Department of Surgery, University of Maryland School of Medicine, Baltimore, Maryland.
Fischell Department of Bioengineering, A. James Clark School of Engineering, University of Maryland, College Park, Maryland.
Artif Organs. 2019 Sep;43(9):897-908. doi: 10.1111/aor.13465. Epub 2019 May 14.
The PI3K/Akt signaling pathway has been implicated in playing an important role in platelet activation during hemostasis and thrombosis involving platelet-matrix interaction and platelet aggregation. Its role in non-physiological shear stress (NPSS)-induced platelet activation relevant to high-shear blood contacting medical devices (BCMDs) is unclear. In the context of blood cells flowing in BCMDs, platelets are subjected to NPSS (>100 Pa) with very short exposure time (<1 s). In this study, we investigated whether NPSS with short exposure time induces platelet activation through the PI3K/Akt signaling pathway. Healthy donor blood treated with or without PI3K inhibitor was subjected to NPSS (150 Pa) with short exposure time (0.5 s). Platelet activation indicated by the surface P-selectin expression and activated glycoprotein (GP) IIb/IIIa was quantified using flow cytometry. The phosphorylation of Akt, activation of the PI3K signaling, was characterized by western blotting. Changes in adhesion behavior of NPSS-sheared platelets on fibrinogen, collagen, and von Willebrand factor (vWF) were quantified with fluorescent microscopy by perfusing the NPSS-sheared and PI3K inhibitor-treated blood through fibrinogen, collagen, and vWF-coated microcapillary tubes. The results showed that the PI3K/Akt signaling was involved with both NPSS-induced platelet activation and platelet-matrix interaction. NPSS-sheared platelets exhibited exacerbated platelet adhesion on fibrinogen, but had diminished platelet adhesion on collagen and vWF. The inhibition of PI3K signaling reduced P-selectin expression and GPIIb/IIIa activation with suppressed Akt phosphorylation and abolished NPSS-enhanced platelet adhesion on fibrinogen in NPSS-sheared blood. The inhibition of PI3K signaling can attenuate the adhesion of unsheared platelets (baseline) on collagen and vWF, while had no impact on adhesion of NPSS-sheared platelets on collagen and vWF. This study confirmed the important role of PI3K/Akt signaling pathway in NPSS-induced platelet activation. The finding of this study suggests that blocking PI3K/Akt signaling pathway could be a potential method to treat thrombosis in patients implanted with BCMDs.
PI3K/Akt 信号通路在止血和血栓形成过程中血小板的激活中起着重要作用，涉及血小板与基质的相互作用和血小板聚集。但其在与高剪切力接触的医疗器械（BCMDs）相关的非生理剪切力（NPSS）诱导的血小板激活中的作用尚不清楚。在 BCMD 中血流的细胞环境下，血小板受到 NPSS（>100 Pa）的作用，且暴露时间极短（<1 s）。在这项研究中，我们研究了短时间暴露于 NPSS 是否通过 PI3K/Akt 信号通路诱导血小板激活。用或不用 PI3K 抑制剂处理的健康供体血液暴露于短时间（0.5 s）的 NPSS（150 Pa）下。通过流式细胞术定量血小板表面 P-选择素表达和激活糖蛋白（GP）IIb/IIIa 来指示血小板激活。通过 Western blot 分析 Akt 的磷酸化和 PI3K 信号的激活来描述 PI3K 信号的变化。通过在 NPSS 剪切和 PI3K 抑制剂处理的血液中灌注纤维蛋白原、胶原蛋白和血管性血友病因子（vWF）包被的微毛细管管，荧光显微镜量化 NPSS 剪切血小板在纤维蛋白原、胶原蛋白和 vWF 上的黏附行为。结果表明，PI3K/Akt 信号参与了 NPSS 诱导的血小板激活和血小板与基质的相互作用。NPSS 剪切的血小板在纤维蛋白原上表现出加剧的血小板黏附，但在胶原蛋白和 vWF 上的黏附减少。PI3K 信号的抑制减少了 P-选择素的表达和 GPIIb/IIIa 的激活，抑制了 Akt 的磷酸化，并消除了 NPSS 增强的 NPSS 剪切血小板在纤维蛋白原上的黏附。PI3K 信号的抑制可以减弱未剪切血小板（基线）在胶原蛋白和 vWF 上的黏附，但对 NPSS 剪切血小板在胶原蛋白和 vWF 上的黏附没有影响。这项研究证实了 PI3K/Akt 信号通路在 NPSS 诱导的血小板激活中的重要作用。该研究结果表明，阻断 PI3K/Akt 信号通路可能是治疗植入 BCMDs 的患者血栓形成的一种潜在方法。
