PLATELET RECEPTOR RECOGNITION SITE ON HUMAN-FIBRINOGEN - SYNTHESIS AND STRUCTURE-FUNCTION RELATIONSHIP OF PEPTIDES CORRESPONDING TO THE CARBOXY-TERMINAL SEGMENT OF THE GAMMA-CHAIN
PLATELET RECEPTOR RECOGNITION SITE ON HUMAN-FIBRINOGEN - SYNTHESIS AND STRUCTURE-FUNCTION RELATIONSHIP OF PEPTIDES CORRESPONDING TO THE CARBOXY-TERMINAL SEGMENT OF THE GAMMA-CHAIN
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DOI：
10.1021/bi00303a028
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发表时间：
1984-01-01
期刊：
影响因子：
2.9
通讯作者：
HAWIGER, J
中科院分区：
文献类型：
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作者：
KLOCZEWIAK, M;TIMMONS, S;HAWIGER, J
纤维蛋白原与人血小板的结合取决于γ-链羧基末端片段与不同激动剂如ADP、肾上腺素和凝血酶暴露的特异性受体。一系列合成肽的功能包括γ-氨基丁酸的15个羧基末端残基的序列。链进行了研究。十五肽（γ. 397-411）和十二肽（γ 400 -411）抑制125 I-纤维蛋白原与ADP处理的血小板的结合，引起50%抑制的浓度（IC 50）为28 μ M。十肽（γ 402 -411）的活性几乎低4倍（IC 50 = 106 μ M），因此表明完全抑制作用需要2个组氨酸残基（γ 400 -401）。七肽（γ 405 -411）具有类似的作用（IC 50 = 102 μ M），而五肽（γ 407 -411）的抑制性甚至更低（IC 50 = 190 μ M），表明赖氨酸（γ 406）的缺乏进一步降低了γ上血小板识别位点的反应性。人纤维蛋白原链。含有2个组氨酸残基并通过胰蛋白酶蛋白水解降解从十二肽衍生的七肽（γ 400 -406）具有非常低的抑制活性。合成肽抑制纤维蛋白原支持的血小板聚集的反应性降低的顺序相同：十五肽=十二肽>十肽=七肽>五肽。制备氨基末端带有酪氨酸或半胱氨酰酪氨酸的修饰的合成十五肽，以提供用于放射性标记和形成更高化合价的分子的手段。酪氨酰-γ 397 -411和通过在2个单肽之间形成二硫键而获得的二聚体胱氨酸-（酪氨酰-γ 397 -411）2对血小板上的纤维蛋白原受体具有相同的抑制活性。放射性标记的酪氨酰-十五肽表现出与人血小板的特异性结合，其被十二肽（γ 400 -411）抑制。半胱氨酰-酪氨酰-γ 397 -411与人血清白蛋白的多价缀合物能够诱导ADP-刺激的血小板的聚集，其被十五肽（γ 397 -411）或十二肽（γ 397 -411）阻断。400-411）。针对该肽的单特异性抗体Fab片段，包括残基γ 385 -411，部分抑制合成十五肽-白蛋白缀合物的血小板聚集功能。因此，多价肽缀合物在血小板聚集系统中充当合成的纤维蛋白原替代物。总之，羧基末端的12个氨基酸残基的连续序列构成γ的血小板识别位点。人纤维蛋白原链。该片段与特异性血小板受体结合并参与血小板的聚集。
Binding of fibrinogen to human platelets depends on the interaction of the .gamma.-chain carboxy-terminal segment with specific receptors exposed by different agonists such as ADP, epinephrine and thrombin. The functions of a series of synthetic peptides encompassing the sequence of the 15 carboxy-terminal residues of the .gamma. chain were investigated in this study. Both pentadecapeptide (.gamma. 397-411) and dodecapeptide (.gamma.400-411) inhibited binding of 125I-fibrinogen to ADP-treated platelets, with the concentration causing 50% inhibition (IC50) being 28 .mu.M. Decapeptide (.gamma.402-411) was almost 4 times less active (IC50 = 106 .mu.M), thus suggesting that the 2 histidine residues (.gamma.400-401) are required for a full inhibitory effect. A heptapeptide (.gamma.405-411) had a similar effect (IC50 = 102 .mu.M) whereas a pentapeptide (.gamma.407-411) was even less inhibitory (IC50 = 190 .mu.M), indicating that the lack of lysine (.gamma.406) further diminishes the reactivity of the platelet recognition site on the .gamma. chain of human fibrinogen. The heptapeptide (.gamma.400-406) containing 2 histidine residues and derived from the dodecapeptide by proteolytic degradation with trypsin had very low inhibitory activity. The synthetic peptides inhibited fibrinogen-supported platelet aggregation in the same order of decreasing reactivity: pentadecapeptide = dodecapeptide > decapeptide = heptapeptide > pentapeptide. Modified synthetic pentadecapeptides bearing tyrosine or cysteinyltyrosine at the amino terminal were prepared to provide a means for radiolabeling and for formation of molecules of higher valency. Tyrosyl-.gamma.397-411 and the dimer cystinyl-(tyrosyl-.gamma.397-411)2 obtained by the formation of a disulfide bond between 2 single peptides had the same inhibitory activity toward the fibrinogen receptor on platelets. Radiolabeled tyrosyl-pentadecapeptide exhibited specific binding to human platelets which was inhibited by the dodecapeptide (.gamma.400-411). A polyvalent conjugate of cysteinyl-tyrosyl-.gamma.397-411 with human serum albumin was able to induce aggregating of ADP-stimulated platelets which was blocked by pentadecapeptide (.gamma.397-411) or dodecapeptide (.gamma.-400-411). Monospecific antibody Fab fragment directed against the peptide, encompassing residues .gamma.385-411, partially inhibited the platelet-aggregating function of the synthetic pentadecapeptide-albumin conjugate. Thus a polyvalent peptide conjugate functioned as a synthetic fibrinogen substitute in the platelet aggregation system. In conclusion, the continuous sequence of the 12 amino acid residues at the carboxy-terminal end constitutes the platelet recognition site for the .gamma. chain of human fibrinogen. This segment binds to specific platelet receptors and is involved in the aggregation of platelets.