MOLECULAR-CLONING, EXPRESSION, AND PARTIAL CHARACTERIZATION OF A 2ND HUMAN TISSUE-FACTOR-PATHWAY INHIBITOR
MOLECULAR-CLONING, EXPRESSION, AND PARTIAL CHARACTERIZATION OF A 2ND HUMAN TISSUE-FACTOR-PATHWAY INHIBITOR
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DOI：
10.1073/pnas.91.8.3353
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发表时间：
1994-04-12
影响因子：
11.1
通讯作者：
FOSTER, DC
中科院分区：
文献类型：
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作者：
SPRECHER, CA;KISIEL, W;FOSTER, DC
先前的研究表明，组织因子途径抑制剂（TFPI）通过抑制 Xa 因子和 VIIa 因子 - 组织因子活性的能力，成为凝血外源途径的重要调节剂。我们描述了编码分子（命名为 TFPI-2）的全长 cDNA 的分子克隆和表达，该分子与 TFPI 具有相似的整体结构域组织和相当大的一级氨基酸序列同源性。在包含 22 个残基的信号肽之后，成熟蛋白包含 213 个氨基酸、18 个半胱氨酸和两个典型的 N 连接糖基化位点。成熟 TFPI-2 的推导序列揭示了一个短的酸性氨基末端区域、三个串联的 Kunitz 型结构域和一个富含碱性氨基酸的羧基末端尾部。 Northern 分析表明 TFPI-2 在脐静脉内皮细胞、肝脏和胎盘中转录。 TFPI-2 在幼仓鼠肾细胞中表达，并通过肝素-琼脂糖色谱、Mono Q FPLC、Mono S FPLC 和 Superose 12 FPLC 的组合从无血清条件培养基中纯化。纯化的 TFPI-2 在 SDS/PAGE 中作为单条带迁移，并且在存在和不存在还原剂的情况下表现出 32 kDa 的分子量。重组TFPI-2的氨基末端序列与cDNA预测的相同。尽管其结构与 TFPI 相似，但纯化的重组 TFPI-2 未能与多克隆抗 TFPI IgG 发生反应。初步研究表明，纯化的重组TFPI-2强烈抑制胰蛋白酶和VIIa因子-组织因子复合物的酰胺分解活性。此外，在肝素存在下，重组TFPI-2对因子VIIa-组织因子酰胺分解活性的抑制显着增强。高浓度的TFPI-2微弱地抑制人因子Xa的酰胺分解活性，但对人凝血酶的酰胺分解活性没有可测量的影响。
Previous studies have shown that tissue-factor-pathway inhibitor (TFPI) is an important regulator of the extrinsic pathway of blood coagulation through its ability to inhibit factor Xa and factor VIIa-tissue factor activity. We describe the molecular cloning and expression of a full-length cDNA that encodes a molecule, designated TFPI-2, that has a similar overall domain organization and considerable primary amino acid sequence homology to TFPI. After a 22-residue signal peptide, the mature protein contains 213 amino acids with 18 cysteines and two canonical N-linked glycosylation sites. The deduced sequence of mature TFPI-2 revealed a short acidic amino-terminal region, three tandem Kunitz-type domains, and a carboxyl-terminal tail highly enriched in basic amino acids. Northern analysis indicates that TFPI-2 is transcribed in umbilical vein endothelial cells, liver, and placenta. TFPI-2 was expressed in baby hamster kidney cells and purified from the serum-free conditioned medium by a combination of heparin-agarose chromatography, Mono Q FPLC, Mono S FPLC, and Superose 12 FPLC. Purified TFPI-2 migrated as a single band in SDS/PAGE and exhibited a molecular mass of 32 kDa in the presence and absence of reducing agent. The amino-terminal sequence of recombinant TFPI-2 was identical to that predicted from the cDNA. Despite its structural similarity to TFPI, the purified recombinant TFPI-2 failed to react with polyclonal anti-TFPI IgG. Preliminary studies indicated that purified recombinant TFPI-2 strongly inhibited the amidolytic activities of trypsin and the factor VIIa-tissue factor complex. In addition, the inhibition of factor VIIa-tissue factor amidolytic activity by recombinant TFPI-2 was markedly enhanced in the presence of heparin. TFPI-2 at high concentrations weakly inhibited the amidolytic activity of human factor Xa, but had no measurable effect on the amidolytic activity of human thrombin.