Røjkjaer R, Hasan A A, Motta G, Schousboe I, Schmaier A H
Department of Medical Biochemistry and Genetics, The Panum Institute, University of Copenhagen, Denmark.
Thromb Haemost. 1998 Jul;80(1):74-81.
It is well known that on artificial surfaces, binding and autoactivation of factor XII (FXII) is the initiating event of plasma prekallikrein (PK) activation. We performed investigations to examine whether this mechanism was true for FXII activation on endothelial cells (HUVEC). Activation of PK on HUVEC required an optimal substrate and Zn2+ concentration, the latter of which varied with the buffer's carrier protein. Maximal PK activation required the addition of 250 microM or 10 microM Zn2+ to buffers containing bovine serum albumin (BSA) or gelatin, respectively. However, the actual free Zn2+ concentration in these buffers was the same at 8 microM. In both BSA- and gelatin-containing buffers and using two different chromogenic substrates for FXII, no autoactivation of FXII on HUVEC was seen when incubated for up to 60 min. Rather, initiation of FXII enzymatic activity required the presence of PK. FXII activation after PK activation contributed to the extent of measured enzymatic activity, but its role was secondary because treatment with corn trypsin inhibitor or a neutralizing antibody to FXIIa did not abolish the measured enzymatic activity. They also reduced the activity to the level seen with PK activation alone. Alternatively, soybean trypsin inhibitor abolished the proteolytic activity associated with PK and FXII activation on HUVEC. Further, only normal human and FXII-deficient plasmas, not PK-deficient plasma, had the ability to generate proteolytic activity when incubated over endothelial cells. In a purified system, maximal PK activation was measured after a 10-15 min incubation depending upon the concentration of reactants. When FXII was added with the PK, maximal activation occurred within 7.5-10 min. In normal human or FXII-deficient plasmas, but not in PK-deficient plasma, maximal activation was seen in 4 min. These data indicate that on HUVEC, unlike artificial surfaces, PK activation when bound to HK is the initiating activation event in this system. FXII activation is secondary to PK activation and contributes to the extent of measured enzymatic activity. These data challenge the accepted dogmas of "contact activation" and suggest that on biologic membranes a new notion as to how this system is activated needs to be considered.
众所周知，在人工表面上，因子 XII（FXII）的结合和自激活是血浆前激肽释放酶（PK）激活的起始事件。我们进行了研究，以检验这种机制在内皮细胞（HUVEC）上对 FXII 激活是否成立。HUVEC 上 PK 的激活需要最佳的底物和 Zn2+浓度，其中后者会随缓冲液的载体蛋白而变化。最大程度的 PK 激活分别需要向含有牛血清白蛋白（BSA）或明胶的缓冲液中添加 250 microM 或 10 microM Zn2+。然而，这些缓冲液中实际的游离 Zn2+浓度相同，均为 8 microM。在含有 BSA 和明胶的缓冲液中，使用两种不同的 FXII 显色底物，孵育长达 60 分钟时，未观察到 HUVEC 上 FXII 的自激活。相反，FXII 酶活性的起始需要 PK 的存在。PK 激活后 FXII 的激活对测得的酶活性程度有贡献，但其作用是次要的，因为用玉米胰蛋白酶抑制剂或针对 FXIIa 的中和抗体处理并未消除测得的酶活性。它们还将活性降低到仅 PK 激活时所见的水平。另外，大豆胰蛋白酶抑制剂消除了与 HUVEC 上 PK 和 FXII 激活相关的蛋白水解活性。此外，只有正常人血浆和 FXII 缺陷血浆，而不是 PK 缺陷血浆，在内皮细胞上孵育时具有产生蛋白水解活性的能力。在纯化系统中，根据反应物浓度，孵育 10 - 15 分钟后测得最大程度的 PK 激活。当将 FXII 与 PK 一起添加时，最大激活在 7.5 - 10 分钟内发生。在正常人或 FXII 缺陷血浆中，但不在 PK 缺陷血浆中，4 分钟时可见最大激活。这些数据表明，在 HUVEC 上，与人工表面不同，与 HK 结合时 PK 的激活是该系统中的起始激活事件。FXII 的激活继发于 PK 的激活，并对测得的酶活性程度有贡献。这些数据挑战了公认的“接触激活”教条，并表明在生物膜上需要考虑关于该系统如何被激活的新观念。
