THROMBOSIS AND HEMOSTASIS
Alina Hategan,1Kathryn C. Gersh,1Daniel Safer,2and John W. Weisel1
1Department of Cell and Developmental Biology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA; and2Pennsylvania Muscle Institute,
University of Pennsylvania, Philadelphia, PA
• Fibrin polymerization was ob-
served for the first time at the
single-molecule level by total
internal reflection fluores-
• Live observation of fibrin po-
lymerization with a single-
molecule fluorescence inten-
sity calibration revealed the
real-time growth kinetics.
Individual fluorescently labeled fibrin(ogen) molecules and their assembly to make a
clot were observed by total internal reflection fluorescence microscopy (TIRFM). We
used the bleaching of the fluorescent labels to determine the number of active
fluorophores attached nonspecifically to each molecule. From the total intensity of
bleaching steps, as single-molecule signature events, and the distribution of active
labeling, we developed a new single-molecule intensity calibration, which accounts for
all molecules, including those “not seen.” Live observation of fibrin polymerization in
TIRFM by diffusive mixing of thrombin and plasma revealed the real-time growth kinetics
of individual fibrin fibers quantitatively at the molecular level. Some fibers thickened in
time to thousands of molecules across, equivalent to hundreds of nanometers in
diameter, whereas others reached an early stationary state at smaller diameters. This
new approach to determine the molecular dynamics of fiber growth provides informa-
tion important for understanding clotting mechanisms and the associated clinical
implications. (Blood. 2013;121(8):1455-1458)
Much is known about the properties and functions of fibrinogen,
including fibrin polymerization. Most studies of fibrin clotting
visualized large clot structures either at low resolution by confocal
microscopy or at higher resolution by electron microscopy (at
certain time points), or used indirect biophysical methods.1-6As a
result, little is known about the microscopic and molecular
structural origins of the clot, especially about the mechanisms in
the early stages of polymerization and the molecular kinetics.1,7
More recently, some aspects of assembly before the gel point were
visualized by deconvolution and spinning disk confocal micros-
copy,8,9but the smallest structures observed were protofibrils.
Information about clotting, especially in the early stages that
determine clot properties and the gel point, has clinical relevance,
because the gel point is used diagnostically and disorders of
clotting or fibrinolysis accompany or cause many pathologic
conditions, including myocardial infarction and stroke.
Here we visualize and characterize individual fibrinogen mol-
and investigate the dynamics of fibrin assembly and fibrin fiber
properties at the single-molecule level. In TIRFM, because of low
background, sensitivity is high for events near the coverslip, and
single molecules can be detected.10-12By analyzing the bleaching
of single fibrinogen molecules, we characterize the distribution of
labeling and develop a new molecular calibration for the fluores-
cence intensity of fibers, revealing aspects of the real-time growth
of individual fibers at the molecular level.
Approval was obtained from the University of Pennsylvania Institutional
Review Board for these studies.
Purified human fibrinogen from Hyphen BioMed was labeled nonspe-
cifically with tetramethyl-rhodamine and Alexa Fluor 488 (supplemental
Methods, available on the Blood Web site; see supplemental Materials link
at the top of the online article) in parallel samples to assure that none of the
observations were the result of artifacts because of dye attachment.
For single-molecule TIRFM observation, 1 mg/mL fibrinogen stocks
were diluted 1000? in 20mM HEPES, 150mM NaCl, pH 7.4 (HBS), and
ultracentrifuged for 30 minutes at 100 000g to sediment aggregates, leaving
only single molecules in the supernatant. The supernatant was diluted to
0.1 ?g/mLbefore introduction into the TIRFM chamber.
To study fibrin fibers in later stages of polymerization, we mixed
platelet-poor plasma (PPP; preparation described in supplemental Methods)
with fluorescently labeled fibrinogen (1 vol fluorescent fibrinogen to 17 vol
PPP) with HBS containing thrombin and CaCl2for a final concentration of
0.5 U/mL thrombin and 20mM CaCl2. The mixture was immediately
introduced into the observation chamber.
Submitted August 21, 2012; accepted November 4, 2012. Prepublished online
as Blood First Edition paper, December 11, 2012; DOI 10.1182/blood-2012-08-
There is an Inside Blood commentary on this article in this issue.
The online version of this article contains a data supplement.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
© 2013 by TheAmerican Society of Hematology
1455 BLOOD, 21 FEBRUARY 2013?VOLUME 121, NUMBER 8
To study the real-time polymerization of fibrin in plasma, PPP spiked
with fluorescent fibrinogen (1 vol fluorescent fibrinogen to 10 vol PPP) was
introduced into one side of the observation chamber and HBS containing
5 U/mL thrombin and 200mM CaCl2was introduced into the other side to
induce polymerization through diffusion at the interface of the 2 liquids.
Data processing was done with an ImageJ plug-in that measures the
time evolution of total intensity of single molecules, by recording intensity
in regions of interest centered automatically on the bright areas in the first
image of a time-series stack and repeating the measurement throughout the
stack (ImageJ Version 1.36b software). To measure total intensity of fibrin
fibers, similar but larger regions of interest, between branch points of fibers,
were selected manually when using the same plug-in. After background
subtraction, bleaching correction was performed using the bleaching time
constant of each dye (supplemental Methods).Anew molecular calibration
was developed to determine M, the number of molecules in each fiber
(including fluorescent and unlabeled molecules; see supplemental Meth-
ods). Using M and considering the fiber structure of parallel rows1,13,14we
calculated the number of molecules in the cross-section of each fiber, c, and
the corresponding diameter, D, of the hydrated fiber (see supplemental
Results and discussion
Individual fluorescently labeled fibrinogen molecules that attached
to the glass chamber surface were visualized (Figure 1A). Because
of the low fibrinogen concentration, prior removal of aggregates,
and low probability of attachment to the surface, most fluorescent
signals observed represented single molecules, as previously
demonstrated (see supplemental Methods).12Time sequences of
fluorescence images showed that most of the signals bleached in
1 step (Figure 1B), some bleached in multiple steps (Figure 1C-E)
or presented an exponential decay (Figure 1F). Bleaching in 1 step
is the signature of a single molecule labeled with 1 fluorescent
probe.10Bleaching in several steps indicates the presence of several
dyes attached to a fibrinogen molecule. The intensity of the
bleaching steps was in the same range for all bleaching events
tion of signals from several dyes that bleach over time, and their
number can be calculated from the intensity analysis. With
increasing bulk-labeling ratio, the number of 2-, 3-, and 4-step
bleaching events increased slightly; however, the most frequent
were the 1-step bleaching events, even at higher ratios (Figure 1H).
Similar results were obtained with both dyes.
Using this molecular distribution of active fluorophores per
fibrinogen molecule (Figure 1H) and the total intensity of a
bleaching step, while taking into account the labeling probability,
and considering the unlabeled plasma fibrinogen, a single-molecule
calibration for the fluorescent fibrin fibers seen in TIRFM was devel-
Two different approaches were used to observe aspects of fibrin
polymerization. Fluorescent fibrin fibers were observed in TIRFM
after induction of polymerization in a vial by adding thrombin into
recalcified citrated plasma and transferring the mixture to the
chamber by flow (Figure 2A). The flow did not orient the fibers
parallel to the flow direction, showing that within seconds, the
network was already formed and stable. However, the flow did
bend many of the fibers, indicating a deformable network.
Figure 1. Bleaching of single fluorescently labeled fibrinogen molecules in TIRFM. (A) TIRFM image showing single fibrinogen molecules that adhered to the chamber
glass surface from bulk solution. Time sequences of images like this showed the irreversible bleaching of the attached fluorophores in (B) 1 step, (C) 2 steps, (D) 3 steps,
(E) 4 steps, or (F) showed sometimes an exponential decay. (G) Intensities of steps were in the same range for all individual bleaching events. Data plotted are from 1 single set
of measurements and all the other datasets present similar values. Each data point represents a mean of at least 5 values and error bars are standard deviations. Because the
magnitudes of the bleaching steps are all multiples of a single bleaching step, the number of bleaching steps accounts for the number of fluorophores attached, whereas the
exponential decay curve accounts for a larger number of dyes attached to a fibrinogen molecule (number that can be determined from the intensity analysis). (H) Bleaching
event distributions for several bulk-labeling ratios using tetramethyl-rhodamine–labeled fibrinogen show that by increasing the bulk-labeling ratio, the 1-step bleaching events
decrease in favor of multiple steps events; however, 1-step bleaching events remain predominant. This distribution of active labeling we used for the molecular calibration. At
least 90 bleaching events were collected for each ratio. The lines are linear fits of 1-step bleaching events, 2-step bleaching events, and so on. We used an Olympus PlanApo
60?/1.45 oil TIRFM objective and an ORCA-ER Hamamatsu (Japan) camera. Images were acquired with National Instruments IMAQ Vision Builder 6 and image sequences
were recorded at 1 frame/s. Resolution in the TIRFM image was 80 nm/pixel.
1456 HATEGAN et al BLOOD, 21 FEBRUARY 2013?VOLUME 121, NUMBER 8
To observe polymerization ab initio, fluorescently labeled
fibrinogen in plasma was introduced at one side of the microfluidic
observation chamber and thrombin was introduced at the other side
to induce polymerization at the interface between the 2 liquids
(Figure 2B). Because at the ?m dimensions of the TIRFM
chamber, liquids that make contact do not mix,15polymerization
was due exclusively to the diffusion of thrombin molecules into the
fibrinogen region, and the rate of fiber growth was therefore
diffusion dependent. As expected for the low concentration of
thrombin reaching the fibrinogen region, the fibers were thick.16,17
Lateral growth of fibers continued for minutes (Figure 2C) and the
single-molecule calibration showed that growth increases to thou-
sands of molecules across (Figure 2D), but some fiber regions
reach an early stationary state at lower values (Figure 2D inset).
Fiber lateral growth is probably determined by the local kinetics of
fibrinopeptide cleavage and oligomerization, as well as physical
factors, such as the twisting of fibers.16,18The calculated diameters
of the hydrated fibers are variable along the length of the fibers and
can grow to hundreds of nanometers (Figure 2E) being in the same
range as those measured in unbleached fluorescent images.
In conclusion, we demonstrated that the dynamics of fibrin
polymerization can be studied at the single-molecule level using
TIRFM. Through analysis of bleaching individual fibrinogen
molecules, we obtained the molecular distribution of fibrinogen
labeling, which was used to obtain a single-molecule intensity
calibration. We observed live growth kinetics with molecular
accuracy, determined for the first time the number of molecules
within individual fibrin fibers, and calculated their diameters,
bridging in this way the gap from molecular to assembled
structures.This new methodology can be used for further studies of
the assembly of fibrin or other biopolymers.
The authors thank Dr Henry Shuman and Dr Yale Goldman for use
of their TIRFM setup.
This work was supported by National Institutes of Health grants
HL030954 and HL090774.
Contribution: A.H. designed the research, performed experiments,
analyzed and discussed results, prepared the figures, and wrote the
paper; K.C.G. and D.S. performed the fluorescence labeling of
Figure 2. Fluorescent fibrin fibers observed and characterized in TIRFM. (A) Typical TIRFM image of fluorescently labeled fibrin fibers formed by mixing thrombin into
recalcified plasma and then insertion into the flow chamber. There was no preferential orientation of the fibers because of flow in the chamber, but most fibers were curved
because of shear forces. (B) TIRFM observation chamber (2 clean glass coverslips on top of each other, separated laterally by double-stick tape to create a 75-?m thick
chamber) allowed real-time observation of fibrin polymerization in TIRFM by introducing thrombin solution at one side of the chamber (green region) and allowing its diffusion
into the region with fluorescent fibrinogen molecules (red region). (C) TIRFM images of the kinetics of growth of fibrin clot 1 molecule at a time. Growth of fibers continued for
tens of minutes in the observation region, arbitrarily chosen in the center of the chamber close to the interface of thrombin and plasma. Frame 1 shows fluorescent fibrinogen in
the central area of the chamber before introducing thrombin, and frames 2 and 3 were taken at 4.30 and 21 minutes after thrombin insertion. The sample was kept in the dark
between frames 2 and 3 to avoid bleaching and thus show real lateral growth. (D-E) Graphs of fiber growth as a function of time, plotted as either number of molecules in the
cross section of fiber, c, or hydrated fiber diameter, D. The 1-second time-point is the first moment of observation of the network after contact of thrombin with fibrinogen.
According to the single-molecule calibration, lateral growth of fibers went up to thousands of molecules across (D), but some fiber regions reached an early stationary state
(inset). Calculated diameters of the hydrated fibers (E) showed growth of up to 950 nm in 250 seconds for the largest fibers, and are in the same range as fiber diameters
measured in unbleached fluorescence images, but more accurate. Regions of fibers analyzed were between branch points or intersections of fibers and had lengths from 2 to
10 ?m. Image sequences were recorded at 1 frame/s. Resolution in allTIRFM images was 80 nm/pixel. Images presented are raw data with no bleaching correction performed
for the frames in (C). Scale bar is 3 ?m in panelAand 2.5 ?m in panel C.
SINGLE-MOLECULE DYNAMICS OF FIBRIN POLYMERIZATION1457 BLOOD, 21 FEBRUARY 2013?VOLUME 121, NUMBER 8
fibrinogen; and J.W.W. discussed results, analyzed data, and wrote Download full-text
Conflict-of-interest disclosure: The authors declare no compet-
ing financial interests.
tal Biology, University of Pennsylvania Perelman School of
Medicine, 421 Curie Blvd, 1054 BRB II/III, Philadelphia, PA
19104; e-mail: firstname.lastname@example.org.
1. Weisel JW. Fibrinogen and fibrin. Adv Protein
2. Hantgan RR, Hermans J.Assembly of fibrin.A
light scattering study. J Biol Chem. 1979;254(22):
3. Hantgan R, Fowler W, Erickson H, Hermans J.
Fibrin assembly: a comparison of electron micro-
scopic and light scattering results. Thromb Hae-
4. Janmey PA, Ferry JD. Gel formation by fibrin oli-
gomers without addition of monomers. Biopoly-
5. Blomback B, Carlsson K, Hessel B, LiljeborgA,
Procyk R, Aslund N. Native fibrin gel networks
observed by 3D microscopy, permeation and tur-
bidity. Biochim Biophys Acta. 1989;997(1-2):96-
Structure of fibrin gels studied by elastic light
scattering techniques: dependence of fractal di-
mension, gel crossover length, fiber diameter,
and fiber density on monomer concentration.
Phys Rev E Stat Nonlin Soft Matter Phys. 2002;
7. FogelsonAL, Keener JP. Toward an understand-
ing of fibrin branching structure. Phys Rev E Stat
Nonlin Soft Matter Phys. 2010;81(5 Pt 1):051922.
8. Chernysh IN, Weisel JW. Dynamic imaging of
fibrin network formation correlated with other
measures of polymerization. Blood. 2008;111(10):
9. Chernysh IN, Nagaswami C, Weisel JW. Visual-
ization and identification of the structures formed
during early stages of fibrin polymerization.
10. YildizA, Forkey JN, McKinney SA, Ha T,
Goldman YE, Selvin PR. Myosin V walks hand-
over-hand: single fluorophore imaging with
1.5-nm localization. Science. 2003;300(5628):
11. Gordon MP, Ha T, Selvin PR. Single-molecule
high-resolution imaging with photobleaching.
Proc Natl Acad Sci U S A. 2004;101:6462-6465.
12. Park H, Toprak E, Selvin PR. Single-molecule
fluorescence to study molecular motors. Q Rev
Lateral packing of protofibrils in fibrin fibers and
fibrinogen polymers. Biopolymers. 1986;25(12):
14. Voter WA, Lucaveche C, Erickson HP. Concentra-
tion of protein in fibrin fibers and fibrinogen poly-
mers determined by refractive index matching.
15. Ottino JM, Wiggins S. Designing optimal micro-
mixers. Science. 2004;305(5683):485-486.
16. Weisel JW, Nagaswami C. Computer modeling of
fibrin polymerization kinetics correlated with elec-
tron microscope and turbidity observations: clot
structure and assembly are kinetically controlled.
Biophys J. 1992;63:111-128.
Weisel JW. Influence of fibrin network conforma-
tion and fibrin fiber diameter on fibrinolysis speed:
dynamic and structural approaches by confocal
microscopy. Arterioscler Thromb Vasc Biol. 2000;
18. Weisel JW, Nagaswami C, Makowski L. Twisting
of fibrin fibers limits their radial growth. Proc Natl
Acad Sci U S A. 1987;84:8991-8995.
1458HATEGAN et alBLOOD, 21 FEBRUARY 2013?VOLUME 121, NUMBER 8