Kumar R Anand, Dong Jing-fei, Thaggard Jenny A, Cruz Miguel A, López José A, McIntire Larry V
Department of Bioengineering, Rice University, Houston, Texas, USA.
Biophys J. 2003 Dec;85(6):4099-109. doi: 10.1016/S0006-3495(03)74822-X.
The interaction between platelet glycoprotein (GP) Ib-IX-V complex and von Willebrand factor (vWF) is the first step of the hemostatic response to vessel injury. In platelet-type von Willebrand disease, two mutations, G233V and M239V, have been described within the Cys209-Cys248 disulfide loop of GPIbalpha that compromise hemostasis by increasing the affinity for vWF. We have earlier shown that converting other residues in this region to valine alters the affinity of GPIbalpha for vWF, with mutations K237V and Q232V, respectively, showing the greatest increase and decrease in affinity. Here, we investigated further the effect of these two mutations on the kinetics of the GPIbalpha interaction with the vWF-A1 domain under dynamic flow conditions. We measured the cellular on- and off-rate constants of Chinese hamster ovary cells expressing GPIb-IX complexes containing wild-type or mutant GPIbalpha interacting with vWF-A1-coated surfaces at different shear stresses. We found that the gain-of-function mutant, K237V, rolled very slowly and continuously on vWF-A1 surface while the loss-of-function mutant, Q232V, showed fast, saltatory movement compared to the wild-type (WT). The off-rate constants, calculated based on the analysis of lifetimes of transient tethers formed on surfaces coated with limiting densities of vWF-A1, revealed that the Q232V and K237V dissociated 1.25-fold faster and 2.2-fold slower than the WT. The cellular on-rate constant of WT, measured in terms of tethering frequency, was threefold more and threefold less than Q232V and K237V, respectively. Thus, the gain- and loss-of-function mutations in GPIbalpha affect both the association and dissociation kinetics of the GPIbalpha-vWF-A1 bond. These findings are in contrast to the functionally similar selectin bonds where some of the mutations have been reported to affect only the dissociation rate.
血小板糖蛋白（GP）Ib-IX-V复合物与血管性血友病因子（vWF）之间的相互作用是对血管损伤产生止血反应的第一步。在血小板型血管性血友病中，已在GPIbalpha的Cys209-Cys248二硫键环内发现了两种突变，即G233V和M239V，它们通过增加对vWF的亲和力而损害止血功能。我们之前已经表明，将该区域的其他残基转换为缬氨酸会改变GPIbalpha对vWF的亲和力，其中K237V和Q232V突变分别显示出亲和力的最大增加和降低。在此，我们进一步研究了这两种突变在动态流动条件下对GPIbalpha与vWF-A1结构域相互作用动力学的影响。我们测量了表达含有野生型或突变型GPIbalpha的GPIb-IX复合物的中国仓鼠卵巢细胞在不同剪切应力下与vWF-A1包被表面相互作用的细胞结合和解离速率常数。我们发现，功能获得性突变体K237V在vWF-A1表面上滚动非常缓慢且持续，而功能丧失性突变体Q232V与野生型（WT）相比表现出快速、跳跃式的运动。基于对在vWF-A1有限密度包被表面上形成的瞬时连接寿命的分析计算得出的解离速率常数表明，Q232V和K237V的解离速度分别比WT快1.25倍和慢2.2倍。以连接频率衡量的WT细胞结合速率常数分别比Q232V和K237V多三倍和少三倍。因此，GPIbalpha中的功能获得性和功能丧失性突变均影响GPIbalpha-vWF-A1键的缔合和解离动力学。这些发现与功能相似的选择素键形成对比，在选择素键中，一些突变据报道仅影响解离速率。
