EXPRESSION OF RECOMBINANT PLATELET GLYCOPROTEIN IB/IX AND EFFECT OF POST-TRANSLATIONAL MODIFICATION ON ITS FUNCTIONS

血管性血友病因子（vWF）与其血小板受体之一糖蛋白（GP） Ib/IX复合物的相互作用触发血小板聚集，在血小板血栓形成的初始阶段起着关键的调节作用。为了描述这种相互作用的调节机制，我们在哺乳动物细胞中表达了重组GP Ib/IX蛋白，并研究了氨基酸取代或翻译后修饰的变化对受体功能的影响。(1)我们对血小板型血管性血友病患者的基因组DNA进行了分析，发现GPIb/IX基因异常，对vWF的亲和力异常高，在受体α链上发现了一个点突变。此外，我们还在CHO细胞中表达了突变蛋白，该蛋白具有该疾病的表型异常，与配体的相互作用增强。(2)用苯丙氨酸残基取代可能被硫酸盐酸化的三个酪氨酸残基导致受体vWF结合功能丧失。在阻断蛋白酪氨酸硫酸化的条件下，即在铬酸钠存在的情况下，CHO细胞中表达的重组蛋白损害了vWF的结合功能。(3)我们还改进了细胞培养技术，获得了大量的重组蛋白，因为这种可溶性重组蛋白可以抑制正常的vWF与血小板的结合，从而具有预防血小板血栓形成的潜在作用。我们的结果为动脉血栓形成的基本机制和新的抗血小板疗法的发展提供了重要的信息。

The interaction of von Willebrand factor (vWF) and its one of the platelet receptorsglycoprotein (GP) Ib/IX complex, triggers platelet aggregation and plays a key role in regulating the initial step of platelet thrombus formation. In order tocharacterize the mechanisms that regualate the interaction, we have expressed a recombinant GP Ib/IX protein in mammalian cells and investigated the effect of amino acid substitutions or the changes in post-translational modifications on the receptor function. (1) We have analyzed the genomic DNA of patients with platelet-type von Willebrand disease, which is characterized by an abberant GPIb/IX with abnormally high affinity for vWF,and have found a point mutation in the alpha chain of the receptor. Furthemory, we have expressed in CHO cells the mutant pretein, which possessed the phenotypic abnormality of the disorder, the enhenced interaction with the ligand. (2) Substitutiuon of potentially sulfated three tyrosine residues with phenylalanine residues resulted in the loss of vWF binding function of the rteceptor. Recombinant protein expressed in CHO cells cultured in a condition that blocks protein tyrosine sulfation, i. e., in the presence of sodium chrolate, had impaired vWF binding function. (3) We have also improved the cell culture technique to obtain large amount of the recombinant protein, since this soluble recombinant protein inhibits the normal vWF binding to platelets thus having the potential role in the prevention of platelet thrombus formation. Our results provides essential informations for the basic mechanisms of arterial thrombosis and for the development of newantiplatelet therapies.