Hoylaerts M F, Yamamoto H, Nuyts K, Vreys I, Deckmyn H, Vermylen J
Center for Molecular and Vascular Biology, University of Leuven, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium.
Biochem J. 1997 May 15;324 ( Pt 1)(Pt 1):185-91. doi: 10.1042/bj3240185.
Collagen VI is abundant in the arterial subendothelium. To investigate its mechanism of interaction with von Willebrand factor (vWF), collagen VI was isolated from human placenta and from the extracellular matrix of the human lung fibroblast cell line MRC-5. Purified vWF bound to non-digested collagen VI with moderately high affinity (EC50 approximately 5 nM) and could be inhibited by the Hirudo medicinalis collagen inhibitor calin. The anti-(human vWF A1 domain) monoclonal antibody (AJvW-2), as well as aurin tricarboxylic acid (ATA), at concentrations that saturate the vWF A1 domain, also inhibited this binding. In contrast, the monoclonal anti-(human vWF A3 domain) antibody (82D6A3) inhibited vWF binding to collagens I, III and IV, but had no effect on vWF binding to collagen VI. Likewise, vWF binding to collagen VI was not inhibited by the recombinant vWF domain D4. Polyclonal anti-(collagen VI) antibodies, specifically neutralizing the binding of vWF to collagen VI, confirmed that in the intact endothelial cell extracellular matrix, collagen VI was accessible for interaction with vWF. This binding was only marginally affected by 82D6A3 but was dose-dependently inhibited by AJvW-2, ATA and the A1 domain analogue VCL (recombinant A1 domain of vWF), with IC50 values comparable to those found for the inhibition of vWF binding to isolated collagen VI. The weak interaction of isolated human platelets with collagen VI was mediated via the platelet collagen receptor (GPIa/IIa) and was competitively inhibited by vWF but not by VCL, suggesting that vWF and GPIa/IIa bind to neighbouring but distinct sites on collagen VI. We conclude that vWF binds to collagen VI primarily via its A1 domain, which distinguishes it from the vWF A3 domain-mediated binding to fibrillar collagens.
胶原蛋白VI在动脉内皮下层含量丰富。为了研究其与血管性血友病因子（vWF）相互作用的机制，从人胎盘以及人肺成纤维细胞系MRC - 5的细胞外基质中分离出胶原蛋白VI。纯化的vWF以中等偏高的亲和力（EC50约为5 nM）与未消化的胶原蛋白VI结合，并且可被水蛭胶原蛋白抑制剂卡林抑制。抗（人vWF A1结构域）单克隆抗体（AJvW - 2）以及在饱和vWF A1结构域的浓度下的金精三羧酸（ATA）也抑制这种结合。相比之下，抗（人vWF A3结构域）单克隆抗体（82D6A3）抑制vWF与I型、III型和IV型胶原蛋白的结合，但对vWF与胶原蛋白VI的结合没有影响。同样，vWF与胶原蛋白VI的结合也未被重组vWF结构域D4抑制。多克隆抗（胶原蛋白VI）抗体特异性中和vWF与胶原蛋白VI的结合，证实了在完整的内皮细胞外基质中，胶原蛋白VI可与vWF相互作用。这种结合仅受到82D6A3的轻微影响，但被AJvW - 2、ATA和A1结构域类似物VCL（vWF的重组A1结构域）剂量依赖性地抑制，其IC50值与抑制vWF与分离的胶原蛋白VI结合时的值相当。分离的人血小板与胶原蛋白VI的弱相互作用是通过血小板胶原蛋白受体（GPIa/IIa）介导的，并且被vWF竞争性抑制，但未被VCL抑制，这表明vWF和GPIa/IIa结合在胶原蛋白VI上相邻但不同的位点。我们得出结论，vWF主要通过其A1结构域与胶原蛋白VI结合，这使其与vWF A3结构域介导的与纤维状胶原蛋白的结合有所不同。
