整合素αⅡbβ3缺陷对血小板由内到外信号传导的影响

目的：探讨整合素αⅡbβ3缺陷对血小板由内到外信号传导的影响。方法：采用DNA序列分析、流式细胞术、RT－PCR和Western blotting等检测细胞中转染cDNA的存在、表达及细胞结合PAC－1、纤维蛋白原（fibrinogen,Fb)的能力。结果：血小板无力症（GT）患者整合素αⅡbβ3明显低下，且血小板在ADP激活后不能结合PAC－1，转染β3和αⅡb（突变αⅡb^R995A或正常αⅡb）cDNA的CHO细胞株分别表达αⅡb^R995Aβ3和αⅡbβ3，并且突变株中国仓鼠卵巢细胞（Chinese hamster ovary,CHO)细胞αⅡb基因30外显子上第3077和3078位碱基由CG突变为GC，导致第995位精氨酸被替换为丙氨酸；在未活化的情况下，αⅡbβ3 CHO细胞几乎不结合PAC－1，但能结合Fb，而αⅡb^R995Aβ3 CHO细胞能结合PAC－1，且Fb明显增加。结论：αⅡb^R995A突变能诱导血小板内到外信号传导，使αⅡb^R995Aβ3表现为激活型受体；而GT患者却因整合素αⅡbβ3缺陷使αⅡbβ3介导的血小板由内到外信号传导受阻，使该GT患者表现出血小板聚集异常。

Effect of the defect of integrin alpha II b beta 3 on the inside-out signal transduction in platelets]

OBJECTIVE: To investigate the effect of the defect of integrin alpha II b beta 3 on the inside-out signal transduction in platelets. METHODS: The transfected cDNA, its expression and the ability of cells binding to PAC-1 and fibrinogen were investigated by RT-PCR, DNA sequence analysis, flow cytometry and Western blotting. RESULTS: The integrin alpha II b beta 3 level in the patients with Glanzmann's thrombasthenia was significantly lower than that of the normal subjects and the platelets of the patients failed to bind PAC-1 activated by ADP. There were both C3077G and G3078C mutations in exon 30 of mutant alpha II bR995A beta 3 cDNA, which resulted in an amino acid substitution arginine (R) 995 to alanine (A). CHO cells transfected with wild-type alpha II b beta 3 or mutant alpha II bR995A beta 3 cDNA respectively expressed normal alpha II b beta 3 and mutant alpha II bR995A beta 3. When cells were not activated, wild-type alpha II b beta 3 CHO cells failed to bind PAC-1, but could adhere to fibrinogen, but mutant chimera alpha II bR995A beta 3 CHO cells were able to bind PAC-1 and exhibited high affinity binding fibrinogen. CONCLUSION: The point mutation in integrin alpha II bR995A can induce the inside-out signal transduction in platelets, and have integrin alpha II bR995A beta 3 locked into an activation state; the defect of alpha II b beta 3 in patients with GT impairs the inside-out signal transduction mediated by alpha II b beta 3 in platelets and the adhesive functions of platelets.