The role of TFPI in regulation of TF-induced thrombogenicity on the surface of human monocytes
The role of TFPI in regulation of TF-induced thrombogenicity on the surface of human monocytes
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DOI：
10.1016/j.thromres.2010.07.014
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发表时间：
2010-11-01
影响因子：
7.5
通讯作者：
Hansen, John-Bjarne
中科院分区：
文献类型：
--
作者：
Basavaraj, Manjunath Goolyam;Gruber, Franz X.;Hansen, John-Bjarne
简介：虽然单核细胞的促凝反应性很大程度上取决于组织因子（TF）的表达和细胞表面呈递，但组织因子途径抑制剂（TFPI）对单核细胞表面TF功能调节的影响知之甚少。 材料和方法：从健康受试者的血液中分离外周血单核细胞（PBMC）并冷冻保存。我们通过逆转录定量实时 PCR (RT-qPCR) 研究了 TF 和 TFPI mRNA 表达，通过流式细胞术和共聚焦显微镜研究了表面呈现，并通过 TF 活性测定和校准自动血栓图 (CAT) 测定研究了 TFPI 介导的静息和 LPS 刺激的 PBMC 表面上 TF 功能活性的调节。结果：未刺激的 PBMC 几乎不含 TF，但可检测到 TFPI 蛋白水平。 TFPI mRNA 水平比 TF 高 2 倍，并且 TFPI α mRNA 亚型表达高于 TFPI beta。 LPS 刺激导致两种 TFPI 同工型平行且持续的上调，同时 TFPI 抗原的表面呈递增加。 LPS 刺激 6 小时时观察到 TF mRNA 和表面抗原更强但短暂的上调。 LPS 刺激后，TF 和 TFPI 共定位于单核细胞膜的相同区域。将 PBMC 与抗 TFPI IgG 预孵育可显着增强 TF 活性、缩短滞后时间并增加凝血酶生成。 TFPI 依赖性的 TF 抑制在静息细胞中比在 LPS 刺激的细胞中更为显着。结论：我们的结果支持表面 TFPI 是人单核细胞促凝血反应性的重要调节剂的概念。 (C) 2010 Elsevier Ltd. 保留所有权利。
Introduction: Although the procoagulant reactivity of monocytes largely depends on expression and cell surface presentation of tissue factor (TF), little is known about the impact of tissue factor pathway inhibitor (TFPI) on regulation of TF function on the monocyte surface.Materials and methods: Peripheral blood mononuclear cells (PBMCs) were isolated from blood of healthy subjects and cryopreserved. We investigated TF and TFPI mRNA expression by reverse transcription-quantitative real-time PCR (RT-qPCR), surface presentation by flow cytometry and confocal microscopy, and TFPI-mediated regulation of TF functional activity on the surface of resting and LPS-stimulated PBMCs by TF activity assay and Calibrated Automated Thrombogram (CAT) assay.Results: Unstimulated PBMCs contained nearly no TF, but detectable TFPI protein levels. TFPI mRNA levels were 2-fold higher than TF, and the TFPI alpha mRNA isoform expression was higher than TFPI beta. LPS stimulation caused a parallel and sustained upregulation of both TFPI isoforms, concomitant with increased surface presentation of TFPI antigen. Stronger, but transient upregulation of TF mRNA and surface antigen was observed at 6 hrs of LPS stimulation. After LPS stimulation TF and TFPI were co-localized in the same areas of the monocyte membrane. Pre-incubation of PBMCs with anti-TFPI IgG significantly enhanced TF activity, shortened Lag-time, and increased thrombin generation. TFPI-dependent inhibition of TF was more prominent in resting than in LPS-stimulated cells.Conclusions: Our results support the concept that surface TFPI is an important regulator of procoagulant reactivity of human monocytes. (C) 2010 Elsevier Ltd. All rights reserved.