Congress of the International Society on Thrombosis and Haemostasis

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Strategy for the use of single molecule force spectroscopy to investigate binding of integrin integrin alpha(2)beta(1) and collagen

Abstract number: PP-MO-058

Simpson¹ A.M.C., Attwood² S.J., Bihan¹ D., Hamaia¹ S.W., Welland² M.E., Farndale¹ R.W.

¹¹Department of Biochemistry ²²Nanoscience Centre, University of Cambridge, Cambridge, UK

How-to-cite Simpson AMC, Attwood SJ, Bihan D, Hamaia SW, Welland ME, Farndale RW. Strategy for the use of single molecule force spectroscopy to investigate binding of integrin integrin alpha(2)beta(1) and collagen. Journal of Thrombosis and Haemostasis 2009; Volume 7, Supplement 2: Abstract PP-MO-058

The interaction between platelets and collagen is essential in haemostasis. Adhesion via integrin alpha2beta1 is a key component in this interaction. Single molecule force spectroscopy using the atomic force microscope is a powerful tool that can be used to investigate the force of unbinding of a ligand and its receptor. Recombinant alpha2 I-domain and a synthetic triple-helical collagen-like peptide containing the high affinity recognition motif GFOGER were used to investigate integrin-collagen binding. Gold surfaces were chosen for functionalisation in order to utilise the properties of thiols which covalently bind to and form a self-assembled monolayer (SAM) on gold. Both I domain and GFOGER peptide contain cysteine, providing free thiol. In the initial strategy GFOGER peptide was covalently bound to a gold coated AFM tip and I-domain adsorbed to mica. A mean force of 225pN was reduced to 58pN on addition of EDTA. Similar results were obtained when force curves were performed with a bare gold tip (222pN reduced to 92pN) indicating initial measurements were in fact non-specific. This highlights the importance of minimising non-specific interactions. Gold tips functionalised with a compound known to reduce protein absorption (HS(CH2)11-PEG6-OH) show dramatically reduced non-specific forces. This has led to the creation of a new strategy for functionalisation; a SAM consisting of this compound mixed with a similar but amine-terminating compound. A heterobifunctional PEG cross linker containing amine and thiol reactivity is used to link I domain and peptide to the SAM. The PEG linker allows free rotation of molecules in solution and its length means specific interactions will occur at a detectable distance from non-specific tip-surface forces. The mixed SAM results in low background adhesion and sparse coverage of ligand and receptor allowing single molecule unbinding events to be measured. Functionalisation of gold has been confirmed by AFM imaging.

Disclosure of interest: none declared.

To cite this abstract use the following format:

Journal of Thrombosis and Haemostasis 2007; Volume 5, Supplement 2: abstract number

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Date:	Unpresented
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Session name:	ISTH2009
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