Elsevier

Acta Biomaterialia

Volume 104, 1 March 2020, Pages 39-52
Acta Biomaterialia

Full length article
Revealing the molecular origins of fibrin's elastomeric properties by in situ X-ray scattering

https://doi.org/10.1016/j.actbio.2020.01.002Get rights and content

Abstract

Fibrin is an elastomeric protein forming highly extensible fiber networks that provide the scaffold of blood clots. Here we reveal the molecular mechanisms that explain the large extensibility of fibrin networks by performing in situ small angle X-ray scattering measurements while applying a shear deformation. We simultaneously measure shear-induced alignment of the fibers and changes in their axially ordered molecular packing structure. We show that fibrin networks exhibit distinct structural responses that set in consecutively as the shear strain is increased. They exhibit an entropic response at small strains (<5%), followed by progressive fiber alignment (>25% strain) and finally changes in the fiber packing structure at high strain (>100%). Stretching reduces the fiber packing order and slightly increases the axial periodicity, indicative of molecular unfolding. However, the axial periodicity changes only by 0.7%, much less than the 80% length increase of the fibers, suggesting that fiber elongation mainly stems from uncoiling of the natively disordered αC-peptide linkers that laterally bond the molecules. Upon removal of the load, the network structure returns to the original isotropic state, but the fiber structure becomes more ordered and adopts a smaller packing periodicity compared to the original state. We conclude that the hierarchical packing structure of fibrin fibers, with built-in disorder, makes the fibers extensible and allows for mechanical annealing. Our results provide a basis for interpreting the molecular basis of haemostatic and thrombotic disorders associated with clotting and provide inspiration to design resilient bio-mimicking materials.

Statement of Significance

Fibrin provides structural integrity to blood clots and is also widely used as a scaffold for tissue engineering. To fulfill their biological functions, fibrin networks have to be simultaneously compliant like skin and resilient against rupture. Here, we unravel the structural origin underlying this remarkable mechanical behaviour. To this end, we performed in situ measurements of fibrin structure across multiple length scales by combining X-ray scattering with shear rheology. Our findings show that fibrin sustains large strains by undergoing a sequence of structural changes on different scales with increasing strain levels. This demonstrates new mechanistic aspects of an important biomaterial's structure and its mechanical function, and serves as an example in the design of biomimicking materials.

Section snippets

1. Introduction

In Nature, many examples are found of elastomeric materials made of protein fibers. Prominent elastomeric proteins in the human body are the intermediate filament cytoskeleton of cells [1], elastin and fibronectin in tissues [2,3], and fibrin networks in blood clots and wounds [4]. These proteins all form filaments that can reversibly stretch up to tensile strains of around 150% and resist strains of several hundred percent without breaking [5,6]. Two main molecular mechanisms have been

Fibrin network assembly

Human plasma fibrinogen (Plasminogen, von Willebrand Factor and Fibronectin depleted) and human α-thrombin were obtained in lyophilized form from Enzyme Research Laboratories (Swansea, United Kingdom). All chemicals were obtained from Sigma Aldrich (Zwijndrecht, The Netherlands). Fibrinogen (lyophilized in 20 mM sodium citrate-HCl buffer at pH 7.4) was dissolved in water at 37 °C for 15 min to its original concentration (ca. 13 mg/ml) and extensively dialysed against fibrin buffer containing

Shear-induced structural changes at the network and fiber level

In order to probe the effects of shear strain on fibrin network structure, we combined rheology experiments on fibrin networks in a Couette cell with in situ SAXS measurements (Fig. 1a,b). We subjected the networks to a stepwise increase in shear strain γ interspersed with intervals where the strain was brought back to zero to test for reversibility (Fig. 1c). Fig. 1d shows the background-subtracted scattering images of a 4 mg/ml fibrin gel at strains of 0%, 100% and 300%, and the same network

Discussion

The aim of our work was to elucidate how fibrin gels obtain their remarkable strain-stiffening behaviour, which spans a wide range of shear strains up to 250% (Fig. 3d). We therefore combined macroscopic measurements of the shear rheology of fibrin networks with in situ structural measurements by SAXS. We found that the networks exhibit multiple distinct mechanical regimes that are closely correlated with distinct changes in network structure on different length scales.

At low strains (γ < 25%),

Conclusion

We performed X-ray measurements coupled with shear rheology to reveal the structural mechanisms that mediate the nonlinear elastic response and large resilience of fibrin networks. We showed that increasing levels of shear strain induce distinct changes in elasticity that coincide with a sequence of structural responses. There were no observable changes in the fiber or network structure up to strains of around 25%, consistent with entropic elasticity. Above 25% strain the fibers progressively

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We gratefully acknowledge Paul Kouwer (Radboud University Nijmegen, the Netherlands) for kindly lending us his TA HR2-rheometer during beamtime BM26-02–797. We also acknowledge the beamline staff, in particular Daniel Hermida Merino, Giuseppe Portale and Wim Bras, of BM26 at the ESRF for their help with the experiments and data analysis. We thank Bela Mulder (AMOLF, Amsterdam, the Netherlands) for discussions on the calculation of the nematic order parameter, and Karin Jansen and Lucia Baldauf

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