Cranmer S L, Ulsemer P, Cooke B M, Salem H H, de la Salle C, Lanza F, Jackson S P
Australian Centre for Blood Diseases, Department of Medicine, Monash Medical School, Monash University, Victoria 3128, Australia.
J Biol Chem. 1999 Mar 5;274(10):6097-106. doi: 10.1074/jbc.274.10.6097.
Adhesion of platelets to sites of vascular injury is critical for hemostasis and thrombosis and is dependent on the binding of the vascular adhesive protein von Willebrand factor (vWf) to the glycoprotein (GP) Ib-V-IX complex on the platelet surface. A unique but poorly defined characteristic of this receptor/ligand interaction is its ability to support platelet adhesion under conditions of high shear stress. To examine the structural domains of the GPIb-V-IX complex involved in mediating cell adhesion under flow, we have expressed partial (GPIb-IX), complete (GPIb-V-IX), and mutant (GPIbalpha cytoplasmic tail mutants) receptor complexes on the surface of Chinese hamster ovary (CHO) cells and examined their ability to adhere to a vWf matrix in flow-based adhesion assays. Our studies demonstrate that the partial receptor complex (GPIb-IX) supports CHO cell tethering and rolling on a bovine or human vWf matrix under flow. The adhesion was specifically inhibited by an anti-GPIbalpha blocking antibody (AK2) and was not observed with CHO cells expressing GPIbbeta and GPIX alone. The velocity of rolling was dependent on the level of shear stress, receptor density, and matrix concentration and was not altered by the presence of GPV. In contrast to selectins, which mediate cell rolling under conditions of low shear (20-200 s-1), GPIb-IX was able to support cell rolling at both venous (150 s-1) and arterial (1500-10,500 s-1) shear rates. Studies with a mutant GPIbalpha receptor subunit lacking the binding domain for actin-binding protein demonstrated that the association of the receptor complex with the membrane skeleton is not essential for cell tethering or rolling under low shear conditions, but is critical for maintaining adhesion at high shear rates (3000-6000 s-1). These studies demonstrate that the GPIb-IX complex is sufficient to mediate cell rolling on a vWf matrix at both venous and arterial levels of shear independent of other platelet adhesion receptors. Furthermore, our results suggest that the association between GPIbalpha and actin-binding protein plays an important role in enabling cells to remain tethered to a vWf matrix under conditions of high shear stress.
血小板黏附于血管损伤部位对于止血和血栓形成至关重要，且依赖于血管黏附蛋白血管性血友病因子（vWf）与血小板表面糖蛋白（GP）Ib-V-IX复合物的结合。这种受体/配体相互作用的一个独特但定义不明确的特征是其在高剪切应力条件下支持血小板黏附的能力。为了研究在流动状态下介导细胞黏附的GPIb-V-IX复合物的结构域，我们在中国仓鼠卵巢（CHO）细胞表面表达了部分（GPIb-IX）、完整（GPIb-V-IX）和突变型（GPIbalpha胞质尾突变体）受体复合物，并在基于流动的黏附试验中检测了它们黏附于vWf基质的能力。我们的研究表明，部分受体复合物（GPIb-IX）在流动状态下支持CHO细胞在牛或人vWf基质上的系留和滚动。这种黏附被抗GPIbalpha阻断抗体（AK2）特异性抑制，并且单独表达GPIbbeta和GPIX的CHO细胞未观察到这种黏附。滚动速度取决于剪切应力水平、受体密度和基质浓度，并且不受GPV存在的影响。与在低剪切（20-200 s-1）条件下介导细胞滚动的选择素不同，GPIb-IX能够在静脉（150 s-1）和动脉（1500-10500 s-1）剪切速率下支持细胞滚动。对缺乏肌动蛋白结合蛋白结合结构域的突变型GPIbalpha受体亚基的研究表明，受体复合物与膜骨架的结合对于低剪切条件下的细胞系留或滚动并非必需，但对于在高剪切速率（3000-6000 s-1）下维持黏附至关重要。这些研究表明，GPIb-IX复合物足以在静脉和动脉剪切水平下介导细胞在vWf基质上的滚动，而不依赖于其他血小板黏附受体。此外，我们的结果表明，GPIbalpha与肌动蛋白结合蛋白之间的关联在使细胞在高剪切应力条件下保持系留于vWf基质中起重要作用。
