Inhibitory properties of full-length and truncated recombinant tissue factor pathway inhibitor (TFPI). Evidence that the third Kunitz-type domain of TFPI is not essential for the inhibition of factor VIIa-tissue factor complexes on cell surfaces.
Inhibitory properties of full-length and truncated recombinant tissue factor pathway inhibitor (TFPI). Evidence that the third Kunitz-type domain of TFPI is not essential for the inhibition of factor VIIa-tissue factor complexes on cell surfaces.
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DOI：
10.1016/s0021-9258(18)52932-x
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发表时间：
1993-04
期刊：
影响因子：
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通讯作者：
T. Hamamoto;M. Yamamoto;O. Nordfang;J. Petersen;D. Foster;W. Kisiel
中科院分区：
文献类型：
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作者：
T. Hamamoto;M. Yamamoto;O. Nordfang;J. Petersen;D. Foster;W. Kisiel
人组织因子途径抑制剂 (TFPI) 是一种血浆蛋白酶抑制剂，由三个串联的 Kunitz 型抑制剂结构域组成，两侧是带负电的 NH2 末端和带正电的 COOH 末端尾部。先前的研究表明，TFPI 中的第一个和第二个 Kunitz 型结构域分别参与抑制因子 VIIa 和因子 Xa 活性。在本研究中，我们比较了全长重组 TFPI 和缺乏第三个 Kunitz 型结构域和 COOH 末端尾部的截短形式 TFPI (TFPI1-161) 对人膀胱癌细胞系 J82 表面上的因子 VIIa-组织因子复合物的抑制特性。在缺乏因子 Xa 的情况下，全长 TFPI 和 TFPI1-161 在中和 J82 细胞表面上预先形成的因子 VIIa-组织因子复合物的蛋白水解活性方面在动力学上无法区分。等摩尔量的因子Xa使两种TFPI制剂的抗凝血活性增强到相同程度，并且两种TFPI制剂在溶液相中抑制因子VIIa-组织因子酰胺分解活性的效果相同。此外，两种形式的 TFPI 与因子 Xa 形成化学计量复合物的血浆浓度，即使在存在 1 单位/ml 肝素的情况下，对细胞表面因子 VIIa-组织因子蛋白水解活性的抑制也明显快于抗凝血酶 III 的血浆水平。置换研究的结果表明，两种 TFPI 分子对细胞表面的亲和力略有差异，J82 细胞上约 5% 的 VIIa.TF.Xa.TFPI1-161 四元复合物可被高浓度的因子 VIIa (10-100 nM) 从细胞表面置换，而在可比条件下，只有 1-2% 的 VIIa.TF.Xa.TFPI 复合物可置换。单独用 TFPI/Xa 或与 R152E 因子 VII 一起预处理细胞，然后用因子 VIIa 处理，显示两种 TFPI 形式在提供因子 VIIa 恢复细胞表面因子 X 活化的程度方面存在显着差异。尽管存在这些差异，我们的集体研究结果表明，TFPI 的第三个 Kunitz 型结构域和/或 COOH 末端尾部对于抑制细胞表面因子 VIIa-组织因子复合物并不是必需的，并且表明 TFPI1-161 可能是治疗血栓栓塞发作的有用治疗剂。
Human tissue factor pathway inhibitor (TFPI) is a plasma protease inhibitor that consists of three tandem Kunitz-type inhibitor domains flanked by a negatively charged NH2 terminus and a positively charged COOH-terminal tail. Previous studies have shown that the first and second Kunitz-type domains in TFPI are involved in the inhibition of factor VIIa and factor Xa activity, respectively. In the present study, we have compared the inhibitory properties of full-length recombinant TFPI and a truncated form of TFPI lacking the third Kunitz-type domain and COOH-terminal tail (TFPI1-161) with respect to inhibition of factor VIIa-tissue factor complexes on the surface of a human bladder carcinoma cell line J82. Full-length TFPI and TFPI1-161 were kinetically indistinguishable with respect to neutralization of the proteolytic activity of preformed complexes of factor VIIa-tissue factor on the J82 cell surface in the absence of factor Xa. Equimolar amounts of factor Xa augmented the anticoagulant activity of both preparations of TFPI to the same extent, and both preparations of TFPI were equally effective in inhibiting factor VIIa-tissue factor amidolytic activity in solution phase. In addition, plasma concentrations of both forms of TFPI, in stoichiometric complex with factor Xa, inhibited cell surface factor VIIa-tissue factor proteolytic activity markedly faster than plasma levels of antithrombin III, even in the presence of 1 unit/ml heparin. The results of displacement studies suggested slight differences in the affinity of the two TFPI molecules for the cell surface in that approximately 5% of a VIIa.TF.Xa.TFPI1-161 quaternary complex on J82 cells was displaceable from the cell surface by high concentrations of factor VIIa (10-100 nM), whereas only 1-2% of a VIIa.TF.Xa.TFPI complex was displaceable under comparable conditions. Pretreatment of the cells with TFPI/Xa alone or together with R152E factor VII, followed by factor VIIa treatment, revealed significant differences in the two TFPI forms with respect to the degree with which offered factor VIIa could restore factor X activation on the cell surface. These differences notwithstanding, our collective findings indicate that the third Kunitz-type domain and/or COOH-terminal tail of TFPI is not essential for the inhibition of cell surface factor VIIa-tissue factor complexes and suggests that TFPI1-161 may be a useful therapeutic agent in the treatment of thromboembolic episodes.