在此，我们详细描述了一种能够定量血浆中FV1和FV2水平的检测方法。用凝血酶激活高度稀释血浆样本中存在的FV，随后在两个基于凝血酶原酶的检测系统中对产生的FVa进行定量。在第一个检测中，在FXa和具有高摩尔分数磷脂酰丝氨酸的磷脂囊泡饱和量下进行，FVa1和FVa2在凝血酶原激活中表现出相同的辅因子活性。因此，该检测定量血浆中存在的总FV水平（FV1 + FV2）。在第二个检测中，在FXa和具有低摩尔分数磷脂酰丝氨酸的磷脂囊泡次优量下进行，FVa2的辅因子活性比FVa1高约8倍。因此，该检测中的反应取决于血浆样本中FV1和FV2的相对量。随后用含有已知浓度纯化FVa1和FVa2的样本制作校准曲线，从而能够计算出血浆中FV1和FV2的量。
