Rossi F, Rossi E, Pareti F I, Colli S, Tremoli E, Gallo L
Department of Pharmacologic Sciences, E. Grossi Paletti Center, University of Milan, via Balzaretti 9, 20133 Milan, Italy.
Haematologica. 2001 Feb;86(2):192-8.
Inhibition of soluble fibrinogen binding to activated platelets represents the target of pharmacologic approach with antagonists of the glycoprotein IIb/IIIa (GPIIb/IIIa) complex. In this study we assessed the effects of abciximab, a recombinant chimeric Fab fraction of the antibody against GPIIb/IIIa, on several markers of platelet activation.
The platelet surface expression of GPIIb/IIIa was measured by a flow cytometry technique using a two-color assay. GPIIb/IIIa was detected by FITC-conjugated antibodies in whole blood, either unstimulated or exposed to platelet stimuli. The following antibodies were used: CD41, which recognizes the IIb/IIIa complex both in activated and non-activated conformers, and PAC-1, which is directed toward the activated conformer of GPIIb/IIIa. In addition, the same blood sample was incubated with CD62 antibody to measure P-selectin, as a marker of a-granule degranulation. The effect of abciximab was also assessed by experiments carried out on shear stress-induced platelet aggregation, a test that appears to be a predictor of platelet hemostatic function.
Abciximab inhibited CD41 binding to glycoprotein IIb (GPIIb) in a concentration-dependent manner and also inhibited the binding of PAC-1 to active GPIIb/IIIa. In contrast, membrane-associated P-selectin was significantly increased by the drug, which suggests that blockade of GPIIb/IIIa receptors results in an increased platelet degranulation in response to agonists. Shear stress-induced platelet aggregation was inhibited by abciximab, with a more pronounced effect on blood filtration, which represents an index of platelet aggregate formation.
Our results indicate that GPIIb/IIIa blockade by abciximab is accompanied by an increase of a-granule secretion, suggesting that different mechanisms regulate these aspects of platelet activation. The described flow cytometry technique, that allows the simultaneous in vitro detection of several platelet markers, is a suitable method for assessing the effects of agents which interfere with platelet function.
抑制可溶性纤维蛋白原与活化血小板的结合是使用糖蛋白IIb/IIIa（GPIIb/IIIa）复合物拮抗剂进行药物治疗的靶点。在本研究中，我们评估了阿昔单抗（一种针对GPIIb/IIIa的重组嵌合Fab片段抗体）对几种血小板活化标志物的影响。
采用双色流式细胞术检测GPIIb/IIIa在血小板表面的表达。在全血中，使用异硫氰酸荧光素（FITC）偶联抗体检测未刺激或经血小板刺激后的GPIIb/IIIa。使用了以下抗体：CD41，可识别活化和未活化构象的IIb/IIIa复合物；PAC-1，针对GPIIb/IIIa的活化构象。此外，将同一血样与CD62抗体孵育以测量P-选择素，作为α-颗粒脱颗粒的标志物。还通过剪切应力诱导的血小板聚集实验评估阿昔单抗的作用，该实验似乎是血小板止血功能的预测指标。
阿昔单抗以浓度依赖方式抑制CD41与糖蛋白IIb（GPIIb）的结合，也抑制PAC-1与活性GPIIb/IIIa的结合。相反，该药物使膜相关P-选择素显著增加，这表明GPIIb/IIIa受体的阻断导致血小板对激动剂的脱颗粒增加。阿昔单抗抑制了剪切应力诱导的血小板聚集，对血液过滤有更明显的影响，血液过滤是血小板聚集形成的指标。
我们的结果表明，阿昔单抗阻断GPIIb/IIIa伴随着α-颗粒分泌增加，提示不同机制调节血小板活化的这些方面。所描述 的流式细胞术技术可在体外同时检测多种血小板标志物，是评估干扰血小板功能药物作用的合适方法。
