Lockett J Marcus, Mast Alan E
Research and Pathology Services, Department of Veterans Affairs, Memphis, Tennessee 38104, USA.
Biochemistry. 2002 Apr 16;41(15):4989-97. doi: 10.1021/bi016058n.
The functions of the first two Kunitz domains of tissue factor pathway inhibitor (TFPI) are well defined as active site-directed inhibitors of factor VIIa and factor Xa. The anticoagulant properties of the third Kunitz domain and C-terminal region were probed using altered forms of TFPI. TFPI-160 contains the first two Kunitz domains. K1K2C contains the first two Kunitz domains and the basic C-terminus. Neither TFPI-160 nor K1K2C contains the third Kunitz domain. In amidolytic assays containing calcium, TFPI-160 is a less potent inhibitor of factor Xa than TFPI. However, addition of the C-terminus in K1K2C nearly restores inhibitory activity to that of TFPI, indicating that the third Kunitz domain is not required for direct inhibition of factor Xa. When compared in assays containing phospholipids and factor Va, K1K2C and TFPI-160 are poor inhibitors compared to TFPI, demonstrating that the third Kunitz domain is required for the full anticoagulant activity of TFPI. TFPI was further characterized in amidolytic assays performed with Gla-domainless factor Xa and in prothrombin activation assays using submicellar concentrations of short-chain phospholipids (C6PS). TFPI and K1K2C are worse inhibitors of Gla-domainless factor Xa, compared to wild-type factor Xa, while TFPI-160 inhibits both forms of factor Xa equally, suggesting a C-terminus/Gla domain interaction. TFPI is a potent inhibitor of thrombin generation by prothrombinase assembled with C6PS, while TFPI-160 and K1K2C are not. Conversely, TFPI does not inhibit prothrombin activation by prothrombinase assembled on a two-dimensional lipid bilayer. Together, the data indicate that the region between Gly-160 and the end of the third Kunitz domain contributes to TFPI function by orienting the second Kunitz domain so that it can bind the active site of phospholipid-associated factor Xa prior to prothrombinase assembly and/or by slowing formation of the prothrombinase complex.
组织因子途径抑制剂（TFPI）前两个Kunitz结构域的功能已明确为因子VIIa和因子Xa的活性位点定向抑制剂。使用TFPI的变体形式探究了第三个Kunitz结构域和C末端区域的抗凝特性。TFPI - 160包含前两个Kunitz结构域。K1K2C包含前两个Kunitz结构域和碱性C末端。TFPI - 160和K1K2C均不包含第三个Kunitz结构域。在含钙的酰胺水解测定中，TFPI - 160对因子Xa的抑制作用比TFPI弱。然而，在K1K2C中添加C末端几乎可将抑制活性恢复至TFPI的水平，表明直接抑制因子Xa不需要第三个Kunitz结构域。在含有磷脂和因子Va的测定中进行比较时，与TFPI相比，K1K2C和TFPI - 160是较差的抑制剂，这表明第三个Kunitz结构域是TFPI充分抗凝活性所必需的。在使用无Gla结构域的因子Xa进行的酰胺水解测定以及使用亚微胶束浓度的短链磷脂（C6PS）进行的凝血酶原激活测定中，对TFPI进行了进一步表征。与野生型因子Xa相比，TFPI和K1K2C对无Gla结构域的因子Xa的抑制作用更弱，而TFPI - 160对两种形式的因子Xa的抑制作用相同，这表明存在C末端/Gla结构域相互作用。TFPI是由与C6PS组装的凝血酶原酶产生凝血酶的有效抑制剂，而TFPI - 160和K1K2C则不是。相反，TFPI不抑制在二维脂质双层上组装的凝血酶原酶对凝血酶原的激活。总之，数据表明，Gly - 160与第三个Kunitz结构域末端之间的区域通过使第二个Kunitz结构域定向，使其能够在凝血酶原酶组装之前结合磷脂相关因子Xa的活性位点和/或通过减缓凝血酶原酶复合物的形成，从而对TFPI的功能有贡献。
