A mechanically stabilized receptor–ligand flex-bond important in the vasculature

Abstract

Haemostasis in the arteriolar circulation mediated by von Willebrand factor (VWF) binding to platelets is an example of an adhesive interaction that must withstand strong hydrodynamic forces acting on cells. VWF is a concatenated, multifunctional protein that has binding sites for platelets as well as subendothelial collagen1,2. Binding of the A1 domain in VWF to the glycoprotein Ib α subunit (GPIbα) on the surface of platelets mediates crosslinking of platelets to one another and the formation of a platelet plug for arterioles3,4. The importance of VWF is illustrated by its mutation in von Willebrand disease, a bleeding diathesis1. Here, we describe a novel mechanochemical specialization of the A1–GPIbα bond for force-resistance. We have developed a method that enables, for the first time, repeated measurements of the binding and unbinding of a receptor and ligand in a single molecule (ReaLiSM). We demonstrate two states of the receptor–ligand bond, that is, a flex-bond. One state is seen at low force; a second state begins to engage at 10 pN with a 20-fold longer lifetime and greater force resistance. The lifetimes of the two states, how force exponentiates lifetime, and the kinetics of switching between the two states are all measured. For the first time, single-molecule measurements on this system are in agreement with bulk phase measurements. The results have important implications not only for how platelets bound to VWF are able to resist force to plug arterioles, but also how increased flow activates platelet plug formation.

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Figure 1: The A1 and GP1bα single-molecule construct and change in extension on unbinding and rebinding.
Figure 2: Force spectroscopy and bond lifetime.
Figure 3: Force-clamp experiments.
Figure 4: The flex-bond.

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Acknowledgements

Supported by NIH HL-48675 (TAS). The authors are indebted to S. B. Smith and C. Bustamante for help with laser tweezers construction and insightful discussion. We thank B. Coller, O. K. Dudko and C. Lu for reagents and insightful discussions, and J. Dill for software for data analysis.

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T.A.S. designed and supervised the project. X.Z. cloned the ReaLiSM construct. J.K. designed experiments and collected and analysed data. C.-Z.Z. analysed data. T.A.S, J.K. and C.-Z.Z. wrote the paper.

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Correspondence to Timothy A. Springer.

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The authors declare no competing financial interests.

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This file contains Supplementary Results, Supplementary Figures 1- 6 with legends, Supplementary Tables 1-2 and References. (PDF 774 kb)

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Kim, J., Zhang, C., Zhang, X. et al. A mechanically stabilized receptor–ligand flex-bond important in the vasculature. Nature 466, 992–995 (2010). https://doi.org/10.1038/nature09295

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