The a-Helix to b-Sheet Transition in Stretched and Compressed Hydrated
Rustem I. Litvinov,†Dzhigangir A. Faizullin,‡Yuriy F. Zuev,‡and John W. Weisel†*
†Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; and
‡Kazan Institute of Biochemistry and Biophysics, Russian Academy of Sciences, Kazan, Russia
tance 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 b-sheets and a cor-
responding reduction of the a-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?1was characteristic of the nascent
interchain b-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 a-helix to b-sheet conversion of the coiled-coils
occurs in the fibrin clot as a part of forced protein unfolding.
Fibrin is a proteinpolymer thatforms the viscoelastic scaffoldof bloodclots and thrombi.Despite thecritical impor-
Fibrin is an insoluble protein polymer that forms in the
blood at sites of vascular injury. Together with platelets,
fibrin builds a plug that stops bleeding. Fibrin also provides
the scaffold for pathological obstructive thrombi that block
blood vessels, seize blood flow, and cause myocardial
infarction and ischemic stroke (1).
Fibrin is formed from its soluble precursor, fibrinogen, at
the final stage of blood clotting. Fibrinogen is a 340-kDa
blood plasma protein 45 nm in length and 2–3 nm in diam-
eter, consisting of three pairs of polypeptide chains, desig-
nated Aa, Bb, and g, linked by 29 S-S bonds. Two distal
and one central globular parts of fibrinogen are connected
by two 17-nm-long triple (and partially quadruple) a-helical
coiled-coils comprising 25% of amino acid residues of the
whole molecule and 34% of its structured portion visualized
using x-ray crystallography (2) (Fig. 1 A, inset). Fibrin
formation is initiated by the cleavage of the small fibrino-
peptides A and B from the N-termini of the Aa and
Bb chains, respectively, converting fibrinogen to fibrin
monomer, which maintains major structural features of
fibrinogen, including the coiled-coils. Monomers then
self-assemble, resulting in a three-dimensional filamentous
network. The fibrin clot is finally stabilized by intermolec-
ular covalent cross-linking by a plasma transglutaminase,
Factor XIIIa, rendering the clot more resilient and resistant
to enzymatic cleavage (1,3).
Because in vivo clots are formed in flowing blood or in
the dynamic wound environment, the mechanical and rheo-
logical properties of fibrin are necessary for their function.
The structural changes underlying deformation of a fibrin
clot occur at different spatial scales, including molecular
unfolding (4), which remains unclear despite recent insights
(5). Based on the existence of two relatively long and coax-
ially aligned coiled-coils in rod-like fibrin(ogen), it has been
hypothesized that the a-helix to b-strand conversion of the
coiled-coils accompanies molecular extension of fibrin (6).
By means of Congo red staining to detect the b-structures,
which is commonly used to identify stacks of b-sheets in
amyloid proteins, we revealed the formation of congophilic
material, presumably new b-sheets, in stretched fibrin fibers
(7), but the specificity of this method is not fully justified.
The only direct experimental observation for an a-b trans-
formation in fibrin is the low-resolution wide-angle x-ray
scattering of squeezed fibrin films published back in 1943
(8), providing dubious evidence for the transition from
a-helix to b-sheet upon fibrin stretching. Obviously, this
quite important structural mechanism of fibrin nanome-
chanics needs to be probed using modern high-resolution
methodologies for various types and degrees of deformation
in physiologically relevant settings.
This work has been aimed at the role of secondary
structure changes during deformations of fibrin polymers.
In addition to tension, which hasbeenused tostudy mechan-
ical properties of fibrin, we applied pressure towatch molec-
ular transitions in fibrin clots in response to compression,
which is physiologically as relevant in relation to fibrin as
extension and could occur under the pressure of arterial
blood flow. Here, we present experimental data showing
that both extension and compression of a hydrated fibrin
clot are accompanied by changes in the a-helical coiled-
coils, namely the transitionofa-helix tob-sheet, determined
using Fourier transform infrared (FTIR) spectroscopy.
Submitted May 4, 2012, and accepted for publication July 24, 2012.
Editor: Patricia Clark.
? 2012 by the Biophysical Society
1020Biophysical JournalVolume 103September 2012 1020–1027
MATERIALS AND METHODS
Formation of fibrin clots
To form fibrin clots, 9 volumes of human citrated blood plasma (Blood
Transfusion Center, Kazan, Russia) were mixed with 1 volume of 0.25M
CaCl2in 5-ml plastic syringes. The clots were formed for 1–2 h at room
temperature followed by thorough washing in 20 mM Tris-HCl/150 mM
NaCl, pH 7.4, until the absorbance of the washing buffer at 280 nm reached
zero. Duringformation the clots were naturally cross-linked by Factor XIIIa
(Fig. S1 in the Supporting Material) because Factor XIII normally present
in plasmais activated by thrombin,makingtheir propertiesrelevant to fibrin
networks formed in vivo. The clots were kept in buffer before use and were
continuously irrigated during measurements to prevent drying.
Among a number of current techniques to study secondary structure of
proteins, FTIR spectroscopy has been widely used for structural character-
ization of many polypeptides and proteins, including fibrinogen (9–13),
fibrin (14,15), and fibrinogen fragments (14); however, it has never been
applied to study structural dynamics of fibrin clots upon deformation.
The clots before and after deformation were analyzed in a TENSOR 27
FTIR spectrometer (Bruker Optik GmbH, Ettlingen, Germany). For
inducing strain, a clot sample was clamped in a custom-built stretching
device (Fig. S2) and elongated manually at a speed of ~20 mm/min up to
fourfold before it ruptured. The strained clot was positioned on the PIKE
MIRacle attenuated total reflectance (ATR) device (Ge crystal, single-
bounce beam path, 45?incident angle) and spectra were measured while
in the stretched state. To account for potential partial light polarization
on the ATR device and its interaction with the elongated anisotropic clots
(16), we performed control measurements of the same stretched fibrin clots
in two perpendicular orientations relative to the ATR prism without
observing any difference in the FTIR spectra. To study effects of compres-
sion, a clot sample was pressed directly over a 2-mm detecting spot of the
ATR crystal (Fig. S3). The pressurevaried up to 300 bar but, because it was
not precisely controlled, the degree of compression was roughly designated
relatively small (10–50 bar), intermediate (100–150 bar), or large (200–
250 bar). Spectra were recorded with 4.0 cm?1resolution and corrected
for the water vapor and the buffer. For each measurement 128 scans were
averaged. An unperturbed fibrin clot has a pronounced amide I band
(1600–1700 cm?1) as well as amide II and III bands with lower absorption
intensity (Fig. 1 A). Multiple measurements were performed on freshly
prepared fibrin clots, including 58 unperturbed, 39 stretched, and 33 com-
pressed samples, each at different degrees of deformation.
Deconvolution of amide I
Quantitative analysis of the amide I band contour was done using
curve fitting, second derivative, and Fourier self-deconvolution methods
(17,18). Spectra were processed using Bruker OPUS software. After
subtraction of the buffer and water vapor absorbencies, the resulting
spectra were smoothed by a 7–13-point Savitsky-Golay function, depend-
ing on the quality of the data (19). Second-derivative spectral analysis was
applied to locate the position of the overlapping components of the amide
I band to assign them to different secondary structure elements (17,18,20).
Fourier self-deconvolution (21) provides band narrowing through multipli-
cation of the Fourier transform by a lineshape function and an apodization
function. Generally, a value of 13 cm?1for the full bandwidth at
half-height and a resolution enhancement factor of 2.4 are adequate
(18). Deconvolution using a too narrow full bandwidth at half-height
will not separate all the widest bands, whereas a value that is too large
will overemphasize the spectrum’s narrower components leading to arti-
facts and possible misinterpretation. We have found optimal deconvolution
to be employing Lorentzian band shape, resolution enhancement factor 2,
and bandwidth 13 cm?1(Bruker OPUS 6.5 software). The applied param-
eters resulted in a relatively moderate resolution enhancement that
prevents large spectral distortions. The fractions of each component in
the resolution-enhanced amide I band were estimated quantitatively by
a nonlinear least-squares fitting program Fityk (22), iterating the curve-
fitting process according to the specific functions. Because Fourier self-de-
convolution performed with the previous parameters results in depression
of the Lorentzian contribution, the Gaussian function was selected for the
best fitting. The number of components and their positions on the wave-
number scale were determined using both second derivatives and differ-
ence FTIR spectra collected during deformation of the same sample.
Only spectral components exhibiting reproducible positions and consistent
response to the deformation were taken into account. Based on the char-
acteristic minima and shoulders of the second derivative curves, the spec-
tral range of 1600–1700 cm?1was decomposed in 10 bands including two
small marginal bands (1604 sn?1and 1592 sn?1) due to side-chain
absorption (Fig. 1 B) (23). They were included in the deconvolution to
avoid unreliable straight baseline subtraction, but were excluded from
the secondary structure quantification. A proportion of each component
in the amide I band was computed as a fractional area of the correspond-
ing peak divided by the sum of the areas of the peaks belonging to the
amide I band (17,18).
1200 130014001500 1600 1700
Wavenumber (cm )
Wavenumber (cm )
and the crystallographic structure of human fibrin(ogen) (2) (inset).
(B) Deconvolution of resolution-enhanced amide I band and assignment
to the secondary structure elements.
(A) Representative FTIR spectrum of a hydrated fibrin clot
Biophysical Journal 103(5) 1020–1027
Mechanical a-b Transition in Hydrated Fibrin 1021
Assignment of the secondary structure elements
The peaks obtained by deconvolution of the amide I band can be assigned
to specifictypesofsecondarystructurebasedon theestablishedcorrelations
between crystallographic structures of proteins and their FTIR spectra
(17,18,20). For the assignments made in this work, we also took into
account theprevious FTIR spectroscopy
(9,11,14,15), with results shown in Fig. 1 B, Fig. S6, and Tables S1–S8.
To make quantitative characteristics of nondeformed and deformed clots
fully comparable, we always fitted spectra with the same set of parameters.
During the fitting and assignment only the amplitudes of components were
allowed to vary freely, but the frequency and bandwidth variations were
constrained to 1–2 cm?1. Fitting quality was verified by calculating the
differences between fitted spectra at various deformations and comparing
them with the differences between the corresponding original spectra at
the same deformations. Fitting was accepted only if the spectral differences
and derivatives both from the original and computed spectra coincided
within the noise level. Following this approach we distinguished one major
peak corresponding to a-helices at 1651 5 2 cm?1and three different types
of b-structure peaking at 1614 5 2, 1622 5 2, and 1636 5 2 cm?1
(Fig. 1 B). The peak at 1614 cm?1partly contains absorbance from tyrosine
side chain but itsrelativecontribution is negligiblysmall (23). We tested the
precision of our fitting and sample to sample variability by calculating
a coefficient of variation for the fractions of secondary structures from
FTIR spectra of 11 independent samples of nondeformed fibrin (Table
S1, summarized content). The values of coefficient of variation obtained
were ~10%, very close to those obtained with the same method for fibrin
spectra from various sources (15).
Scanning electron microscopy
Clots prepared for scanning electron microscopy (SEM) were thoroughly
washed with 50 mM sodium cacodylate-HCl buffer, pH 7.4, to remove
excess salt and fixed overnight in 2% glutaraldehyde. Stretched clots
were washed and fixed by immersion into the washing buffer or fixative
while being clamped in the stretching device right after they reached
a certain length. Compressed clots were immersed into the fixative within
10 s after decompression. Clots were then cut into small pieces, washed
in deionized water, dehydrated in a graded series of increasing ethanol
concentrations (30–100%), and impregnated with hexamethyldisilazane
followed by air-drying. The specimens were mounted, sputter coated with
gold-palladium in a Sputter Coating Unit E5100 (Polaron Equipment,
Hertfordshire, UK) at 2.2 kVand 20 mA for 1.5 min, and examined in an
XL20 scanning electron microscope (FEI, Hillsboro, OR). Several fields
on each clot were examined before choosing fields that were characteristic
of the entire clot. Digital electron micrographs were taken at different
magnifications between 2,000? and 10,000?.
RESULTS AND DISCUSSION
Deformation-induced molecular transformations
in fibrin clots
The main finding of this study is that deformations of
hydrated fibrin clots, either elongation or compression,
induce distinct rearrangement of the protein secondary
structure reflected by characteristic changes of FTIR
spectra. At the qualitative level, these changes could be
described as a redistribution of absorption intensities from
higher to lower wavenumbers. This shift was confirmed
by peak positions of the difference spectra and second
derivative spectra observed in various peptide vibration
modes: amide I (decrease at 1649–1651 cm?1and increase
at 1620–1630 cm?1), amide II (decrease at 1540–1550 cm?1
and increase at 1520–1540 cm?1), and amide III (decrease
at 1290–1320 cm?1and increase at 1220–1240 cm?1)
(Fig. 2, Fig. 3, Fig. S4, and Fig. S5). It has been shown in
FTIR studies of various proteins that absorbance at the
higher wavenumbers (1649–1651 cm?1, 1540–1550 cm?1,
and 1290–1320 cm?1) is predominantly due to a-helical
structures, whereas thelower
1630 cm?1, 1520–1540 cm?1, and 1220—1240 cm?1) are
largely characteristic of b-structures (17,24). Because the
shift of absorbance intensity in the amide I band was the
most pronounced and well defined in terms of correspon-
dence to secondary structure elements, we used the intensity
ratio at 1622/1651 cm?1to estimate the deformation-
induced changes in FTIR spectra and also as a semiquantita-
tive determination of the b-sheet/a-helix content ratio. This
parameter displayed a clear dependence on the degree of
deformation both for elongation (Fig. 2, inset; Fig. S6)
and compression (Fig. S6), corroborating that the observed
changes in FTIRspectra and corresponding secondary struc-
ture transitions from a-helices to b-sheets are inherently
coupled to the force-induced deformations.
To better quantify the observed changes in FTIR spectra,
we performed a determination of secondary structure
elements using deconvolution of the amide I band, which
is primarily governed by the stretching vibration of C¼O
and C–N bonds and is mainly used for secondary structure
determinations (17). Curve fitting of Fourier self-decon-
volved FTIR spectra with underlying band positions
determined from second derivatives was performed in
Wavenumber (cm )
(normalized by amide I and II peaks area)
Strain = 1
Strain = 2
Strain = 3
(black line) and the same clot after twofold (strain ¼ 1, green solid line),
threefold (strain ¼ 2, red solid line), and fourfold (strain ¼ 3, blue solid
line) elongation. Corresponding difference spectra (1-0, 2-0, and 3-0) ob-
tained by subtraction of the initial spectrum at strain ¼ 0 are shown as
the dashed lines of the corresponding color. (Inset) The absorbance inten-
sity ratio at 1622/1651 cm?1as a function of strain. Strain is defined as
stretched length/initial length-1.
Normalized FTIR ATR spectra of unstretched plasma clot
Biophysical Journal 103(5) 1020–1027
1022Litvinov et al.
accordance with the procedure used earlier to analyze FTIR
spectra of fibrin(ogen) (9,11,14,15,25,26) as well as other
fibrillar proteins, such as a-keratin (27), silk fibroin (28),
and fibronectin (29). The results of curve fitting for the
secondary structure elements are shown in Fig. S6 and
Tables S1–S8. On the basis of this analysis, changes in the
absolute content of different secondary structure elements
in response to elongation and compression of fibrin clots
are summarized in Table 1.
Repeated and reproducible measurements performed on
unperturbed clots show that human fibrin contains 30 5
3% a-helices, 37 5 4% b-sheets, and 32 5 3% turns, loops,
and random coils (M 5 SD, n ¼ 11). Notwithstanding some
degree of uncertainty inherently present in deconvolution of
FTIR spectra and assignment of secondary structures (17),
the numbers obtained for these structures are very close to
real because of at least two supporting arguments. First,
the results are in good agreement with the numbers obtained
from other FTIR (14,15) or Raman (30) spectroscopy
studies of nondeformed fibrin, despite distinctions in the
modes of measurement and analyses. Second, we confirmed
our results by comparing them with the available crystallo-
graphic data, the gold standard for protein structure.
Because there is no crystallographic data for the entire fibrin
molecule, we validated our secondary structure analysis by
comparing the FTIR spectroscopy determined a-helical
content in unperturbed fibrin with the content of a-helices
revealed in the x-ray crystallographic structure of human
fibrinogen (PDB entry: 3GHG (2)). Taking into account
some additional a-helices predicted but crystallographically
unresolved in the flexible region of the fibrinogen Aa chain
(31), our experimental estimation (~30%) is quite close to
the crystallographic data (27%), well within a 10% global
error of secondary structure prediction based on the FTIR
spectra band fitting algorithm (32).
The elongation and compression of fibrin clots was
followed by a decrease of the a-helix content down to
16% upon fourfold elongation or maximal compression.
Remarkably, the observed reduction in the a-helix content
upon stretching or compression was approximately equal
to the corresponding 14–15% increase in the fraction of
b-sheets without appreciable changes in the content of turns,
loops, and random structures. Similar quantitative changes
in the secondary structure observed after extreme elonga-
tion and compression suggest that the clots have reached
the level of deformation at which they start to become
physically damaged or ruptured rather than undergo further
protein unfolding. These data support the conclusion drawn
from the raw spectra that both elongation and compres-
sion of fibrin clots are followed by rearrangement of the
secondary structure, comprising mainly the a-helix to
b-sheet transition. The strong correspondence between an
increase of the b-structures and a reduction of the a-helix
content suggests that secondary structure alterations upon
stretching and compression occur in the coiled-coil regions
of a fibrin molecule.
Taken together these results indicate that forced fibrin
elongation and compression are accompanied by a signifi-
cant a-b conversion under relatively high deformations. It
is noteworthy that the a-b transition in response to stress
has been demonstrated for a number of filamentous proteins,
such as a-keratin (27,33), keratin-like intermediate fila-
ments (34), desmin (35), and vimentin (36). Inasmuch as
the occurrence of the a-b transition directly depends on
the length of a-helices with a 3.8-nm threshold (37), the
17-nm-long a-helical coiled-coils in fibrin are fully compat-
ible with this transformation.
Deformation-induced b-sheet-mediated protein
aggregation in fibrin clots
The rising degree of deformation in stretched/compressed
fibrin was followed by a progressively increasing shoulder
in the absorbance spectra peaking at 1622–1624 cm?1
(Figs. 2 and 3). The second derivative split that shoulder
into two components: 1622–1624 cm?1
1618 cm?1, the latter being almost absent in the unper-
turbed fibrin. Marked deformation was accompanied by a
and after stretching or compression
Secondary structure of hydrated fibrin clots before
Types of secondary
01230 Small Interm. Large
31% 30% 25% 16% 31%
37% 40% 46% 52% 37%
32% 30% 29% 32% 32%
*Strain is defined as stretched length/initial length-1.
Wavenumber (cm )
1500 1600 1700
(normilized by amide I intensity)
line) and the same clot after compression (green line). The difference spec-
trum is shown as a black dashed line. (Inset) Changes in the amide III band.
Normalized FTIR spectra of uncompressed plasma clot (red
Biophysical Journal 103(5) 1020–1027
Mechanical a-b Transition in Hydrated Fibrin1023
substantial increase of 1612–1618 cm?1at the expense of
a 1651-cm?1a-helical component along with a decrease
of intensity at 1637–1638 cm?1and 1622–1624 cm?1
(Fig. S4 and Fig. S5). Because all three peaks in the
1612–1638 cm?1range reflect b-structures, the shift to
the smaller wavenumbers suggests that there is a deforma-
tion-dependent redistribution in the types of b-structures,
most likely due to emergence of newly formed intermolec-
ular b-sheets associated with protein aggregation (38).
Deconvolution of the amide I band contour confirmed
that deformation induced not only an overall quantitative
increase of the b-structures, but also caused their qualitative
rearrangement. Fig. 1 B, Fig. S6, and Tables S1–S8 show
that there are three major types of b-structures in fibrin
reflected by deconvolution components at 1612–1614,
b-structures responded differently to extension and com-
pression of a fibrin clot (Fig. S7). The content of the
1636–1638-cm?1type of b-structures remained almost
unchanged, whereas two other fractions increased according
to the degree of deformation. The fraction peaking at 1612–
1614 cm?1increased two- to threefold in the maximally
deformed clots, whereas the fraction peaking at 1624–
1627 cm?1increased moderately by 18–35%. These data
indicate that some types of b-structures remain unperturbed
during fibrin deformation, whereas others are newly formed
as a result of conversion from a-helices. The infrared-
spectral parameters of the nascent b-structures are charac-
teristic of the intermolecular b-sheets involved in protein
To see what might happen to fibrin fibers under elonga-
tion and pressure, we performed SEM of stretched and
compressed fibrin clots in comparison with unperturbed
fibrin (Fig. 4). What can be unambiguously gleaned from
the scanning electron micrographs is that both deformed
clots, stretched and compressed, clearly undergo dramatic
fiber bundling. The phenomenon of fiber bundling in
response to elongation of a clot was demonstrated previ-
ously (4), whereas, to the best of our knowledge, this effect
has never been observed before for compressed fibrin clots.
Essentially, the bundling of fibrin fibers observed using
SEM is a microscopic manifestation of protein aggregation
that occurs within and between filaments and protofibrils.
This aggregation is followed by water expulsion underlying
volume shrinkage of fibrin clots during deformation (4). On
the basis of our results and the literature, it is quite possible
that protein aggregation during extension and compression
of fibrin fibers is associated with the a-b transition followed
by b-sheet-mediated protein aggregation. It has been
shown that aggregation of protein molecules is driven by
the formation of intermolecular hydrogen bonds responsible
for intermolecular b-sheet structure (40); therefore, the
decrease of a-helix in favor of b-sheet aggregates have
been proposed as a general mechanism of oligomerization
of bovine serum albumin (41). Progressive transformation
from a-helix to b-sheets in bovine serum albumin was
followed by an increasing shoulder at 1620 cm?1in the
FTIR spectrum attributed to the intermolecular b-sheet
structure (42). With respect to protein-specific variation in
the peaks of vibration spectra, this finding is similar to the
changes that we observed as a deformation-dependent
increase in intensity at 1612–1614 sn?1and 1624–
1627 cm?1(Fig. S7), which comprises formation of inter-
molecular b-sheets (17,38,39). Thus, the a-b transition
followed by formation of an intermolecular b-sheet struc-
ture and protein aggregation could be a common mechanism
underlying the different types of fibrin deformation.
Reversibility and time course of the a-b transition
To see whether the deformation-induced a-b transition is
reversible or not, the clots were examined 15 min after
they were released from the stretcher or decompressed
while being kept in the buffer. Fig. 5 A shows that at strains
1 and 2 the relaxed fibrin clots partially restored the absor-
bance intensity ratio at 1622/1651 cm?1, which roughly
10 10 μ μm m
strongly compressed (C) fibrin clots. Magnification bar ¼ 10 mm. Note fiber
bundling both in the stretched and compressed fibrin clots.
SEM images of unperturbed (A), fourfold stretched (B), and
Biophysical Journal 103(5) 1020–1027
1024Litvinov et al.
corresponds to the b-sheet/a-helix content ratio, indicating
partial reversibility of the a-b transition in the experimental
timescale. There was a clear inverse relationship between
the degree of reversibility and the strain (inset of
Fig. 5 A). It is noteworthy that the ability of stretched fibrin
clots to reverse their FTIR spectra upon relaxation (assessed
by the shift of intensity ratio at 1622/1651 cm?1) corre-
sponded to partial recovery of the macroscopic length of
hydrated clots (correlation coefficient R ¼ 0.75, p <
0.05). The changes in FTIR spectra induced by compression
of fibrin clots were also partially reversible (Fig. 5 B). The
residual 1622/1651 cm?1intensity ratio after decompres-
sion correlated well with the values observed in the same
samples at the peak of compression (R ¼ 0.85, p < 0.05)
(Fig. 5 B, inset), indicating that the ability to recover (lower
values of the 1622/1651 cm?1intensity ratio upon decom-
pression) displayed an inverse relationship with the degree
of compression (higher values of the 1622/1651 cm?1inten-
sity ratio upon compression).
The partial macroscopic reversibility of the fibrin clots
fits with the deformation-induced a-b transition in fibrin
(Fig. 5); however it does not exclude other mechanisms of
fibrin elasticity, such as straightening and unfolding of the
aC regions (43). If we think of the a-b transition as
a biphasic process with elastic and plastic components,
then it would be reasonable to assume that the force-induced
transition of a-helix to b-sheet is reversible, whereas sub-
sequent intermolecular aggregation of b-structures is irre-
versible, making the reversibility of the entire deformation
dependent on the fraction of b-sheets, which correlates
directly with the degree of deformation (Table 1).
To estimate how fast and stable are the observed changes
in the secondary structure, FTIR spectra of the same clots
were measured over time, starting as soon as possible after
considerable stretching or compression and followed by
repeated measurements with ~10-min intervals. Because
of technical limitations, it took 3–10 min after clot deforma-
tion to start data acquisition and 3 min to record a spectrum
at each time point. To avoid effects of drying, the clots were
kept wet in the stretched or compressed state by continu-
ously irrigating them with buffer. Three different fibrin clots
were watched over time under each experimental condition.
Fig. S8 shows the time course of the intensity ratio at 1622/
1651 cm?1(from second derivative spectra), which was
quite sensitivetoalterations in the rawFTIR spectrainduced
by deformation of fibrin clots. The results demonstrate that
the FTIR spectra remained unchanged for at least ~1 h after
deformation as the clots were kept under strain or pressure,
implying that they were observed under equilibrium con-
ditions. In other words, in our experimental settings the
rate of nonequilibrium a-b transition was too fast to be
followed in the real timescale. In other words, the a-b tran-
sition occurred sometime between deformation and the first
measurement (within 3–10 min) and remained steady under
the action of a constant force.
Effect of dehydration of fibrin clots
on the FTIR spectra
Because plasma clots contain >99% liquid (2.5 g/L of
fibrinogen in plasma corresponds to 0.25% protein) and
deformation of fibrin clots is followed by a dramatic loss
of water due to mechanical squeezing as well as expulsion
during protein aggregation (4), we tested whether the
observed changes in the secondary structure were induced
solely or substantially by the loss of water, we studied
whether mechanical dehydration per se can account for
the observed changes in the secondary structure. When an
unperturbed hydrated fibrin clot was dried, either partially
Intensity ratio at 1622/1651 cm-1
Intensity ratio at 1622/1651 cm- -1
Clot under pressure
R = 0.85
ATR spectra. (A) The average ratio of second derivative of absorbance
intensities at 1622/1651 cm?1in unstretched, stretched at strains 1 (n ¼
9) and 2 (n ¼ 4), and relaxed fibrin clots. (Inset) Relative reversibility of
the average ratio of absorbance intensities at 1622/1651 cm?1as a function
of strain. The reversibility was calculated as 100*(average value in the
stretched state – average value after relaxation)/(average value in the
stretched state – average initial value). (B) The average ratio of second
derivative of absorbance intensities at 1622/1651 cm?1in uncompressed,
compressed, and decompressed fibrin clots (n ¼ 10) at various pressures.
(Inset) The residual ratio of absorbance intensities at 1622/1651 cm?1in
individual decompressed fibrin clots plotted against the ratio measured in
the same fibrin clots at the peak of compression. Error bars represent stan-
Reversibility of the deformation-induced changes in FTIR
Biophysical Journal 103(5) 1020–1027
Mechanical a-b Transition in Hydrated Fibrin1025
in the air or almost completely over P2O5, the measurable
changes in FTIR spectra had nothing to do with transition
of the a-helices into b-sheets (Fig. S9). Instead, loss of
loosely bound water was detected as the clot shifted from
the wet to air-dried states. Much more pronounced changes
occurred upon deeper drying over P2O5. Positive peaks on
the difference trace at 1510, 1664, 1676, and 1692 cm?1
suggest an increase of turns and unhydrated peptide groups.
Negative peaks at 1556, 1621, and 1643 cm?1point to the
breakup of some a-helices, b-sheets, and dehydration of
random structures (18,20,24). The data indicate that dehy-
dration cannot account for the observed secondary structure
changes upon fibrin deformation.
CONCLUSIONS AND POTENTIAL IMPLICATIONS
In conclusion, FTIR spectroscopy of wet washed human
plasma clots allowed us to demonstrate the a-helix to b-
sheet transition that occurred in coiled-coils during substan-
tial extension and compression of fibrin gels. We showed
that a decrease of a-helix was equivalent to an increase of
b-structures without changes in other secondary structure
elements, suggesting that the change occurred in the
coiled-coils. The extent of the a-b transition was directly
proportional to the degree of deformation and was a pure
mechanical effect without a significant role of dehydration.
The observed a-b transition both in elongation and com-
pression was associated with b-sheet-mediated protein
aggregation as revealed by fiber bundling observed in the
scanning electron micrographs and characteristic changes
of FTIR spectra, indicating formation of interchain b-sheets.
The alterations of secondary structures were partially
reversible with the degree of reversibility being inversely
proportional to the strain or pressure applied. The results
provide additional evidence for the a-b transition as a
universal mechanism of forced unfolding of filamentous
proteins with relatively high a-helical content. The data
also shed light at the molecular structural origins of the
elasticity and plasticity of the fibrin polymer, underlying its
mechanical role in preventing bleeding and in thrombosis.
Taken together, our results provide insight into the molec-
ular basis of fibrin clot mechanics and a general mechanism
for deformability of proteins, but they also have important
physiological and medical implications. First, the a-b
transition may toughen fibrin at large deformations because
b-sheets are more resistant to shear than a-helices (37), and
clot stiffness has been known to correlate directly with the
incidence of myocardial infarction and other cardiovascular
diseases. Second, the ability of fibrin to undergo the a-b
transition and aggregation may result in formation of tightly
packed b-sheets, analogous to those of amyloid structures.
Of importance, fibrin clots displayed amyloid-like features
upon extension revealed by staining by Congo red (7), and
accumulation of b-amyloid peptide (Ab) makes fibrin clots
more resistant to proteolytic degradation (44). There is also
genetically determined fibrin(ogen)-related amyloidosis
in some dysfibrinogenemias (45). Third, controlling the
a-b transition could potentially lead to new strategies for
elimination of thrombi by either stabilizing or destabilizing
the coiled-coil, rendering clots more sensitive to treatment.
Nine figures, eight tables, and three references are available at http://www.
The authors thank Drs. Feng Gai and Prashant K. Purohit (University of
Pennsylvania) for reading the manuscript and helpful suggestions and
Chandrasekaran Nagaswami for assistance with electron microscopy.
This work was supported by the National Institutes of Health, grants
HL030954 and HL090774 (J.W.W.), and the Russian Academy of Sciences
under the Program "Molecular and Cellular Biology’’ (D.A.F. and Y.F.Z.).
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