Baugh R J, Broze G J, Krishnaswamy S
Department of Medicine, Division of Hematology/Oncology, Emory University, Atlanta, Georgia 30322, USA.
J Biol Chem. 1998 Feb 20;273(8):4378-86. doi: 10.1074/jbc.273.8.4378.
Tissue factor (TF) pathway inhibitor (TFPI) regulates factor X activation through the sequential inhibition of factor Xa and the VIIa.TF complex. Factor Xa formation was studied in a purified, reconstituted system, at plasma concentrations of factor X and TFPI, saturating concentrations of factor VIIa, and increasing concentrations of TF reconstituted into phosphatidylcholine:phosphatidylserine membranes (TF/PCPS) or PC membranes (TF/PC). The initial rate of factor Xa formation was equivalent in the presence or absence of 2.4 nM TFPI. However, reaction extent was small (<20%) relative to that observed in the absence of TFPI, implying the rapid inhibition of VIIa.TF during factor X activation. Initiation of factor Xa formation using increasing concentrations of TF/PCPS or TF/PC in the presence of TFPI yielded families of progress curves where both initial rate and reaction extent were linearly proportional to the concentration of VIIa.TF. These observations were consistent with a kinetic model in which the rate-limiting step represents the initial inhibition of newly formed factor Xa. Numerical analyses of progress curves yielded a rate constant for inhibition of VIIa.TF by Xa.TFPI (>10(8) M-1.s-1) that was substantially greater than the value (7.34 +/- 0.8 x 10(6) M-1.s-1) directly measured. Thus, VIIa.TF is inhibited at near diffusion-limited rates by Xa.TFPI formed during catalysis which cannot be explained by studies of the isolated reaction. We propose that the predominant inhibitory pathway during factor X activation may involve the initial inhibition of factor Xa either bound to or in the near vicinity of VIIa.TF on the membrane surface. As a result, VIIa.TF inhibition is unexpectedly rapid, and the concentration of active factor Xa that escapes regulation is linearly dependent on the availability of TF.
组织因子（TF）途径抑制剂（TFPI）通过依次抑制因子Xa和VIIa·TF复合物来调节因子X的激活。在纯化的重组系统中，研究了在血浆中因子X和TFPI的浓度、因子VIIa饱和浓度以及重构于磷脂酰胆碱：磷脂酰丝氨酸膜（TF/PCPS）或PC膜（TF/PC）中且浓度不断增加的TF存在的情况下因子Xa的形成。在存在或不存在2.4 nM TFPI的情况下，因子Xa形成的初始速率相当。然而，相对于在不存在TFPI时观察到的反应程度，反应程度较小（<20%），这意味着在因子X激活过程中VIIa·TF被快速抑制。在存在TFPI的情况下，使用浓度不断增加的TF/PCPS或TF/PC启动因子Xa的形成，得到了一系列进程曲线，其中初始速率和反应程度均与VIIa·TF的浓度呈线性比例关系。这些观察结果与一个动力学模型一致，在该模型中限速步骤代表对新形成的因子Xa的初始抑制。对进程曲线的数值分析得出Xa·TFPI对VIIa·TF的抑制速率常数（>10⁸ M⁻¹·s⁻¹），该值远大于直接测量的值（7.34±0.8×10⁶ M⁻¹·s⁻¹）。因此，VIIa·TF在催化过程中形成的Xa·TFPI以接近扩散限制的速率被抑制，这无法通过对孤立反应的研究来解释。我们提出，在因子X激活过程中主要的抑制途径可能涉及对结合于膜表面的VIIa·TF或其附近的因子Xa的初始抑制。结果，VIIa·TF的抑制出乎意料地迅速，且逃脱调节的活性因子Xa的浓度与TF的可用性呈线性相关。
