The α-Helix to β-Sheet Transition in Stretched and Compressed Hydrated Fibrin Clots.

Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania.
Biophysical Journal (Impact Factor: 3.83). 09/2012; 103(5):1020-7. DOI: 10.1016/j.bpj.2012.07.046
Source: PubMed

ABSTRACT Fibrin is a protein polymer that forms the viscoelastic scaffold of blood clots and thrombi. Despite the critical importance of fibrin deformability for outcomes of bleeding and thrombosis, the structural origins of the clot's elasticity and plasticity remain largely unknown. However, there is substantial evidence that unfolding of fibrin is an important part of the mechanism. We used Fourier transform infrared spectroscopy to reveal force-induced changes in the secondary structure of hydrated fibrin clots made of human blood plasma in vitro. When extended or compressed, fibrin showed a shift of absorbance intensity mainly in the amide I band (1600-1700 cm(-1)) as well as in the amide II and III bands, indicating an increase of the β-sheets and a corresponding reduction of the α-helices. The structural conversions correlated directly with the strain or pressure and were partially reversible at the conditions applied. The additional absorbance observed at 1612-1624 cm(-1) was characteristic of the nascent interchain β-sheets, consistent with protein aggregation and fiber bundling during clot deformation observed using scanning electron microscopy. We conclude that under extension and/or compression an α-helix to β-sheet conversion of the coiled-coils occurs in the fibrin clot as a part of forced protein unfolding.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Fibrin is a protein polymer that forms a 3D filamentous network, a major structural component of protective physiological blood clots as well as life threatening pathological thrombi. It plays an important role in wound healing, tissue regeneration and is widely employed in surgery as a sealant and in tissue engineering as a scaffold. The goal of this study was to establish correlations between structural changes and mechanical responses of fibrin networks exposed to compressive loads. Rheological measurements revealed nonlinear changes of fibrin network viscoelastic properties under dynamic compression, resulting in network softening followed by its dramatic hardening. Repeated compression/decompression enhanced fibrin clot stiffening. Combining fibrin network rheology with simultaneous confocal microscopy provided direct evidence of structural modulations underlying nonlinear viscoelasticity of compressed fibrin networks. Fibrin clot softening in response to compression strongly correlated with fiber buckling and bending, while hardening was associated with fibrin network densification. Our results suggest a complex interplay of entropic and enthalpic mechanisms accompanying structural changes and accounting for the nonlinear mechanical response in fibrin networks undergoing compressive deformations. These findings provide new insight into the fibrin clot structural mechanics and can be useful for designing fibrin-based biomaterials with modulated viscoelastic properties.
    Biomaterials 05/2014; 35(25). DOI:10.1016/j.biomaterials.2014.04.056 · 8.31 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The purpose of this study was to develop and characterize a novel photo-cross-linkable chitosan-lactide-fibrinogen (CLF) hydrogel and evaluate the efficacy of bone morphogenetic protein-2 (BMP-2) containing CLF hydrogel for osteogenesis in vitro and in vivo. We synthesized the CLF hydrogels and characterized their chemical structure, degradation rate, compressive modulus, and in vitro BMP-2 release kinetics. We evaluated bioactivities of the BMP-2 containing CLF hydrogels (0, 50, 100, and 500 ng/ml) in vitro using W-20-17 preosteoblast mouse bone marrow stromal cells and C2C12 mouse myoblast cells. The effect of BMP-2 containing CLF gels (0, 0.5, 1, 2, and 5μg) on bone formation was evaluated using rat critical size segmental bone defects for 4 weeks. FTIR spectra and SEM images showed chemical and structural changes by addition of fibrinogen into chitosan-lactide copolymer. Incorporation of fibrinogen molecules significantly increased compressive modulus of the hydrogels. In vitro BMP-2 release study showed initial burst releases from the CLF hydrogels followed by sustained releases, regardless of the concentration of the BMP-2 over 4 weeks. Cells in all groups were viable in the presence of the hydrogels regardless of BMP-2 doses, indicating non-cytotoxicity of hydrogels. Alkaline phosphate activity and mineralization of cells exhibited dose dependence on BMP-2 containing CLF hydrogels. Radiographs, microcomputed tomography, and histology confirmed that the BMP-2 containing CLF hydrogels prompted neo-osteogenesis and accelerated healing of the defects in a dose-dependent manner. Thus the CLF hydrogel is a promising delivery system of growth factors for bone regeneration.
    Acta Biomaterialia 08/2014; DOI:10.1016/j.actbio.2014.08.028 · 5.68 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: When PEG (M.W.∼5000 Daltons) is conjugated to poly(l-alanine), the polymer aqueous solutions (<10.0 wt.%) undergo sol-to-gel (thermal gelation), whereas it is conjugated to poly(l-lactic acid), the polymer aqueous solutions (>30.0 wt.%) undergo gel-to-sol (gel melting) as the temperature increases. In the search for molecular origins of such a quite different phase behavior, poly(ethylene glycol)-poly(l-alanine) (PEG-PA; EG113-A12) and poly(ethylene glycol)-poly(l-lactic acid) (PEG-PLA; EG113-LA12) are synthesized and their aqueous solution behavior is investigated. PEG-PAs with an α-helical core assemble into micelles with a broad size distribution, and the dehydration of PEG drives the aggregation of the micelles, leading to thermal gelation, whereas increased molecular motion of the PLA core overwhelms the partial dehydration of PEG, thus gel melting of the PEG-PLA aqueous solutions occurs. The core-rigidity of micelles must be one of the key factors in determining whether a polymer aqueous solution undergoes sol-to-gel or gel-to-sol transition, as the temperature increases. © 2014 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2014
    Journal of Polymer Science Part A Polymer Chemistry 09/2014; 52(17). DOI:10.1002/pola.27254 · 3.54 Impact Factor