Humbert M, Nurden P, Bihour C, Pasquet J M, Winckler J, Heilmann E, Savi P, Herbert J M, Kunicki T J, Nurden A T
UMR 5533 CNRS, Hôpital Cardiologique, Pessac, France.
Arterioscler Thromb Vasc Biol. 1996 Dec;16(12):1532-43. doi: 10.1161/01.atv.16.12.1532.
Our study investigated the effect of the antithrombotic drug clopidogrel (75 mg/d for 7 days) on the ultrastructure of platelet aggregates induced by ADP or 2-methylthio-ADP (2-MeS-ADP) in citrated platelet-rich plasma and examined the activation state of the GP IIb/IIIa complexes. Results were compared with those obtained for patient M.L., who has a congenital disorder characterized by a reduced and reversible platelet response to ADP. When untreated normal platelets were stimulated with high-dose ADP, electron microscopy revealed large and stable aggregates often surrounded by a layer of what appeared to be degranulated platelets. The reversible aggregates of platelets from subjects receiving clopidogrel or from patient M.L. did not show this layer. Electron microscopy showed that in both situations, the aggregates were composed of loosely bound platelets with few contact points. Immunogold labeling of ultrathin sections of Lowicryl-embedded aggregates formed by ADP or 2-MeS-ADP showed a much decreased platelet surface staining by (1) a polyclonal anti-fibrinogen antibody and (2) AP-6, a murine anti-ligand-induced binding site monoclonal antibody specific for GP IIb/IIIa complexes occupied with fibrinogen. Similar findings were seen after disaggregation, when many single platelets were present that showed no signs of secretion. Flow cytometry confirmed that the number of ligand-occupied GP IIb/IIIa complexes was much lower on platelets stimulated with ADP or 2-MeS-ADP after clopidogrel treatment. As expected from previous studies, ADP-induced platelet shape change and Ca2+ influx were unaffected by clopidogrel. These results agree with the hypothesis that platelet activation by ADP is biphasic and highlight a receptor-induced activation pathway affected by clopidogrel (or congenitally impaired in patient M.L.) that is necessary for the full activation of GP IIb/IIIa and the formation of stable macroaggregates.
我们的研究调查了抗血栓药物氯吡格雷（75毫克/天，持续7天）对枸橼酸化富血小板血浆中由ADP或2-甲硫基-ADP（2-MeS-ADP）诱导的血小板聚集体超微结构的影响，并检测了GP IIb/IIIa复合物的活化状态。将结果与患者M.L.的结果进行比较，患者M.L.患有先天性疾病，其特征是对ADP的血小板反应降低且可逆。当用高剂量ADP刺激未处理的正常血小板时，电子显微镜显示出大的、稳定的聚集体，其周围通常有一层似乎已脱颗粒的血小板。接受氯吡格雷治疗的受试者或患者M.L.的血小板可逆聚集体未显示出这一层。电子显微镜显示，在这两种情况下，聚集体均由结合松散、接触点少的血小板组成。对由ADP或2-MeS-ADP形成的用Lowicryl包埋的聚集体超薄切片进行免疫金标记显示，（1）多克隆抗纤维蛋白原抗体和（2）AP-6（一种针对被纤维蛋白原占据的GP IIb/IIIa复合物的鼠抗配体诱导结合位点单克隆抗体）对血小板表面的染色明显减少。在解聚后也观察到类似的结果，此时存在许多未显示分泌迹象的单个血小板。流式细胞术证实，氯吡格雷治疗后，用ADP或2-MeS-ADP刺激的血小板上被配体占据的GP IIb/IIIa复合物数量要低得多。正如先前研究所预期的，ADP诱导的血小板形状变化和Ca2+内流不受氯吡格雷影响。这些结果与ADP诱导的血小板活化是双相的这一假设一致，并突出了一条受氯吡格雷影响（或在患者M.L.中先天性受损）的受体诱导活化途径，该途径对于GP IIb/IIIa的完全活化和稳定大聚集体的形成是必需的。
