Hansen J B, Svensson B, Olsen R, Ezban M, Osterud B, Paulssen R H
Department of Medicine, Institute of Clinical Medicine, University of Tromsø, Norway.
Thromb Haemost. 2000 Jun;83(6):937-43.
TFPI is a potent inhibitor of the extrinsic coagulation system constitutively synthesized by endothelial cells. A major portion of intravascular TFPI is stored associated with endothelial cells. and administration of unfractionated heparin (UFH) in vivo causes a prompt mobilization of TFPI into the circulation. The present study was conducted to investigate how UFH affected the synthesis, secretion and anticoagulant potency of TFPI in endothelial cells in vitro. A spontaneously transformed immortal endothelial cell line was used (ECV304). Stimulation of ECV304 cells with UFH caused a prompt dose-dependent (0-5 IU UFH/ml) release of TFPI to the medium accompanied by no change of TFPI at the surface membrane assessed by immunocytochemical methods. Northern blot analysis revealed two mRNA transcripts for TFPI with a molecular size of 1.4 kb and 4.4 kb, respectively. Stimulation of ECV304 cells for 24 hrs with various concentrations of UFH caused a dose-dependent increase of TFPI in the medium (6.2-29.6 ng/10(6) cells within the concentration range 0-10 IU/ml). A similar dose-dependent increase in the expression of both TFPI mRNA species was observed. Long-term incubation of ECV304 cells with 5.0 IU/ml UFH caused a 5-10 fold increase in the TFPI concentration accumulated in the medium over 48 hrs. The increased TFPI mRNA expression induced by UFH appeared already after 10 min, peaked after 2-4 hrs, remained augmented throughout the entire period of UFH exposure, and preceded the synthesis-dependent increase in TFPI release by 2-4 hrs. The procoagulant activity of the cells was downregulated by 36% and the contribution of TFPI to the anticoagulant potency of ECV304 cells was moderately increased after 24 hrs heparin stimulation. It is suggested that these mechanisms are of major importance for the anticoagulant function of heparins.
组织因子途径抑制物（TFPI）是内皮细胞持续合成的外源性凝血系统的一种强效抑制剂。血管内TFPI的主要部分与内皮细胞结合储存，体内给予普通肝素（UFH）会使TFPI迅速释放入循环。本研究旨在探讨UFH如何在体外影响内皮细胞中TFPI的合成、分泌及抗凝活性。使用了一种自发转化的永生内皮细胞系（ECV304）。用UFH刺激ECV304细胞会导致TFPI迅速以剂量依赖方式（0 - 5 IU UFH/ml）释放到培养基中，通过免疫细胞化学方法评估，表面膜上的TFPI无变化。Northern印迹分析显示TFPI有两种mRNA转录本，分子大小分别为1.4 kb和4.4 kb。用不同浓度的UFH刺激ECV304细胞24小时会导致培养基中TFPI呈剂量依赖性增加（在0 - 10 IU/ml浓度范围内，每10^6个细胞中为6.2 - 29.6 ng）。两种TFPI mRNA种类的表达也有类似的剂量依赖性增加。用5.0 IU/ml UFH长期培养ECV304细胞48小时会使培养基中积累的TFPI浓度增加5 - 10倍。UFH诱导的TFPI mRNA表达增加在10分钟后即出现，2 - 4小时达到峰值，在UFH暴露的整个期间持续增强，并比依赖合成的TFPI释放增加提前2 - 4小时。肝素刺激24小时后，细胞的促凝活性下调36%，TFPI对ECV304细胞抗凝活性的贡献适度增加。提示这些机制对肝素的抗凝功能至关重要。
