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一种新型的血栓弹力描记术中包含黏附内皮细胞的凝血分析。

A novel coagulation assay incorporating adherent endothelial cells in thromboelastometry.

机构信息

Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, AUVA Research Centre, Donaueschingenstrasse 13, Vienna, Austria.

出版信息

Thromb Haemost. 2013 May;109(5):869-77. doi: 10.1160/TH12-10-0767. Epub 2013 Mar 14.

Abstract

Following vascular injury or activation, endothelial cells (ECs) participate in the modulation of haemostasis and fibrinolysis. Viscoelastic tests (VETs) are a potent bedside monitoring tool that reports haemostatic parameters in real time. However, VETs neglect the influence of the surrounding endothelium. Our aim was therefore to establish an assay that incorporates ECs in a whole blood VET and to assess the impact of ECs on coagulation parameters. Outgrowth endothelial cells (OECs) and human umbilical vein endothelial cells (HUVECs) were seeded onto microbeads to create transferable EC-microcarriers. Microbeads were then added to citrated whole blood in the measurement cup of a thromboelastometry device (ROTEM). After the addition of CaCl2 (star-TEM®) to the blood sample (NATEM assay), standard ROTEM parameters were analysed. Scanning electron microscopy (SEM) was carried out to visualise the interactions of the beads, whole blood components and the ROTEM pin after clotting. SEM showed that the added microbeads were effectively incorporated into the final blood clot. In the presence of activated ECs, the clotting time (CT) of the blood was shortened fourfold compared to that in uncoated control beads. A significant reduction in CT was also observed in the presence of unstimulated ECs. Interestingly, CT was also reduced by the addition of purified EC culture supernatant. CT shortening was prevented by incubating the supernatant with an inhibiting antibody against tissue factor (TF). Our findings demonstrate that ECs can be incorporated into a ROTEM assay via coated microbeads, and whole blood clotting initiation is accelerated by non-activated and activated ECs.

摘要

血管损伤或激活后,内皮细胞(EC)参与止血和纤溶的调节。粘弹性测试(VET)是一种强大的床边监测工具,可实时报告止血参数。然而,VET 忽略了周围内皮的影响。因此,我们的目的是建立一种将 EC 纳入全血 VET 的测定方法,并评估 EC 对凝血参数的影响。贴壁生长的内皮细胞(OEC)和人脐静脉内皮细胞(HUVEC)被接种到微珠上,以制成可转移的 EC-微载体。然后将微珠添加到血栓弹性描记仪(ROTEM)测量杯中的枸橼酸盐全血中。向血液样本中加入 CaCl2(Star-TEM®)(NATEM 测定)后,分析标准 ROTEM 参数。进行扫描电子显微镜(SEM)以可视化珠粒、全血成分和血凝后 ROTEM 针之间的相互作用。SEM 显示,添加的微珠有效地整合到最终的血凝块中。在激活的 EC 存在下,与未涂覆对照珠相比,血液的凝血时间(CT)缩短了四倍。在未刺激的 EC 存在下,CT 也明显缩短。有趣的是,添加纯化的 EC 培养上清液也会缩短 CT。通过用组织因子(TF)抑制抗体孵育上清液,可防止 CT 缩短。我们的研究结果表明,EC 可以通过涂覆的微珠纳入 ROTEM 测定,非激活和激活的 EC 可加速全血凝血的启动。

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