Ammit A J, O'Neill C
Human Reproduction Unit, Royal North Shore Hospital of Sydney, New South Wales, Australia.
J Pharmacol Methods. 1991 Aug;26(1):7-21. doi: 10.1016/0160-5402(91)90050-f.
A bioassay for the measurement of platelet-activating factor (PAF) based on the quantification of platelet aggregation was developed. The method used a platelet analyzer in conjunction with a multiwelled micromixing device and whole blood collected from male New Zealand White rabbits. This bioassay can be nonselective and used to quantitate platelet aggregation induced by any activator. The EC50 for platelet-activating factor, arachidonic acid, and adenosine diphosphate were 0.0232, 55, and 10 microM, and at these concentrations the half maximal aggregation response occurred at 7.5, 10.0, and 12.5 min, respectively. The bioassay was capable of the sensitive quantification of platelet aggregation in small volumes (50 microL) of citrated rabbit whole blood, using a short (i.e., 15-min) incubation period. This enabled multiple bioassays to be performed without the need for a large volume of whole blood to be collected from the rabbit. Selective measurement of platelet-activating factor was achieved by adding inhibitors of arachidonic acid- and adenosine diphosphate-dependent pathways of platelet activation, that is, acetylsalicyclic acid and phosphoenolpyruvate/pyruvate kinase, respectively, to the citrated rabbit whole blood immediately before bioassay. These inhibited arachidonic acid- and adenosine diphosphate-induced platelet aggregation, but had no effect on platelet aggregation induced by platelet-activating factor. Platelet-activating factor was selectively inhibited by its receptor antagonist BN 52021. This method for measuring platelet-activating factor was reproducible; at the EC50, the inter- and intrabioassay coefficients of variation were within acceptable limits at 13.17% and 9.75%, respectively.
开发了一种基于血小板聚集定量的血小板活化因子（PAF）生物测定法。该方法使用血小板分析仪结合多孔微混合装置以及从雄性新西兰白兔采集的全血。这种生物测定法可以是非选择性的，用于定量任何激活剂诱导的血小板聚集。血小板活化因子、花生四烯酸和二磷酸腺苷的半数有效浓度（EC50）分别为0.0232、55和10微摩尔，在这些浓度下，最大聚集反应的一半分别在7.5、10.0和12.5分钟出现。该生物测定法能够在短（即15分钟）孵育期内，对小体积（50微升）枸橼酸化兔全血中的血小板聚集进行灵敏定量。这使得无需从兔子采集大量全血就能进行多次生物测定。通过在生物测定前立即向枸橼酸化兔全血中加入血小板活化的花生四烯酸依赖性途径和二磷酸腺苷依赖性途径的抑制剂，即分别加入乙酰水杨酸和磷酸烯醇丙酮酸/丙酮酸激酶，实现了对血小板活化因子的选择性测量。这些抑制剂抑制了花生四烯酸和二磷酸腺苷诱导的血小板聚集，但对血小板活化因子诱导的血小板聚集没有影响。血小板活化因子被其受体拮抗剂BN 52021选择性抑制。这种测量血小板活化因子的方法具有可重复性；在EC50时，生物测定间和生物测定内的变异系数分别在可接受范围内，为13.17%和9.75%。
