Osterlund Maria, Persson Egon, Carlsson Uno, Freskgård Per-Ola, Svensson Magdalena
IFM-Department of Chemistry, Linköping University, SE-581 83 Linkoping, Sweden.
Biochem Biophys Res Commun. 2005 Dec 2;337(4):1276-82. doi: 10.1016/j.bbrc.2005.09.190. Epub 2005 Oct 10.
Vessel wall tissue factor (TF) is exposed to blood upon vascular damage which enables association with factor VIIa (FVIIa). This leads to initiation of the blood coagulation cascade through localization and allosteric induction of FVIIa procoagulant activity. To examine the docking pathway of the FVIIa-TF complex, various residues in the extracellular part of TF (sTF) that are known to interact with FVIIa were replaced with cysteines labelled with a fluorescent probe. By using stopped-flow fluorescence kinetic measurements in combination with surface plasmon resonance analysis, we studied the association of the resulting sTF variants with FVIIa. We found the docking trajectory to be a sequence of events in which the protease domain of FVIIa initiates contact with sTF. Thereafter, the two proteins are tethered via the first epidermal growth factor-like and finally the gamma-carboxyglutamic acid (Gla) domain. The two labelled sTF residues interacting with the protease domain of FVIIa bind or become eventually ordered at different rates, revealing kinetic details pertinent to the allosteric activation of FVIIa by sTF. Moreover, when the Gla domain of FVIIa is removed the difference in the rate of association for the remaining domains is much more pronounced.
血管壁组织因子（TF）在血管损伤时暴露于血液中，从而能够与因子VIIa（FVIIa）结合。这通过FVIIa促凝血活性的定位和变构诱导引发血液凝固级联反应。为了研究FVIIa-TF复合物的对接途径，将TF细胞外部分（sTF）中已知与FVIIa相互作用的各种残基替换为用荧光探针标记的半胱氨酸。通过结合使用停流荧光动力学测量和表面等离子体共振分析，我们研究了所得sTF变体与FVIIa的结合。我们发现对接轨迹是一系列事件，其中FVIIa的蛋白酶结构域开始与sTF接触。此后，这两种蛋白质通过第一个表皮生长因子样结构域，最终通过γ-羧基谷氨酸（Gla）结构域连接在一起。与FVIIa蛋白酶结构域相互作用的两个标记的sTF残基以不同的速率结合或最终变得有序，揭示了与sTF对FVIIa变构激活相关的动力学细节。此外，当去除FVIIa的Gla结构域时，其余结构域结合速率的差异更加明显。
