Isolation and characterization of a collagen binding domain in human von Willebrand factor.
Isolation and characterization of a collagen binding domain in human von Willebrand factor.
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DOI：
10.1016/s0021-9258(18)66869-3
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发表时间：
1986-11
期刊：
影响因子：
--
通讯作者：
F. Pareti;Y. Fujimura;J. Dent;L. Holland;T. Zimmerman;Z. Ruggeri
中科院分区：
文献类型：
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作者：
F. Pareti;Y. Fujimura;J. Dent;L. Holland;T. Zimmerman;Z. Ruggeri
血管性血友病因子以快速、温度非依赖性、可逆、特异性和可饱和的方式与纤维状I型胶原结合。通过Scatchard型分析评价结合等温线表明，每毫克胶原蛋白结合6-18微克血管性血友病因子，Ka在2和8 × 10（8）M-1之间。五个不同的胰蛋白酶片段，在变性和还原条件下纯化，并代表超过75%的分子量的血管性血友病因子亚基，测试其抑制血管性血友病因子-胶原蛋白相互作用的能力。用浓度约为1 μ M的52/48-kDa片段获得完全抑制。该片段在亚基中的位置被确定为在瓦尔-449和Lys-728之间。针对52/48-kDa片段的15种单克隆抗体抑制血管性血友病因子与胶原的结合。六个抗体对其他部分的血管性血友病因子亚基没有抑制作用。胰蛋白酶片段是血管性血友病因子与胶原蛋白结合的竞争性抑制剂，因此，识别与完整分子相同的相互作用位点。这些研究精确地定义了血管性血友病因子亚基中与I型胶原相互作用的结构域。
von Willebrand factor binds to fibrillar type I collagen in a rapid, temperature-independent, reversible, specific, and saturable manner. Evaluation of binding isotherms by Scatchard-type analysis demonstrated that 6-18 micrograms of von Willebrand factor bind per mg of collagen, with Ka between 2 and 8 X 10(8) M-1. Five distinct tryptic fragments, purified under denaturing and reducing conditions and representing over 75% of the molecular mass of the von Willebrand factor subunit, were tested for their capacity to inhibit the von Willebrand factor-collagen interaction. Complete inhibition was obtained with a 52/48-kDa fragment at a concentration of approximately 1 microM. The location of this fragment in the subunit was established to be between Val-449 and Lys-728. Fifteen monoclonal antibodies against the 52/48-kDa fragment inhibited von Willebrand factor binding to collagen. Six antibodies against other portions of the von Willebrand factor subunit had no inhibitory effect. The tryptic fragment was a competitive inhibitor of von Willebrand factor binding to collagen and, therefore, recognizes the same interaction site as the intact molecule. These studies precisely define a domain in the von Willebrand factor subunit that interacts with type I collagen.