Heparin modulates the conformation and signaling of platelet integrin 伪IIb尾3

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• 作者：Mayumi Yagi^a ; ^myagi@uw.edu ; Jacqueline Murray^a ; ^b ; Kurt Strand^a ; Scott Blystone^c ; Gianluca Interlandi^d ; Yasuo Suda^e ; Michael Sobel^a ; ^b
• 关键词：GlcNS6S-IdoA2S ; glucosamine N-sulfate (6-O-sulfate)-iduronic acid (2-O-sulfate) ; mAb ; monoclonal antibody ; PBS ; phosphate-buffered saline ; PRP ; platelet-rich plasma ; pY ; phosphotyrosine ; SPR ; surface plasmon resonance
• 刊名：Thrombosis Research
• 出版年：2012
• 期刊代码：247_00493848
• 类别：med
• 出版时间：June, 2012
• 卷：129
• 期：6
• 页码：743-749
• 文件大小：909 K

摘要

Introduction

The glycosaminoglycan heparin has been shown to bind to platelet integrin 伪IIb尾3 and induce platelet activation and aggregation, although the relationship between binding and activation is unclear. We analyzed the interaction of heparin and 伪IIb尾3 in detail, to obtain a better understanding of the mechanism by which heparin acts on platelets.

Methods

We assessed conformational changes in 伪IIb尾3 by flow cytometry of platelets exposed to unfractionated heparin. In human platelets and K562 cells engineered to express 伪IIb尾3, we assayed the effect of heparin on key steps in integrin signaling: phosphorylation of the 尾3 chain cytoplasmic tail, and activation of src kinase. We measured the heparin binding affinity of purified 伪IIb尾3, and of recombinant fragments of 伪IIb and 尾3, by surface plasmon resonance.

Results and conclusions

Heparin binding results in conformational changes in 伪IIb尾3, similar to those observed upon ligand binding. Heparin binding alone is not sufficient to induce tyrosine phosphorylation of the integrin 尾3 cytoplasmic domain, but the presence of heparin increased both 尾3 phosphorylation and src kinase activation in response to ligand binding. Specific recombinant fragments derived from 伪IIb bound heparin, while recombinant 尾3 did not bind. This pattern of heparin binding, compared to the crystal structure of 伪IIb尾3, suggests that heparin-binding sites are located in clusters of basic amino acids in the headpiece and/or leg domains of 伪IIb. Binding of heparin to these clusters may stabilize the transition of 伪IIb尾3 to an open conformation with enhanced affinity for ligand, facilitating outside-in signaling and platelet activation.