Central Laboratory of Yong-chuan Hospital, Chongqing Medical University, Chongqing, China.
Department of Blood Transfusion of Yong-chuan Hospital, Chongqing Medical University, Chongqing, China.
Clin Hemorheol Microcirc. 2023;84(2):125-139. doi: 10.3233/CH-221567.
Circulating platelets are sometimes exposed to high shear rate environments due to vascular stenosis, and the effect of transiently elevated pathological high shear rates on platelet activation and aggregation function has not been clarified. The aim of this study was to investigate the effect of pathological high shear rate (8302s-1) exposure time (3.16-25.3 ms) on platelet activation and aggregation function. In addition, by adding active ingredients of antiplatelet drugs such as ASA (an active ingredient of aspirin), Ticagrelor, Tirofiban and GP1BA (platelet membrane protein GPIb inhibitor) in vitro, we studied TXA2, P2Y12-ADP, GPIIb/IIIa-fibrinogen and GPIb /IX/V-vWF receptor pathways to determine platelet activation function mediated by pathological high shear rate. In this study, we designed a set of microfluidic chips with stenosis lengths of 0.5 mm, 1 mm, 2 mm, 3 mm, and 4 mm, all with 80% stenosis, to generate pathological high shear forces that can act at different times. The whole blood flowing through the microchannels was collected by perfusion of sodium citrate anticoagulated whole blood at a physiological arterial shear rate (1500 s-1), and the expression levels of platelet surface activation markers (P-selectin and GP IIb/IIIa) and the degree of platelet aggregation were analyzed by flow cytometry; platelet aggregation patterns were observed by microscopic examination of blood smears. The results showed that shearing significantly increased platelet activation and aggregation levels compared to un-sheared whole blood, and the activation and aggregation levels increased with increasing duration of pathological high shear rate. In vitro inhibition studies showed that ASA barely inhibited the expression of P-selectin and PAC-1 on the platelet surface; Ticagrelor effectively inhibited the expression of both P-selectin and PAC-1; Tirofiban significantly inhibited the expression of PAC-1 on the platelet surface and slightly inhibited the expression of P-selectin; GP1BA significantly inhibited the expression of both. Our results suggest that transient pathological high shear rate (8302s-1) exposure can induce platelet activation in a time-dependent manner; however, the mechanism is more complex and may be due to the following reasons: transient elevated pathological high shear rate activates platelets through the GPIb/IX/V-vWF receptor pathway, and after platelet activation, its surface membrane protein GPIIb/IIIa receptors activate platelets through fibrinogen to form platelet-platelet aggregates, and further activation of active substances such as ADP and TXA2 released by platelet alpha particles, which contribute to the formation of irreversible platelet aggregation.
循环血小板有时由于血管狭窄而暴露于高剪切率环境中，而短暂升高的病理性高剪切率对血小板激活和聚集功能的影响尚未阐明。本研究旨在探讨病理性高剪切率（8302s-1）暴露时间（3.16-25.3ms）对血小板激活和聚集功能的影响。此外，通过在体外添加抗血小板药物的活性成分，如阿司匹林（阿司匹林的活性成分）、替格瑞洛、替罗非班和 GP1BA（血小板膜蛋白 GPIb 抑制剂），我们研究了 TXA2、P2Y12-ADP、GPIIb/IIIa-纤维蛋白原和 GPIb/IX/V-vWF 受体途径，以确定由病理性高剪切率介导的血小板激活功能。在这项研究中，我们设计了一组狭窄长度为 0.5mm、1mm、2mm、3mm 和 4mm 的微流控芯片，均为 80%狭窄，以产生可在不同时间起作用的病理性高剪切力。通过以生理动脉剪切率（1500s-1）灌注柠檬酸钠抗凝全血来收集流过微通道的全血，并通过流式细胞术分析血小板表面活化标志物（P-选择素和 GP IIb/IIIa）的表达水平和血小板聚集程度；通过血液涂片的显微镜检查观察血小板聚集模式。结果表明，与未剪切的全血相比，剪切明显增加了血小板的激活和聚集水平，并且随着病理性高剪切率持续时间的增加，激活和聚集水平增加。体外抑制研究表明，ASA 几乎不能抑制血小板表面 P-选择素和 PAC-1 的表达；替格瑞洛有效抑制 P-选择素和 PAC-1 的表达；替罗非班显著抑制血小板表面 PAC-1 的表达，轻微抑制 P-选择素的表达；GP1BA 显著抑制两者的表达。我们的结果表明，短暂的病理性高剪切率（8302s-1）暴露可以以时间依赖性方式诱导血小板激活；然而，其机制更为复杂，可能是由于以下原因：短暂升高的病理性高剪切率通过 GPIb/IX/V-vWF 受体途径激活血小板，血小板活化后，其表面膜蛋白 GPIIb/IIIa 受体通过纤维蛋白原激活血小板，形成血小板-血小板聚集物，进一步激活血小板α颗粒释放的 ADP 和 TXA2 等活性物质，有助于不可逆血小板聚集的形成。
