Laboratory of Electron Microscopy, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan.
Thromb Res. 2011 Dec;128(6):552-9. doi: 10.1016/j.thromres.2011.07.031. Epub 2011 Aug 15.
The dodecapeptide HHLGGAKQAGDV (H12) in the carboxy-terminus of the fibrinogen γ-chain is a specific binding site of the ligand for platelet GPIIb/IIIa complex. We have evaluated liposomes carrying fibrinogen γ-chain dodecapeptide as a synthetic platelet substitute.
We examined the interaction between human platelets and H12-liposomes during thrombin-induced activation using flow cytometry and electron microscopy (EM).
After thrombin-activation, a remarkable time-dependent increase in binding of the H12-liposomes to platelets was found by flow cytometry. A large-sized swollen open canalicular system (OCS) was observed in the spheroidal platelets from 60 sec to 5 min after thrombin-activation, but intact H12-liposomes were not evident by conventional EM. Cryoultramicrotomy and immunogold staining with anti-H12 antibody were successful in identifying the liposomes; they appeared as small particles with a unit membrane around 0.2 to 0.4 μm in diameter, and gold labels representing H12 were distributed homogeneously on the surface. Abundant H12-liposomes were localized not only on the surface membrane but also in the lumen of the large-sized swollen OCS in the platelets at 60 sec after thrombin-activation. The formation of the large-sized swollen OCS was inhibited by pre-incubation with unbound H12, EDTA or anti-GPIIb/IIIa antibody. In thrombin-induced platelet aggregates we observed electron-transparent areas between adherent platelets, in which abundant H12-liposomes were distributed.
We demonstrate morphologically that H12-liposomes bind to thrombin-activated platelets and accumulate between adherent platelets like fibrinogen, leading to large-scale aggregation.
纤维蛋白原 γ 链羧基末端的十二肽 HHLGGAKQAGDV（H12）是血小板 GPIIb/IIIa 复合物配体的特异性结合位点。我们已经评估了携带纤维蛋白原 γ 链十二肽的脂质体作为合成血小板替代物。
我们使用流式细胞术和电子显微镜（EM）检查了凝血酶诱导激活期间人血小板与 H12-脂质体之间的相互作用。
在凝血酶激活后，通过流式细胞术发现 H12-脂质体与血小板的结合显著随时间增加。在凝血酶激活后 60 秒至 5 分钟，球形血小板中观察到大型肿胀的开放小管系统（OCS），但通过常规 EM 未发现完整的 H12-脂质体。低温超微切割和用抗 H12 抗体进行免疫金染色成功地鉴定了脂质体；它们表现为带有 0.2 至 0.4μm 直径的单位膜的小颗粒，并且表面均匀分布着代表 H12 的金标记。在凝血酶激活后 60 秒，不仅在血小板的大型肿胀 OCS 的腔内部，而且在表面膜上都存在大量的 H12-脂质体。通过预孵育未结合的 H12、EDTA 或抗 GPIIb/IIIa 抗体可抑制大型肿胀 OCS 的形成。在凝血酶诱导的血小板聚集物中，我们观察到在黏附的血小板之间有电子透明区域，其中分布着大量的 H12-脂质体。
我们从形态学上证明 H12-脂质体与凝血酶激活的血小板结合，并像纤维蛋白原一样在黏附的血小板之间积聚，导致大规模聚集。
