Glycoprotein (GP) VI dimer as a major collagen-binding site of native platelets: direct evidence obtained with dimeric GPVI-specific Fabs
Glycoprotein (GP) VI dimer as a major collagen-binding site of native platelets: direct evidence obtained with dimeric GPVI-specific Fabs
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DOI：
10.1111/j.1538-7836.2009.03496.x
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发表时间：
2009-08-01
影响因子：
10.4
通讯作者：
Moroi, M.
中科院分区：
文献类型：
--
作者：
Jung, S. M.;Tsuji, K.;Moroi, M.
背景：血小板胶原受体糖蛋白(GP)VI被认为是以二聚体形式存在于血小板表面，但尚无直接证据证明二聚体形成的功能重要性。目的：为了获得血小板膜上存在GPVI二聚体及其功能重要性的直接证据，通过噬菌体展示方法制备了与GPVI二聚体(GPVI-Fc(2))结合而不与GPVI单体(GPVIex)结合的Fab抗体。结果：发现6种Fab：B-F，仅与GPVI-Fc(2)反应；A，主要与GPVI-Fc(2)反应，与GPVIex有一定反应；每个Fab(Fab-dHLX-MH)通过两个Fab分子的dHLX结构域二聚形成二价二聚体(b-Fab)。通过删除Fab F的dHLX结构域，将其亚克隆为单价形式。所有的b-Fab都能诱导血小板聚集，但单体形式的Fab F(m-Fab-F)特异性地抑制胶原诱导的聚集。所有b-Fabs和m-Fab-F均抑制GPVI-Fc(2)与纤维性胶原的结合。免疫印迹显示b-Fab-F和m-Fab-F与GPVI-Fc(2)结合较弱。加入抗GPVI单抗204-11增加了m-Fab-F与GPVI-Fc(2)结合的Bmax，这表明204-11结合到尚未处于识别Fab的适当构象的GPVI-Fc(2)分子上，将它们转化为对Fab起反应的构象。结论：GPVI通过二聚化形成一种特殊的结构，该结构是该受体与胶原纤维结合所必需的。M-Fab-F与血小板的结合直接证明了GPVI作为一种功能相关的二聚体存在于血小板表面。
Background: The platelet collagen receptor glycoprotein (GP) VI is suggested to exist as a dimer on the platelet surface, but no direct proof of the functional importance of dimer formation has been provided. Objectives: To obtain direct evidence for GPVI dimers on the platelet membrane and their functional importance, Fab antibodies were developed that bind to GPVI dimer (GPVI-Fc(2)) but not to GPVI monomer (GPVIex) through a phage display method. Results: Ssix Fabs were found: B-F, only reactive with GPVI-Fc(2), and A, mainly reactive with GPVI-Fc(2), with some reactivity towards GPVIex; each Fab (Fab-dHLX-MH) forms a bivalent dimer (b-Fab) by dimerizing the dHLX domains from two Fab molecules. Fab F was subcloned to a monovalent format by deleting its dHLX domain. All b-Fabs induced platelet aggregation, but the monomeric form of Fab F (m-Fab-F) specifically inhibited collagen-induced aggregation. All b-Fabs and m-Fab-F inhibited GPVI-Fc(2) binding to fibrous collagen. Immunoblotting showed that b-Fab-F and m-Fab-F bound weakly to GPVI-Fc(2). Adding the anti-GPVI monoclonal antibody 204-11 increased the B-max of m-Fab-F binding to GPVI-Fc(2), suggesting that 204-11 binds to GPVI-Fc(2) molecules not already in the appropriate conformation to recognize the Fab, converting them to a conformation reactive to the Fab. Conclusions: GPVI forms a specific structure by dimerization that is necessary for the binding of this receptor to collagen fibrils. The binding of m-Fab-F to platelets directly demonstrates that GPVI is present as a functionally relevant dimer on the platelet surface.