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Coagulation Factor X and Apixaban
St. Dominic Middle School SMART Team
Jennifer Austin, Mia Bando, Christopher Brzozowski, Giovanni Faraciano, Caroline Flyke, Nicholas Hilbert, Isabella Huschitt, Thomas Hyde, Elizabeth Klingsporn, Janna Lieungh
Emily Maslowski, Kaitlyn O’Hair, Mary Rose Otten, Isabelle Phan, Sophia Pittman, Ryan Reilly, Alex Reinbold, Catherine Simmons, Michael Sohn, Emma Urban, Rachel Vasan
Teacher: Donna LaFlamme
St. Dominic Catholic School
18105 W. Capitol Drive, Brookfield, WI 53045
Mentors: Michael Pickart, Ph.D. and Xiaoang Xing
Concordia University School of Pharmacy, 12800 N. Lake Shore Dr., Mequon WI 53097
I. Introduction
Factor X is a clotting protein that is made in the liver and found in the blood. Pharmaceutical companies have
recently developed new oral anticoagulants (NOACs) that inhibit Factor X, such as apixaban (Eliquis), to treat
and prevent life threatening blood clots. An older anticoagulant, warfarin, is extremely difficult to dose,
requires constant blood level monitoring, and has many drug and food interactions. Apixaban has none of
these drawbacks, but it does lack an antidote to stop bleeding in emergencies. Our mentors are using zebrafish
as animal models to search for antidotes to the NOAC, apixaban.
II. Factor X in the Coagulation Cascade
Factor X is a serine protease that acts at the end of both the intrinsic and extrinsic clotting cascades.
Factor X cleaves prothrombin to form thrombin. Thrombin then cleaves fibrinogen to form the strands of
fibrin that stabilize and complete the blood clot.
VI. Apixaban versus Warfarin
IV. Catalytic Domain of Activated Factor X
Bound to Apixaban
The blood-clotting protein Factor X is composed of a heavy protein chain and a light protein chain. The
heavy chain contains the active site where prothrombin is cleaved during the last steps of the coagulation
cascade. The light chain (not shown) anchors Factor X to the phospholipid membrane of platelets during
the last steps of the blood clot formation. The St. Dominic SMART Team modeled the catalytic domain of
activated Factor X using 3D printing technology to explore how the new oral anticoagulant apixaban
(Eliquis) blocks the active site. Our mentors are in the process of searching for an antidote to apixaban
using zebrafish as a model organism. Anticoagulants like apixaban are used to treat and prevent deep vein
thrombosis (DVT), pulmonary embolisms (PEs), and stroke in atrial fibrillation patients.
In the September 15, 2011 issue of The New England Journal of Medicine, Christopher B.
Granger et. al. reported on the Aristotle Clinical Trials involving over 18,000 patients with
atrial fibrillation. Apixaban was found to be superior to warfarin in preventing stroke or
systemic embolism, caused less bleeding, and resulted in lower mortality. (Funded by
Bristol-Myers Squibb and Pfizer; ARISTOTLE ClinicalTrials.gov number, NCT0041)
Percent of Afib Patients Experiencing
Major Bleeding
Percent of AFib Patients Experiencing
Stroke or Systemic Embolism
Theoretical model of the assembly of
the prothrombinase complex on the
platelet phospholipid membrane.
[8]
[8]
Asp102
Trp215 Tyr99
His57 Ser195
Coagulation
Cascade
Warfarin vs Apixaban in Medical Practice
For patients and physicians,
apixaban has many advantages
over warfarin including easier
dosing, no continual monitoring
of blood anticoagulant ability,
and much fewer food and drug
interactions. The major
disadvantage for apixaban,
however, is the lack of an
antidote for emergency bleeding.
Phe174
[2]
[1]
Coagulation Cascade
Prothrombinase Complex
The intrinsic series of reactions
is triggered by surface damage
to a blood vessel, and the
extrinsic cascade of reactions is
triggered by trauma to both the
blood vessel and the
surrounding tissue.
Factor Xa (activated Factor X) and Factor Va
(activated Factor V) combine to form the
prothrombinase complex on the platelet
phospholipid membrane. Prothrombin then
binds to the complex, and Factor Xa cleaves
prothrombin to produce thrombin. The
prothrombinase complex cleaves
prothrombin at 300,000 times the rate that
Factor Xa does alone. [1]
Stabilized Blood Clot
Thrombin cleaves
fibrinogen to form fibrin
strands, which stabilize
the clot by trapping
platelets and blood cells
in a fibrin net.
Pulmonary Embolism
Apixaban
Glu146
Physical Model of the Catalytic Domain of Activated
Factor X Based on PDB ID: 2P16 [7]
Atrial Fibrillation and Stroke
Factor X Active Site without Apixaban
• Factor X is a serine protease that uses the
catalytic triad formed from Asp102, His57, and
Ser195 to cleave the clotting protein thrombin
by hydrolysis of a peptide bond.
• Trp215, Phe174, and Tyr99 on the left side of
the active site surround the phenyllactam ring
of apixaban.
• The backbone N-H of Gly216 shown in the
lower portion of the active site interacts with
the carbonyl oxygen of scaffold carboxamide.
• Apixaban does not interact with the catalytic
triad; however, it does block the substrate
prothrombin from binding correctly.
• Glu146 interacts with the N-H of apixaban’s
carboxamide group.
Glu146 is show at the bottom right
of the image above.
• The catalytic triad of Factor X cleaves
prothrombin in two places (after Arg271 and
after Arg32) to produce activated thrombin,
which is a serine protease that cleaves
fibrinogen.
[5]
[3]
Deep vein thrombosis (DVT) is
the formation of a blood clot in
the deep veins of the leg or
pelvis. Symptoms can include leg
pain and swelling; however, there
may be no symptoms. DVT
requires immediate medical
attention as clots can travel to
the lungs and cause a lifethreatening pulmonary
embolism. The NOAC apixaban
(Eliquis) is FDA approved to treat
and prevent deep vein
thrombosis, which it does by
inhibiting Factor X.
VII. Using Zebrafish to Find an Apixaban Antidote
Lack of an antidote for apixaban can cause serious problems if a patient needs to undergo
immediate surgery or has been overdosed. Our mentors are using zebrafish to screen 10,000
compounds for possible antidotes to apixaban. Zebrafish are a good model organism because they
can be bred in the laboratory and can be used for testing as embryos when apixaban and antidote
compounds can be added to their water. If the antidote is not effective, hemorrhaging is visible in
the embryo under the microscope. (See figure below.) In addition, zebrafish have genes for all of the
coagulation proteins found in mammals (Hanumanthaiah et. al. (2002). A Blast alignment comparing
the amino acid sequence of Human Factor X to zebrafish Factor X showed 47% identity (the same
amino acid at the same positon) and 63% positivity (a similar amino acid at the same position).
Factor X, Zebrafish vs Human Protein
Sequence Alignment
V. Structure of Apixaban
Hemorrhage in Embryo
[4]
A pulmonary embolism (PE) is
caused by a blood clot that
travels through the heart to
the lungs from the legs or, in
rare cases, other parts of the
body. The clot damages the
lung by blocking blood flow.
The dark area in the diagram
above indicates tissue damage.
Pulmonary embolism can
result in death. Anticoagulants
are used to prevent and treat
pulmonary embolisms.
Apixaban is FDA approved to
treat PE.
PDB ID: 2P16
Donald J. Pinto, Michael J. Orwat, Stephanie Koch, Karen A. Rossi, Richard S. Alexander, Angela
Smallwood, Pancras C. Wong, Alan R. Rendina, Joseph M. Luettgen, Robert M. Knabb, Kan
He, Baomin Xin, Ruth R. Wexler, and Patrick Y. S. Lam. (2007). Discovery of 1-(4Methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo[3,4c]pyridine-3-carboxamide (Apixaban, BMS-562247), a Highly Potent, Selective, Efficacious, and
Orally Bioavailable Inhibitor of Blood Coagulation Factor Xa. J.Med.Chem. 50: 5339-5356
1. James C. Whisstock. (2013). Assembling the Machinery of Coagulation. Blood 122: 2773- 2774.
2. https://www.mhhe.com/biosci/esp/2002_general/Esp/folder_structure/tr/m1/s7 trm1s7_3.htm
3. Society of Interventional Radiiology
4. http://www.drugdangers.com/nuvaring/pulmonary-embolism.htm
[11]
[9]
Factor X Active Site with Apixaban
III. Medical Conditions Treated with Anticoagulants
Deep Vein Thrombosis
Gly216
[10]
[6]
Atrial fibrillation (AFib) occurs
when the two upper chambers of
the heart, the atria, beat
irregularly (fibrillate) slowing
blood flow. Slower flow causes
clots to form. If a clot enters a
blood vessel leading to the brain,
a stroke will occur. Patients with
AFib are at increased risk of
stroke and take anticoagulants to
prevent the formation of clots.
Apixaban is FDA approved to
prevent stroke in AFib patients.
5. http://www.cdc.gov/dhdsp/data_statistics/fact_sheets/fs_atrial_fibrillation.htm
6. National Heart, Blood and Lung Institute, National Institutes of Health
7. PDB ID 2P16 Primary Citation
8. Christopher B. Granger, et.al. (2011). Apixaban versus Warfarin in Patients with Atrial Fibrillation N Engl J Med. 365:981-99
9 http://files.www.clotconnect.org/Comparison_of_oral_anticoagulants_Final.pdf
10. http://www.mmm-online.com/features/eliquis-aims-to-amp-up-its-sales-push/article/348082
11. http://america.pink/images/4/7/3/5/3/6/3/en/1-warfarin.jpg
12. Xiaoang Xing, Concordia University School of Pharmacy
13.S. Eisa-Beygi, RL Macdonald RL, XY Wen (2014) Regulatory pathways affecting vascular stabilization via VE-cadherin dynamics: insights
from zebrafish (Danio rerio). Journal of Cerebral Blood Flow & Metabolism 34(9):1430-1433
14.Ravikumar Hanumanthaiah, Kenneth Day, Pudar Jagadeeswaran (2002) Comprehensive Analysis of Blood Coagulation Pathways
in Teleostei: Evolution of Coagulation Factor Genes and Identification of Zebrafish Factor VIIi. Blood Cells, Molecules, and
Diseases 29(1):57-68
The N-H of the
carboxamide group
interacts with the
peptide bond
carbonyl of Glu146
(Magnet)
Pyrazole ring has a
carboxamide group
on carbon three.
The p-methoxyphenyl
group does not interact
with any specific active site
amino acid residues.
Carbonyl of scaffold
carboxamide interacts with
backbone N-H of Gly216.
(Magnet)
The phenyllactam group is
positioned in a pocket
bound by Trp215, Phe174,
and Tyr99
[13]
Jmol Image of apixaban.
PDB 2P16
[12]
Alignment of Human (AAA5242.1) and
zebrafish (NP_968870) Factor X
The human amino acid sequence is
above the zebrafish sequence.
Asterisks identify the amino acids that
are identical and in the same position
in the human and zebrafish protein.
Colons indicate similar amino acids.
3D physical model of
apixaban. PDB 2P16
[7]
The SMART Team Program is supported by the National Center for Advancing Translational Sciences, National
Institutes of Health, through Grant Number 8UL1TR000055. It’s contents are solely the responsibility of the authors
and do not necessarily represent the official views of the NIH.
Hemorrhage after warfarin
treatment as shown in this
embryo would be evidence
of ineffective antidote.
Apixaban antidotes will be
tested in the same way.
Zebrafish Embryonic
Development
VIII. Summary
Factor X coagulation protein is the target of new oral anticoagulants like apixaban (Eliquis). Apixaban
.
inhibits
activated Factor X in the coagulation cascade to prevent clot formation and has been
approved by the FDA to treat and prevent deep vein thrombosis and pulmonary embolism as well as
to prevent strokes in atrial fibrillation patients. Apixaban’s major drawback is lack of an antidote. Our
mentors are using zebrafish as a model organism to test thousands of possible antidote compounds.