Fibrinogen and b-Amyloid Association Alters
Thrombosis and Fibrinolysis: A Possible
Contributing Factor to Alzheimer’s Disease
Marta Cortes-Canteli,1,3Justin Paul,1,3Erin H. Norris,1Robert Bronstein,1Hyung Jin Ahn,1Daria Zamolodchikov,1
Shivaprasad Bhuvanendran,2Katherine M. Fenz,1and Sidney Strickland1,*
1Laboratory of Neurobiology and Genetics
2Bio-Imaging Resource Center
The Rockefeller University, New York, NY 10065, USA
3These authors contributed equally to this work
Alzheimer’s disease (AD) is a neurodegenerative
disorder in which vascular pathology plays an impor-
factor in this disease, we examined its relationship to
fibrin clot formation in AD. In vitro and in vivo exper-
iments showed that fibrin clots formed in the pres-
ence of Ab are structurally abnormal and resistant
to degradation. Fibrin(ogen) was observed in blood
vessels positive for amyloid in mouse and human
AD samples, and intravital brain imaging of clot
formation and dissolution revealed abnormal throm-
bosis and fibrinolysis in AD mice. Moreover, deple-
tion of fibrinogen lessened cerebral amyloid angiop-
athy pathology and reduced cognitive impairment in
tant contribution of Ab to AD is via its effects on fibrin
clots, implicating fibrin(ogen) as a potential critical
factor in this disease.
One common pathology in Alzheimer’s disease (AD) patients is
the deposition of the b-amyloid peptide (Ab) in the walls of capil-
laries, arteries, and arterioles, known as cerebral amyloid angi-
opathy (CAA) (Vinters, 1987). CAA is an important factor in the
severity of AD pathology (Nicoll et al., 2004), as it provokes the
degeneration of vessel wall components, affects cerebral blood
flow (Thal et al., 2008b), and worsens cognitive decline (Esiri
et al., 1999; Greenberg et al., 2004).
In addition to CAA being a contributing factor to the vascular
pathology in AD, there is other evidence suggesting that AD
has a strong vascular component. Epidemiology links vascular
diseases such as stroke (Honig et al., 2003; Kalaria and Ballard,
2001), atherosclerosis (Hofman et al., 1997; Roher et al., 2003),
atrial fibrillation (Mielke et al., 2007; Ott et al., 1997), and hyper-
tension (Mielke et al., 2007; Skoog et al., 1996) with an increased
risk for dementia and AD (Breteler et al., 1998; de la Torre, 2002).
The combined presence of these vascular risk factors further
increases the risk for AD (Luchsinger et al., 2005). In addition,
cerebrovascular dysfunction takes place in AD (Farkas and
Luiten, 2001; Iadecola, 2004; Niwa et al., 2002); there is
decreased and altered cerebral blood flow in AD patients
(Johnson et al., 2005; Staffen et al., 2009), and chronic brain hy-
poperfusion is associated with the development of AD (de la
Torre, 2006). Compromised blood flow can lead to pathological
synaptic changes typical of AD (Wen et al., 2004), and thus
circulatory deficiencies could play an important role in its patho-
genesis, with neuronal loss and memory deficits being
secondary to vascular problems (de la Torre, 2004; Farkas and
Luiten, 2001; Iadecola, 2004). A mechanism for how Ab could
alter thrombosis and hemostasis is not known, although several
characteristics of this peptide suggest that it may be involved in
blood flow (Hardy, 2007) and blood vessel function (Smith and
Fibrinogen is a large glycoprotein that circulates in the blood at
micromolar concentrations and can be converted to insoluble
fibrin, which is essential for coagulation (Weisel, 2005). Elevated
fibrinogen levels are correlated with increased risk for AD (van
Oijen et al., 2005; Xu et al., 2008), and the level of fibrinogen-g-A
chain precursor in cerebral spinal fluid has been proposed as
a biomarker for AD (Lee et al., 2007). Fibrinogen is normally
excluded from the brain, but it has been found to accumulate in
the extravascular space in AD (Fiala et al., 2002; Lipinski and Saj-
The inhibition of tissue plasminogen activator (tPA)/plasmin fibri-
nolytic activity (Ledesma et al., 2000; Melchor et al., 2003), the
increased levels of prothrombotic molecules (Grammas et al.,
barrier (BBB) disorder found in AD brains (Bowman et al., 2007;
Kalaria, 1999; Paul et al., 2007; Ujiie et al., 2003; Zipser et al.,
2007) could contribute to the accumulation of fibrinogen or fibrin
(designated fibrin(ogen)). Furthermore, previous studies demon-
strated that reducing fibrinogen levels decreased BBB perme-
ability in AD mouse models (Paul et al., 2007). Therefore, since
fibrin(ogen) may play a critical role in AD (Cortes-Canteli and
Strickland, 2009), we analyzed its participation in this disease.
Here, we report that Ab induces the formation of abnormal,
degradation-resistant blood clots. We also demonstrate that
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AD mice with decreased levels of fibrinogen in their blood
present less CAA burden and perform better in memory tasks.
We suggest that the association between Ab and fibrinogen
causesaltered fibrin clotting, and thisaberranthemostasis could
lead to compromised blood flow and increased inflammation,
thereby contributing to cognitive decline in AD.
Fibrin(ogen) Clearance Is Delayed in the Brain of
Fibrin(ogen) accumulates in the brains of AD patients (Fiala et al.,
2002; Ryu and McLarnon, 2009) and TgCRND8 mice transgenic
for human amyloid precursor protein (APP) (Paul et al., 2007).
Since one explanation for fibrin(ogen) accumulation in mice is
a higher level of fibrinogen in the blood, we measured fibrinogen
levels in wild-type (WT) and TgCRND8 mice and found no
difference (see Figure S1A available online). A second possibility
was that blood is hypercoagulable in TgCRND8 mice, but the
coagulation times for WT and TgCRND8 mice were the same
(Figure S1B). A third option is increased persistence of
fibrin(ogen) in AD mice. Therefore, we investigated whether
these mice had increased stability of fibrinogen injected into
the brain. Nine-week- and six-month-old TgCRND8 mice and
their littermates were injected with fluorescently-labeled human
fibrinogen into thehippocampus andsacrificedthefollowing day
(Figures 1A–1C). Using two different ages of this transgenic line
allowed us to determine whether any differences in fibrinogen
clearance correlated with the degree of pathology. At 9 weeks
of age, TgCRND8 mice have not yet fully developed AD
pathology and present very low levels of Ab, while at 6 months
they have abundant amyloid plaques and CAA (Chishti et al.,
Figure 1. AD Mouse Brains Do Not Clear Fibrin
TgCRND8 and their nontransgenic littermates (WT) were
stereotaxically injected with fluorescently-labeled fibrin-
of FBG-488 (green) at the site of the injection in 6-month-
old WT (A) and TgCRND8 (B) mice one day after injection.
Nuclei were counterstained with DAPI (blue). Scale bar,
200 mm. Quantification of FBG-488 area in 9-week and
6-month-old injected mice (C) showed the amyloid burden
and degree of pathology affect fibrin(ogen) clearance.
A control molecule, tetramethylrhodamine-BSA (BSA-
Rhod), was also injected and quantified in another set of
6-month-old mice (D). Values represent the mean ± SEM
from 3–4 mice/group. *p < 0.05; TgCRND8 versus WT.
See also Figure S1.
2001). Both 9-week-old TgCRND8 mice and
their WT littermates showed almost complete
clearance of the injected fibrinogen compared
to the older mice (Figure 1C). However, the in-
jected fibrin(ogen) persisted longer in the
6-month-old mice, and its clearance was de-
compared to WT (Figures 1A–1C). In contrast to fibrin(ogen),
the rate of clearance of bovine serum albumin (BSA) was similar
in 6-month-old TgCRND8 and WT mice (Figure 1D), demon-
strating that the impaired clearance and increased persistence
of fibrin(ogen) in the AD mouse brain were specific for fibrinogen.
To examine if the fluorescent labeling of fibrinogen interfered
was injected into 6-month-old TgCRND8 and WT mice and de-
tected by immunostaining. The unlabeled fibrin(ogen) also per-
sisted longer in TgCRND8 than in WT mice (Figures S1C–S1E).
Conversion of injected fibrinogen to fibrin was demonstrated
by detection of D-dimer in brain homogenates (Figure S1F), indi-
cating that the injected fibrinogen was polymerized and cross-
linked by transglutaminase prior to proteolytic cleavage. There-
fore, injected fibrinogen was converted to fibrin in the TgCRND8
hippocampus, and this fibrin persisted longer in TgCRND8 than
These results indicate that there is an impairment in fibrin
(ogen) clearance that is dependent on the degree of pathology
and amyloid burden.
Ab42 Affects Clot Formation and Degradation In Vitro
Elevated levels of fibrin(ogen) could be due to increased forma-
tion or decreased clearance. To examine these possibilities, we
performed clot formation/degradation experiments with purified
human fibrinogen, thrombin, tPA, and plasminogen in the pres-
ence or absence of Ab42 (Merkle et al., 1996). As thrombin
converts fibrinogen to fibrin, the newly formed fibers scatter light
theybecomesubstrates fortPA-activated plasmin,andtheequi-
librium shifts from formation to dissolution, reducing the turbidity
as the clot dissolves. Ab42 was incubated overnight at room
temperature to promote nucleation and growth of oligomers
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(Chauhan et al., 2001) and form a heterogeneous mixture of Ab
fibrils and soluble prefibrillar amyloid assemblies (Figure S2A).
When the clot experiments were performed in the presence of
Ab42, the dissolution of the fibrin clot was delayed (Figures 2A
and 2B). To examine if Ab42 affects tPA and plasminogen inter-
actions (Hoylaerts et al., 1982), the experiment was repeated
using streptokinase, and the purified fibrin clot lysis was also
delayed in the presence of Ab (data not shown). It was possible
that Ab promoted transglutaminase activity, since increased
cross-linking could strengthen the fibrin clot and reduce lysis
speed. However, there was no difference in D-dimer in the pres-
ence or absence of Ab as determined by the analysis of fibrin
degradation products (data not shown). To test this effect in
the presence of all components involved in hemostasis, we
clotted recalcified human plasma in the presence of tPA and,
similar to pure fibrin clots, fibrinolysis was delayed in the pres-
ence of Ab42 (data not shown).
Previous results have shown that Ab can be a scaffold for effi-
cient conversion of plasminogen to plasmin by tPA (Kranenburg
et al., 2002), although Ab42 had no direct enhancement of tPA
proteolytic activity (Figure S2B). Increased plasmin generation
is inconsistent with delayed fibrinolysis. Therefore, Ab must
play a role in clot lysis that counteracts its effect on increasing
plasmin generation. Additionally, there was no effect of Ab42
on thrombin or plasmin activity (Figures S2C and S2D).
To separate possible consequences of Ab on clot formation
and dissolution, we tested the effect of Ab42 on fibrin during
the formation phase only. The presence of Ab42 during
thrombin-induced clot formation from pure fibrinogen produced
a dose-dependent decrease in the normal rise in turbidity (Fig-
ure 2C), which could reflect incomplete clotting. However, in
both the presence and absence of Ab42, all fibrinogen was
removed from solution and incorporated into the clot (data not
shown), indicating complete clotting in both cases. Therefore,
the lower turbidity suggested that the fibrin clot formed in the
presence of Ab42 was structurally abnormal. Structurally altered
clots can be resistant to fibrinolysis (Collet et al., 2000), which
may explain the persistence of fibrin in the presence of Ab42.
The experiment was repeated in the presence of other types of
amyloid peptides known to be associated with other human
diseases. Amylin (Clark et al., 1987) and calcitonin (Arvinte
et al., 1993) did not affect the formation of the fibrin clot as the
turbiditywas notdifferent fromthe control (Figure2D).Thisresult
showed that the effect of Ab42 on fibrin clot formation is specific
for this peptide.
Clot Structure Is Affected In Vitro by Ab42
The decreased turbidity during clot formation in the presence of
Ab42 (Figure 2C) prompted examination of the clot structure. To
visualize the fibrin network, we clotted human plasma in the
presence of fluorescent fibrinogen. Confocal microscopy
showed that clots were structurally altered in the presence of
Ab42, with fibrils arranged in a nonhomogeneous network. Areas
of normal clotting were interrupted by irregular regions of clus-
tering in Ab42-influenced fibrin (Figures 3A and 3B). Immunos-
fibrinogen produced identical aggregates (data not shown).
Fibrin aggregates stained positive with Congo red (Figures 3C
and 3D), suggesting they contained both fibrin and fibrillar forms
of Ab. Neither fibrin fluorescence nor Congo red-positive Ab
aggregates were observed when thrombin was omitted from
the reaction mix (data not shown). To further investigate the co-
localization of fibrin with aggregated Ab, we used biotinylated
Ab42 in the clotting reaction and then stained with fluorophore-
conjugated streptavidin. Staining for Ab was only observed in
the aggregates, confirming that the peptide was confined to
these areas (data not shown). Ab incubated without fibrinogen
did not produce any cluster formation (data not shown).
and 3F). Adding equal amounts of Ab42 to collagen did not intro-
duce any irregularities into the network of collagen fibrils (Figures
Figure 2. Ab Alters the Development of Fibrin Clot
Turbidity and Slows Degradation
(A) Combined fibrin formation/degradation assays. Clot-
ting of purified fibrinogen was initiated with thrombin in
the presence of tPA and plasminogen and either 10 mM
of Ab42 or vehicle (control) (n = 4/group).
(B) Total formation/degradation time for pure fibrin clots in
(A) determined from initiation to complete dissolution;
**p < 0.01. Bar graphs represent the mean ± SEM.
(C) Dose response of the effect of Ab42 on clot formation
by adding human thrombin to fibrinogen at time 0. Control
(vehicle); Ab42 100 nM; Ab42 500 nM; Ab42 5 mM.
(D) The addition of other b-pleated sheet amyloids (calci-
tonin and amylin; 5 mM) does not affect the turbidity of
the clot like Ab42 (5 mM). The lower general turbidity
obtained in (D) is due to DMSO added in each curve, as
explained in Experimental Procedures.
Curves in (C) and (D) are representative of four
experiments. See also Figure S2.
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S3A and S3B), indicating specificity in the fibrin-Ab interaction.
Scanning electron microscopy (SEM) images of purified fibrin
clots also showed aggregate formation in the presence of Ab42
that were fixed and postprocessed, fibrils appeared tangled and
up to 10 times thinner than in normal hydrated clots. Ab can
induce platelet aggregation (Kowalska and Badellino, 1994), but
the aggregates observed in our experiments were not platelets
since we used platelet-deficient plasma (Figures 3A–3F) as well
as purified fibrinogen (Figures 3G, 3H, S3C, and S3D).
To observe the degradation process alone, we added tPA to
a clot previously formed from plasma and observed fibrin(ogen)
degradation using confocal time-lapse image acquisition (Collet
et al., 2000) (Figure 3I). The lysis front retreated as the clot was
degraded by plasmin, and the retreat rate was calculated. In the
presence of Ab42, lysis was delayed and retreat of the lysis front
was slowed (Figure 3J). During clot lysis, aggregates often
aggregates that were Congo red-positive remained after the
orthose formedin thepresence ofscrambledAb42peptide (data
not shown). All these results indicate that Ab42 has an effect on
fibrin clot structure and on its formation and degradation in vitro.
Fibrin(ogen) Is Deposited in CAA-Positive Vessels
Wenext analyzedwhetherfibrin(ogen) andAbwereinteracting in
brain blood vessels, since excess brain-derived Ab is actively
drained through the vasculature (Shibata et al., 2000). We exam-
ined fibrin(ogen) deposition in CAA-positive vessels in the cortex
and hippocampus of 6-month-old TgCRND8 mice. Congo red
stainingand fibrin(ogen) immunohistochemistry
amyloid-laden vessels containing fibrin(ogen) deposited intra-
and extravascularly (Figures 4A–4F). To investigate the clinical
Figure 3. Ab Alters Clot Structure
Confocal images (inverted gray levels) at low magnification of a control fibrin clot (A) or an Ab42-influenced clot (B) using platelet-deficient human plasma and
thrombin in the presence of fluorescent-conjugated fibrinogen. Scale bar in (A) and (B), 36.5 mm.
(C) Fluorescent fibrin forms a network with aggregates in the presence of Ab42.
(D)Congo redfluorescenceinthesamefieldasin(C).Scalebar in(C)and(D),36.5mm.Confocalimage offibrinclotshowing aggregates acquired 15(E)and90(F)
min after thrombin addition. Scale bar in (E) and (F), 8.75 mm. SEM image obtained from control fibrin clot formed from pure fibrinogen and thrombin (G) or in the
presence of Ab42 (H). Scale bar in (G) and (H), 1.25 mm. Inset, 1 mm 3 1 mm.
(I) Red and green show the edge of aclot formed from plasma inthe presence of Ab42 before and 5 min after the addition of tPA, respectively. Scale bar, 36.5 mm.
(J) The lysis front retreat rate (mm/min) was determined from 5 min time-lapse confocal acquisitions (n = 4); ***p < 0.001. Bar graphs represent the mean ± SEM.
Aggregates remaining after fibrinolysis contain fibrin(ogen) (K) and Ab42 (L).
See also Figure S3 and Movie S1.
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Figure 4. Fibrin(ogen) Deposition in the CAA-Positive Vessels of TgCRND8 Mice and Human AD Patients
Perfused TgCRND8 mice at 6 months-of-age have fibrin(ogen) deposits at sites of CAA, detected by Congo red fluorescence, in the cortex (A–C) and hippo-
campus (D–F). Fibrin(ogen) immunohistochemistry was performed on human postmortem sections from the frontal cortex of 5 control and 9 AD patients.
The number of parenchymal vessels >20 mm that contained fibrin(ogen) was quantified (G), and patients diagnosed with AD presented nearly twice the number
of fibrin(ogen)-positive vessels compared to control subjects; **p < 0.01. Values represent the mean ± SEM. Representative 203 images showing fibrin(ogen)-
positive vessels (arrows) in controls (H) and AD patients (I). High-power magnification showed that fibrin(ogen) in AD patients colocalized with CAA (green, via
Thioflavin S staining; J–L), lined the interior of the vessel wall (M–O), and filled the lumen (P-R) in addition to being deposited in the tunica media of CAA-positive
vessels (S–U). Scale bars, 20 mm.
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human postmortem brain sections from control and AD patients.
We found that AD patients presented significantly more large
parenchymal vessels (>20 mm) affected with fibrin(ogen)
deposits than nondemented controls (Figures 4G–4I). We used
Thioflavin S staining to detect CAA and found vessels where
fibrin(ogen) colocalized with Ab deposits in the vessel wall and
the tunica media (Figures 4J–4L), as well as CAA-positive
vessels where fibrin(ogen) lined the interior (Figures 4M–4O) or
occupied the entire lumen of the vessel (Figures 4P–4R). We
also found CAA-positive vessels completely occluded by
fibrin(ogen) deposits with additional fibrin(ogen) accumulation
in the vessel wall (Figure 4S–4U).
Decreasing Fibrin(ogen) Levels Lessens CAA Pathology
One proposed hypothesis for the formation of CAA is that Ab
clearance is impaired throughout the vasculature, leading to its
accumulation in the vessel wall (Nicoll et al., 2004; Thal et al.,
2008b). Since we detected fibrin(ogen) deposition in mouse
and human CAA-positive vessels, we analyzed whether this
deposition could be playing a direct role in CAA pathogenesis.
We depleted fibrin(ogen) from the blood of 3-month-old
TgCRND8 mice by administering ancrod, a serine protease puri-
toma, which has been used to alleviate fibrin-mediated
pathology (Akassoglou et al., 2004; Busso et al., 1998). We
implanted mice with pumps to deliver ancrod or saline for
4 weeks, and we quantified the total area of CAA via Thioflavin
S staining. We found that treatment with ancrod produced
a significant decrease in vascular amyloid relative to saline treat-
ment (Figure 5A). As previously described (Paul et al., 2007),
fibrinogen levels in the plasma of ancrod-treated mice
icant effect on the total amount of Ab42 in the brains of the
implanted mice (see below for more detail). These data suggest
on Ab production but to a decrease in fibrin(ogen) levels.
To complement this pharmacological experiment, we crossed
TgCRND8 mice with mice deficient in one copy of the fibrinogen
Aa chain gene (TgCRND8-fbg+/?mice) and determined if CAA
was also decreased. We found that genetic reduction of fibrin-
ogen also lessens CAA pathology (Figure 5B). These results
demonstrate that Ab deposition as CAA is affected by the
amount of fibrinogen in blood, since depleting fibrinogen levels,
Given the importance that reducing CAA could have on the
progression of AD, we examined whether decreasing fibrinogen
levels would have the same effect in a different AD transgenic
mouse model. Tg6799 mice carry five different familial AD muta-
tions, present amyloid deposition as early as 2 months of age,
and exhibit neuronal death, memory deficits, and decreased
levels of synaptic markers (Oakley et al., 2006). Since no studies
had been performed on their CAA pathology, we analyzed the
brains of 2.5-, 4.5-, and 7.5-month-old Tg6799 mice. Because
Thioflavin S staining showed CAA-positive vessels starting at
4.5 months (data not shown), we implanted pumps filled with an-
crod or saline in Tg6799 mice of that age. We analyzed the total
amount of CAA after 4 weeks of treatment and, as with the
TgCRND8 mice, found a significant reduction of total CAA area
in ancrod-treated Tg6799 mice compared to the saline-treated
mice (Figure 5C). Given the involvement of CAA in AD patho-
genesis (Nicoll et al., 2004; Thal et al., 2008b), the finding that
decreasing fibrin(ogen) levels lessens CAA pathology in two
different transgenic AD mouse lines implicates fibrin(ogen)
as a contributing factor to CAA pathogenesis and therefore
Figure 5. Decrease in Fibrinogen Levels Reduces CAA in AD Mice
Fibrinogen levels were pharmacologically (A) or genetically (B) reduced
in TgCRND8 mice and Tg6799 (C) AD mice. The distribution of amyloidosis
in the brain was analyzed after treatment. The decrease in fibrinogen levels
provoked a significant decrease in the total amount of CAA in both transgenic
AD mouse lines. CAA was determined by Thioflavin S staining in 2–4 sections
from 4–7 mice/group. Bars represent mean ± SEM. *p < 0.05.
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TgCRND8 Mice Present an Increased Tendency
and 3) and fibrin(ogen) was found deposited in CAA-positive
vessels (Figure 4), we investigated clot formation in vivo. We
thrombosis in real time in 6-month-old TgCRND8 mice and WT
littermates. A cranial window was created, and blood flow was
monitored via injected fluorescent-conjugateddextran. Weused
two different yet complementary procedures to induce clot
formation in different sets of mice. The first method for inducing
thrombosis was the topical application of FeCl3, which provokes
oxidative injury, endothelial damage, and subsequent formation
of occluding thrombi rich in platelets. It has been used broadly
to induce arterial thrombosis in rodents (Westrick et al., 2007).
The addition of increasing concentrations of FeCl3to the brain
surface caused clot formation as revealed by the appearance
of an enlarging shadow superimposed over normal blood flow
(Figures 6A–6D; Movie S2). We counted the number of visibly
occluded large vessels (>20 mm) over time for both TgCRND8
and WT mice and found that TgCRND8 mice often thrombosed
spontaneously, even before the addition of FeCl3, and lower
doseswere needed to occlude vesselsof similar size(Figure 6E).
As a second method to induce clot formation, weused a laser-
microscope (Nishimura et al., 2006). In contrast with the general-
ized damage that occurs after the addition of FeCl3, this method
allows for the induction of clot formation in individual vessels
(Movie S3; Figure 6F–6J), and the content of amyloid in these
specific vessels can be visualized using the fluorescent dye
Methoxy-X04 (Klunk et al., 2002) (Figures 6G and 6H). The quan-
tification of the area occupied by the clot in each of the targeted
vessels showed that WT mice presented an average of 46% ±
7% vessel occlusion while TgCRND8 had 79% ± 5% blockage
(Figure 6K). The laser conditions used in these experiments
were chosen after testing pulses of different wavelengths, laser
intensities, and number of repetitions adequate to induce repro-
ducible damage in the TgCRND8 mice. However, the same
conditions were not enough to induce any occlusion in some
of the vessels of the nontransgenic WT littermates (Figure 6K).
This finding, combined with the increased number of occluded
vessels after FeCl3application (Figure 6E) and the higher degree
of occlusion after laser-induced thrombosis in TgCRND8 mice
is more prone to clot.
To determine whether the amyloid content in the vessel wall
was affecting clot propensity in the vessels of the TgCRND8
mice, we used Methoxy-X04 staining (Klunk et al., 2002) in vivo
to visualize cerebrovascular amyloid as ring-like structures sur-
rounding the vessels (Figures 6G and 6H). Replotting the injured
AD vessels from Figure 6K as positive or negative for CAA
showed that the laser-targeted CAA-positive vessels occluded
almost completely (83% ± 5%), while the degree of occlusion in
CAA-negative vessels (74% ± 10%) did not reach a statistically
significant difference (p = 0.075) compared to WT mice (Fig-
ure 6L). This result indicates that the deposition of Ab in the
vessels as CAA promotes a higher degree of occlusion after a
thrombotic event. However, it should be noted that vessels lack-
ing CAA also tend to present an elevated degree of occlusion
when compared to WT vessels. Therefore, it is possible that
factors other than CAA could affect clotting in the AD brain.
Laser-induced thrombosis experiments were also carried out
in 9-week-old predepositing mice. We found no difference
between the average percentage of vessel occlusion in 9-
week-old TgCRND8 (60% ± 23%) and WT mice (57% ± 15%),
suggesting that the increased tendency to clot in TgCRND8
mice is dependent on the degree of AD progression.
Clot Degradation Is Inhibited in TgCRND8 Mice
Because the clot degradation process was affected in vitro
(Figures 2 and 3), we examined this process in vivo. The rate of
fibrinolysis was determined by topically administering tPA to
clots formed with 10% FeCl3as described in Figure 6. Clot lysis
was observed as the shrinking and eventual disappearance of
the shadow over the fluorescently labeled blood flow (Movie
Clots formed in TgCRND8 mice maintained similar size for more
than 5 min, while WT clots lysed quickly, often dissolving within
1 min (Figures 7A–7C). The lack of clot lysis in TgCRND8 animals
is not due to the increased levels of plasminogen activator inhib-
itor in the AD brain (Melchor et al., 2003), since the activity of the
added tPA was comparable between TgCRND8 mice and WT
littermates (data not shown). These experiments show that clot
degradation is inhibited in the brain of a living AD mouse.
Fibrin(ogen) Levels Affect the Memory and Cognitive
Performance of Mice
To analyze the physiological significance of fibrin(ogen) deposi-
tion in brain vessels (Figure 4) and the increased clotting propen-
sity in the TgCRND8 mice (Figure 6), we examined the cognitive
performance of TgCRND8 mice with reduced fibrinogen levels.
WT and TgCRND8 mice were infused for 4 weeks with ancrod
to decrease fibrinogen levels and tested in the Morris water
maze, a learning and spatial memory task. Mice infused with
during the hidden platform test showed that the four groups of
mice learned to locate the platform (data not shown). TgCRND8
mice had profound memory impairment; they spent significantly
less time in the target quadrant than WT littermates (Figures 8A
better performance, as they spent significantly more time in the
target quadrant than TgCRND8 infused with saline (Figures 8A
and S4A), demonstrating a higher retention of spatial memory
than TgCRND8 controls.
Fibrin(ogen) levels in plasma were measured at three different
times (before, during, and end of ancrod treatment), with an
average 46% reduction in fibrin(ogen) (*p = 0.0112 compared
to saline-infused). Mice that did not reach a significant decrease
in fibrin(ogen) levels at any point were excluded from the anal-
ysis. To determine if ancrod affects Ab levels, we measured
the total Ab42 content in brain homogenates of the TgCRND8
was no significant difference in Ab42 content (25.9 ± 5.2 mg/g
of tissue TgCRND8-saline versus 21.6 ± 8.2 mg/g of tissue
TgCRND8-ancrod; p = 0.668). These results indicate that the
effect of ancrod treatment in reducing memory impairment and
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cognitive deficits of TgCRND8 mice is due to a reduction in
fibrin(ogen) levels and not the total amyloid burden.
To complement the results obtained with the pharmacological
reduction in fibrin(ogen) levels, we performed water maze exper-
iments with TgCRND8-fbg+/?
mice and their littermates.
TgCRND8-fbg+/?mice presented a tendency to spend more
time in the target quadrant than TgCRND8, although the results
were not significant (time in target quadrant: 38.8% ± 4.2%
TgCRND8 versus 46.6% ± 3.05% TgCRND8-fbg+/?; p = 0.169).
The improvement in spatial memory of the TgCRND8-fbg+/?
Figure 6. Altered Thrombosis in TgCRND8 Mice
A cranial window was opened in 6-month-old TgCRND8 and WT littermates, and in vivo imaging and clot formation were carried out. Representative images of
time series of clot formation before (A) and after the addition of 2.5% (B), 10% (C), and 20% (D) FeCl3. Fluorescent-labeled blood flow (gray) is interrupted with
dark zones representing clot formation (black).
(E) The number of occluded vessels after FeCl3treatment at incremental doses was recorded in TgCRND8 mice and WT littermates over time. TgCRND8 mice
show occlusion earlier and with lower doses of FeCl3. Clot formation was also induced using the near-infrared laser of a two-photon laser scanning fluorescence
microscope. Representative images of maximum projections (Z stack) from the same area taken before (F–H) and after (I and J) the laser-induced injury in
a TgCRND8 mouse. Methoxy-X04 was injected to label Ab deposits and identify CAA-positive vessels (G, pseudocolored red). The white boxes show the region
of interest where the laser was focused to form a clot (arrow in J). Scale bars, 40 mm.
that under the same conditions as TgCRND8 mice, some of the vessels in WT mice did not occlude.
(L) The same vessels quantified in (K) were classified and plotted as CAA-positive and CAA-negative based on the Methoxy-X04 staining. *p < 0.05, **p < 0.01,
***p < 0.001; TgCRND8 versus WT mice. Values represent the mean ± SEM.
See also Movie S2 and Movie S3.
Altered Hemostasis in Alzheimer’s Disease
702 Neuron 66, 695–709, June 10, 2010 ª2010 Elsevier Inc.
mice was not as robust as TgCRND8 mice treated with ancrod
(Figure 8A). Thisdifference can be explained by the level of fibrin-
ogen in the animals, as treatment with ancrod provokes ?50%
decrease in fibrin(ogen) levels, while genetic depletion of one
copy of the fibrinogen gene only produces ?35% reduction
compared to the mice bearing two copies of the gene (Suh
et al., 1995). Also, we found that the background of fbg+/?mice
interferes with the water maze analysis, since the difference
between WT and TgCRND8 mice on this background was
reduced. Therefore, we tested the mice in the novel arm version
of the Y maze, another memory task. We found that the
TgCRND8-fbg+/?mice spent significantly more time in the novel
mentour previous resultsshowingthat reducing fibrinogenlevels
pharmacologically improves memory of TgCRND8 mice.
If fibrin(ogen) deposition and the increased tendency to clot in
the AD brain are factors contributing to poor cognitive function in
AD, then mice with an increased rate of thrombosis will have
memory deficits. Plasminogen-deficient mice (plg?/?) present
fibrin(ogen) deposits in different tissues and are predisposed to
thrombosis (Bugge etal.,1995; Ploplis etal.,1995).We therefore
tested 3-month-old plg?/?mice in the water maze. Although
these mice present progressive wasting during their lives (Bugge
(Bugge et al., 1996) and their swim speed was similar to that of
WT mice during the probe trial (20.1 ± 0.3 cm/s WT versus 18.4 ±
times during the hidden platform test showed that plg?/?mice
were not impaired in the training session (data not shown), indi-
cating no motor or visual problems at this age. However, during
compared to WT mice (Figures 8C and S4B). The removal of
fibrinogen from plg?/?mice corrects manyof their abnormalities,
pathology (Bugge et al., 1996; Degen et al., 2001). Therefore, the
fibrinolysis, demonstrating a role for fibrin(ogen) in this process.
Figure 7. Delayed Fibrinolysis in TgCRND8
(A) Clots formed after topical application of 10%
FeCl3in TgCRND8 and WT mice were treated
with tPA, and clot size was followed and deter-
mined over time. Representative images of the
time series of clot dissolution after tPA treatment
of a preformed clot in WT (B) and TgCRND8
mice (C). Blood flow is interrupted with dark zones
representing clot formation (arrows). Scale bar,
50 mm. *p < 0.05, **p < 0.01, ***p < 0.001;
TgCRND8 versus WT mice. Bar graphs represent
the mean ± SEM. See also Movie S4.
The work presented here is especially
relevant to two critical features of AD: (1)
the slow, progressive loss of nervous
system function and cognitive decline
and (2) a role for Ab. We will discuss our results with these
features in mind.
Altered Hemostasis in Alzheimer’s Disease
mised vascular system, since all cells require oxygen and nutri-
entsforsurvival.This requirement isespecially acuteinthebrain,
20% of oxygen used by mammals (Squire et al., 2003). Our work
shows that the hemostatic system is abnormal in AD mouse
models. Clots are formed more rapidly (Figure 6) and are more
difficult to lyse (Figures 3 and 7) in the brains of AD mice. This
that could compromise cerebral blood flow and hence neuronal
function and survival.
The AD brain is known to have altered blood flow (Farkas and
Luiten, 2001), impaired vascular function (Smith and Greenberg,
2009), and to constitute a prothrombotic environment. There are
increased levels of prothrombin (Zipser et al., 2007), thrombin
(Grammas et al., 2006), and transglutaminase (Johnson et al.,
1997) and decreased fibrinolytic activity (Ledesma et al., 2000;
Melchor et al., 2003). In fact, subcortical infarcts play a role in
the cognitive impairment present in AD (Schneider et al., 2007);
there are white matter lesions in AD brains resembling those
observed after ischemia (Brun and Englund, 1986), and ischemia
has been shown to induce tauopathy (Wen et al., 2004). Further-
more, cerebral emboli have been detected in patients with AD
and associated with cognitive decline (Purandare and Burns,
Circulatory deficits are notoriously insidious and can cause
increased propensity to clot, coupled with the clots being harder
to clear, could lead to a slow decline in neuronal function.
A Possible New Role for Ab in Alzheimer’s Disease
Ab has been strongly implicated in AD due to analysis of early
onset cases, which are genetic in nature. All mutations known
Altered Hemostasis in Alzheimer’s Disease
Neuron 66, 695–709, June 10, 2010 ª2010 Elsevier Inc. 703
to increase risk of AD influence the amount of Ab that can be
generated, and this peptide may also play a critical role in
sporadic forms of the disease (Dawbarn and Allen, 2007). Since
Ab is a component of plaques that are often associated with AD,
factor (Thal et al., 2008a). However, the lack of correlation
between plaque burden and cognitive impairment (Arriagada
et al., 1992) suggests that soluble Ab (Shankar et al., 2008) or
its deposition in vessels (Greenberg et al., 2004) may be more
Our work identifies a new role for Ab that ties it to potential
circulatory deficiencies. The association of Ab with fibrinogen
leads to abnormal clots that are more resistant to lysis, which
could explain the observations that plaques accumulate fibrin(-
ogen) (Paul et al., 2007; Ryu and McLarnon, 2009). The persis-
tence of fibrin could have multiple deleterious consequences:
first, it could lead to occlusion of blood vessels and impede
flammatory protein and could promote chronic inflammation
in the brain (Adams et al., 2004; Paul et al., 2007), resulting in
neuronal dysfunction. In fact, fibrinogen is closely associated
with the inflammatory response in AD (Ryu and McLarnon,
2009), and intriguingly, microgliosis is reduced when fibrinogen
is depleted (Paul et al., 2007; Ryu and McLarnon, 2009).
A Possible Role for Fibrinogen in Alzheimer’s Disease
There is evidence for a role of fibrinogen in AD pathology.
Fibrin(ogen) deposition is observed in vessels (Figure 4) but
also in the parenchyma of AD mice (Paul et al., 2007) and AD
patients (Fiala et al., 2002; Ryu and McLarnon, 2009). However,
other molecules do not cross the BBB in AD (DeMattos et al.,
2001; Sagare et al., 2007), suggesting that fibrinogen leakage
into the brain parenchyma might be specific for this molecule.
In fact, high levels of fibrinogen have been associated with an
increased risk for AD (van Oijen et al., 2005) and with an elevated
risk for dementia conversion in patients with mild cognitive
impairment (Xu et al., 2008). A causative role for fibrinogen is
indicated by our experiments that show that decreasing
fibrin(ogen) levels lessens CAA pathology and cognitive decline
in transgenic AD mouse models (Figures 5 and 8).
CAA has been defined as a risk factor for ischemic cerebral
infarction (Cadavid et al., 2000) and cognitive dysfunction
(Greenberg et al., 2004). It is considered responsible for many
of the abnormalities present in the vessel wall, alteration of blood
flow, and impairment of vascular function in AD (Smith and
Greenberg, 2009). Increasing evidence suggests that impaired
Ab clearance through the vasculature is one of the main mecha-
nisms of Ab accumulation in the cerebral vessels (Bell and
Zlokovic, 2009). Our results provide a connection between the
vascular deposition of amyloid, first described over 100 years
ago, and the hemostatic system.
Additional evidence supporting
fibrin(ogen) and clot formation in AD are the positive results
obtained with anticoagulant therapy for this disease (Ratner
et al., 1972; Walsh et al., 1978). Although those studies were
small, the anticoagulant treatment either stopped the deteriora-
tion or provoked an improvement of the disease (Walsh, 1996).
More recently, a study carried out in patients with atrial fibrilla-
tion, a known vascular risk factor for AD (Mielke et al., 2007;
Ott et al., 1997), showed that subjects on anticoagulant treat-
ment present less cognitive impairment than those who were
not treated (Barber et al., 2004). Also, an association between
Figure 8. The Modulation of Fibrinogen Levels in Mice Affects
(A) TgCRND8 mice were implanted with pumps delivering ancrod or saline for
4 weeks and tested in the Morris water maze. TgCRND8 mice with reduced
fibrinogen levels spent significantly more time in the target quadrant than
TgCRND8 controls, indicating that the pharmacological reduction of fibrin-
ogen improves spatial memory retention.
(B) TgCRND8 mice heterozygous for fibrinogen (TgCRND8-fbg+/?) and their
littermates were tested in the Y maze. TgCRND8-fbg+/?mice spent signif-
icantly more time exploring the novel arm than TgCRND8 control mice,
indicating that the genetic reduction in fibrinogen improves working
(C) In contrast, mice deficient in plasminogen (plg?/?), which are predisposed
to severe thrombosis and present fibrinogen deposits in different organs,
showed memory impairment compared to WT mice in the Morris water
maze. Bars represent the mean ± SEM. *p < 0.05, **p < 0.01.
See also Figure S4.
Altered Hemostasis in Alzheimer’s Disease
704 Neuron 66, 695–709, June 10, 2010 ª2010 Elsevier Inc.
AD and hypercoagulability disorders such as hyperhomocystei-
nemia (Seshadri et al., 2002) and Factor V Leiden (Bots et al.,
1998) has been postulated. Combined, these studies support
In general terms, the deleterious effects of persistent fibrin
have been dramatically shown in mice. Plasmin generated
from plasminogen is the primary fibrinolytic enzyme, and plg?/?
mice have multiorgan failure and die around 6 months of age
(Bugge et al., 1995). This wasting is due to accumulation of
fibrin, since genetic reduction of fibrinogen rescues plg?/?mice
(Buggeetal.,1996).This resultdemonstrates thatfibrinaccumu-
lation has multitudinous effects that compromise normal physi-
ology. Specific examples of this concept include arthritis (Busso
et al., 1998), in which fibrin persistence worsens joint pathology,
and peripheral nerve regeneration (Akassoglou et al., 2002),
where fibrin inhibits the process.
Hypothetical Model of Alzheimer’s Disease
Putting our results together with previous findings, we propose
a model that could explain many aspects of AD pathology: Ab
accumulates in the brain where it could associate with fibrinogen
in the parenchyma, around blood vessels, or inside vessels. The
prothrombotic environment would lead toclot formation, and the
presenceof Abwouldmake theseclots abnormal andlysis resis-
tant. Fibrin persistence could obstruct blood flow or provoke
inflammation that would damage neurons and lead to their
dysfunction. It is also possible that fibrinogen in blood vessels
of AD patients entraps Ab, impeding its clearance through the
vasculature and potentiating the formation of CAA, decreasing
blood circulation, and eventually leading to cognitive decline.
lopathology will help the identification of candidate treatments
for this disease (Greenberg et al., 2004). One aspect of our
proposed mechanism is that it could be specifically targeted
therapeutically. A drug that could interfere with the effects of
Ab on fibrin clot formation would in theory normalize any blood
clots formed in the brain and increase their lysis, hence
improving cerebral blood flow and neuronal function and
survival. Such a drug would have little effect on general clotting
in other locations where Ab levels are low. Therefore, this
approach, perhaps in conjunction with other strategies, could
have significant therapeutic benefit for the treatment of AD.
TgCRND8 transgenic mice (Chishti et al., 2001) express a double mutant form
of APP695 (K670N/M671L + V717F) driven by the human prion protein
promoter, are on a mixed background (C57XC3H/C57), develop Ab-associ-
ated pathology, and exhibit defects in memory as early as 3 months of age
(provided by A. Chishti and D. Westaway, University of Toronto, Canada).
Tg6799 mice (Jackson Laboratory) are double transgenic for APP/Presenilin
1 and coexpress five early onset familial AD mutations and rapidly accumulate
Ab42by 2monthsof age(Oakley etal.,2006).Miceheterozygous for thefibrin-
for CAA determination and behavioral experiments. Nontransgenic (WT) litter-
mates were used as controls in all experiments. Plasminogen-deficient mice
(plg?/?) (Bugge et al., 1995; Ploplis et al., 1995), backcrossed onto a C57/
BL6 background, and age-matched WT C57/BL6 mice (Charles River) were
used in this study. Genotypes were double-checked by taking a tail tissue
sample the day of sacrifice. Both genders were used in all experiments, and
Mice were maintained in The Rockefeller University’s Comparative Biosci-
ences Center and treated in accordance with IACUC-approved protocols.
A 500 nl solution containing 500 ng of fluorescently labeled human fibrinogen
(Alexa Fluor 488-fibrinogen, Molecular Probes) was stereotactically injected
into the right hemisphere (posterior ?2.0 mm, lateral 1.8 mm, depth 1.2 mm;
Franklin and Paxinos, 2008) of 6-month- and 9-week-old (predepositing)
TgCRND8 and WT mice. As control, the same amount of tetramethylrhod-
amine-BSA (Molecular Probes) was injected in another set of 6-month-old
mice. Mice were perfused with a saline/heparin solution after 24 hr, and
20 mm thick coronal brain sections from the injected hemisphere were fixed
in ethanol, washed, and mounted with Vectashield containing DAPI (Vector
Labs, Burlingame, CA). Ten sections/mouse (n = 3–4 mice/group) were exam-
ined for the fluorescent compounds using an inverted LSM 510 laser scanning
confocal microscope (Zeiss) equipped with a motorized stage. To quantify the
area where fluorescence was present, a tile-scan (4 3 4) was obtained using
a Plan-NeoFluor 253/0.80 objective and thresholded using Image J (NIH).
The results are normalized to the area obtained in WT mice.
Clot Formation/Degradation and Turbidity Experiments
Fibrin clots were formed by mixing purified human fibrinogen (10 mM; Calbio-
chem) or citrated normal human plasma (New York Blood Center, centrifuged
at 10,000 3 g for 15 min to obtain platelet-deficient plasma), with human
thrombin (1 U/ml; Sigma) in the presence of Ab42 peptide (100 nM–10 mM;
Anaspec) or vehicle (PBS). CaCl2was adjusted to 5 mM. Absorbance was
measured for 10 min at 450 nm. Clots were also formed in the presence of
the amyloid peptides calcitonin and amylin (5 mM; Anaspec). Human calcitonin
was reconstituted in dH2O, and human amylin (1–37) in DMSO. To allow
comparison among all three amyloid peptides, clots formed in the presence
of Ab42, amylin, or calcitonin were controlled for the final concentration of
vehicle present. For formation/degradation curves, clots were formed under
thesameconditionswithtPA(14nM;Genentech)and purified humanplasmin-
ogen (100 nM; Sigma). Amyloid solutions were shaken for 24–48 hr at room
temperature before use.
Confocal Image Analysis and Lysis Front Retreat Rates
Clots were formed as described above on a glass-bottomed dish with Alexa
Fluor 488-fibrinogen (50 mg/ml; Molecular Probes) in the presence or absence
of 500 nM Ab42. Some clots contained Congo red (10 mM; Sigma). For lysis
experiments, tPA was injected into the center of the preformed clot (20 min
after mixing), and time-lapse image stacks were recorded at 15 s intervals
for 5 min as the lysis front retreated from the center. Initial and final images
were overlayed, and the distance between lysis fronts was divided by the
5 min collection period (n = 3–4 random lysis fronts in 4 separate experiments).
Images were obtained with an inverted Axiovert 200 microscope, acquired
with LSM 510 v. 3.2 confocal software (Carl Zeiss, Mannheim, Germany),
and analyzed with MetaMorph software (Universal Imaging).
Fibrin clots were formed from purified fibrinogen on glass coverslips. After
20 min, clots were washed with sodium cacodylate buffer, fixed with 2%
glutaraldehyde, dehydrated, critical point dried, and sputter-coated with
gold palladium. Images were obtained using a LEO 1550 scanning electron
Six-month-old TgCRND8 mice were saline/heparin-perfused, and 20 mm
coronal brain sections were prepared, ethanol-fixed, and stained with FITC-
conjugated fibrin(ogen) antibody (Dako). Tissue was counterstained for
30minatroomtemperaturewith0.2% Congo Red(Sigma)in70%isopropanol
to detect CAA. Immunofluorescence images were acquired using an inverted
Zeiss Axiovert 200 microscope.
Altered Hemostasis in Alzheimer’s Disease
Neuron 66, 695–709, June 10, 2010 ª2010 Elsevier Inc. 705
Human Brain Immunohistochemistry
Human postmortem brain tissue was provided by the Harvard Brain Tissue
Resource Center and the Washington University AD Research Center. Paraffin
sections (7 mm) from the frontal cortex of 5 control (52–90 years of age) and
9 AD (77–91 years of age) cases were deparaffinized, treated with proteinase
K (Dako), and immersed in methanol/H2O2to inactivate endogenous peroxi-
dases. Immunohistochemistry was carried out using a rabbit polyclonal anti-
fibrin(ogen) antibody (Dako) and the Tyramide Signal Amplification system
(Perkin Elmer) according to manufacturer’s instructions. Sections were devel-
oped using diaminobenzidine, costained for 30 min with 1% Thioflavin S
(Sigma) in 70% ethanol, dehydrated, and mounted. Twenty to twenty-five
fields per section were acquired using an inverted Zeiss Axiovert 200 micro-
scope, and vessels >20 mm that contained fibrin(ogen) were quantified.
Ancrod Treatment and CAA Determination
Tg6799, TgCRND8, and their WT littermates were treated with ancrod as
described (Paul et al., 2007). To calculate total amyloidosis, 3-month-old
TgCRND8 and 4.5-month-old Tg6799 mice implanted with pumps delivering
saline or ancrod, and 7- to 11-month-old TgCRND8-fbg+/?mice were used.
Mice were saline/heparin-perfused, and 20 mm coronal brain cryostat sections
70% ethanol. Pictures of all the areas with CAA were acquired, thresholded
using Image J, and the totalCAA area per sectionwas calculated. The average
of 2–4 different sections from 4–7 mice per group was determined and plotted
relative to the corresponding control group.
Intravital Imaging of Thrombosis
To observe blood circulation and to induce thrombosis, a cranial window was
prepared over the parietal cortex of 6-month- and 9-week-old TgCRND8 mice
and WT littermates following the surgical procedure described (Mostany and
Portera-Cailliau, 2008) with some modifications (see Supplemental Experi-
mental Procedures). Two different methods were used to induce clot forma-
Topical Application of FeCl3
Increasing concentrations of FeCl3(2.5%–20%) were added directly to the
brain surface with an interval of ?15 min, and thrombosis was recorded using
real-time video acquisition with a video camera fitted to an upright Zeiss
Axiovert 200 epifluorescence microscope (Metavue software). The whole
procedure lasted approximately 60 min per mouse (n = 2–4 mice/group and
up to 10 vessels > 20 mm were thrombosed per animal). After treatment with
10% FeCl3, some mice were topically administered with recombinant tPA
and brainswereprocessedforinsituzymographyasdescribed (Melchoretal.,
2003). Clot formation and dissolution were observed using time-stamped
image stacks. Clot size was traced by hand using Metamorph software to
calculate the area of the dark zone representing the clot.
Laser-induced thrombosis was provoked and imaged using a Fluoview
1000MPE two-photon laser scanning fluorescence microscope (Olympus)
equipped with a SpectraPhysics MaiTai DeepSee laser (with a tuneable range
of 690–1040 nm) and a 253/1.05 NA objective. To identify CAA-positive
vessels, Methoxy-X04 (Klunk et al., 2002; Neuroptix Corporation) was admin-
istered via tail vein injection 1 hr prior to imaging (3 mg/kg dissolved as
described in Garcia-Alloza et al., 2009). The procedure to induce localized
laser thrombosis was adjusted from the one described (Nishimura et al.,
2006). See Supplemental Experimental Procedures for details. We targeted
3–4 vessels/mouse with a diameter greater than 20 mm (n = 5–6 at 6 months
of age; n = 2 at 9 weeks of age TgCRND8 and WT). A 23 zoom Z stack of
the area where the clot was formed was acquired for vessel occlusion quanti-
fication using Image J (diameter of the clot versus diameter of the vessel in all
Morris Water Maze
TgCRND8 and WT mice infused with saline or ancrod (n = 7, 3–5 month old),
TgCRND8-fbg+/?mice and their littermates (n = 5–10, ?4 month old), and
maze to evaluate cognitive function (Chishti et al., 2001) with some minor
modifications (see Supplemental Experimental Procedures for details). Spatial
memory was measured by quantifying the percent time spent in the target
quadrant and the number of platform crossings during the probe trial test.
The experiment was recorded and analyzed using Ethovision video tracking
TgCRND8-fbg+/?mice and their littermates (n = 8–16, 7- to 11-month-old)
room under soft illumination, and visual clues were placed on walls of the
testing room. Each trial consisted of two 5 min periods, separated by
a 2 min intertrial interval in which the mouse was placed in its home cage.
During the first 5 min period, one of the three arms was blocked by an opaque
Plexiglas insert; this arm acts as the novel arm in the subsequent 5 min testing
period. The entire experiment was recorded, and the first 2 min of the testing
period were analyzed using Ethovision (Noldus). Time spent in the novel arm
was averaged and compared between groups.
Ab42 and Fibrinogen ELISA
To calculate the total Ab42 brain content, tissue was weighed and homoge-
nized in 5 M guanidine HCl/50 mM Tris-HCl (pH 8) buffer, agitated for 4 hr at
room temperature, and centrifuged for 20 min at 16,000 3 g to extract total
Ab (Chishti et al., 2001). Ab42 concentration was determined by the BetaMark
x-42 ELISA kit according to manufacturer’s instructions (Covance). Tail prick
blood samples were taken before, during, and after pump implantation in
mice treated with ancrod, and the decrease in fibrinogen levels in plasma
was measured by ELISA (GenWay Biotech).
All numerical values presented in graphs are mean ± SEM. Statistical signifi-
cance was determined using two-tailed t test analysis comparing control to
Supplemental Information includes four figures, four movies, and Supple-
mental Experimental Procedures and can be found with this article online at
This work was supported by grants from the National Institutes of Health
(NS50537 and GM66699), Institute for the Study of Aging (261104), Alz-
heimer’s Drug Discovery Foundation (281203), Alzheimer’s Association
(IIRG-04-1356), Woodbourne Foundation, Blanchette Hooker Rockefeller
Fund, May and Samuel Rudin Family Foundation, Bridges to Better Medicine
Technology Fund, and NIH Medical Scientist Training Program (GM07739).
M.C.-C. was supported by The Rockefeller University Women & Science
Fellowship Program and by the American Health Assistance Foundation. We
thank the Alzheimer’s Disease Research Center at Washington University
(P50 AG05681 grant) and the Harvard Brain Tissue Resource Center (PHS
grant R24-MH 068855) for providing human samples, and the support of the
two-photon Olympus microscope by the Empire StateStem Cell Fund through
NYSDOH Contract #C023046. We thank Anita Ramnarain for assistance in
mouse genotyping, Alexander Bounoutas, Maxime Kinet, Barry Coller,
Marketa Jirouskova, and members of the Strickland laboratory for helpful
discussions, and Alison J. North and Kunihiro Uryu at The Rockefeller Univer-
sity’s Bio-Imaging and Electron Microscopy Resource Centers. We greatly
appreciate assistance from Sarah Bhagat and the Bruce McEwen laboratory
with behavioral experiments. We thank M. Azhar Chishti and David Westaway
for the TgCRND8 mice, Genentech for recombinant tPA, the New York Blood
Center for human plasma, and David E. Levy, Neurobiological Technologies,
Accepted: May 5, 2010
Published: June 9, 2010
Altered Hemostasis in Alzheimer’s Disease
706 Neuron 66, 695–709, June 10, 2010 ª2010 Elsevier Inc.
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