Wood Jeremy P, Petersen Helle H, Yu Bingke, Wu Xiaoai, Hilden Ida, Mast Alan E
Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, WI.
Global Research, Novo Nordisk, Maaloev, Denmark.
Blood Adv. 2017 Dec 26;1(27):2692-2702. doi: 10.1182/bloodadvances.2017011098.
Tissue factor pathway inhibitor α (TFPIα) inhibits prothrombinase, the thrombin-generating complex of factor Xa (FXa) and factor Va (FVa), during the initiation of coagulation. This inhibition requires binding of a conserved basic region within TFPIα to a conserved acidic region in FXa-activated and platelet-released FVa. In this study, the contribution of interactions between TFPIα and the FXa active site and FVa heavy chain to prothrombinase inhibition were examined to further define the inhibitory biochemistry. Removal of FXa active site binding by mutation or by deletion of the second Kunitz domain (K2) of TFPIα produced 17- or 34-fold weaker prothrombinase inhibition, respectively, establishing that K2 binding to the FXa active site is required for efficient inhibition. Substitution of the TFPIα basic region uncharged residues (Leu252, Ile253, Thr255) with Ala (TFPI-AAKA) produced 5.8-fold decreased inhibition. This finding was confirmed using a basic region peptide (Leu252-Lys261) and Ala substitution peptides, which established that the uncharged residues are required for prothrombinase inhibitory activity but not for binding the FVa acidic region. This suggests that the uncharged residues mediate a secondary interaction with FVa subsequent to acidic region binding. This secondary interaction seems to be with the FVa heavy chain, because the FV Leiden mutation weakened prothrombinase inhibition by TFPIα but did not alter TFPI-AAKA inhibitory activity. Thus, efficient inhibition of prothrombinase by TFPIα requires at least 3 intermolecular interactions: (1) the TFPIα basic region binds the FVa acidic region, (2) K2 binds the FXa active site, and (3) Leu252-Thr255 binds the FVa heavy chain.
组织因子途径抑制剂α（TFPIα）在凝血起始阶段抑制凝血酶原酶，即因子Xa（FXa）和因子Va（FVa）形成的凝血酶生成复合物。这种抑制作用需要TFPIα内一个保守的碱性区域与FXa激活并由血小板释放的FVa中一个保守的酸性区域相结合。在本研究中，对TFPIα与FXa活性位点以及FVa重链之间的相互作用对凝血酶原酶抑制的贡献进行了研究，以进一步明确抑制的生化机制。通过突变或删除TFPIα的第二个Kunitz结构域（K2）来消除FXa活性位点结合，分别使凝血酶原酶抑制作用减弱17倍或34倍，这表明K2与FXa活性位点的结合是有效抑制所必需的。用丙氨酸取代TFPIα碱性区域的不带电荷残基（Leu252、Ile253、Thr255）（TFPI - AAKA）使抑制作用降低了5.8倍。使用碱性区域肽（Leu252 - Lys261）和丙氨酸取代肽证实了这一发现，结果表明不带电荷残基是凝血酶原酶抑制活性所必需的，但不是与FVa酸性区域结合所必需的。这表明不带电荷残基在酸性区域结合后介导了与FVa的二级相互作用。这种二级相互作用似乎是与FVa重链的相互作用，因为FV Leiden突变减弱了TFPIα对凝血酶原酶的抑制作用，但未改变TFPI - AAKA的抑制活性。因此，TFPIα对凝血酶原酶的有效抑制至少需要3种分子间相互作用：（1）TFPIα碱性区域结合FVa酸性区域；（2）K2结合FXa活性位点；（3）Leu252 - Thr255结合FVa重链。
