MECHANISMS OF PLATELET ACTIVATION BY A STIMULATORY ANTIBODY - CROSS-LINKING OF A NOVEL PLATELET RECEPTOR FOR MONOCLONAL-ANTIBODY F11 WITH THE FC-GAMMA-RII RECEPTOR
MECHANISMS OF PLATELET ACTIVATION BY A STIMULATORY ANTIBODY - CROSS-LINKING OF A NOVEL PLATELET RECEPTOR FOR MONOCLONAL-ANTIBODY F11 WITH THE FC-GAMMA-RII RECEPTOR
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DOI：
10.1042/bj3100155
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发表时间：
1995-08-15
影响因子：
4.1
通讯作者：
KORNECKI, E
中科院分区：
文献类型：
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作者：
NAIK, UP;EHRLICH, YH;KORNECKI, E
刺激性单克隆抗体（mAb）（称为mAb F11）诱导人血小板颗粒分泌和聚集的机制已得到表征。发现mAb F11的Fab片段以及针对血小板Fc γ RII受体的mAb（mAb IV.3）可抑制mAb F11诱导的血小板分泌和聚集，表明mAb F11 IgG分子通过其Fc结构域与Fc γ RII受体相互作用，并通过其Fab结构域与其自身抗原相互作用。F11单克隆抗体识别血小板膜表面的两个血小板蛋白的32和35 kDa，通过Western印迹分析确定。我们使用DEAE-Sepharose层析，然后使用mAb F11亲和层析从人血小板膜中纯化这两种蛋白。当添加到富血小板血浆中时，纯化的蛋白质剂量依赖性地抑制mAb F11诱导的血小板聚集。纯化的蛋白质制剂也竞争性抑制I-125标记的mAb F11与完整血小板的结合。32和35 kDa蛋白质的N-末端26个氨基酸序列是相同的，并且在N-末端位置含有单个未封闭的丝氨酸。当用N-聚糖酶消化时，32和35 kDa的蛋白质被转化成类似于29 kDa的单一蛋白质，表明这两种蛋白质来源于相同的核心蛋白质，但其糖基化程度不同。F11抗原的内部氨基酸序列分析提供了关于68个氨基酸的信息，并提出了蛋白激酶C（PKC）的两个共识磷酸化位点。用佛波醇12-肉豆蔻酸酯13-乙酸酯刺激分离的F11抗原后，观察到PKC的磷酸化。F11抗原的N-末端和内部氨基酸序列的数据库分析表明，N-末端序列与人T细胞受体（TCR）α链可变区具有最高程度的相似性。相反，F11内部序列与TCR没有任何相似性。我们的研究结果表明，F11抗原是一种新型的血小板膜表面糖蛋白，当血小板被刺激性mAb F11激活时，它与Fc γ RII受体交联。这些机制可能与血小板活化抗体产生免疫性血小板减少症有关。
The mechanisms by which a stimulatory monoclonal antibody (mAb), called mAb F11, induces granular secretion and aggregation in human platelets have been characterized. Fab fragments of mAb F11, as well as an mAb directed against the platelet Fc gamma RII receptor (mAb IV.3) were found to inhibit mAb F11-induced platelet secretion and aggregation, indicating that the mAb F11 IgG molecule interacts with the Fc gamma RII receptor through its Fc domain and with its own antigen through its Fab domain. The mAb F11 recognized two platelet proteins of 32 and 35 kDa on the platelet membrane surface, as identified by Western blot analysis. We purified both proteins from human platelet membranes using DEAE-Sepharose chromatography followed by mAb F11 affinity chromatography. When added to platelet-rich plasma, the purified proteins dose-dependently inhibited mAb F11-induced platelet aggregation. The purified protein preparation also competitively inhibited the binding of I-125-labelled mAb F11 to intact platelets. The N-terminal 26 amino acid sequences of both the 32 and 35 kDa proteins were identical and contained a single unblocked serine in the N-terminal position. When digested with N-glycanase, the 32 and 35 kDa proteins were converted into a single similar to 29 kDa protein, indicating that these two proteins are derived from the same core protein but differ in their degree of glycosylation. Internal amino acid sequence analysis of the F11 antigen provided information concerning 68 amino acids and suggested two consensus phosphorylation sites for protein kinase C (PKC). The phosphorylation by PKC of the isolated F11 antigen was observed following stimulation by phorbol 12-myristate 13-acetate. Databank analysis of the N-terminal and internal amino acid sequences of the F11 antigen indicated that the N-terminal sequence exhibited the highest degree of similarity to the variable region of the alpha-chain of human T-cell receptors (TCR). In contrast, the F11 internal sequences did not exhibit any similarity to the TCR. Our results demonstrate that the F11 antigen is a novel platelet membrane surface glycoprotein which becomes cross-linked with the Fc gamma RII receptor when platelets are activated by the stimulatory mAb F11. These mechanisms may be relevant to the production of immune thrombocytopenia by platelet-activating antibodies.