Research ArticleBIOPHYSICS

Microscale spatial heterogeneity of protein structural transitions in fibrin matrices

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Science Advances  08 Jul 2016:
Vol. 2, no. 7, e1501778
DOI: 10.1126/sciadv.1501778
  • Fig. 1 Strain-dependent elasticity of fibrin hydrogels.

    (A) Shear rheology of partially cross-linked and cross-linked fibrin (7.5 mg/ml). Three hydrogel samples were averaged per strain point. Error bars are SEM. (B) Tensile tests for the same types of samples. Force-strain curves were normalized to the rupture force. The average (black and red) represents five measurements from independent samples; SEM is depicted as gray area.

  • Fig. 2 Phase-retrieved and CH3-normalized BCARS spectrum of fibrin.

    (A and B) Never-loaded (A) and 80% strained (B) fibrin hydrogel. The amide I band was decomposed with a sum of five Lorentzians: green, blue, and red peaks that represent structural species indicated and two smaller ring modes (depicted in gray) that are related to tyrosine rings. The contribution of each species to the amide I band was determined by the fractional area under each component. Black lines indicate the raw data, and orange lines represent the Lorentzian fits.

  • Fig. 3 Polar plots show the orientation of the different secondary structure motifs in a never-loaded and strained fibrin gel.

    Thirty spectra from a single gel (at rest and then strained) with the most intense CH3 peak (2934 cm−1) were averaged to generate each point in these plots. Error bars are SEM. (A) At 0% strain, both motifs show an isotropic distribution, as expected. (B) At 60% strain, the two motifs exhibit elliptical shapes, indicating a preferred direction of the load with respect to the laser polarization (double-headed arrow). The α helix peak shows a maximum intensity when the load aligned to the polarization of the lasers, indicated by the arrow, whereas the β sheet peak maximum is rotated 80° with respect to the α helix. Lines show fits to a sine function, Embedded Image. For each secondary structure motif, the contribution to the amide I spectrum was calculated by integrating the fit function over the entire polar range.

  • Fig. 4 Secondary structure content for increasing strain in partially cross-linked and cross-linked hydrogels.

    (A and B) Percentage contribution of β sheets (A) and percentage contribution of α helices (B) to the amide I band at increasing strain. Each data point represents the average of 150 spectra (top 30 from five independent experiments), with SEM as error bars.

  • Fig. 5 Fibrin gel secondary structure becomes more heterogenous under deformation.

    (A to F) Images and histogram plots showing the β sheet contribution as percentage content in partially cross-linked fibrin gels at different strains: never-loaded (A and B), 56% vertical strain (C and D), and 100% vertical strain (E and F). The direction of load is indicated by the arrow. Pixels with CH3 values below threshold and pixels that showed polystyrene signal were excluded from further analysis and are shown in gray. Scale bars, 4 μm. Histogram plots of all three samples had a bin size of 1%.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/7/e1501778/DC1

    Supplementary Data

    Supplementary Methods

    fig. S1. SDS-PAGE of reduced fibrin gels without (Fib) and with additional cross-linking by FXIIIa (Fib+)

    fig. S2. Differential storage modulus measured as a function of prestrain for three different mixtures of fibrin hydrogels.

    fig. S3. Creep recovery tests of partially cross-linked and fully cross-linked fibrin gels.

    fig. S4. Curves showing average α helix, β sheet, and random coil content of a typical relaxed hydrogel as a function of number of pixels included in the calculation, sorted from maximum protein content (CH value) to minimum protein content.

    fig. S5. Fibrin protein concentration under strain.

    fig. S6. Random coil content for increasing strain in three different hydrogels.

    fig. S7. SD of CH3 intensity (2930 cm−1) within the top 30 highest protein signal pixels normalized to the average CH3 intensity of the experiment.

    fig. S8. Spectral maps for never-loaded partially cross-linked hydrogels (7.5 mg/ml).

    fig. S9. Images and histogram plots showing the α helix peak contribution as percent content within partially cross-linked fibrin gels at different strains.

    fig. S10. Images and histogram plots showing the random coil peak contribution as percent content within partially cross-linked fibrin gels at different strains.

    fig. S11. Normalized spatial autocorrelation of β sheet content maps along direction orthogonal to loading axis.

    fig. S12. Spectral maps for 85% vertical strained cross-linked hydrogel.

    fig. S13. Confocal fluorescence microscopy of fibrin gels with different fibrinogen concentrations (total field of view, 48 μm × 48 μm).

    fig. S14. Contribution of β sheets to the amide I band for increasing strain in low- and high-concentration fibrin.

    fig. S15. SDS-PAGE of the supernatant from different hydrogels to check the complete polymerization of fibrinogen monomers.

    fig. S16. Schematic of the BCARS microscope setup.

    table S1. Results from creep recovery experiments for partially cross-linked and fully cross-linked fibrin.

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Data
    • Supplementary Methods
    • fig. S1. SDS-PAGE of reduced fibrin gels without (Fib) and with additional cross-linking by FXIIIa (Fib+).
    • fig. S2. Differential storage modulus measured as a function of prestrain for three different mixtures of fibrin hydrogels.
    • fig. S3. Creep recovery tests of partially cross-linked and fully cross-linked fibrin gels.
    • fig. S4. Curves showing average α helix, β sheet, and random coil content of a typical relaxed hydrogel as a function of number of pixels included in the calculation, sorted from maximum protein content (CH value) to minimum protein content.
    • fig. S5. Fibrin protein concentration under strain.
    • fig. S6. Random coil content for increasing strain in three different hydrogels.
    • fig. S7. SD of CH3 intensity (2930 cm−1) within the top 30 highest protein signal pixels normalized to the average CH3 intensity of the experiment.
    • fig. S8. Spectral maps for never loaded partially cross-linked hydrogels (7.5 mg/ml).
    • fig. S9. Images and histogram plots showing the α helix peak contribution as percent content within partially cross-linked fibrin gels at different strains.
    • fig. S10. Images and histogram plots showing the random coil peak contribution as percent content within partially cross-linked fibrin gels at different strains.
    • fig. S11. Normalized spatial autocorrelation of β sheet content maps along direction orthogonal to loading axis.
    • fig. S12. Spectral maps for 85% vertical strained cross-linked hydrogel.
    • fig. S13. Confocal fluorescence microscopy of fibrin gels with different fibrinogen concentrations (total field of view, 48 μm ? 48 μm).
    • fig. S14. Contribution of β sheets to the amide I band for increasing strain in low- and high-concentration fibrin.
    • fig. S15. SDS-PAGE of the supernatant from different hydrogels to check the complete polymerization of fibrinogen monomers.
    • fig. S16. Schematic of the BCARS microscope setup.
    • table S1. Results from creep recovery experiments for partially cross-linked and fully cross-linked fibrin.

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