Vascular Biology Laboratory, Baker Medical Research Institute, Prahran, Victoria, Australia.
Platelets. 1995;6(5):245-51. doi: 10.3109/09537109509023562.
von Willebrand Factor (vWF) is a multifunctional glycoprotein in plasma and vascular subendothelial matrix which plays a major role in cellular adhesion. vWFdependent adhesion of platelets to the subendothelium at high shear rates involves a specific platelet membrane receptor, the glycoprotein (GP) Ib-IX complex. We have previously purified a 39/34-kiloDalton (kDa) dispase fragment of vWF (Leu-480/Val-481 to Gly-718) and demonstrated that this fragment contains the binding site for the GP Ib-IX complex [Andrews R K, et al. Biochemistry 1989; 28: 8326-83361. vWF also mediates agglutination of erythrocytes by a mechanism that appears to involve binding to membrane sulfatides. In this study, we demonstrate that the 39/34-kDa vWF fragment also contains an exclusive discrete binding domain for membrane sulfatides and that the sulfatide-binding sequence also mediates binding of vWF to equine tendon collagen. Specific binding of (125)I-vWF to sulfatides immobilized on microtiter wells was completely inhibited by unlabeled vWF (IC(50)∼0.02 μ;M) and by the isolated 39/34-kDa vWF fragment (IC(50)∼0.8 μ;M). A specific anti-39/34-kDa fragment rabbit polyclonal antibody, but not nonimmune immunoglobulin, also strongly inhibited the vWF-sulfatide interaction in this assay. Using synthetic peptides corresponding to hydrophilic sequences from within the 39/34-kDa vWF fragment, a positively-charged sequence, Gln-628 to Val-646, was identified as mediating specific binding of vWF to sulfatides, since it competitively inhibited this interaction (IC(50)∼0.6 μ;M) comparable on a molar basis to the 39/34-kDa vWF fragment (IC, -0.8 μ;M). The inhibition by the Gln-626 to Val-646 peptide was specific since neither other peptides from the 39/34-kDa domain of vWF nor another highly basic peptide, polylysine, at comparable concentrations to the Gln-628 to Val-646 peptide blocked vWF binding to sulfatides. Similarly, the Gln-628 to Val-646 peptide blocked binding of vWF to equine tendon type I collagen (IC(50) of 0.6 μ;M) suggesting that this interaction probably involves recognition of a sulfatide-like impurity in the collagen preparation. The specific binding of vWF to sulfatides via a discrete peptide sequence, Gln-628 to Val-646, within the A1 repeat domain suggests the potential for involvement of sulfatides as a class of receptors for vWF in cellular adhesion.
血管性血友病因子（vWF）是血浆和血管内皮基质中的一种多功能糖蛋白，在细胞黏附中起主要作用。vWF 依赖的血小板在高剪切率下与血管内皮的黏附涉及特定的血小板膜受体，糖蛋白（GP）Ib-IX 复合物。我们之前已经纯化了 vWF 的一个 39/34 千道尔顿（kDa）糜蛋白酶片段（Leu-480/Val-481 到 Gly-718），并证明该片段包含与 GP Ib-IX 复合物的结合位点[Andrews R K，等。生物化学 1989；28：8326-8336]。vWF 还通过一种似乎涉及与膜硫酸脑苷脂结合的机制介导红细胞的聚集。在这项研究中，我们证明 39/34 kDa vWF 片段还包含一个用于膜硫酸脑苷脂的独特离散结合域，并且硫酸脑苷脂结合序列也介导 vWF 与马腱型 I 胶原的结合。（125）I-vWF 与固定在微量滴定孔中的硫酸脑苷脂的特异性结合完全被未标记的 vWF（IC50∼0.02 μ;M）和分离的 39/34 kDa vWF 片段（IC50∼0.8 μ;M）抑制。一种特异性抗 39/34 kDa 片段兔多克隆抗体，但不是非免疫球蛋白，也强烈抑制了在该测定中的 vWF-硫酸脑苷脂相互作用。使用对应于 39/34 kDa vWF 片段内亲水性序列的合成肽，鉴定到一个带正电荷的序列，Gln-628 到 Val-646，作为介导 vWF 与硫酸脑苷脂特异性结合的序列，因为它竞争性地抑制了这种相互作用（IC50∼0.6 μ;M），与 39/34 kDa vWF 片段（IC50∼0.8 μ;M）相当。由于其他来自 vWF 的 39/34 kDa 结构域的肽或另一种高度碱性肽，聚赖氨酸，在与 Gln-628 到 Val-646 肽相当的浓度下，不能阻断 vWF 与硫酸脑苷脂的结合，因此抑制是特异性的。类似地，Gln-628 到 Val-646 肽阻断了 vWF 与马腱型 I 胶原的结合（IC50 的 0.6 μ;M），表明这种相互作用可能涉及到胶原制备中硫酸脑苷脂样杂质的识别。通过位于 A1 重复结构域内的离散肽序列 Gln-628 到 Val-646，vWF 与硫酸脑苷脂的特异性结合提示硫酸脑苷脂作为 vWF 在细胞黏附中的一类受体的潜在作用。
