Hematological Research Group (HERG), Department of Clinical Medicine, University of Tromsø, Tromsø, Norway.
Thromb Res. 2010 Nov;126(5):418-25. doi: 10.1016/j.thromres.2010.07.014. Epub 2010 Aug 17.
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.
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.
Unstimulated PBMCs contained nearly no TF, but detectable TFPI protein levels. TFPI mRNA levels were 2-fold higher than TF, and the TFPIα mRNA isoform expression was higher than TFPIβ. 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 6hrs 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.
Our results support the concept that surface TFPI is an important regulator of procoagulant reactivity of human monocytes.
单核细胞的促凝反应在很大程度上取决于组织因子（TF）的表达和细胞表面呈现，但是关于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。
从健康受试者的血液中分离外周血单核细胞（PBMCs）并进行冷冻保存。我们通过逆转录定量实时 PCR（RT-qPCR）检测 TF 和 TFPI 的 mRNA 表达，通过流式细胞术和共聚焦显微镜检测表面呈现，通过 TF 活性测定和校准自动血栓图（CAT）测定法检测 TFPI 对静息和 LPS 刺激的 PBMC 表面 TF 功能活性的调节作用。
未刺激的 PBMC 几乎不含 TF，但可检测到 TFPI 蛋白水平。TFPI mRNA 水平是 TF 的 2 倍，TFPIα mRNA 异构体的表达高于 TFPIβ。LPS 刺激引起两种 TFPI 异构体的平行和持续上调，同时伴随 TFPI 抗原的表面呈现增加。在 LPS 刺激的 6 小时，观察到 TF mRNA 和表面抗原的更强但短暂的上调。在 LPS 刺激后，TF 和 TFPI 共同定位于单核细胞膜的相同区域。在 PBMCs 预孵育时，用抗 TFPI IgG 显著增强了 TF 活性，缩短了 Lag-time，并增加了凝血酶生成。在静息细胞中，TFPI 依赖性抑制 TF 比在 LPS 刺激的细胞中更为显著。
我们的结果支持这样的概念，即表面 TFPI 是调节人单核细胞促凝反应的重要调节剂。
