凝血因子VIII与低密度脂蛋白受体家族的几个成员相互作用,包括低密度脂蛋白受体相关蛋白,低密度脂蛋白受体,和极低密度脂蛋白受体。本研究旨在比较VIII因子与低密度脂蛋白受体相关蛋白相互作用的机制。megalin,低密度脂蛋白受体,和极低密度脂蛋白受体,以揭示这些相互作用的一般模式。在固相和表面等离子体共振测定中研究了血浆衍生因子VIII及其片段与低密度脂蛋白受体(sLDLR1-7)的重组可溶性配体结合域和纯化的megalin的结合。全长因子VIII及其轻链以相似的亲和力与受体结合(KD=260+/-9和156+/-4nmol/l,分别,对于megalin和KD=210+/-3和174+/-13nmol/l,分别,对于sLDLR1-7)。血管性血友病因子抑制因子VIII与两种受体的结合。与轻链相反,重链内高亲和力受体结合位点的暴露(对于megalin,KD=22/-4nmol/l,对于sLDLR1-7,KD=17/-3nmol/l)需要凝血酶的蛋白水解裂解。基于抗A2单克隆抗体413的抑制作用,该位点被定位到A2结构域残基484-509,并且由所有四种受体共享。使用一组A2突变体,我们确定了关键氨基酸残基-带正电荷的K466,R471,R489和R490,以及亲水残基Y487和S488-它们形成了该“共有”结合位点的框架。我们得出结论,VIII因子与低密度脂蛋白受体家族成员的相互作用遵循一般模式,需要因子VIII与血管性血友病因子分离,并且是激活敏感的。
Coagulation factor VIII interacts with several members of the low-density lipoprotein receptor family including low-density lipoprotein receptor-related protein, low-density lipoprotein receptor, and very low-density lipoprotein receptor. The present study was aimed to compare the mechanisms of factor VIII interaction with low-density lipoprotein receptor-related protein, megalin, low-density lipoprotein receptor, and very low-density lipoprotein receptor in order to reveal a general mode of these interactions. Binding of plasma-derived factor VIII and its fragments to recombinant soluble ligand-binding domain of low-density lipoprotein receptor (sLDLR1-7) and purified megalin was studied in solid phase and surface plasmon resonance assays. Full-length factor VIII and its light chain bound to the receptors with similar affinities (KD = 260 +/- 9 and 156 +/- 4 nmol/l, respectively, for megalin and KD = 210 +/- 3 and 174 +/- 13 nmol/l, respectively, for sLDLR1-7). Von Willebrand factor inhibited factor VIII binding to both receptors. In contrast to the light chain, exposure of the high-affinity receptor-binding site within the heavy chain (KD = 22 +/- 4 nmol/l for megalin and 17 +/- 3 nmol/l for sLDLR1-7) required proteolytic cleavage by thrombin. This site was mapped to the A2 domain residues 484-509, based on the inhibitory effects of anti-A2 monoclonal antibody 413, and is shared by all four receptors. Using a panel of A2 mutants, we identified key amino acid residues- positively charged K466, R471, R489 and R490, and hydrophilic residues Y487 and S488- which form the frame of this \'
consensus\' binding site. We conclude that interaction of factor VIII with the members of the low-density lipoprotein receptor family follows the general mode, requires dissociation of factor VIII from von Willebrand factor, and is activation sensitive.