Bajt M L, Ginsberg M H, Frelinger A L, Berndt M C, Loftus J C
Committee on Vascular Biology, Scripps Research Institute, La Jolla, California 92037.
J Biol Chem. 1992 Feb 25;267(6):3789-94.
This work characterizes a mutant integrin alpha IIb beta 3 (glycoprotein (GP) IIb-IIIa) from a thrombasthenic patient, ET, whose platelets fail to aggregate in response to stimuli. The nature of defect was defined by the reduced ability of synthetic peptide ligands, corresponding to the carboxyl terminus of the fibrinogen gamma chain (gamma 402-411) and Arg-Gly-Asp (RGD), to increase the binding of the occupancy-dependent anti-LIBS1 antibody to mutant alpha IIb beta 3 and the reduced binding of mutant alpha IIb beta 3 to an immobilized RGD peptide. In addition, ET's platelets failed to bind the ligand-mimetic monoclonal anti-alpha IIb beta 3, PAC1. DNA sequence analysis of amplified ET genomic DNA revealed a single G----A base change which encoded substitution of R214 by Q in mature beta 3. Introduction of this point mutation into recombinant wild type alpha IIb beta 3 expressed in Chinese hamster ovary cells reproduced the ET platelet alpha IIb beta 3 deficits in binding of fibrinogen, mAb PAC1, and synthetic peptide ligands. Furthermore, substitution of R214 by Q in the synthetic peptide containing the sequence of beta 3(211-222) resulted in decreased ability of this peptide to block fibrinogen binding to purified alpha IIb beta 3. These findings suggest that substitution of beta 3 R214 by Q is responsible for the functional defect in alpha IIb beta 3 and that R214 is proximal to or part of a ligand binding domain in alpha IIb beta 3.
这项研究对一名血小板无力症患者ET的突变整合素αIIbβ3（糖蛋白（GP）IIb-IIIa）进行了特征分析，该患者的血小板在受到刺激时无法聚集。通过合成肽配体（对应于纤维蛋白原γ链的羧基末端（γ402-411）和精氨酸-甘氨酸-天冬氨酸（RGD））增强占据依赖性抗LIBS1抗体与突变αIIbβ3结合的能力降低，以及突变αIIbβ3与固定化RGD肽结合能力降低，确定了缺陷的性质。此外，ET的血小板无法结合模拟配体的单克隆抗αIIbβ3抗体PAC1。对扩增的ET基因组DNA进行的DNA序列分析显示，有一个单一的G→A碱基变化，该变化编码成熟β3中R214被Q取代。将此点突变引入在中国仓鼠卵巢细胞中表达的重组野生型αIIbβ3，再现了ET血小板αIIbβ3在纤维蛋白原、单克隆抗体PAC1和合成肽配体结合方面的缺陷。此外，在包含β3（211-222）序列的合成肽中，R214被Q取代导致该肽阻断纤维蛋白原与纯化的αIIbβ3结合的能力下降。这些发现表明，β3的R214被Q取代是αIIbβ3功能缺陷的原因，并且R214靠近αIIbβ3中配体结合域或为其一部分。
