Influence of Hematocrit Level and Integrin αIIbβIII Function on vWF-Mediated Platelet Adhesion and Shear-Induced Platelet Aggregation in a Sudden Expansion.
Influence of Hematocrit Level and Integrin αIIbβIII Function on vWF-Mediated Platelet Adhesion and Shear-Induced Platelet Aggregation in a Sudden Expansion.
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血细胞比容水平和整合素αIIbβIII 功能对vWF 介导的血小板粘附和突然扩张中剪切诱导的血小板聚集的影响。
DOI：
10.1007/s12195-024-00796-0
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发表时间：
2024
影响因子：
2.8
通讯作者：
Manning,KeefeB
中科院分区：
文献类型：
--
作者：
Watson,ConnorT;Ward,ShaneC;Rizzo,StefanoA;Redaelli,Alberto;Manning,KeefeB
目的剪切介导的血栓形成是一种临床相关现象，是过度动脉血栓形成和装置诱导血栓形成的基础。众所周知，红细胞通过血小板和 vWF 的边缘化，促进 vWF 多聚体的展开，并增加血栓接触血小板的比例，从而机械地促进生理止血。剪切力也在这种现象中发挥作用，增加了 vWF 和血小板的边缘化程度和近壁力，导致展开和激活。尽管如此，红细胞在剪切诱导的血小板聚集中的作用尚未得到充分研究，特别是血细胞比容升高的影响尚未得到证实。方法在此，提出了突然扩张的微流体模型作为研究血小板在血细胞比容范围为 0 至 60% 和剪切速率范围为 1000 至 10,000 s−1 时的粘附的平台。突然膨胀的几何形状模拟了机械循环支持装置的非生理流动分离特性，以及 FDA 基准喷嘴的验证框架。 PDMS 微通道被制造并涂有人类胶原蛋白。血小板被荧光标记，并且在灌注实验之前以可变的血细胞比容重建血液。封闭抗体抑制所选血液样本的整合素功能，并监测灌注过程中的血小板粘附和聚集。结果在生理和升高的血细胞比容水平下增加剪切速率有利于血小板的牢固粘附和大聚集体的形成。剪切诱导的血小板聚集被证明依赖于 αIIbβIII 功能和红细胞的存在。抑制 αIIbβIII 可使总体血小板粘附减少 86.4%，在血细胞比容为 20-60% 时，血栓大小减少 85.7%。 20% 的血细胞比容水平不足以有效进行血小板边缘化和随后的 vWF 束缚，与 40% 和 60% 相比，导致 5000 和 10,000 s-1 时血小板粘附显着下降。 αIIbβIII 的抑制引发整体血栓覆盖率和大聚集体形成的急剧减少。 vWF 束缚的血小板稳定性被证明是 αIIbβIII 依赖性的，因为用 A2A9（一种抗 αIIbβIII 阻断抗体）处理的单个血小板的粘附是短暂的，不会导致持续的血栓形成。结论本研究强调了 vWF 介导的血小板粘附的驱动因素，这些因素与剪切诱导血栓形成的临床抑制和血小板粘附的体外测定相关。首先，增加血细胞比容会促进血小板边缘化，从而在超生理剪切速率下通过 αIIbβIII 介导的粘附来允许剪切诱导的血小板聚集。
PurposeShear-mediated thrombosis is a clinically relevant phenomenon that underlies excessive arterial thrombosis and device-induced thrombosis. Red blood cells are known to mechanically contribute to physiological hemostasis through margination of platelets and vWF, facilitating the unfurling of vWF multimers, and increasing the fraction of thrombus-contacting platelets. Shear also plays a role in this phenomenon, increasing both the degree of margination and the near-wall forces experienced by vWF and platelets leading to unfurling and activation. Despite this, the contribution of red blood cells in shear-induced platelet aggregation has not been fully investigated—specifically the effect of elevated hematocrit has not yet been demonstrated.MethodsHere, a microfluidic model of a sudden expansion is presented as a platform for investigating platelet adhesion at hematocrits ranging from 0 to 60% and shear rates ranging from 1000 to 10,000 s−1. The sudden expansion geometry models nonphysiological flow separation characteristic to mechanical circulatory support devices, and the validatory framework of the FDA benchmark nozzle. PDMS microchannels were fabricated and coated with human collagen. Platelets were fluorescently tagged, and blood was reconstituted at variable hematocrit prior to perfusion experiments. Integrin function of selected blood samples was inhibited by a blocking antibody, and platelet adhesion and aggregation over the course of perfusion was monitored.ResultsIncreasing shear rates at physiological and elevated hematocrit levels facilitate robust platelet adhesion and formation of large aggregates. Shear-induced platelet aggregation is demonstrated to be dependent on both αIIbβIIIfunction and the presence of red blood cells. Inhibition of αIIbβIIIresults in an 86.4% reduction in overall platelet adhesion and an 85.7% reduction in thrombus size at 20-60% hematocrit. Hematocrit levels of 20% are inadequate for effective platelet margination and subsequent vWF tethering, resulting in notable decreases in platelet adhesion at 5000 and 10,000 s-1compared to 40% and 60%. Inhibition of αIIbβIIItriggered dramatic reductions in overall thrombus coverage and large aggregate formation. Stability of platelets tethered by vWF are demonstrated to be αIIbβIII-dependent, as adhesion of single platelets treated with A2A9, an anti-αIIbβIIIblocking antibody, is transient and did not lead to sustained thrombus formation.ConclusionsThis study highlights driving factors in vWF-mediated platelet adhesion that are relevant to clinical suppression of shear-induced thrombosis and in vitro assays of platelet adhesion. Primarily, increasing hematocrit promotes platelet margination, permitting shear-induced platelet aggregation through αIIbβIII-mediated adhesion at supraphysiological shear rates.