Download Resolving the multifaceted mechanisms of the ferric chloride

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THROMBOSIS AND HEMOSTASIS
Resolving the multifaceted mechanisms of the ferric chloride thrombosis
model using an interdisciplinary microfluidic approach
Jordan C. Ciciliano,1-4 Yumiko Sakurai,2-4 David R. Myers,2-4 Meredith E. Fay,2-4 Beatrice Hechler,5 Shannon Meeks,3
Renhao Li,3 J. Brandon Dixon,1,2 L. Andrew Lyon,6 Christian Gachet,5 and Wilbur A. Lam2-4
1
Woodruff School of Mechanical Engineering, Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA; 2Wallace C
Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA; 3Aflac Cancer and Blood Disorders
Center, Department of Pediatrics, Children’s Healthcare of Atlanta and Emory University School of Medicine, Atlanta, GA; 4Institute of Electronics and
Nanotechnology, Georgia Institute of Technology, Atlanta, GA; 5INSERM, Unité Mixte de Recherche S949, Etablissement Français du Sang-Alsace,
Université de Strasbourg, Strasbourg, France; and 6School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA
The mechanism of action of the widely used in vivo ferric chloride (FeCl3) thrombosis
model remains poorly understood; although endothelial cell denudation is historically
cited, a recent study refutes this and implicates a role for erythrocytes. Given the com• The multivariate mechanism
of FeCl3-induced thrombosis is plexity of the in vivo environment, an in vitro reductionist approach is required to systematically isolate and analyze the biochemical, mass transfer, and biological phenomena
rooted in colloidal chemistry,
that govern the system. To this end, we designed an “endothelial-ized” microfluidic device
mass transfer, and biological
to introduce controlled FeCl3 concentrations to the molecular and cellular components of
clotting.
blood and vasculature. FeCl3 induces aggregation of all plasma proteins and blood cells,
• FeCl3-induced thrombosis is
independent of endothelial cells, by colloidal chemistry principles: initial aggregation is due
mediated by charge-based
to binding of negatively charged blood components to positively charged iron, independent
binding of proteins (cell
of biological receptor/ligand interactions. Full occlusion of the microchannel proceeds
surface bound and soluble)
by conventional pathways, and can be attenuated by antithrombotic agents and loss-ofto the Fe31 ion.
function proteins (as in IL4-R/Iba mice). As elevated FeCl3 concentrations overcome protective effects, the overlap between charge-based aggregation and clotting is a function of
mass transfer. Our physiologically relevant in vitro system allows us to discern the multifaceted mechanism of FeCl3-induced thrombosis, thereby reconciling literature findings and cautioning researchers in using the FeCl3 model. (Blood. 2015;126(6):817-824)
Key Points
Introduction
In vivo animal models of thrombosis are used extensively to study clot
formation and propagation, of which the ferric chloride (FeCl3) thrombosis model is the most commonly used. In this model, filter paper
saturated with FeCl3 is applied to vessel adventitia of small mammals,
typically mice, resulting in occlusive thrombosis.1-5 As the FeCl3 diffuses into the vascular space, clot formation is commonly monitored
by Doppler flow and intravital microscopy. Although the FeCl3 injury
model is popular due to ease of implementation and robust thrombus
formation, the underlying mechanisms of FeCl3-induced clotting remain unclear.
Clot initiation in this model is historically attributed to free ironinduced denudation of endothelial cells and the subsequent exposure
of the subendothelium that triggers platelet adhesion/aggregation and
activation of the coagulation cascade.6 However, studies using mice deficient in collagen receptor glycoprotein VI have produced contradictory results, reporting both normal and altered thrombus formation.7,8
Eckly et al observed that although endothelial cells appear denuded and
platelets are present in the occlusive thrombus, there is limited collagen
exposure in the vessel.9 Furthermore, an elegantly designed scanning
electron microscope (SEM) analysis revealed that endothelial cells are
not denuded, and surprisingly, that erythrocytes mediate the adherence
of platelets to the endothelium by an unknown mechanism.10 In addition to the uncertain status of the endothelium, the effects of FeCl3
vary based on the vessel to which it is applied: in mice lacking the extracellular domain of glycoprotein Iba (GPIba), the FeCl3 model results in occlusion in mesenteric venules but not in the carotid artery.7,11
These inconsistencies, in conjunction with the wide range of published FeCl3 concentrations (10 mM-2 M; for percentage, see supplemental Table 1, available on the Blood Web site) and application
times (1-20 minutes) used to elicit a clotting response, raise questions
about the direct chemical effects of FeCl3 on blood components and
the role that mass transfer plays in this thrombosis model.12
These questions inspired us to systematically investigate the
mechanisms of FeCl3-induced thrombosis via an in vitro reductionist
approach: interrogating the effect of FeCl3 on individual blood components in the presence and absence of endothelial cells. To this end, we
designed a microfluidic device that enables precise control over FeCl3
influx while allowing for direct visualization and quantification of
Submitted February 17, 2015; accepted April 24, 2015. Prepublished online as
Blood First Edition paper, April 30, 2015; DOI 10.1182/blood-2015-02-628594.
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.
The online version of this article contains a data supplement.
There is an Inside Blood Commentary on this article in this issue.
BLOOD, 6 AUGUST 2015 x VOLUME 126, NUMBER 6
© 2015 by The American Society of Hematology
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CICILIANO et al
FeCl3-induced aggregation of isolated blood components. Our laboratory has shown that vascular phenomenon relevant to hematologic
processes can be studied in vitro by culturing endothelial cells to
a confluent monolayer in microchannels.13,14 These “endothelial-ized”
microfluidics allow us to recapitulate the in vivo endothelium-bloodFeCl3 interface and monitor the status of the endothelium during FeCl3
application, while controlling the flow conditions and cellular and molecular “inputs” to the system. We find that all isolated blood components and molecules aggregate in a FeCl3 concentration-dependent
manner, with and without endothelial cells. We therefore posit a novel
2-stage mechanism for the action of FeCl3 in thrombus formation. The
first stage is based on colloidal chemistry principles whereby cells and
plasma proteins adhere and aggregate due to the binding of negatively
charged proteins to positively charged iron species. The extent of this
stage is dependent on effective FeCl3 concentration, which is governed
by intraluminal mass transport. In the second-stage, clot formation proceeds via platelet aggregation and activation of the coagulation cascade
in response to both endothelial cell death and arrested blood components. Experimentally separating the first and second phases is nontrivial, as they are visually indistinguishable and occur on a similar time
scale. This novel multifaceted mechanism reconciles conflicting
findings in the literature and cautions the use of the FeCl3 model,
especially in interpreting the initial stages of clotting. Our 2-phase
description of the underlying mechanisms of this model complements
and further explains Barr et al’s findings that red blood cells (RBCs)
mediate FeCl3-induced thrombosis.10
Methods
Human blood was drawn according to institutional review board–approved
protocols per the Declaration of Helsinki into sodium citrate (Becton Dickinson).
To recapitulate the endothelium-blood-FeCl3 interface, a polydimethylsiloxanebased T-shaped microfluidic was designed with a 250-mm-width main channel,
50-mm-width side channel, and overall device height of 50 mm. When appropriate, channels were endothelialized as previously described.15 To study whole
blood, RBCs were diluted in platelet-rich plasma (PRP) to 12.5% to allow for
improved microscopy and visualization of clot components. To recalcify blood,
CaCl2 (Sigma) was added to a final concentration of 0.01 M directly prior to every
experiment, unless otherwise noted. To analyze drug treatment data, we used a
2-tailed Student t test with a 95% confidence interval (with statistical significance
when P , .05). See supplemental Methods for blood separation protocol, in vivo
iron measurements, diffusion calculations, and flocculation experiment and drug
treatment/mouse experiment protocol.
Results
Conceptual framework of in vivo FeCl3 model
To recapitulate the in vivo FeCl3 model in a controlled in vitro system,
the FeCl3 concentration that the endothelial cells and blood are exposed
to was determined. Although the FeCl3 concentration at the vessel adventitia is that applied to the filter paper, the luminal endothelial cell
concentration (after the FeCl3 diffuses through the vessel layers) is the
unknown, but calculable, value of interest (Figure 1A). As diffusion of
FeCl3 through the vessel wall is complex and difficult to experimentally
validate, we instead measured the bulk iron concentration downstream
of the application site in vivo and used Fick’s laws of diffusion to calculate the concentration at the endothelial cell/blood interface. Postapplication of 1 M FeCl3 to the adventitia of a mouse carotid artery,
the bulk iron concentration in blood was found to be ;9 mg/mL or
BLOOD, 6 AUGUST 2015 x VOLUME 126, NUMBER 6
160 mM. Steady-state FeCl3 flux into the vessel is reached by 5 minutes
and maintained at 10 minutes; the patch concentration is thus assumed
to be constant over the relevant time scales. By equating flux of FeCl3
into the vessel to flux out of the vessel, a conservative estimate (that
does not account for system losses) of concentration at the wall of the
lumen is calculated to be ∼50 mM (supplemental Methods). This FeCl3
concentration can be introduced to blood components via perfusion into
an in vitro microfluidic system, thereby giving us spatiotemporal control over FeCl3 concentration, and the ability to monitor its effects on
blood and the vasculature.
In vitro endothelialized microfluidic FeCl3 thrombosis model
To visualize the effects of FeCl3 on blood components in a reductionist
manner, microfluidic channels were designed to introduce FeCl3 to
individual blood components in a fixed shear environment. In vivo,
FeCl3 continuously enters the vessel from the patch and interfaces with
endothelial cells and blood. As convection dominates in this system, the
FeCl3 concentration remains high near the vessel wall and flows downstream with the blood. The flow environment is aptly recapitulated
using a T-shaped microfluidic; the influx of FeCl3 through a small side
channel gives spatiotemporal control over the concentration, ensuring
that the blood cells and endothelial cells at the streams’ interface are
exposed to an exact input concentration. The 2 solutions interface near
the channel wall, as it occurs in vivo (Figure 1C). The main channel of
the microfluidic can be seeded with endothelial cells, which are cultured
to a confluent monolayer on the channel surfaces, further recapitulating
in vivo physiologic conditions (Figure 1B-C), though endothelial cells
do not span the gap where the main channel joins the side channel.
Endothelialized microfluidics enable us to concurrently visualize the
effects of FeCl3 on blood and endothelial cells using standard brightfield and fluorescence video microscopy.
Effects of FeCl3 on in vitro “vasculature”
We recreated the in vivo FeCl3 model by introducing FeCl3 to blood
in an endothelialized microfluidic system. When exposed to 50 mM
FeCl3, endothelial cells remain adherent and whole blood forms an
occlusive aggregate (Figure 2A; supplemental Video 1). Platelets and
fibrinogen are present within the effective “clot,” but interestingly,
erythrocytes constitute the bulk of the aggregate (Figure 2A). Furthermore, when individually introduced, washed RBCs, platelets,
platelet-poor plasma (PPP), PRP, and even 5% bovine serum albumin
(BSA) form aggregates at the FeCl3 interface (Figure 2A; supplemental
Figure 1A, supplemental Video 2). The effect of FeCl3 on endothelial
cell viability is concentration-dependent: endothelial cells are viable at
low concentrations, but as concentrations increase from 5 mM to 1 M,
endothelial cell death is more pervasive (Figure 2B). Dead endothelial
cells remain adherent to the channels at all tested concentrations and
shear stresses (up to 20 dyne/cm2), corroborating the results of Barr et al
(data not shown).10
Effects of FeCl3 on blood in the absence of endothelial cells
To characterize the effects of FeCl3 on blood, independent of the
endothelium, blood cells and proteins were individually exposed to
FeCl3 in a “bare,” nonendothelialized, microfluidic device (Figure 1B),
and time to aggregate initiation (adherence of components) and stabilization (complete or unchanging occlusion) were recorded. In the
absence of endothelial cells, blood components form aggregates in a
FeCl3 concentration–dependent manner, shown at 0.05, 0.5, and 1 M,
as did 5% BSA (Figure 3; supplemental Figure 1B, supplemental
Videos 3-4). At 50 mM, washed erythrocytes and platelets are slowest
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BLOOD, 6 AUGUST 2015 x VOLUME 126, NUMBER 6
MECHANISMS OF FERRIC CHLORIDE-INDUCED THROMBOSIS
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Figure 1. Recreating the in vivo FeCl3 model in a
microfluidic platform. (A) Schematic of FeCl3 application (not to scale) in which a saturated 1 M FeCl3
patch is placed on the vessel adventitia. As the FeCl3
diffuses through the layers of the vessel wall, apparent
concentration decreases, and at the luminal surface of
the endothelial cells, the FeCl3 concentration is $50 mM.
The dashed box at the luminal surface of the endothelial cells represents the location of interface of the
solutions. (B) Endothelial cells are seeded in the large
channel (250 mm) of a T-shaped microfluidic to recapitulate a blood vessel. FeCl3 is infused in the side
channel (50 mm) and the blood/FeCl3 interface is
visualized; shear stress 4 dyne/cm2. (C) Left to right,
Bare microfluidic channel, endothelialized channel, and
an example of fluid interface using fluorescent dyes
and infusion rates of 2.7 mL per minute in large channel
and 0.4 mL per minute in side channel. The dashed box
represents the fluid interface that would be seen at the
vessel wall in vivo. All scale bars represent 50 mm.
to aggregate, and proteins form sheet-like structures that appear to enhance cell binding and aggregation (Figure 3B). As the concentration
of FeCl3 increases, the time to aggregate initiation decreases, and the
time to aggregate stabilization generally decreases (Figure 3A). Full
channel occlusion and flow cessation occur at 1 M FeCl3 in the absence
of endothelial cells for all blood components.
Proposed mechanism of FeCl3-induced aggregation
The universal aggregation effect of FeCl3 on blood cells and proteins in
the absence of endothelial cells led us to posit a charge-based mechanism for aggregation (Figure 4A). Blood is a colloidal suspension
of negatively charged cells and proteins; the stability, or resistance
to aggregation, of colloidal particles in suspension is determined by
the particle z potential, where a higher z potential corresponds to increased stability.16 In aqueous solutions, ferric ions undergo continuous
hydrolysis, complexation, polymerization, and precipitation.17 At equilibrium, over a range of pH and FeCl3 concentrations, a number of species are present, predominantly Fe31, Fe(OH)21, and Fe2(OH)241.18,19
Fe31 ions can bind directly to negatively charged proteins (both soluble
and cell surface localized), resulting in destabilization of those proteins
with respect to flocculation, and furthermore resulting in cell aggregation.20 The cationic, amorphous hydroxide precipitates also deposit on negatively charged surfaces to form a large particulate matrix
that entraps cells.18,21 Aggregation of colloidal components in the
presence of metal salts proceeds by a combination of factors: an
increase in ionic strength that reduces repulsion between similarly
charged particles; adsorption of ions that neutralize charge and allow for
particle bridging; and cell entrapment in a hydroxide precipitate.17,21
Interestingly, both FeCl3 and aluminum (III) chloride (AlCl3) are used
in wastewater treatment to induce aggregation of colloidal suspensions
of negatively charged toxins and biologics.21,22
Figure 2. Endothelialized microfluidics. (A) Aggregates form in endothelialized microfluidics infused with
50 mM FeCl3 and whole blood, washed RBCs, washed
platelets, PPP, and PRP. Endothelial cells (blue: Hoescht),
platelets (green: CD41), fibrinogen (red). (B) Aggregate
formation over the length of the microchannel when
whole blood is exposed to 50 mM FeCl3. (C) Endothelial
cell viability is concentration dependent with increasing
cell death at higher concentrations; left to right: 5 mM,
50 mM, 500 mM, 1 M (green/live: calcien, red/dead:
propidium iodide). All scale bars represent 50 mm; N 5 5.
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CICILIANO et al
BLOOD, 6 AUGUST 2015 x VOLUME 126, NUMBER 6
Figure 3. Proposed mechanism of FeCl3-induced
thrombosis. (A) Aggregates of washed RBCs, washed
platelets, PPP, PRP, and whole blood (WB) form in
microfluidic channels at all concentrations ranging from
50 mM to 1 M. Time (in seconds) to initial adherence
of blood components and stable aggregate formation
(unchanging or complete occlusion) is shown, where complete channel occlusion is denoted by cross-hatched bars.
(B) Representative images of blood component aggregates in nonendothelialized microfluidics are shown at
50 mM. The presence of sheet-like protein aggregates
is clearly visible in PPP. All scale bars represent 50 mm;
N 5 5.
In support of our hypothesized mechanism, introduction of Al31,
an ion similar to Fe31 with respect to binding,20 results in erythrocyte
aggregation in microchannels (Figure 4B). A recent publication
likewise showed that the in vivo application of AlCl3 to a mouse
carotid produces occlusive thrombosis.23 On the other hand, erythrocyte suspensions do not aggregate when exposed to Cr31 because
the ion has slow substitution kinetics and preferentially binds water
instead of negatively charged species (Figure 4C).16,20 This suggests
that to induce aggregation, a metal ion must be able to form inner
sphere complexes (bind directly without intervening water molecules) with negatively charged moieties to form stable aggregates.
Furthermore, exposure to both AlCl3 and chromium (III) chloride
(CrCl3) results in endothelial cell death, suggesting that cell death
alone is insufficient to initiate aggregation (Figure 4B-C).
Blood components aggregate in static conditions when exposed
to FeCl3. From 0.5 mM to 50 mM FeCl3, FeOH precipitates are
visible and RBC membranes appear deformed; at concentrations
.50 mM, proteins form sheet-like structures and RBCs form large
aggregates (Figure 5A). Both FeCl3 and AlCl3 cause aggregation of
recalcified PRP and PPP in the presence of FeCl3 as measured by
dynamic light scattering, with aggregation generally increasing with
Figure 4. Charge-based mechanism of aggregation.
(A) In healthy physiological conditions, blood cells and
proteins are suspended in a stable colloidal suspension
in water, separated due to electrostatic repulsion (left).
Upon introduction of an ion (Fe31 (aq), Al31 (aq)) that
can bind directly (form inner sphere complexes) to
negatively charged proteins on cell surfaces or in
suspension, blood cells and proteins are arrested on
endothelial surface and form aggregates (middle).
Upon introduction of a kinetically inert ion (Cr31 ), the
ion binds directly to water molecules and thus only
has transient interactions with blood cells and proteins,
allowing the cells to maintain their colloidal suspension as in the control condition (right). (B) The introduction of AlCl3 causes RBCs to aggregate at the
fluidic interface in bare (left) and endothelialized
(middle) microfluidics. AlCl3 causes endothelial cell
death, but the cells remain adherent (right) (green/live:
calcein; red/dead: propidium iodide). (C) The introduction of CrCl3 does not result in RBC aggregation in bare
(left) or endothelialized (middle) microfluidics. CrCl3
causes endothelial cell death, but the cells remain
adherent (right) (green/live: calcein; red/dead: propidium iodide). All scale bars represent 50 mm; N 5 3.
increasing concentrations (Figure 5B-C; supplemental Figure 2).
Higher concentrations of AlCl3, relative to FeCl3, are necessary to
initiate aggregation in all solutions; this is expected as AlCl3-induced
colloid aggregation occurs over a narrow concentration and pH
range.21 At concentrations of FeCl3 . 500 mM and AlCl3 . 1 M, the
suspensions form a solid mass (supplemental Figure 2). Lightscattering measurements at these conditions are complicated by the
nonergodic nature of the solution; that is, the size of the aggregates
is large compared with the incident beam, so as the aggregates precipitate out, the solution is not temporally or spatially homogenous,
and light scattering measurements are inaccurate.24 Measurements
of aggregation of washed RBCs are likewise hampered by the
solution’s nonergodicity, but microscopic and macroscopic examination show definitive aggregation, with solid masses forming at
.50 mM FeCl3 (supplemental Figure 2B). As expected, CrCl3 does
not induce aggregation, and there is no increase in light scattering
over the controls at any tested concentration (Figure 5B-C).
The presence of physiologic levels of divalent cations, Mg21 and
21
Ca , does not have a pronounced effect on aggregation parameters,
as there are minimal changes in light scattering between samples
with and without these cations (supplemental Figure 3A). After
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BLOOD, 6 AUGUST 2015 x VOLUME 126, NUMBER 6
MECHANISMS OF FERRIC CHLORIDE-INDUCED THROMBOSIS
821
Figure 5. Flocculation activity of FeCl3. (A) The
concentration-dependent ability of FeCl3 to aggregate
blood components can be seen microscopically. At
mid-range concentrations of FeCl 3 , amorphous iron
hydroxide precipitates can be seen. Platelets (green:
CD41); fibrinogen (red). (B) Light scattering of PRP
(left) when exposed to a range of FeCl3, AlCl3, and
CrCl3 concentrations. Values are expressed as fold
increase over control PRP, where increased scatter
corresponds to increased aggregation. Light scattering
of PPP (right) exposed to a range of FeCl3, AlCl3, and
CrCl3 concentrations. Values are expressed as fold
increase over control PPP. All scale bars represent
50 mm; N 5 3.
20 minutes, recalcified PRP forms a stable, intrinsic coagulation
pathway-derived clot with a sixfold increase in light scattering over
the control. Light scatter of recalcified PRP solution exposed to
500 mM FeCl3 increases a further twofold after 20 minutes, increasing the scatter to that of the physiological clot (supplemental
Figure 3B). This suggests that physiological clotting can occur in
addition to metal ion–based aggregation.
Our findings suggest that initial adherence and aggregation of
blood components in this model is due to inner-sphere complexation of Fe31 to plasma proteins and cell membrane–localized
proteins, as well as binding of cells to FeOH precipitates. Sheet-like
protein aggregates also act as Fe31 linkers that enhance cell binding
(Figure 4A).
FeCl3-induced aggregate formation in altered
clotting environments
Although the underlying mechanisms of the FeCl3 thrombosis model
remain in question, its utility as an in vivo model to study clotting is
well established. Numerous publications have shown that FeCl3induced vascular occlusion is mitigated in knockout mice10,25,26 and
in the presence of antithrombotic agents.27-31 Multiple platelet receptor defects have also been shown to modulate thrombus formation in the FeCl3 injury model.32 We monitored occlusion in both
bare and endothelialized channels when blood was treated with
aspirin (platelet inhibitor), heparin (thrombin inhibitor), hirudin
(thrombin inhibitor), and eptifibatide (GPIIb/IIIa inhibitor), as well
as blood reconstituted with von Willebrand factor (VWF)–free
plasma obtained from a patient with type 3 von Willebrand disease.
After 15 minutes, in both endothelialized and bare channels, all inhibitors significantly reduced occlusion (P , .05) when blood was
exposed to 50 mM FeCl3; alternatively, when 500 mM FeCl3 was
used, aspirin, heparin, and hirudin could not maintain vessel patency
(Figure 6A-B; supplemental Figure 3). In control conditions, RBCs
adhere at the FeCl3/blood interface, platelets stick to adherent RBCs,
and a protein matrix forms; additional cells are entrapped within the
growing protein/cell matrix and the dense aggregate expands across
the full width of the channel. The aggregate is finally bordered by
a layer of fibrinogen that “seals” the clot from blood flow, and flow
within the channel ceases within 3 minutes (supplemental Figure 4).
Cells enter the aggregate mass, but do not traverse it, and blood flow
is restricted to the portion of the channel distal to the FeCl3 inlet.
Alternatively, at 50 mM FeCl3, all inhibitors except aspirin prevent
the extensive growth of aggregates past the interfacial line; aggregates do not have a large protein component and cells traverse the
loose aggregate matrix, adhere transiently, and then resume transit
through the channel. In one heparin experiment, aggregates initially
span the channel, but blood flow dislodges the blockage and the
vessel remains patent (supplemental Video 5). Aspirin-exposed
blood, on the other hand, gives rise to disjointed, unstable protein/
cell aggregates that readily embolize in response to fluid shear and
lodge downstream (Figure 6C). When the concentration of FeCl3 is
increased to 500 mM, aggregate formation is extensive in all conditions. Microchannel patency is partly retained in eptifibatide and
VWF-free conditions because of the loose aggregate matrix, though
blood flow is visibly decreased. At increasing FeCl3 concentrations,
pharmacologic inhibition does not effectively prevent channel occlusion because, although the clotting cascade is compromised, the
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CICILIANO et al
Figure 6. Aggregation of blood in altered clotting environments. (A) In bare microfluidics, all drugs and phenotypic abnormalities lead to significant decreases in channel
occlusion at 50 mM (P , .05) compared with the control solution, but 500 mM FeCl3 overcomes the protective effect of aspirin, heparin, and hirudin. Solid bars represent
patent channels while the slant pattern represents flow cessation. Significance is denoted by an asterisk (*), and was measured by a 2-tailed, Student t test between the
treatment condition and its paired control (eg, 50 mM control and 50 mM aspirin). There was not a significant difference in occlusion between untreated control blood exposed
to 50 mM and that exposed to 500 mM. (B) In endothelialized microfluidics, drugs and phenotypic abnormalities lead to significant decreases in occlusion at 50 mM (P , .5),
but 500 mM FeCl3 overcomes the protective effect. Solid bars represent patent channels whereas the slant pattern represents flow cessation. Significance is denoted by an
asterisk (*), and was measured by a 2-tailed, Student t test between the treatment condition and its paired control (eg, 50 mM control and 50 mM aspirin). There was not
a significant difference in occlusion between untreated control blood exposed to 50 mM and that exposed to 500 mM. (C) Representative images from drug treatments in bare
and endothelialized microfluidics, at 50 mM and 500 mM. Platelets (green: CD41), fibrinogen (red). (D) In bare and endothelialized channels, IL4-R/Iba blood forms fewer
aggregates at 50 mM FeCl3 than WT blood, and the channel remains patent. Significance is denoted by an asterisk (*), and was measured by a 2-tailed, Student t test.
Representative image of WT blood and IL4-R/Iba blood aggregation in the microfluidic channel. All scale bars represent 50 mm; N 5 4.
FeCl3 concentration is sufficiently high across the channel to induce
charge-based aggregation.
Furthermore, FeCl3-induced aggregation in blood from wild-type
mice was compared with that of mice lacking the extracellular domain
of GPIb-a (IL4-R/Iba mice). As expected, wild-type blood exposed
to 50 mM FeCl3 forms aggregates that fully occlude the channel in
a manner similar to human blood (Figure 6D). GPIb-a–deficient blood
results in significantly reduced occlusion with vessel patency maintained after 15 minutes. Unlike the extensive, continuous dense cell
aggregate in the wild-type condition, IL4-R/Iba mouse blood forms
disjointed aggregates that comprise regions of dense RBCs and sparse
RBCs, allowing blood flow to continue through and around the aggregates (Figure 6D; supplemental Videos 6-7). The aggregates are
primarily composed of RBCs as previously shown in vivo.10
The marked effects that mouse genotype and pharmacologic
agents have on FeCl3-induced thrombus formation are congruous
with our proposed mechanism of FeCl3 action, and our findings help
to explain inconsistencies in thrombus formation between injury
models, as well those between studies using the same injury model.
Although the initial adhesion and aggregation of blood proteins and
cells is mediated by concentration-dependent, charged-based interactions between FeCl3 and blood components, the progression to
complete occlusion occurs via conventional clotting mechanisms
involving platelet activation and the coagulation cascade. Similarly,
Eckly et al’s findings suggest that initial platelet adhesion in the
FeCl3 model is atypical of the clotting cascade as there is not
extensive collagen exposure in the vessel.9
Discussion
The extensive use of the FeCl3 model in hematologic research
and the lack of a well-defined mechanism of action led us to take
a reductionist, yet interdisciplinary, microfluidic approach to study
FeCl3-induced thrombosis. Our study reveals that FeCl3 has multiple
dose-dependent effects on blood, in the presence and absence of
endothelial cells, leading us to a novel 2-phase mechanism of action.
In the first phase, the charge-based complexation of positively
charged iron species and negatively charged proteins (cell surface
bound and in solution) initiates FeCl3-induced thrombosis independent of biological ligand-receptor interactions. RBCs are the first
adherent blood component, and form the bulk of the initial aggregate.
Platelets and proteins are visible within the initial aggregate to
a lesser degree (supplemental Figure 4A). We also recreate the
findings that the extent of thrombus formation in this model is
dependent on physiological clotting processes by introducing anticlotting agents and blood with phenotypic clotting deficiencies. This
leads to the second phase of our proposed mechanism in which endothelial cell death and iron-bound, adherent blood components
initiate the conventional biological clotting cascade. The final clot
is RBC, platelet, and protein rich and expands across the width of
the channel (supplemental Figure 4B). The inability to tightly control
the FeCl3 concentration in vivo makes the distinction between
chemically induced aggregation and physiological clotting impossible to assess. Likewise, there may be multifactorial effects from
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BLOOD, 6 AUGUST 2015 x VOLUME 126, NUMBER 6
drugs such as heparin, whose high negative charge density likely affects the activity of FeCl3.
RBCs, platelets, PRP, and PPP all aggregate when exposed to
FeCl3, tending toward increased aggregation as concentration increases. Aggregation is visible both macroscopically and microscopically, independent of endothelial cells, and with and without
shear or mixing. FeCl3 is known to cause aggregation of colloidal
solutions by z potential reduction, by directly binding to negatively
charged particles, and by forming amorphous cationic hydroxide
precipitates that can entrap and bind negatively charged particles.21
Furthermore, the binding of FeCl3 to negative proteins on blood cell
surfaces, known as the “colloidal iron method,” was historically used
to determine the distribution of anionic cites on cells, such as RBCs
and endothelial cells.33-35 Aqueous iron species (hydroxides) precipitate on the surface of endothelial cells, as seen in our in vitro studies,
and as corroborated by an in vivo FeCl3 mechanism study in which
SEM revealed endothelial cell localized hydroxide deposits.36 We
propose that RBCs are the first and most numerous cell species in the
initial FeCl3-induced aggregate due to their membrane’s high negative charge density (sialic acid on the surface) and the concentration
of erythrocytes in blood. Barr et al’s findings that RBC binding in the
FeCl3 model occurs at arterial, venous, and mesenteric shear rates, is
well explained by charge-based binding.10 In the absence of RBCs,
platelets too form aggregates on the channel surface, though interactions are more transient and occlusion proceeds more slowly.
FeCl3 is an oxidizing agent, and at high concentrations is a strong
Lewis acid; this may contribute to the concentration-dependent endothelial cell death seen in our system. The acidity alone, however,
does not cause cell aggregation: introducing acetic acid at pH 1 in
endothelialized devices causes visible endothelial cell death but not
RBC aggregation (not shown). The “charge-based” mechanism we
present is further supported because an ion with similar binding
activity, AlCl3, also causes cell aggregation, and in vivo thrombus
formation.23 Inert CrCl3, however, causes endothelial cell death, but
not cell aggregation.
At low concentrations of FeCl3 and high drug concentrations, extent
of aggregation is mitigated. Aspirin-treated blood gives rise to an unstable, frequently embolizing thrombus; heparin and hirudin reduce the
area of the channel covered by aggregates; eptifibatide leads to a loose
matrix; and VWF deficiency increases time to aggregation and results
in unstable occlusion. As seen in vivo, increasing the FeCl3 concentration can overcome the protective effect of these agents: there is a
narrow range of FeCl3 concentrations for which there are differences
between the treated and untreated conditions.
The dependency of thrombus initiation and extent of occlusion on
FeCl3 concentration is well explained by our proposed mechanisms:
there is a critical concentration of FeCl3 required at the lumen for
aggregate initiation and propagation. Furthermore, the differences
between occlusion in varying injury models and vessel types32 is
well explained, as the sensitivity of the system to FeCl3 concentration
is in part due to the intraluminal mass transport of FeCl3, which is
a function of blood velocity and vessel diameter; simulations show
that intraluminal flow conditions determine the percentage of the
vessel that is exposed to FeCl3 concentrations that induce aggregation (.0.5 mM) (supplemental Figure 5). The role that mass
transfer plays in the FeCl3 thrombosis model explains the variability
in application techniques and results.
Full venous occlusion by FeCl3 in mice lacking the extracellular
domain of GPIba led researchers to propose that there is another
ligand for VWF at venous shear rates; and similarly unusual
RBC/platelet/ligand interactions have been proposed to explain
unanticipated findings when using FeCl3.10,11,25,37 Our findings
MECHANISMS OF FERRIC CHLORIDE-INDUCED THROMBOSIS
823
suggest that initial blood aggregation is independent of biological
ligand/receptor interactions. Furthermore, in small vessels, the effective concentration of FeCl3 is higher over a larger area of the
channel and charge-based binding is likely more extensive (supplemental Figure 5). We surmise that vessel wall composition and
thickness, which vary by vessel type as well as by sex, age, weight,
and genotype of mouse, will also affect the intraluminal FeCl3 concentration. However, investigation of these variables is beyond the
scope of this article.
Our concentration-dependent mechanisms suggest that researchers should take caution when using the FeCl3 injury model and that
the use of this model is not appropriate for studies in clot initiation,
namely platelet-endothelium interactions. Likewise, FeCl3 concentration should be expressed as a molarity, as opposed to a percentage,
for accurate comparisons between studies using hexahydrate and
anhydrous FeCl3 species. FeCl3 should be also be reconstituted/
diluted directly before use as spontaneous FeOH formation may
change the diffusion properties of FeCl3, and thus its thrombotic
activity, especially at low concentrations (high pH) where FeCl3
is unstable.
The controlled reductionist environment afforded by our in vitro
endothelialized microfluidic system allows for precise control over
FeCl3-induced thrombosis and led us to propose a novel, 2-phase
mechanism for the action of FeCl3 in thrombus formation. The first
phase is independent of typical biological “clotting,” and consists
of the FeCl3 concentration-dependent binding of negatively charged
blood cells and proteins to positively charged iron species. The
adherent cells from the first phase initiate the second phase, the
standard biological clotting cascade. Where one phase ends and the
other begins is nontrivial to determine, as the effective FeCl3 concentration in the channel (which determines the extent of the first
phase) is dependent on mass transfer phenomena. Researchers
should take caution that this will affect their findings, especially in
small vessels, and that aggregation phenomena that seemingly suggest a novel ligand/receptor interaction may in fact be due to nonspecific charge-based binding effects.
Acknowledgments
This work was supported by National Science Foundation (NSF)
CAREER award 1150235 (W.A.L), and National Institutes of Health,
National Heart, Lung, and Blood Institute grants U54-HL112309
(J.C.C. and W.A.L.), U01-HL117721 (W.A.L.), and R01-HL121264
(W.A.L.).
Authorship
Contribution: J.C.C., R.L., L.A.L., S.M., C.G., and W.A.L. contributed to the conception, design, analysis, and interpretation of
the research; J.C.C. and W.A.L. wrote the manuscript; J.C.C., Y.S.,
M.E.F., D.R.M., and B.H. performed research and collected and analyzed data; J.B.D. performed diffusion calculations; and all authors
approved the final version of the manuscript.
Conflict-of-interest disclosure: The authors declare no competing
financial interests.
Correspondence: Wilbur A. Lam, Georgia Institute of Technology,
345 Ferst Dr NW, Atlanta, GA 30318; e-mail: [email protected];
[email protected].
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
824
BLOOD, 6 AUGUST 2015 x VOLUME 126, NUMBER 6
CICILIANO et al
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From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2015 126: 817-824
doi:10.1182/blood-2015-02-628594 originally published
online April 30, 2015
Resolving the multifaceted mechanisms of the ferric chloride
thrombosis model using an interdisciplinary microfluidic approach
Jordan C. Ciciliano, Yumiko Sakurai, David R. Myers, Meredith E. Fay, Beatrice Hechler, Shannon
Meeks, Renhao Li, J. Brandon Dixon, L. Andrew Lyon, Christian Gachet and Wilbur A. Lam
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