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 VI. Apixaban versus Warfarin 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 IV. Catalytic Domain of Activated Factor X In the September 15, 2011 issue of The New England Journal of Medicine, Christopher B. and prevent life threatening blood clots. An older anticoagulant, warfarin, is extremely difficult to dose, Granger et. al. reported on the Aristotle Clinical Trials involving over 18,000 patients with requires constant blood level monitoring, and has many drug and food interactions. Apixaban has none of Bound to Apixaban atrial fibrillation. Apixaban was found to be superior to warfarin in preventing stroke or these drawbacks, but it does lack an antidote to stop bleeding in emergencies. Our mentors are using zebrafish systemic embolism, caused less bleeding, and resulted in lower mortality. (Funded by The blood-clotting protein Factor X is composed of a heavy protein chain and a light protein chain. The as animal models to search for antidotes to the NOAC, apixaban. Bristol-Myers Squibb and Pfizer; ARISTOTLE ClinicalTrials.gov number, NCT0041) 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 Percent of AFib Patients Experiencing Percent of Afib Patients Experiencing II. Factor X in the Coagulation Cascade the last steps of the blood clot formation. The St. Dominic SMART Team modeled the catalytic domain of Stroke or Systemic Embolism Major Bleeding activated Factor X using 3D printing technology to explore how the new oral anticoagulant apixaban Factor X is a serine protease that acts at the end of both the intrinsic and extrinsic clotting cascades. (Eliquis) blocks the active site. Our mentors are in the process of searching for an antidote to apixaban Factor X cleaves prothrombin to form thrombin. Thrombin then cleaves fibrinogen to form the strands of using zebrafish as a model organism. Anticoagulants like apixaban are used to treat and prevent deep vein fibrin that stabilize and complete the blood clot. thrombosis (DVT), pulmonary embolisms (PEs), and stroke in atrial fibrillation patients.

Theoretical model of the assembly of the prothrombinase complex on the platelet phospholipid membrane. [8] [8] Asp102

His57 Ser195 Trp215 Tyr99 Warfarin vs Apixaban in Medical Practice For patients and physicians, Coagulation apixaban has many advantages Cascade Phe174 over warfarin including easier [1] [2] [10] dosing, no continual monitoring Coagulation Cascade Prothrombinase Complex Stabilized Blood Clot Gly216 of blood anticoagulant ability, The intrinsic series of reactions Factor Xa (activated Factor X) and Factor Va Thrombin cleaves Apixaban and much fewer food and drug is triggered by surface damage (activated Factor V) combine to form the interactions. The major fibrinogen to form fibrin Glu146 to a blood vessel, and the prothrombinase complex on the platelet strands, which stabilize disadvantage for apixaban, extrinsic cascade of reactions is phospholipid membrane. Prothrombin then the clot by trapping however, is the lack of an [11] triggered by trauma to both the binds to the complex, and Factor Xa cleaves platelets and blood cells Physical Model of the Catalytic Domain of Activated antidote for emergency bleeding. blood vessel and the prothrombin to produce thrombin. The in a fibrin net. Factor X Based on PDB ID: 2P16 [7] prothrombinase complex cleaves [9] surrounding tissue. Factor X Active Site with Apixaban prothrombin at 300,000 times the rate that Factor X Active Site without Apixaban Factor Xa does alone. [1] • Factor X is a serine protease that uses the • Trp215, Phe174, and Tyr99 on the left side of catalytic triad formed from Asp102, His57, and the active site surround the phenyllactam ring Ser195 to cleave the clotting protein thrombin of apixaban. by hydrolysis of a peptide bond. VII. Using Zebrafish to Find an Apixaban Antidote III. Medical Conditions Treated with Anticoagulants • The backbone N-H of Gly216 shown in the • Apixaban does not interact with the catalytic Lack of an antidote for apixaban can cause serious problems if a patient needs to undergo lower portion of the active site interacts with triad; however, it does block the substrate immediate surgery or has been overdosed. Our mentors are using zebrafish to screen 10,000 Deep Vein Thrombosis Pulmonary Embolism Atrial Fibrillation and Stroke the carbonyl oxygen of scaffold carboxamide. prothrombin from binding correctly. 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 • The catalytic triad of Factor X cleaves Glu146 interacts with the N-H of apixaban’s carboxamide group. compounds can be added to their water. If the antidote is not effective, hemorrhaging is visible in prothrombin in two places (after Arg271 and the embryo under the microscope. (See figure below.) In addition, zebrafish have genes for all of the after Arg32) to produce activated thrombin, Glu146 is show at the bottom right of the image above. coagulation proteins found in mammals (Hanumanthaiah et. al. (2002). A Blast alignment comparing which is a serine protease that cleaves the amino acid sequence of Human Factor X to zebrafish Factor X showed 47% identity (the same fibrinogen. amino acid at the same positon) and 63% positivity (a similar amino acid at the same position). [5] Factor X, Zebrafish vs Human Protein Hemorrhage in Embryo V. Structure of Apixaban Sequence Alignment [3] [4] The p-methoxyphenyl Deep vein thrombosis (DVT) is A pulmonary embolism (PE) is group does not interact the formation of a blood clot in caused by a blood clot that [13] The N-H of the with any specific active site the deep veins of the leg or travels through the heart to carboxamide group amino acid residues. Hemorrhage after warfarin pelvis. Symptoms can include leg the lungs from the legs or, in interacts with the treatment as shown in this pain and swelling; however, there rare cases, other parts of the [6] peptide bond embryo would be evidence may be no symptoms. DVT body. The clot damages the Atrial fibrillation (AFib) occurs carbonyl of Glu146 of ineffective antidote. requires immediate medical lung by blocking blood flow. when the two upper chambers of (Magnet) Apixaban antidotes will be attention as clots can travel to The dark area in the diagram Carbonyl of scaffold the heart, the atria, beat tested in the same way. the lungs and cause a life- above indicates tissue damage. irregularly (fibrillate) slowing carboxamide interacts with Jmol Image of apixaban. [12] threatening pulmonary Pulmonary embolism can backbone N-H of Gly216. PDB 2P16 blood flow. Slower flow causes Alignment of Human (AAA5242.1) and embolism. The NOAC apixaban result in death. Anticoagulants clots to form. If a clot enters a (Magnet) zebrafish (NP_968870) Factor X (Eliquis) is FDA approved to treat are used to prevent and treat blood vessel leading to the brain, The human amino acid sequence is and prevent deep vein pulmonary embolisms. a stroke will occur. Patients with above the zebrafish sequence. thrombosis, which it does by Apixaban is FDA approved to AFib are at increased risk of Asterisks identify the amino acids that inhibiting Factor X. treat PE. stroke and take anticoagulants to Pyrazole ring has a are identical and in the same position prevent the formation of clots. carboxamide group The phenyllactam group is in the human and zebrafish protein. Apixaban is FDA approved to on carbon three. positioned in a pocket Colons indicate similar amino acids. Zebrafish Embryonic prevent stroke in AFib patients. bound by Trp215, Phe174, 3D physical model of Development and Tyr99 apixaban. PDB 2P16

PDB ID: 2P16 [7] Donald J. Pinto, Michael J. Orwat, Stephanie Koch, Karen A. Rossi, Richard S. Alexander, Angela 5. http://www.cdc.gov/dhdsp/data_statistics/fact_sheets/fs_atrial_fibrillation.htm VIII. Summary Smallwood, Pancras C. Wong, Alan R. Rendina, Joseph M. Luettgen, Robert M. Knabb, Kan 6. National Heart, Blood and Lung Institute, National Institutes of Health He, Baomin Xin, Ruth R. Wexler, and Patrick Y. S. Lam. (2007). Discovery of 1-(4- 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 Methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo[3,4- Factor X coagulation protein is the target of new oral anticoagulants like apixaban (Eliquis). Apixaban 9 http://files.www.clotconnect.org/Comparison_of_oral_anticoagulants_Final.pdf c]pyridine-3-carboxamide (Apixaban, BMS-562247), a Highly Potent, Selective, Efficacious, and 10. http://www.mmm-online.com/features/eliquis-aims-to-amp-up-its-sales-push/article/348082 The SMART Team Program is supported by the National Center for Advancing Translational Sciences, National inhibits. activated Factor X in the coagulation cascade to prevent clot formation and has been Orally Bioavailable Inhibitor of Blood Coagulation Factor Xa. J.Med.Chem. 50: 5339-5356 11. http://america.pink/images/4/7/3/5/3/6/3/en/1-warfarin.jpg 1. James C. Whisstock. (2013). Assembling the Machinery of Coagulation. Blood 122: 2773- 2774. 12. Xiaoang Xing, Concordia University School of Pharmacy Institutes of Health, through Grant Number 8UL1TR000055. It’s contents are solely the responsibility of the authors 2. https://www.mhhe.com/biosci/esp/2002_general/Esp/folder_structure/tr/m1/s7 trm1s7_3.htm 13.S. Eisa-Beygi, RL Macdonald RL, XY Wen (2014) Regulatory pathways affecting vascular stabilization via VE-cadherin dynamics: insights approved by the FDA to treat and prevent deep vein thrombosis and pulmonary embolism as well as 3. Society of Interventional Radiiology from zebrafish (Danio rerio). Journal of Cerebral Blood Flow & Metabolism 34(9):1430-1433 and do not necessarily represent the official views of the NIH. 4. http://www.drugdangers.com/nuvaring/pulmonary-embolism.htm 14.Ravikumar Hanumanthaiah, Kenneth Day, Pudar Jagadeeswaran (2002) Comprehensive Analysis of Blood Coagulation Pathways to prevent strokes in atrial fibrillation patients. Apixaban’s major drawback is lack of an antidote. Our in Teleostei: Evolution of Coagulation Factor Genes and Identification of Zebrafish Factor VIIi. Blood Cells, Molecules, and Diseases 29(1):57-68 mentors are using zebrafish as a model organism to test thousands of possible antidote compounds.