Faraday N, Goldschmidt-Clermont P, Dise K, Bray P F
Division of Hematology, Johns Hopkins University School Of Medicine, Baltimore, MD.
J Lab Clin Med. 1994 May;123(5):728-40.
Soluble fibrinogen binding to agonist-stimulated blood platelets is the essential physiologic function of the glycoprotein IIb-IIIa (GPIIb-IIIa) receptor. We describe a method of quantifying this receptor-ligand interaction by using flow cytometry to detect the binding of fluorescein-labeled fibrinogen to activated platelets. Fibrinogen conjugated with fluorescein isothiocyanate (FITC-FGN) was structurally and functionally indistinguishable from native fibrinogen when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, thrombin clottability, and receptor affinity studies. Platelet samples, at a concentration of 2 x 10(7) ml, were incubated with FITC-FGN and activated with adenosine diphosphate (ADP) before cytometric acquisition of fluorescence and scatter data. ADP-induced binding of soluble FITC-FGN to platelet GPIIb-IIIa was specific, time dependent, and saturable. Cytometric analysis of FITC calibration beads allowed generation of standard curves relating bead fluorescence intensity to the number of fluorescein equivalents per bead. With this information, platelet fluorescence intensity was converted into the number of FITC-FGN molecules bound per platelet. Such quantitative analysis of fibrinogen binding yielded a dissociation constant of 2.48 +/- 0.5 x 10(-7) mol/L and a maximum fibrinogen binding capacity of 42, 124 +/- 5, 628 molecules per platelet (mean +/- SEM), which are comparable to published results with radioligand assays. The simplicity, sensitivity, and quantifiability of this method may make it a useful technique for basic and clinical research involving GPIIb-IIIa function.
可溶性纤维蛋白原与激动剂刺激的血小板结合是糖蛋白IIb-IIIa（GPIIb-IIIa）受体的基本生理功能。我们描述了一种通过使用流式细胞术检测荧光素标记的纤维蛋白原与活化血小板的结合来定量这种受体-配体相互作用的方法。当通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、凝血酶凝固性和受体亲和力研究进行分析时，与异硫氰酸荧光素（FITC-FGN）缀合的纤维蛋白原在结构和功能上与天然纤维蛋白原无法区分。将浓度为2×10⁷/ml的血小板样品与FITC-FGN孵育，并用二磷酸腺苷（ADP）活化，然后进行流式细胞术采集荧光和散射数据。ADP诱导的可溶性FITC-FGN与血小板GPIIb-IIIa的结合具有特异性、时间依赖性且可饱和。对FITC校准微珠的流式细胞术分析允许生成将微珠荧光强度与每个微珠的荧光素当量数相关联的标准曲线。利用这些信息，将血小板荧光强度转换为每个血小板结合的FITC-FGN分子数。这种对纤维蛋白原结合的定量分析产生的解离常数为2.48±0.5×10⁻⁷mol/L，每个血小板的最大纤维蛋白原结合能力为42,124±5,628个分子（平均值±标准误），这与放射性配体测定的已发表结果相当。该方法的简单性、敏感性和可量化性可能使其成为涉及GPIIb-IIIa功能的基础和临床研究的有用技术。
