Lankhof H, Damas C, Schiphorst M E, Ijsseldijk M J, Bracke M, Furlan M, Tsai H M, de Groot P G, Sixma J J, Vink T
Department of Haematology, University Hospital Utrecht, The Netherlands.
Thromb Haemost. 1997 May;77(5):1008-13.
von Willebrand factor (vWF) is a complex multimeric plasma glycoprotein, that plays a critical role in the mediation of platelet adhesion to the damaged vascular wall, and functions as a carrier protein for factor VIII. vWF has a domain structure consisting of repeated A, B, C, and D domains. The A1 domain is involved in binding to the platelet receptor glycoprotein (GP) Ib, and the A3 domain has a binding site for collagen. A function of the A2 domain has not been described, although point mutations identified in von Willebrand disease (vWD) type 2A patients are localized in this domain. To study the role of the A2 domain a deletion mutant was constructed which lacked the A2 domain, delta A2-vWF. Previous studies have shown that this approach is a powerful tool to study the function of a domain in a protein since it does not affect the activity of other domains. After expression in baby hamster kidney (BHK) cells, delta A2-vWF was compared to wild-type (WT) vWF, and to delta A1-vWF (Lankhof et al., Blood 86: 1035, 1995). Ristocetin induced platelet binding was slightly increased but botrocetin induced platelet binding was normal as was binding to heparin and collagen type III. Adhesion studies to surface coated purified delta A2-vWF or to delta A2-vWF preincubated on collagen under flow conditions showed no abnormalities. Incubation with normal human plasma showed that delta A2-vWF like WT-vWF was not sensitive to proteolysis. After addition of urea, WT-vWF becomes sensitive to the protease, indicating that unfolding of the molecule is necessary for exposure of the cleavage site. delta A2-vWF tested under the same conditions was resistant, indicating that the protease sensitive site is located in the A2 domain.
血管性血友病因子（vWF）是一种复杂的多聚体血浆糖蛋白，在介导血小板黏附于受损血管壁过程中起关键作用，并作为因子VIII的载体蛋白发挥功能。vWF具有由重复的A、B、C和D结构域组成的结构域结构。A1结构域参与与血小板受体糖蛋白（GP）Ib的结合，A3结构域具有与胶原蛋白的结合位点。尽管在2A型血管性血友病（vWD）患者中鉴定出的点突变位于该结构域，但A2结构域的功能尚未明确。为了研究A2结构域的作用，构建了一个缺失A2结构域的缺失突变体，即ΔA2-vWF。先前的研究表明，这种方法是研究蛋白质中一个结构域功能的有力工具，因为它不会影响其他结构域的活性。在幼仓鼠肾（BHK）细胞中表达后，将ΔA2-vWF与野生型（WT）vWF以及ΔA1-vWF进行比较（Lankhof等人，《血液》86: 1035，1995）。瑞斯托霉素诱导的血小板结合略有增加，但博托霉素诱导的血小板结合正常，与肝素和III型胶原蛋白的结合也正常。在流动条件下对表面包被的纯化ΔA2-vWF或预孵育在胶原蛋白上的ΔA2-vWF进行的黏附研究未显示异常。与正常人血浆孵育表明，ΔA2-vWF与WT-vWF一样对蛋白水解不敏感。加入尿素后，WT-vWF对蛋白酶变得敏感，表明分子的展开对于切割位点的暴露是必要的。在相同条件下测试的ΔA2-vWF具有抗性，表明蛋白酶敏感位点位于A2结构域。
