From www.bloodjournal.org by guest on July 16, 2017. For personal use only.

PML/RARA is detected in cytoplasmic auto- 9. Isakson P, Bjoras M, Boe SO, Simonsen A. Autophagy 10. Bernardi R, Guernah I, Jin D, et al. PML inhibits
contributes to therapy-induced degradation of the PML/ HIF-1alpha translation and neoangiogenesis through re-
phagic vesicles after treatment with these RARA oncoprotein. Blood. 2010;116(13):2324-2331. pression of mTOR. Nature. 2006;442(7104):779-785.
agents. Functionally, activation of autophagy
by the mammalian target of rapamycin
PLATELETS & THROMBOPOIESIS
(mTOR) inhibitor triggers PML/RARA de-
stabilization in the NB4 APL cell line, result- Comment on Zhang et al, page 2336
ing in enhanced ATRA-induced differentia-
tion. Conversely, the autophagy inhibitor
BafA impedes treatment-induced PML/ Arterial thrombosis: going, gone!
----------------------------------------------------------------------------------------------------------------
RARA degradation and biologic response.
These results are important in several re- Elizabeth E. Gardiner and Michael C. Berndt MONASH UNIVERSITY; DUBLIN CITY UNIVERSITY
spects. They identify a novel cytoplasmic Most antithrombotic approaches target prevention rather than the more clinically
PML/RARA catabolic pathway and explain relevant issue of resolution of an existing thrombus. In this issue of Blood, Zhang
why some studies had observed PML/RARA and colleagues1 describe a novel and effective therapeutic strategy for clearance of
in the cytoplasm. Other oncogenic fusion pro- preexisting arterial thrombus in murine models of ischemic stroke.
teins may share similar features of poor solu-
bility and may be targets of autophagy. This
study also raises interesting questions as to the
possible links between autophagy and PML,
which is highly stress-sensitive and may also
assemble into cytoplasmic bodies.4 Finally,
PML suppresses mTOR activity,10 a key in-
hibitor of the autophagic process. Then, re-
lease of PML from PML/RARA by ATRA or
As2O3 treatment induces a self-perpetuating
cycle accelerating PML/RARA degradation.
The latter could be implicated in enhanced
differentiation, as suggested in the Isakson
study, but also in loss of self-renewal of APL
clonogenic progenitors. Thus, modulation of
autophagy in APL could have important The bifunctional SLK consisting of the scFv-A11, that binds to an epitope on activated IIb3 (GPIIb-IIIa) and
therapeutic consequences. induces platelet lysis, fused to the kringle 1 domain that recognizes fibrin (not fibrinogen). This construct can
target an active thrombus (via activated IIb3/polymerized fibrin) providing both dual antithrombotic activity
Conflict-of-interest disclosure: The authors and a unique therapeutic approach. Monovalent antibody also induces oxidative lysis via IIb3, while
declare no competing financial interests. kringle-containing plasminogen interacts directly with fibrin.

REFERENCES
1. Kogan SC. Curing APL: differentiation or destruction?
Cancer Cell. 2009;15(1):7-8.
2. Nasr R, Guillemin MC, Ferhi O, et al. Eradication of acute
promyelocytic leukemia-initiating cells through PML-RARA
degradation. Nat Med. 2008;14(12):1333-1342.
3. Tallman MS, Altman JK. How I treat acute promyelo-
cytic leukemia. Blood. 2009;114(25):5126-5135.
4. Bernardi R, Pandolfi PP. Structure, dynamics and func-
tions of promyelocytic leukaemia nuclear bodies. Nat Rev
Mol Cell Biol. 2007;8(12):1006-1016.
5. Hu J, Liu YF, Wu CF, et al. Long-term efficacy and
safety of all-trans retinoic acid/arsenic trioxide-based
therapy in newly diagnosed acute promyelocytic leukemia.
Proc Natl Acad Sci U S A. 2009;106(9):3342-3347.
6. Nervi C, Ferrara FF, Fanelli M, et al. Caspases mediate
retinoic acid-induced degradation of the acute promyelo-
cytic leukemia PML/RARalpha fusion protein. Blood.
1998;92(7):2244-2251.
7. Zhu J, Gianni M, Kopf E, et al. Retinoic acid induces
proteasome-dependent degradation of retinoic acid receptor
alpha (RAR alpha) and oncogenic RAR alpha fusion pro-
teins. Proc Natl Acad Sci U S A. 1999;96(26):14807-14812.
8. Lallemand-Breitenbach V, Jeanne M, Benhenda S, et al.
Arsenic degrades PML or PML-RARalpha through a
SUMO-triggered RNF4/ubiquitin-mediated pathway.
Nat Cell Biol. 2008;10(5):547-555.
rterial thrombosis is initiated by platelets
A adhering to the damaged vessel wall.
These adherent platelets become activated,
leading to activation of the platelet integrin
glycoprotein (GP) IIb-IIIa (IIb3), which in
binding fibrinogen and von Willebrand factor
allows formation of a platelet aggregate or mu-
ral thrombus. At the same time, the damaged
vessel wall and the activated platelet surface
provide an acceleration of localized coagulation
leading to thrombus stabilization by fibrin.2
Thrombosis precipitating ischemic stroke
is a major cause of morbidity and death. The
capacity to limit brain infarct formation and
resultant permanent neurologic damage re-
quires resolution of the arterial thrombus,
ideally within a narrow therapeutic window of
up to 4 hours postocclusion. Clinical studies
with GPIIb-IIIa receptor antagonists3 or other
profibrinolytic approaches4 are not optimal
and are typically associated with a high inci-
dence of bleeding. Current treatment of acute
ischemic stroke with tissue plasminogen acti-
vator shows best utility within 4.5 hours of
occlusion and can also be associated with clini-
cally significant bleeding,5 highlighting the
pressing need for better and safer therapeutic
approaches.
Zhang and colleagues have previously de-
scribed a unique antiplatelet autoantibody in
patients with HIV- or hepatitis Crelated
thrombocytopenia that recognizes the
sequence GPIIIa49-66 and induces
complement-independent platelet lysis by
generation of reactive oxygen species and per-
oxide after platelet-reduced nicotinamide
adenine dinucleotide phosphate oxidase acti-
vation.6,7 Subsequently, they identified a
human single-chain fragment variable region
(scFv) antibody that recognized GPIIIa49-66
(A11), with similar functional properties to the
patient autoantibody in that it preferentially
bound to activated platelets and could also lyse
arterial thrombus in vitro. In the current

blood 3 0 S E P T E M B E R 2 0 1 0 I V O L U M E 1 1 6 , N U M B E R 1 3 2201
 From www.bloodjournal.org by guest on July 16, 2017. For personal use only.

study,1 Zhang et al have taken this transla-
tional medicine approach full circle by assess-
ing the capacity of A11 to clear arterial throm-
bus in 2 murine stroke models. Although A11
showed considerable promise in these studies,
likely to activate complement or invoke an
inflammatory phagocytic cytokine response
because it lacks an Fc domain.
The current study did not address SLKs
use as a thrombolytic in other vascular beds,
2. Andrews RK, Gardiner EE, Shen Y, Berndt MC. Plate-
let interactions in thrombosis. IUBMB Life. 2004;56(1):
13-18.
3. Mandava P, Thiagarajan P, Kent TA. Glycoprotein
IIb/IIIa antagonists in acute ischaemic stroke: current sta-
tus and future directions. Drugs. 2008;68(8):1019-1028.

restoring blood flow, for example, when ad-
ministered 2 hours after carotid artery occlu-
sion, the most dramatic and encouraging find-
ings were obtained with a second-generation
derivative of A11 (see figure).
This variant, termed SLK (single-chain
linked first kringle), was produced by coupling
the A11 scFv fragment through a linker pep-
tide to the first kringle domain of plasminogen.
The rationale, experimentally confirmed in
this study, was that the first kringle domain of
plasminogen would allow SLK to home to,
and covalently bond with, terminal lysine resi-
dues within partially degraded fibrin associ-
ated with the platelet thrombus, similar to
the approach recently used to create Delta-
plasmin8 and serving to concentrate the
A11 scFv component directly at the platelet
thrombus where platelet lysis would then occur.
Critically, SLK behaves in mice with many of
the advantages that would be hoped for in an
optimal therapeutic thrombolytic. First, SLK
could reproducibly lyse thrombus and restore
blood flow when administered 4 hours after
cessation of blood flow or induction of stroke,
whereas A11 was ineffective at this time period
after arterial occlusion. SLK showed partial pro-
tection against infarct (34% reduction) and sig-
nificantly preserved neurologic function (as mea-
sured by postural reflex), without evidence of
cerebral bleeding. In contrast, in the same mu-
rine model, one-third of mice treated with tissue
plasminogen activator died of intracranial hem-
orrhage.9 Second, SLK caused minimal throm-
bocytopenia (11% reduction in platelet count
at the 4-hour postadministration nadir), with
restoration of normal count at 24 hours. Third,
SLK bound preferentially to activated platelets
and lysed platelets by the same functional
mechanism as A11. Fourth, SLK had no affect
on platelet aggregation in vitro. Importantly, ex
vivo analysis of blood platelets taken from
mice 4 hours after SLK administration
showed no evidence of platelet activation
and did show normal collagen and thrombin
agonist responses in terms of P-selectin ex-
pression and formation of platelet-monocyte
aggregates. Fifth, SLK had no effect on
bleeding time. Finally, as a human scFv an-
tibody construct, SLK would be less likely
to act as a foreign antigen and would be un-
for example, in coronary artery thrombosis, 4. Gardiner EE, Andrews RK. The cut of the clot(h): snake
venom fibrinogenases as therapeutic agents. J Thromb
where it might act as an alternative in some Haemost. 2008;6(8):1360-1362.
circumstances to coronary artery stenting and 5. Lees KR, Bluhmki E, von Kummer R, et al. Time to
other more interventional approaches. Any treatment with intravenous alteplase and outcome in stroke:
an updated pooled analysis of ECASS, ATLANTIS,
potential effect of A11 and/or SLK on the
NINDS, and EPITHET trials. Lancet. 2010;375(9727):
endothelial integrin v3 sharing the A11 1695-1703.
epitope-bearing 3 chain also remains to be 6. Li Z, Nardi MA, Li Y-S, et al. C-terminal ADAMTS-18
elucidated. If these promising preclinical stud- fragment induces oxidative platelet fragmentation, dissolves
platelet aggregates, and protects against carotid artery oc-
ies can be replicated in first studies in humans, clusion and cerebral stroke. Blood. 2009;113(24):6051-6060.
they are likely to provide better and safer treat- 7. Nardi M, Tomlinson S, Greco MA, Karpatkin S.
ment options in ischemic stroke. Complement-independent, peroxide-induced antibody
Conflict-of-interest disclosure: The authors lysis of platelets in HIV-1-related immune thrombocytope-
nia. Cell. 2001;106(5):551-561.
declare no competing financial interests.
8. Hunt JA, Petteway SR Jr, Scuderi P, Novokhatny V.
Simplified recombinant plasmin: production and functional
REFERENCES comparison of a novel thrombolytic molecule with plasma-
derived plasmin. Thromb Haemost. 2008;100(3):413-419.
1. Zhang W, Li YS, Nardi MA, et al. Dissolution of arterial
platelet thrombi in vivo with a bifunctional platelet 9. Quartermain D, Li YS, Jonas S. Acute enoxaparin treat-
GPIIIa49-66 ligand which specifically targets the platelet ment widens the therapeutic window for tPA in a mouse
thrombus. Blood. 2010;116(13):2336-2344. model of embolic stroke. Neurol Res. 2007;29(5):469-475.
PLATELETS & THROMBOPOIESIS
Comment on Lordier et al, page 2345
Shedding light on endomitosis
----------------------------------------------------------------------------------------------------------------
Amy E. Geddis UNIVERSITY OF CALIFORNIA SAN DIEGO
Endomitosis is an enigma fascinating to hematologists and cell biologists alike.
Whereas aneuploidy can be associated with chromosomal instability and cancer,1
megakaryocytes become polyploid in the course of terminal differentiation. In this
issue of Blood, Lordier and colleagues delve further into the function of Aurora B
kinase in endomitosis.2
Mitotic and endomitotic cycling in early megakaryocytes. Progenitors that will become polyploid proceed
through anaphase and then regress their cleavage furrows and re-enter G1 as 4N cells. Aurora B kinase is
shown in orange, localized to kinetochores in metaphase, the spindle midzone in anaphase, and the midbody in
telophase. Professional illustration by Marie Dauenheimer.
bservational studies have shown that tion of the cleavage furrow.3 Whereas cells
O endomitotic megakaryocytes deviate
from mitosis in late anaphase, after the initia-
undergoing diploid mitoses proceed
through cytokinesis and complete division

2202 30 SEPTEMBER 2010 I VOLUME 116, NUMBER 13 blood

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2010 116: 2201-2202

doi:10.1182/blood-2010-06-291492

Arterial thrombosis: going, gone!

Elizabeth E. Gardiner and Michael C. Berndt

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