Department of Medicine, Sarver Heart Center, University of Arizona, Tucson, Arizona, United States.
Department of Electronics, Information and Bioengineering, Politecnico di Milano, Milan, Italy.
Thromb Haemost. 2020 May;120(5):776-792. doi: 10.1055/s-0040-1709524. Epub 2020 May 5.
Implantable cardiovascular therapeutic devices, while hemodynamically effective, remain limited by thrombosis. A driver of device-associated thrombosis is shear-mediated platelet activation (SMPA). Underlying mechanisms of SMPA, as well as useful biomarkers able to detect and discriminate mechanical versus biochemical platelet activation, are poorly defined. We hypothesized that SMPA induces a differing pattern of biomarkers compared with biochemical agonists.
Gel-filtered human platelets were subjected to mechanical activation via either uniform constant or dynamic shear; or to biochemical activation by adenosine diphosphate (ADP), thrombin receptor-activating peptide 6 (TRAP-6), thrombin, collagen, epinephrine, or arachidonic acid. Markers of platelet activation (P-selectin, integrin αIIbβ3 activation) and apoptosis (mitochondrial membrane potential, caspase 3 activation, and phosphatidylserine externalization [PSE]) were examined using flow cytometry. Platelet procoagulant activity was detected by chromogenic assay measuring thrombin generation. Contribution of platelet calcium flux in SMPA was tested employing calcium chelators, ethylenediaminetetraacetic acid (EDTA), and BAPTA-AM.
Platelet exposure to continuous shear stress, but not biochemical agonists, resulted in a dramatic increase of PSE and procoagulant activity, while no integrin αIIbβ3 activation occurred, and P-selectin levels remained barely elevated. SMPA was associated with dissipation of mitochondrial membrane potential, but no caspase 3 activation was observed. Shear-mediated PSE was significantly decreased by chelation of extracellular calcium with EDTA, while intracellular calcium depletion with BAPTA-AM had no significant effect. In contrast, biochemical agonists ADP, TRAP-6, arachidonic acid, and thrombin were potent inducers of αIIbβ3 activation and/or P-selectin exposure. This differing pattern of biomarkers seen for SMPA for continuous uniform shear was replicated in platelets exposed to dynamic shear stress via circulation through a ventricular assist device-propelled circulatory loop.
Elevated shear stress, but not biochemical agonists, induces a differing pattern of platelet biomarkers-with enhanced PSE and thrombin generation on the platelet surface. This differential biomarker phenotype of SMPA offers the potential for early detection and discrimination from that mediated by biochemical agonists.
尽管植入式心血管治疗设备在血流动力学上有效，但仍受到血栓形成的限制。导致设备相关血栓形成的一个因素是剪切介导的血小板激活（SMPA）。SMPA 的潜在机制以及能够检测和区分机械与生化血小板激活的有用生物标志物定义尚不明确。我们假设 SMPA 诱导的生物标志物模式与生化激动剂不同。
通过均匀恒速或动态剪切对凝胶过滤的人血小板进行机械激活；或通过二磷酸腺苷（ADP）、血栓素受体激活肽 6（TRAP-6）、凝血酶、胶原蛋白、肾上腺素或花生四烯酸进行生化激活。使用流式细胞术检测血小板激活标志物（P-选择素、整合素αIIbβ3 激活）和凋亡标志物（线粒体膜电位、半胱天冬酶 3 激活和磷脂酰丝氨酸外化[PSE]）。通过测定凝血酶生成的显色测定法检测血小板促凝活性。通过使用乙二胺四乙酸（EDTA）和 BAPTA-AM 螯合钙离子来测试 SMPA 中的血小板钙流贡献。
血小板暴露于连续剪切应力，但不是生化激动剂，会导致 PSE 和促凝活性显著增加，而整合素αIIbβ3 激活没有发生，P-选择素水平几乎没有升高。SMPA 与线粒体膜电位耗散相关，但没有观察到半胱天冬酶 3 激活。用 EDTA 螯合细胞外钙可显著降低剪切介导的 PSE，而用 BAPTA-AM 耗尽细胞内钙则没有显著影响。相比之下，ADP、TRAP-6、花生四烯酸和凝血酶等生化激动剂是整合素αIIbβ3 激活和/或 P-选择素暴露的有效诱导剂。在通过心室辅助装置驱动的循环回路循环使血小板暴露于动态剪切应力时，SMPA 引起的这种连续均匀剪切的生物标志物模式在血小板中得到了复制。
升高的剪切应力，而不是生化激动剂，会引起血小板生物标志物的不同模式——血小板表面的 PSE 增加和凝血酶生成。SMPA 的这种差异生物标志物表型提供了早期检测和与生化激动剂介导的检测区分的潜力。
