Purohit Vivek S, Ramani Karthik, Kashi Ramesh S, Durrani Manzer J, Kreiger Timothy J, Balasubramanian Sathyamangalam V
The Department of Pharmaceutical Sciences, University at Buffalo, State University of New York, 541 Cooke Hall, Amherst, NY 14260-1200, USA.
Biochim Biophys Acta. 2003 Oct 31;1617(1-2):31-8. doi: 10.1016/j.bbamem.2003.08.012.
Factor VIII (FVIII), a plasma glycoprotein, is an essential cofactor in the blood coagulation cascade. It is a multidomain protein, known to bind to phosphatidylserine (PS)-containing membranes. Based on X-ray and electron crystallography data, binding of FVIII to PS-containing membranes has been proposed to occur only via the C2 domain. Based on these models, the molecular topology of membrane-bound FVIII can be envisioned as one in which only a small fraction of the protein interacts with the membrane, whereas the majority of the molecule is exposed to an aqueous milieu. We have investigated the topology of the membrane-bound FVIII using biophysical and biochemical techniques. Circular dichroism (CD) and fluorescence studies indicate no significant changes in the secondary and tertiary structure of FVIII associated with the membranes. Acrylamide quenching studies show that the protein is predominantly present on the surface of the membrane, exposed to the aqueous milieu. The light scattering and electron microscopy studies indicate the absence of vesicle aggregation and fusion. Binding studies with antibodies directed against specific epitopes in the A1, A2 and C2 domains suggest that FVIII binds to the membrane primarily via C2 domain including the specific phospholipid binding epitope (2303-2332) and may involve subtle conformational changes in this epitope region.
凝血因子VIII（FVIII）是一种血浆糖蛋白，是血液凝固级联反应中必不可少的辅因子。它是一种多结构域蛋白，已知可与含磷脂酰丝氨酸（PS）的膜结合。根据X射线和电子晶体学数据，FVIII与含PS膜的结合被认为仅通过C2结构域发生。基于这些模型，膜结合FVIII的分子拓扑结构可设想为只有一小部分蛋白质与膜相互作用，而分子的大部分暴露于水相环境中。我们使用生物物理和生化技术研究了膜结合FVIII的拓扑结构。圆二色性（CD）和荧光研究表明，与膜相关的FVIII的二级和三级结构没有显著变化。丙烯酰胺猝灭研究表明，该蛋白主要存在于膜表面，暴露于水相环境中。光散射和电子显微镜研究表明不存在囊泡聚集和融合现象。针对A1、A2和C2结构域中特定表位的抗体结合研究表明，FVIII主要通过包含特定磷脂结合表位（2303 - 2332）的C2结构域与膜结合，并且可能涉及该表位区域的细微构象变化。
