The Residues AGDV of Recombinant γ Chains of Human Fibrinogen Must Be Carboxy-Terminal to Support Human Platelet Aggregation
The Residues AGDV of Recombinant γ Chains of Human Fibrinogen Must Be Carboxy-Terminal to Support Human Platelet Aggregation
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DOI：
10.1055/s-0038-1646347
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发表时间：
1992-12
影响因子：
6.7
通讯作者：
J. Hettasch;M. Bolyard;S. Lord
中科院分区：
文献类型：
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作者：
J. Hettasch;M. Bolyard;S. Lord
纤维蛋白原γ链的羧基末端含有一个序列，据信该序列是与糖蛋白（GP）IIb/IIIa相互作用以支持血小板聚集的结构域之一。存在纤维蛋白原的正常变体，其中4个羧基末端氨基酸被20个氨基酸取代。据报道，这种变体称为“β”，与血小板的结合效率较低。本研究的目的是设计新的蛋白质，以确定γ和γ'链之间的氨基酸序列差异影响羧基端与GPIIb/IIIa的相互作用。在这方面，通过寡核苷酸定向突变来修饰细菌质粒表达载体中的γ链cDNA，以产生羧基末端氨基酸发生变化的重组γ链，这反映了γ和γ '之间的差异。羧基端未修饰的重组γ链与完整的纤维蛋白原在相同程度上支持腺苷二磷酸（ADP）诱导的血小板聚集。相反，γ' 427（重组γ'变体）和γ 427（其中16个氨基酸的γ'延伸[412-427]被添加到γ的羧基末端）支持血小板聚集的能力显著降低。此外，在γ' 411（其中γ中的氨基酸408 - 411被γ'中的氨基酸408 - 411替换）存在下，ADP诱导的血小板聚集的程度降低，而γ 407（其中4个羧基末端氨基酸缺失）不能支持聚集。这些发现表明，四个残基AGDV不仅是必需的，而且必须是羧基末端，以支持血小板聚集。
Summary The carboxy-terminus of the γ chain of fibrinogen contains a sequence which is believed to be one of the domains that interacts with glycoprotein (GP) IIb/IIIa to support platelet aggregation. A normal variant of fibrinogen exists in which the four carboxy-terminal amino acids are replaced by 20 amino acids. This variant, known as γ’, has been reported to bind less effectively to platelets. The purpose of the present study was to engineer novel proteins to determine what differences in amino acid sequence between the γ and γ’ chains influence the interaction of the carboxyterminus with GPIIb/IIIa. In this regard, the γ chain cDNA in a bacterial plasmid expression vector was modified by oligonucleotide-directed mutagenesis to produce recombinant γ chains with amino acid changes in the carboxy-terminus which reflect the differences between γ and γ’. The recombinant γ chain with an unmodified carboxy-terminus supported adenosine diphosphate (ADP)-induced platelet aggregation to the same extent as intact fibrinogen. In contrast, the ability of γ’ 427 (the recombinant γ’ variant) and γ 427 (where the 16 amino acid γ’ extension [412–427] was added to the carboxy-terminus of γ) to support platelet aggregation was markedly reduced. In addition, the extent of ADP-induced platelet aggregation was decreased in the presence of γ’ 411 (where amino acids 408–411 in γ were replaced with amino acids 408–411 in γ’), while γ 407 (where the four carboxy-terminal amino acids were deleted) was not capable of supporting aggregation. These findings demonstrate that the four residues AGDV are not only required but must be carboxy-terminal to support platelet aggregation.