背景:胶原蛋白诱导的血小板活化主要由糖蛋白(GP)VI通过形成受体簇介导,该受体簇与信号分子的积累一致,并被认为驱动强大而持续的血小板活化。
目的:确定剪切下GPVI簇对全血中血栓形成的重要性。
方法:我们利用了全血微流体和抗GPVI纳米抗体(Nb),Nb28,用AlexaFluor(AF)488标记,以评估GPVI在血小板表面上的分布,该血小板粘附于一系列具有不同血小板活化电位的胶原蛋白样底物。
结果:对血小板上GPVI表面分布的自动分析支持了GPVI簇形成之间存在关系的假设,血栓大小和磷脂酰丝氨酸(PS)暴露。支持大团簇形成的底物也诱导了明显更大的聚集体,与未检测到GPVI簇的底物相比,PS暴露血小板的量增加。此外,我们证明,只有直接抑制GPVI结合,但不是下游信令,能够破坏集群的形成。
结论:标记的抗GPVINb28允许在流动条件下可视化GPVI聚类。此外,虽然下游信号的抑制不影响聚集,但它确实阻止了血栓形成.因此,GPVI宏聚类是血栓形成和血小板活化的先决条件,即PS暴露,在高度依赖GPVI的胶原表面。
Collagen-induced platelet activation is predominantly mediated by glycoprotein (GP) VI through formation of receptor clusters that coincide with the accumulation of signaling molecules and are hypothesized to drive strong and sustained platelet activation.
To determine the importance of GPVI clusters for thrombus formation in whole blood under shear.
We utilized whole blood microfluidics and an anti-GPVI nanobody (Nb), Nb28, labeled with AlexaFluor 488, to assess the distribution of GPVI on the surface of platelets adhering to a range of collagen-like substrates with different platelet activation potentials.
Automated analysis of GPVI surface distribution on platelets supported the hypothesis that there is a relationship between GPVI cluster formation, thrombus size, and phosphatidylserine (PS) exposure. Substrates that supported the formation of macroclusters also induced significantly bigger aggregates, with increased amounts of PS-exposing platelets in comparison to substrates where no GPVI clusters were detected. Furthermore, we demonstrate that only direct inhibition of GPVI binding, but not of downstream signaling, is able to disrupt cluster formation.
Labeled anti-GPVI Nb28 permits visualization of GPVI clustering under flow conditions. Furthermore, whilst inhibition of downstream signaling does not affect clustering, it does prevent thrombus formation. Therefore, GPVI macroclustering is a prerequisite for thrombus formation and platelet activation, namely, PS exposure, on highly GPVI-dependent collagen surfaces.