Abrams C S, Ruggeri Z M, Taub R, Hoxie J A, Nagaswami C, Weisel J W, Shattil S J
Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104.
J Biol Chem. 1992 Feb 5;267(4):2775-85.
Binding of the adhesive ligand fibrinogen and the monoclonal antibody PAC1 to platelet glycoprotein (GP) IIb-IIIa is dependent on cell activation and inhibited by Arg-Gly-Asp (RGD)-containing peptides. Previously, we identified a sequence in a hypervariable region of PAC1 (mu-CDR3) that mimics the activity of the antibody. Here we examine whether monoclonal antibodies to this idiotypic determinant in PAC1 can mimic GP IIb-IIIa by binding to fibrinogen. Mice were immunized with a peptide derived from the mu-CDR3 of PAC1. Four antibodies were obtained that recognized fibrinogen as well as a recombinant form of the variable region of PAC1. However, they did not bind to other RGD-containing proteins, including von Willebrand factor, fibronectin, and vitronectin. Several studies suggested that these anti-PAC1 peptide antibodies were specific for GP IIb-IIIa recognition sites in fibrinogen. Three such sites have been proposed: two RGD-containing regions in the A alpha chain, and the COOH terminus of the gamma chain (gamma 400-411). Two of the antibodies inhibited fibrinogen binding to activated platelets, and all four antibodies bound to the fibrinogen A alpha chain on immunoblots. Antibody binding to immobilized fibrinogen was partially inhibited by monoclonal antibodies specific for the two A alpha chain RGD regions. However, the anti-PAC1 peptide antibodies also bound to plasmin-derived fibrinogen fragments X and D100, which contain gamma 400-411 but lack one or both A alpha RGD regions. This binding was inhibited by an antibody specific for gamma 400-411. When fragment D100 was converted to D80, which lacks gamma 400-411, antibody binding was reduced significantly (p less than 0.01). Electron microscopy of fibrinogen-antibody complexes confirmed that each antibody could bind to sites on the A alpha and gamma chains. These studies demonstrate that certain anti-PAC1 peptide antibodies mimic GP IIb-IIIa by binding to platelet recognition sites in fibrinogen. Furthermore, they suggest that the gamma 400-411 region of fibrinogen may exist in a conformation similar to that of an A alpha RGD region of the molecule.
黏附配体纤维蛋白原和单克隆抗体PAC1与血小板糖蛋白（GP）IIb-IIIa的结合依赖于细胞活化，并受到含精氨酸-甘氨酸-天冬氨酸（RGD）的肽的抑制。此前，我们在PAC1的一个高变区（μ-CDR3）中鉴定出一个模拟该抗体活性的序列。在此，我们研究针对PAC1中该独特型决定簇的单克隆抗体是否能通过与纤维蛋白原结合来模拟GP IIb-IIIa。用源自PAC1的μ-CDR3的肽免疫小鼠。获得了四种能识别纤维蛋白原以及PAC1可变区重组形式的抗体。然而，它们不与其他含RGD的蛋白结合，包括血管性血友病因子、纤连蛋白和玻连蛋白。多项研究表明，这些抗PAC1肽抗体对纤维蛋白原中GP IIb-IIIa识别位点具有特异性。已提出三个这样的位点：Aα链中的两个含RGD区域，以及γ链的羧基末端（γ400-411）。其中两种抗体抑制纤维蛋白原与活化血小板的结合，并且所有四种抗体在免疫印迹中均与纤维蛋白原Aα链结合。针对两个Aα链RGD区域的单克隆抗体部分抑制了抗体与固定化纤维蛋白原的结合。然而，抗PAC1肽抗体也与纤溶酶衍生的纤维蛋白原片段X和D100结合，这些片段含有γ400-411但缺少一个或两个Aα RGD区域。这种结合被针对γ400-411的特异性抗体抑制。当片段D100转化为缺少γ400-411的D80时，抗体结合显著减少（p<0.01）。纤维蛋白原-抗体复合物的电子显微镜检查证实，每种抗体都能与Aα链和γ链上的位点结合。这些研究表明，某些抗PAC1肽抗体通过与纤维蛋白原中的血小板识别位点结合来模拟GP IIb-IIIa。此外，它们提示纤维蛋白原的γ400-411区域可能以与该分子Aα RGD区域相似的构象存在。
