Establishment of the stable cell line expressing human platelet GPIb/IX receptor and its utilization for platelet function

血小板GPIb/IX复合体是von Willebrand因子(VWF)的表面受体，由GPIB-α、-β和IX三个独立的基因产物组成。这些成分连接在一起，形成vWF的功能性受体。到目前为止，对Bernard-Soulier综合征患者的遗传分析表明，GPIB-α中的富含亮氨酸重复序列(LRR)对于该复合体的有效表面表达是必不可少的。LRR是一个含有保存完好的亮氨酸残基的24个氨基酸片段，每个GP至少拥有一个这个共识基序。然而，由于缺乏临床病例，人们对LRR在GPIB-beta中的重要性知之甚少。为了评估GPIB-βLRR对GPIB/IX复合体表达的重要性，我们选择了GPIB-βLRR中的四个氨基酸残基，并进行定点突变以获得一系列突变体：Leu35、Leu45和Leu50→Ala、Val或Phe、Asn40→Ala、Thr或Cys。OBTAI…用GPIb-α和Gpix共转染更多的Ned载体，用流式细胞仪和Northern blotting分析GPIb/IX复合体的表达，I.FACS分析：所有突变型Asn40-突变体都显著降低了复合体的表面表达。用Leu→Ala或Phe突变体也得到了类似的结果，但是当用FACS分析时，Leu→val突变体的表面总有40-80%的复合体表达。这些数据表明，GPIB-β的LRR中的亮氨酸残基和Asn40对于GPIB/IX复合体的高效表达是重要的，尽管Leu→Val突变体表现出中等的复合体表达，这可能是由于两者的结构相似。III.人血小板GPIB/IX受体稳定转化子的建立：我们利用上述载体建立了稳定的转化子。转化子具有类似人血小板GPIb/IX复合体的血小板样聚集性。然而，经过长期的培养，他们逐渐失去了这种活力。为了保持转化子的聚合潜力，一些修饰是必要的。较少

The platelet GPIb/IX complex, a surface receptor for von Willebrand factor (vWF), is composed of three-independent gene products of GPIb-alpha, -beta and IX. Those components linked together and form a functional receptor for vWF. So far, genetic analyses of the patients with Bernard-Soulier syndrome have shown that the leucine rich repeat (LRR) in the GPIb-alpha is essential for an efficient surface expression of the complex. LRR is a short 24 amino acid stretch containing well preserved leucine residues, and each GP possesses at least one of this consensus motif. Little is known, however, about the importance of the LRR within GPIb-beta because of the absence of the clinical case. To assess the importance of the GPIb-beta LRR for the expression of GPIb/IX complex, four amino acid residues within the LRR of the GPIb-beta have been chosen and site-directed mutagenesis was performed to obtain series of mutants; Leu35, Leu45 and Leu50 → Ala, Val or Phe, Asn40 → Ala, Thr or Cys. The obtai … More ned plasmids were co-transfected with GPIb-alpha and GPIX, and the expression of GPIb/IX complex was analyzed by FACS and Northern blotting.I. FACS analyses of the GPIb/IX receptor transfected with the mutant plasmids: All Asn40-mutants drastically decreased the surface expression of the complex. Similar results were obtained by using Leu → Ala or Phe mutants, however, Leu → Val mutants always showed 40-80% surface expression of the complex when analyzed by FACS.II. Northern blot analyses of the mutants: Northern blotting analyses showed that GPIb-beta mRNA level of the mutants were almost the same as control, indicating post-transcriptional events were causative for the decreased receptor expression. These data have suggested that Leu residues and Asn40 within the LRR of the GPIb-beta are important for the efficient expression of the GPIb/IX complex, although Leu → Val mutants showed moderate complex expression probably due to the structural similarity of both amino acids.III. Establishment of stable transformant expressing human platelet GPIb/IX receptor: We have utilized the above described plasmids to establish the stable transformants. The transformants possess the platelet-like aggregatory potential by ristocetin, mimic the human platelet GPIb/IX complex. However, they gradually lost the activity by long-term cultivation. Some modifications are necessary to maintain the aggregatory potential of the transformants. Less