Fredrickson B J, Dong J F, McIntire L V, López J A
Cox Laboratory for Biomedical Engineering, Rice University, Houston, TX, USA.
Blood. 1998 Nov 15;92(10):3684-93.
Mural thrombi form on exposed arterial subendothelium by a two-step process of platelet adhesion and aggregation. At high shear stresses such as are found in stenotic arteries, both steps are mediated by von Willebrand factor (vWF). Platelets initially adhere on vWF affixed to the subendothelial matrix through the glycoprotein (GP) Ib-IX-V complex. To examine the role of the GP Ib-IX-V complex under dynamic conditions, we modeled initial platelet adhesion at shear stresses ranging from 2 to 40 dyn/cm2 using vWF-coated glass slides, mammalian cells expressing full or partial GP Ib-IX-V complexes, and a parallel plate flow chamber with phase contrast video microscopy and digital image processing. Mammalian cells expressing the full complex tethered and rolled on the vWF substrate, whereas control cells did not. The rolling was completely inhibited by the monoclonal GP Ib antibody, AK2, or the vWF antibody, 5D2, both shown previously to block vWF-dependent platelet aggregation. Other GP Ib antibodies, WM23 and SZ2, did not significantly change the number or mean velocity of rolling cells. At low levels of GP Ib surface expression, cells expressing the full complex rolled slower than cells expressing the complex without GP V, indicating that GP V strengthens the interactions with the vWF surface under these conditions. Preshearing vWF for 5 minutes at 40 dyn/cm2 immediately before introducing cells into the chamber did not significantly change the number or the mean velocity of rolling cells. Inhibiting sulfation of the tyrosine residues within the GP Ib subunit reduced the number but did not change the mean velocity of the rolling cells. Our results indicate that, under the conditions of these experiments, bonds between vWF and GP Ib constantly form and break under fluid shear stress. Additionally, our results suggest that GP Ib-IX-V complexes behave like selectin receptors in their ability to mediate smooth rolling while cells maintain continuous surface contact. Such a mechanism, in vivo, would allow platelets to slow down and eventually arrest on the blood vessel wall. The system described provides a valuable approach for investigating the structure-function relationship of individual receptors and ligands in the process of platelet adhesion and thrombosis.
壁血栓通过血小板黏附和聚集的两步过程在暴露的动脉内皮下形成。在诸如狭窄动脉中存在的高剪切应力下，这两个步骤均由血管性血友病因子（vWF）介导。血小板最初通过糖蛋白（GP）Ib-IX-V复合物黏附于附着在 subendothelial 基质上的 vWF。为了研究 GP Ib-IX-V 复合物在动态条件下的作用，我们使用 vWF 包被的载玻片、表达完整或部分 GP Ib-IX-V 复合物的哺乳动物细胞以及配备相差视频显微镜和数字图像处理的平行板流动腔，模拟了在 2 至 40 dyn/cm2 的剪切应力下血小板的初始黏附。表达完整复合物的哺乳动物细胞在 vWF 底物上 tethered 并滚动，而对照细胞则没有。滚动被单克隆 GP Ib 抗体 AK2 或 vWF 抗体 5D2 完全抑制，这两种抗体先前均显示可阻断 vWF 依赖性血小板聚集。其他 GP Ib 抗体 WM23 和 SZ2 并未显著改变滚动细胞的数量或平均速度。在 GP Ib 表面表达水平较低时，表达完整复合物的细胞比不表达 GP V 的复合物的细胞滚动得慢，这表明在这些条件下 GP V 增强了与 vWF 表面的相互作用。在将细胞引入腔室之前，立即在 40 dyn/cm2 下对 vWF 进行 5 分钟的预剪切，并未显著改变滚动细胞的数量或平均速度。抑制 GP Ib 亚基内酪氨酸残基的硫酸化减少了滚动细胞的数量，但未改变其平均速度。我们的结果表明，在这些实验条件下，vWF 和 GP Ib 之间的键在流体剪切应力下不断形成和断裂。此外，我们的结果表明，GP Ib-IX-V 复合物在介导平滑滚动的能力方面表现得像选择素受体，同时细胞保持连续的表面接触。在体内，这样一种机制将使血小板能够减慢速度并最终在血管壁上停滞。所描述的系统为研究血小板黏附和血栓形成过程中单个受体和配体的结构-功能关系提供了一种有价值的方法。
