Abstract
The role of the collagen-platelet interaction is of crucial importance to the haemostatic response during both injury and pathogenesis of the blood vessel wall. Of particular interest is the high affinity interaction of the platelet transmembrane receptor, alpha 2 beta 1, responsible for firm attachment of platelets to collagen at and around injury sites. We employ single molecule force spectroscopy (SMFS) using the atomic force microscope (AFM) to study the interaction of the I-domain from integrin alpha 2 beta 1 with a synthetic collagen related triple-helical peptide containing the high-affinity integrin-binding GFOGER motif, and a control peptide lacking this sequence, referred to as GPP. By utilising synthetic peptides in this manner we are able to study at the molecular level subtleties that would otherwise be lost when considering cell-to-collagen matrix interactions using ensemble techniques. We demonstrate for the first time the complexity of this interaction as illustrated by the complex multi-peaked force spectra and confirm specificity using control blocking experiments. In addition we observe specific interaction of the GPP peptide sequence with the I-domain. We propose a model to explain these observations.
Highlights
The interaction of collagen with platelets plays a pivotal role in the haemostatic response [1,2]
I-domain and a triple helical collagen peptide with the GFOGER binding motif using the technique of single molecule force spectroscopy
A decrease in unbinding probability when blocking from 20% to 14% for the GFOGER interaction and from 8% to 2% for the GPP
Summary
The interaction of collagen with platelets plays a pivotal role in the haemostatic response [1,2]. By pressing the cells onto the collagen substrate with a decreased force, they were able to reduce the contact area to the extent that they predict will permit measurement of the rupture of individual integrin receptor-substrate bonds They observed unimodal force spectra, indicated by the close fit to Gaussian distributions. These studies have identified the GFOGER peptide as being a high-affinity ligand for α2 β1 [19], as well as other sequences such as GLOGER, GMOGER and GAOGER of varying affinity [18,20,21] This may suggest that the unimodal force spectra observed reflects an average of several different, but presumably similar interactions of collagen with the receptor. We probed the interaction of the I-domain with both the GFOGER peptide, and a GPP peptide containing no guest residues at the single molecule level
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