Nucleic acid scavengers inhibit thrombosis without increasing bleeding

Article (PDF Available)inProceedings of the National Academy of Sciences 109(32):12938-43 · July 2012with19 Reads
DOI: 10.1073/pnas.1204928109 · Source: PubMed
Abstract
Development of effective, yet safe, antithrombotic agents has been challenging because such agents increase the propensity of patients to bleed. Recently, naturally occurring polyphosphates such as extracellular DNA, RNA, and inorganic polyphosphates have been shown to activate blood coagulation. In this report, we evaluate the anticoagulant and antithrombotic activity of nucleic acid-binding polymers in vitro and in vivo. Such polymers bind to DNA, RNA, and inorganic polyphosphate molecules with high affinity and inhibit RNA- and polyphosphate-induced clotting and the activation of the intrinsic pathway of coagulation in vitro. Moreover, [NH(2)(CH(2))(2)NH(2)](G = 3);dendri PAMAM(NH(2))(32) (PAMAM G-3) prevents thrombosis following carotid artery injury and pulmonary thromboembolism in mice without significantly increasing blood loss from surgically challenged animals. These studies indicate that nucleic acid-binding polymers are able to scavenge effectively prothrombotic nucleic acids and other polyphosphates in vivo and represent a new and potentially safer class of antithrombotic agents.
Nucleic acid scavengers inhibit thrombosis
without increasing bleeding
Shashank Jain
a
, George A. Pitoc
a
, Eda K. Holl
a
, Ying Zhang
b
, Luke Borst
c
, Kam W. Leong
b
, Jaewoo Lee
a
, and
Bruce A. Sullenger
a,1
a
Department of Surgery, Duke University Medical Center, Durham, NC 27710;
b
Department of Biomedical Engineering, Duke University, Durham, NC
27708; and
c
Department of Pathology, North Carolina State University College of Veterinary Medicine, Raleigh, NC 27607
Edited by Robert Langer, Massachusetts Institute of Technology, Cambridge, MA, and approved July 4, 2012 (received for review March 23, 2012)
Development of effective, yet safe, antithrombotic agents has
been challenging because such agents increase the propensity of
patients to bleed. Recently, naturally occurring polyphosphates
such as extracellular DNA, RNA, and inorganic polyphosphates
have been shown to activate blood coagulation. In this report, we
evaluate the anticoagulant and antithrombotic activity of nucleic
acid-binding polymers in vitro and in vivo. Such polymers bind
to DNA, RNA, and inorganic polyphosphate molecules with high
affinity and inhibit RNA- and polyphosphate-induced clotting
and the activation of the intrinsic pathway of coagulation in vitro.
Moreover, ½NH
2
ðCH
2
Þ
2
NH
2
ðG ¼ 3Þ;dendri PAMAMðNH
2
Þ
32
(PA-
MAM G-3) prevents thrombosis following carotid artery injury
and pulmonary thromboembolism in mice without significantly
increasing blood loss from surgically challenged animals. These stu-
dies indicate that nucleic acid-binding polymers are able to sca-
venge effectively prothrombotic nucleic acids and other polypho-
sphates in vivo and represent a new and potentially safer class of
antithrombotic agents.
polyphosphates/DNA/RNA platelet hemorrhage
T
hrombosis remains one of the leading causes of death and dis-
ability in the Western world despite the development of var-
ious anticoagulants for treatment of deep vein thrombosis, stroke,
atherosclerosis, and other cardiovascular diseases, cardiac inter-
ventions, and metastatic cancers (13). Thrombotic episodes dur-
ing these conditions can be managed by various antithrombotic
and anticoagulant drugs, which can also produce moderate to se-
vere side effects (2, 47). Hence, development of an effective, yet
safe, anticoagulant remains a long-sought objective. Recently,
naturally occurring polyphosphates such as extracellular RNA,
DNA, and inorganic polyphosphates have been reported to be
potent activators of the coagulation cascade. Extracellular RNA
activates coagulation though activation of factors XII and XI in
vitro and in vivo (8). In addition, extracellular RNA has also been
found to act as cofactor for the activation of factor VII-activating
protease (FSAP) (9). DNA-rich neutrophil extracellular traps
(NET) have been found to promote thrombosis (10). Inorganic
polyphosphates, which are stored in dense bodies of mammalian
platelets and secreted on platelet activation, can activate the con-
tact pathway of coagulation and strengthen fibrin clots. Polypho-
sphates have been shown to accelerate factor XI activation by
thrombin and factor Xa (11). Polyphosphates can also inhibit
the activity of tissue factor pathway inhibitor (TFPI) and accel-
erate the activity of thrombin-activatable fibrinolysis inhibitor
(TAFI) (12, 13). In the blood of hemophilia A and B patients and
Hermansky-Pudlak syndrome patients, polyphosphates signifi-
cantly reduce the clotting times (14). Moreover, platelet polypho-
sphates have also been reported to be proinflammatory and
polyphosphate-factor XII binding results in the release of the in-
flammatory mediator bradykinin by plasma kallikrein-mediated
kininogen processing (14). Taken together, all these observations
suggest that naturally occurring polyphosphates such as extracel-
lular DNA, RNA, and inorganic polyphosphate are potent acti-
vators of the coagulation cascade and represent a potential
therapeutic target for novel anticoagulation strategies.
Recently, we discovered that nucleic acid-binding polymers
(NABPs) can act as molecular scavengers and counteract the ac-
tivity of any nucleic acid aptamer regardless of its sequence, as
well as inhibit RNA- and DNA-mediated activation of Toll-like
receptors (TLRs) and inflammation (15, 16). The observations
that nucleic acids and other polyphosphates are involved in
thrombosis led us to hypothesize that such scavengers may also
be able to inhibit polyphosphate-mediated thrombosis. There-
fore, we sought polymers that could bind all of these classes of
polyphosphates with high affinity. In this report, we screened a
wide variety of nucleic acid polymers using in vitro clotting assays
for their potential to inhibit activation of coagulation cascade and
to identify the best suitable NABP to act as a potent and safe
anticoagulant and antithrombotic agent. Based on the results of
in vitro experiments, we explored the anticoagulant and antithrom-
botic properties of a widely used NABP: generation-3 PAMAM
G-3, ½NH
2
ðCH
2
Þ
2
NH
2
ðG ¼ 3Þ;dendri PAMAMðNH
2
Þ
32
.
PAMAM G-3 is a polycationic polyamine polymer (MW 6909)
with a core of 1,4-diaminobutane. It has a diameter of 36 Å with
the 32 surface amine groups (17). It has a high degree of molecular
uniformity, narrow molecular weight distribution, well-defined size
and shape characteristics, and a highly functionalized terminal
surface (18). Because of these characteristics, PAMAM has been
proven to be extremely useful for a variety of applications, such as
gene therapy, molecular diagnostics, controlled drug delivery, and
imaging, in the field of biomedical sciences (17, 18).
Results
Nucleic Acid-Binding Polymers Inhibit Polyphosphate-Mediated
Coagulation in Vitro. As previously described, inorganic polypho-
sphates (PolyP) act as strong activators of the coagulation cascade
and can replace the routinely used activator, kaolin, in standard
blood clotting assays (19). We utilized inorganic polyphosphate
with the average chain length of 60 and 130 as activators for
coagulation cascade to screen different NABP for their anticoa-
gulant activity in vitro. Inorganic polyphosphates 60 and 130
decreased the clotting times of normal pooled human plasma
by over 100 s when added at a concentration of 20 μM (Fig. 1).
Then, we evaluated the ability of NABPs CDP (β -cyclodextrin
containing polycation), HDMBr (hexadimethrine bromide),
PAMAM G-1 (polyamidoamine dendrimer, 1,4-diaminobutane
core, generation 1), PAMAM G-3, and PAMAM-G5 to reverse
this procoagulant activity of inorganic polyphosphates (Fig. 1).
All polymers showed anticoagulant activity in a dose-dependent
fashion. At the concentration of 60 μgmL CDP completely in-
hibits the inorganic polyphosphate 60- and 130-mediated activa-
Author contributions: S.J., and B.A.S. designed research; S.J., G.A.P., E.K.H., L.B., and Y.Z.
performed research; S.J., K.W.L., J.L., L.B., and B.A.S. analyzed data; and S.J. and B.A.S.
wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1
To whom correspondence should be addressed. E-mail: bruce.sullenger@duke.edu.
1293812943 PNAS August 7, 2012 vol. 109 no. 32 www.pnas.org/cgi/doi/10.1073/pnas.1204928109
tion of clotting (Fig. 1 A and B), whereas HDMBr inhibited in-
organic polyphosphate 60- and 130-mediated clotting at the con-
centrations of 10 μgmL and 20 μgmL, respectively (Fig. 1 C
and D). Three different generations of PAMAM (G-1, G-3,
and G-5) also displayed significant anticoagulant activity. PA-
MAM G-1 inhibited polyphosphate 60- and 130-mediated clot-
ting at the concentrations of 4 μgmL and 10 μgmL, respec-
tively (Fig. 1 E and F). At concentrations as low as 2 μgmL
and 2.5 μgmL both PAMAM G-3 and G-5 were able to reverse
the procoagulant effects of PolyP 60 and PolyP 130, respectively
(Fig. 1 GJ). These results demonstrate that NABPs can counter-
act polyphosphate-mediated activation of coagulation. Although
all polymers showed anticoagulant activity in vitro, we chose to
focus upon PAMAM G-3 for additional in vitro characterization
and in vivo thrombosis studies because, along with PAMAM G-5,
it was effective at the lowest concentration and has been reported
to have lower toxicity than PAMAM G-5 (20).
PAMAM G-3 Binds with High Affinity to Various Polyphosphates in
Vitro.
Previously, we reported that a PAMAM G-3 binds to
ssRNA, dsRNA, and ssDNA with high affinity (16). Hence, using
isothermal titration calorimetry (ITC), we investigated whether
PAMAM G-3 binds to inorganic polyphosphates and dsDNA with
high affinity. As shown in Tables 1 and 2, we found that PAMAM
G-3 binds PolyP 60 with a higher affinity (K
d
¼ 7.86Eþ08 M
1
)
than ssDNA (CpG) (K
d
¼ 4.12Eþ08 M
1
) and dsRNA (Poly I:
C) (K
d
¼ 1.05Eþ08 M
1
)the larger the number of phosphates
in the inorganic polyphosphate chain (130 versus 60), the higher
the affinity. In addition, we observed that PAMAM G-3 binds
long dsDNA (plasmid) with a similar high binding affinity
(K
d
¼ 6.41Eþ08 M
1
) as inorganic polyphosphate 60 (K
d
¼
7.86Eþ08 M
1
). Thus, PAMAM G-3 binds with high affinity
to prothrombotic polyphosphates such as DNA, RNA, and inor-
ganic polyphosphates.
PAMAM G-3 Inhibits Activation of the Contact Pathway. In addition to
inhibiting PolyP 60- and 130-mediated activation of clotting, PA-
MAM G-3 also inhibits RNA-mediated (Poly I:C) activation of
clotting (Fig. 2A), indicating that NABPs can inhibit activation
of clotting by various types of extracellular polyphosphates. In
addition, we observed that PAMAM G-3 can inhibit activation
of the contact pathway of coagulation. In a standard activated
Fig. 1. NABPs inhibit inorganic polyphosphate-mediated clotting in vitro. Normal human pooled plasma was treated with PolyP 60 (20 μgmL) and PolyP
130 (20 μgmL). Different NABPs were added in various concentrations and clotting times were recorded using a STart® Hemostasis Analyzer. (A and B) CDP;
(C and D) HDMBr; (E and F) PAMAM G-1; (G and H ) PAMAM G-3; and (I and J) PAMAM G-5. Error bars represent standard deviation.
Table 1. Thermodynamic parameters for PAMAM G-3 binding to
various polyphosphates (first-stage binding)
Polyanions N1*
K
d
1
(M
1
)
ΔG1
(kJmole)
ΔH1
§
(kJmole)
TΔS
(kJmole)
PolyP 130 0.004 5.85Eþ09 2.86 0.68 2.18
PolyP 60 0.0113 7.86Eþ08 2.50 0.64 1.87
CpG 0.091 4.12Eþ08 2.58 0.57 2.02
Poly I:C 0.127 1.05Eþ08 2.78 1.19 1.59
Plasmid 0.0181 6.41Eþ08 2.78 1.19 1.59
*N: Stoichiometric ratio of nitrogen to phosphorous in polyphosphates;
K
d
: dissociation constant;
ΔG: free energ y change;
§
ΔH: enthalpy change;
TΔS: entropy change.
Jain et al. PNAS August 7, 2012 vol. 109 no. 32 12939
APPLIED BIOLOGICAL
SCIENCES
partial thromboplastin time (aPTT) clotting assay, which employs
a nonphysiological anionic activator of coagulation kaolin,
PAMAM G-3 inhibited clotting in a dose-dependent fashion
(Fig. 2B). By contrast, PAMAM G-3 did not significantly impact
tissue factor-initiated coagulation as measured in a prothrombin
time (PT) clotting assay (Fig. 2C). These findings indicate that
the NABP PAMAM G-3 inhibits activation of the contact or in-
trinsic pathway of coagulation by polyanions without impacting
activation of the extrinsic pathway of coagulation.
To examine the anticoagulant properties of PAMAM G-3 in
the more relevant physiological setting of human blood, we eval-
uated its effect on clotting in thrombelastography (TEG) assays
(Fig. 2D). Polyphosphate 60 can activate clotting in whole blood
as measured in a TEG assay and shorten the lag time (time to
start clot formation). The lag time (R) in whole blood with poly-
phosphate 60 (140 μM) was 21.9 min as compared to 27.8 min
for whole blood without polyphosphate treatment. Addition of
PAMAM G-3 (200 μgmL) to the blood inhibited the polypho-
sphate-mediated clot formation and increased lag time from
21.9 min to 64.4 min. PAMAM G-3 also slowed down the rate of
clot formation (α) (Fig. 2E). All together, these observations
show that PAMAM G-3 inhibits the formation of extracellular
RNA and inorganic polyphosphate-engendered clots in human
plasma and whole blood in vitro.
PAMAM G-3 Inhibits Thrombosis in Vivo. To evaluate the ability of
the NABP PAMAM G-3 to inhibit thrombosis in vivo, we utilized
two mouse models of thrombosis: the FeCl
3
-induced carotid artery
injury model and collagen/epinephrine-induced pulmonary throm-
boembolism model. We observed that the mean time for the oc-
clusion of the carotid artery after FeCl
3
treatment was 4 min 30 s
for control mice treated with saline (n ¼ 12). By contrast, none of
the vessels in mice treated with PAMAM G-3 (20 mgkg) were
occluded in 5 min, and 11 of the 12 animals showed no occlusion
of the carotid artery for greater than 40 min following FeCl
3
-in-
duced damage (Fig. 3A). Only 50% of animals showed patent ar-
tery after 40 min following FeCl
3
-induced damage at a 15 mgkg
dose (Fig. 3A). Histological analysis of damaged arteries from
FeCl
3
-challenged animals confirmed that large thrombi had
formed in control-treated animals (Fig. 3B), although no clot
was apparent in PAMAM G-3treated animals (Fig. 3C).
An additional mouse model of thrombosis, collagen/epinephr-
ine-induced lethal pulmonary thromboembolism, was also used
to evaluate antithrombotic activity of PAMAM G-3 in vivo in
the microvasculature. None of the control-treated (normal sal-
ine) mice (n ¼ 11) survived beyond 3.5 min after the injection
of collagen/epinephrine, although 83% of mice treated with
Table 2. Thermodynamic parameters for PAMAM G-3 binding to
various polyphosphates (second-stage binding)
Polyanions N2*
K
d
2
(M
1
)
ΔG2
(kJmole)
ΔH2
(kJmole)
§
TΔS
(kJmole)
PolyP 130 0.0039 8.36Eþ06 2.68 0.22 2.91
PolyP 60 0.0.0049 4.97Eþ06 1.38 2.55 3.93
CpG 0.0423 1.64Eþ06 3.61 1.04 2.57
Poly I:C 0.0.0607 1.30Eþ06 3.25 5.83 2.58
Plasmid 0.00482 6.97Eþ06 2.23 1.27 3.50
*N: Stoichiometric ratio of nitrogen to phosphorous in polyphosphates;
K
d
: dissociation constant;
ΔG: free energy change;
§
ΔH: enthalpy change;
TΔS: entropy change.
Fig. 2. Anticoagulant effect of PAMAM G-3. (A) Effect of PAMAM G-3 on Poly I:C-mediated clotting in vitro. Normal human pooled plasma was treated with
Poly I:C and increasing concentrations of PAMAM G-3 were added. Clotting times were recorded using a STart® Hemostasis Analyzer. (B) Effect of PAMAM G-3
on the activation of intrinsic pathway. Normal human pooled plasma was treated with increasing concentrations of PAMAM G-3. aPTT reagent was used to
activate intrinsic pathway. Clotting times were recorded using a STart® Hemostasis Analyzer. (C) Effect of PAMAM G-3 on the activation of extrinsic pathway.
Normal human pooled plasma was treated with increasing concentrations of PAMAM G-3. PT reagent was added to activate extrinsic pathway. Clotting times
were recorded using a STart® Hemostasis Analyzer. (D) Effect of PAMAM G-3 on clotting in a TEG assay: a, whole blood without activator; b, whole blood +
PolyP 60 (140 μM); c, whole blood + PolyP 60 (140 μM) + PAMAM G-3 (200 μgmL). (E) A table showing all coagulation parameters acquired (R, lag time;
K, speed to reach a certain level of clot strength; α angle, rapidity of clot strengthening; MA, maximum amplitude, the ultimate strength of the fibrin clot). Error
bars represent standard deviation.
12940 www.pnas.org/cgi/doi/10.1073/pnas.1204928109 Jain et al.
PAMAM G-3 (20 mgkg; n ¼ 12) survived for more than 30 min
after administration of collagen/epinephrine mixture, indicating
that PAMAM G-3 also has potent antithrombotic activity in
the setting of pulmonary thromboembolism (Fig. 3D). Moreover,
histological analysis demonstrated that microvessels in the lungs
of control-treated animals contain thrombi (arrows in Fig. 3E),
whereas such vessels in animals treated with PAMAM G-3 were
largely patent (arrows in Fig. 3F). These observations demon-
strate that PAMAM G-3 has a strong antithrombotic effect
in two mouse models: a carotid-large artery damage throm-
bosis model and a pulmonary embolism-microvessel thrombosis
model.
PAMAM G-3 Does Not Increase Bleeding. Most of the commonly used
antithrombotic agents come with an inherent risk of severe or
fatal bleeding (2124). To assess the effect of PAMAM G-3 ad-
ministration on bleeding, we surgically challenged mice treated
with the NABP PAMAM G-3 by tail transection and monitored
blood loss. We evaluated the effect of intravenous treatment
of PAMAM G-3 (20 mgkg), heparin (200 Ukg), and saline
on total blood loss caused by tail transection (Fig. 3G). Mean
blood loss caused by tail injury for over 10 min was 18 μLin
normal saline-treated mice (n ¼ 11) versus 19 μL in PAMAM
G-3treated mice (n ¼ 10). No significant difference in blood
loss was observed between saline-treated and PAMAM G-3trea-
ted mice (P ¼ 0.48). Taken together with the observations ob-
tained in carotid artery injury assay, these results suggest that
PAMAM G-3 prevents thrombus formation without increasing
bleeding. By contrast, heparin (200 Ukg) treatment of mice
results in significant bleeding following tail transection (saline
versus heparin treatment, 18 μL versus 69 μL). Elsewhere, it
has been reported that the same concentration of heparin is re-
quired to maintain artery patency in mice treated with FeCl
3
, sug-
gesting that the commonly used anticoagulant heparin can induce
severe bleeding when utilized at the dose required to inhibit
thrombosis in carotid artery damage model (25). These outcomes
suggest that PAMAM G-3 is an anticoagulant that can be used
with a reduced risk of bleeding. These results also underscore
the important role that naturally occurring extracellular polypho-
sphates such as DNA, RNA, and inorganic polyphosphates play
Fig. 3. Effect of PAMAM G-3 on thrombosis and bleeding. FeCl3-induced carotid artery injury: mice were treated with control (normal saline, squares),
PAMAM G-3 (15 mgkg, triangles), and PAMAM G-3 (20 mgkg, circles). Blood flow was observed in the carotid artery after the treatm ent with FeCl
3
,as
described in Materials and Methods.(A) Kaplan-Meier graph showing the percentage of animals with a patent artery after FeCl
3
-induced injury. Representative
H- and E-stained cross-sections of injured carotid artery from mice treated with control (B) and PAMAM G-3 (C). Collagen/Epinephrine induced pulmonary
thromboembolism: (D) Kaplan-Meier graph showing the percentage of animals survived after collagen/epinephrine injection. Mice were treated with control
(normal saline, squares) or PAMAM G-3 (20 mgkg, circles) follow ed by collag en/epinephrine, as described in Materials and Methods. Representative H- and
E-stained cross-sections of lungs from mice treated with (E ) control and (F) PAMAM G-3. Arrows show the vessels in lung sections. Tail-transection assay: (G) Mice
were injected with control (norm al saline), PAMAM G-3 (20 mgkg), and heparin (200 Ukg). After 15 min, 3 mm of distal tail were surgically removed and
blood loss caused by the injury was monitored over 10 min, as described in Materials and Methods. Error bars represent standard deviation.
Jain et al. PNAS August 7, 2012 vol. 109 no. 32 12941
APPLIED BIOLOGICAL
SCIENCES
in thrombosis, but the limited role that they appear to play in
maintaining normal hemostasis.
Discussion
Taken all together, these in vivo observations suggest that the
NABP PAMAM G-3 can inhibit coagulation and thrombosis
without greatly increasing the propensity to bleed. Because
PAMAM G-3 and other existing NABPs were not engineered
to be antithrombotic agents, we fully anticipate that ample op-
portunities now exist to engineer novel NABPs with improved
extracellular polyphosphate-scavenging properties and reduced
toxicities compared to the currently available NABPs. Nucleic
acid-binding polymers have been extensively studied for their
function as carriers of different drugs, nucleic acids, and small
molecules. Because our studies indicate that nucleic acid-binding
polymers such as PAMAM exhibit antithrombotic activity in vivo,
such polymers should be evaluated for their effects on coagula-
tion during their therapeutic development.
Regardless, our results with PAMAM G-3 demonstrate the
potential utility of NABPs as anticoagulants for treating various
thrombotic pathologies as well as their use during various cardiac
interventions. We chose two different animal models of thrombo-
sis, arterial (FeCl
3
-induced carotid artery injury model) and
microvascular (collagen/epinephrine-induced pulmonary throm-
boembolism) to evaluate the antithrombotic effect of PAMAM
G-3. The carotid artery injury model is widely used to assess
thrombosis in large vessels as a model for myocardial infarction
and thrombotic stroke. By contrast, thrombosis in the microvas-
culature is often evaluated in animals using the collagen/
epinephrine-induced pulmonary thromboembolism model we
employed. Therefore, in this manuscript we determined that
PAMAM G-3 not only inhibited thrombosis in large vessels, such
as in a damaged carotid artery, but also had antithrombotic
effects in the microvasculature.
A major unmet clinical need exists for developing improved an-
tithrombotic agents because the anticoagulants currently utilized
may also cause side effects such as severe and fatal bleeding, ad-
verse immunological responses, and thrombocytopenia, as well as
have unpredictable pharmacokinetics (23, 24, 26). We observe that
at concentrations that limit thrombosis, PAMAM G-3 does not sig-
nificantly increase bleeding in a murine tail-transection model. The
most likely explanation for this observation is that PAMAM G-3 is
inhibiting polyphosphates and nucleic acids from inducing throm-
bosis by limiting their ability to activate factors XI and XII (1016).
Recent studies on factor XII- and XI-deficient mice demonstrate
that these factors appear to be important for thrombosis yet less
important for normal hemostasis (27, 28). Thus, PAMAM G-3 may
achieve its anticoagulant effect without greatly increasing bleeding
by limiting activation of factor XII and XI. Though the clinical
development of any novel therapeutic strategy is challenging, it will
be interesting to determine if by scavenging extracellular nucleic
acids and other polyphosphates, NABPs represent a new and safer
approach to control coagulation and limit thrombosis in patients
undergoing cardiac interventions, such as percutaneous coronary
intervention and coronary artery bypass graft surgery, or who re-
quire chronic anticoagulation therapy for limiting pathologic con-
ditions, such as venous thromboembolism, myocardial infarction,
stroke, and cancer-induced thrombosis. Future studies that evalu-
ate the pharmacology and toxicology of NABPs for treating throm-
botic diseases as well as efforts to engineer novel NABPs for such
applications are warranted.
Materials and Methods
Isothermal Titration Calorimetry. ITC was conducted using a MicroCal VP-ITC
calorimeter, as described elsewhere (16).
Clotting Assay. Polyphosphates (approximately 60 mer and 130 mer; Regene-
Tiss Inc.). were added to 50 μL of normal pooled human plasma (George King
Bio-Medical Inc.). and the reaction was incubated at 37 °C for 3 min. Normal
saline or PAMAM G-3 (Sigma-Aldrich) was added and the reaction was incu-
bated at 37 °C for 3 min, followed by the addition of 50 μL CaCl
2
(25 mM).
Clotting times were recorded using STart® Hemostasis Analyzer (Diagnosti-
ca Stago).
aPTT and PT assays. aPTT assays and PT assays were performed using TriniCLOT
aPTTs (TrinityBiotech) and TriniCLOT PT Excel (TrinityBiotech), respectively,
following supplier guidelines. Clotting times were recorded using STart®
Hemostasis Analyzer (Diagnostica Stago).
Thrombelastography. Freshly withdrawn blood (320 μL) from healthy human
donors was incubated with polyphosphates at 37 °C for 5 min, followed by
the addition of dendrimer PAMAM G-3 or normal saline. The reaction was
incubated at 37 °C for 5 min and 20 μL CaCl
2
(200 mM) was added. Clot
formation was recorded using TEG 5000 Thrombelastograph (Haemoscope
Corporation) analyzer for 45 min. The whole procedure was approved by
the Institutional Review Board of Duke University (Durham, NC).
Carotid Artery Injury Assay. Animal procedures were performed using 10
14-wk-old wild-type female C57BL/6J mice (Jackson Laboratory). Mice were
induced by gas inhalation (5% Forane; Baxter). Mice were then intubated
and mechanically ventilated (rodent ventilator 683; Harvard Apparatus) with
maintenance of anesthesia by 22.5% Forane during procedure. Mice were
injected with PAMAM G-3 or normal saline into the lateral tail vein in a total
volume of 200 μL. Right common carotid artery was exposed. A transonic la-
ser 0.5-PSB transit-time flow probe (Transonic Systems Inc.). was placed
around the artery to measure the blood flow. Two small pieces of filter paper
(1 mm by 2 mm) saturated with 2.5% FeCl
3
were placed on both sides of the
carotid artery for 3 min (25). Filter papers were removed and blood flow was
monitored for more than 40 min using TS420 perivascular flowmeter (Transo-
nic Systems) and LabChart software (ADInstruments). All experimental pro-
tocols involving animals were approved by the Duke University Institutional
Animal Care and Use Committee (Durham, NC).
Pulmonary Thromboembolism. Mice were anesthetized using the same meth-
od described above. Mice were injected with PAMAM G-3 or normal saline
into the left retro-orbital plexus. After 30 min, a mixture of collagen
(0.8 mgkg; Chronolog) and epinephrine (60 μgkg; Hospira) was injected
via right retro-orbital plexus. Mice were carefully observed for respiration
to report survival time after the injection of collagen/epinephrine.
Tail-Bleeding Assay. Mice were anesthetized using the same method
described above. PAMAM G-3, heparin (APP Pharmaceuticals), or normal
saline was delivered to mice via retro-orbital plexus. After 15 min, 3 mm
of distal mouse tail was removed and the tail was immediately immersed
in 1 mL isotonic saline (37 °C). Blood was collected for 10 min after tail trans-
ection. The total blood loss was determined by measuring the absorbance of
the blood containing normal saline at 560 nm, as described elsewhere (29). A
standard curve method was used to calculat e the total blood loss caused by
tail transection. All experimental protocols involving animals were approved
by the Duke University Institutional Animal Care and Use Committee
(Durham, NC).
ACKNOWLEDGMENTS. We thank Jens Lohrmann, Maureane Hoffman, and
Shahid Nimjee for technical help and Richard C. Becker for useful discussions.
This study was supported in part by funds from National Heart, Lung, and
Blood Institute (HL065222).
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Jain et al. PNAS August 7, 2012 vol. 109 no. 32 12943
APPLIED BIOLOGICAL
SCIENCES
    • "High levels of cfDNA appear to have high discriminative power to predict mortality in severe sepsis patients [13,20,23,24] . In recent years, DNA-binding polymers have been developed to inhibit coagulation and thrombosis without increasing the propensity to bleed [52]. In addition to activation of coagulation, impairment of the fibrinolytic system also plays an important role in the pathogenesis of microvascular thrombosis and organ dysfunction in DIC [53,54]. "
    [Show abstract] [Hide abstract] ABSTRACT: Disseminated intravascular coagulation (DIC) is a heterogeneous group of disorders, which manifest as a spectrum of haemorrhage and thrombosis complicating many primary conditions including sepsis, trauma and malignancies. The pathophysiology of this condition is complex. In the recent years there is growing evidence that damage associated molecular patterns (DAMPs) play a crucial role in the pathogenesis of DIC. Upon cell-death and/or cell activation of hematopoietic and parenchymal cells extracellular cell-free DNA as well as DNA binding proteins (e.g. histones and high mobility group box 1 protein [HMGB1]) are released into circulation. This release is a highly regulated process mediated among others by serine proteases, such as factor VII-activating protease (FSAP) and DNase1. Circulating cell-free DNA has been demonstrated to influence primary and secondary hemostasis by inducing platelet aggregation, promoting coagulation activation, inhibition of fibrinolysis and directly interfering with clot stability. In this respect cell-free DNA in tissue as well as released into the circulation after neutrophil activation in the form of neutrophil extracellular traps (NETs) has been shown to be cytotoxic and highly procoagulant. DNA-binding proteins such as histones and HMGB1 are also strongly procoagulant and are involved in the pathogenesis of DIC. The present review gives an overview on how extracellular DNA is released into circulation and the structure of circulating DNA. In addition it summarizes the effect of extracellular DNA and DNA-binding proteins on platelet activation, plasmatic coagulation as well as fibrinolysis.
    Full-text · Article · Dec 2015
    • "A recent report showed that topical administration of G3 PAMAM dendrimers could efficiently sequester nucleic acids that cause increase in fibroblast activation and granulation tissue contraction, resulting in decreased pathological scarring during wound healing [91]. In addition, the application of polycations as antithrombotic agents that rapidly remove prothrombotic nucleic acids together with other polyphosphates was also demonstrated in vivo and proposed as a novel strategy to prevent thrombosis after injury [92]. "
    [Show abstract] [Hide abstract] ABSTRACT: Synthetic polymers play a critical role in pharmaceutical discovery and development. Current research and applications of pharmaceutical polymers are mainly focused on their functions as excipients and inert carriers of other pharmacologically active agents. This review article surveys recent advances in alternative pharmaceutical use of polymers as pharmacologically active agents known as polymeric drugs. Emphasis is placed on the benefits of polymeric drugs that are associated with their macromolecular character and their ability to explore biologically relevant multivalency processes. We discuss the main therapeutic uses of polymeric drugs as sequestrants, antimicrobials, antivirals, and anticancer and anti-inflammatory agents.
    Full-text · Article · Sep 2015
    • "Polymers of phosphates, polyphosphate (polyP), discovered in 1888 [1], contain a few to several hundred residues of orthophosphate linked by high-energy phosphoanhydride bonds. PolyP has since been documented in mammalian cells [2], and has been revealed to have physiological roles [3], including regulation of vertebrate skeletal mineralization [4][5][6][7], fibrin clotting [8][9][10], intestinal homeostasis [11] , neuronal excitability [12], innate immunity [13] and to have functions as a P2 receptor agonist [14]. PolyP is also involved in some disease conditions, including tumor proliferation [15,16] , metasta- sis [17], cardiac necrosis or neurodegenerative processes [17][18][19][20]and energy deficiency [21][22][23]. "
    [Show abstract] [Hide abstract] ABSTRACT: Phosphate is essential for all major life processes, especially energy metabolism and signal transduction. A linear phosphate polymer, polyphosphate (polyP), linked by high-energy phosphoanhydride bonds, can interact with various proteins, playing important roles as an energy source and regulatory factor. However, polyP-binding structures are largely unknown. Here we proposed a putative polyP binding site, a positively-charged semi-tunnel (PCST), identified by surface electrostatics analyses in polyP kinases (PPKs) and many other polyP-related proteins.We found that the PCSTs in varied proteins were folded in different secondary structure compositions. Molecular docking calculations revealed a significant value for binding affinity to polyP in PCST-containing proteins. Utilizing the PCST identified in the β subunit of PPK3, we predicted the potential polyP-binding domain of PPK3. The discovery of this feature facilitates future searches for polyP-binding proteins and discovery of the mechanisms for polyP-binding activities. This should greatly enhance the understanding of the many physiological functions of protein-bound polyP and the involvement of polyP and polyP-binding proteins in various human diseases.
    Full-text · Article · Apr 2015
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