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Systems Biology of Blood Coagulation and Platelet Activation University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations Spring 2011 Systems Biology of Blood Coagulation and Platelet Activation Manash S. Chatterjee [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Biochemical and Biomolecular Engineering Commons Recommended Citation Chatterjee, Manash S., "Systems Biology of Blood Coagulation and Platelet Activation" (2011). Publicly Accessible Penn Dissertations. 348. https://repository.upenn.edu/edissertations/348 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/348 For more information, please contact [email protected]. Systems Biology of Blood Coagulation and Platelet Activation Abstract Blood clotting is a highly conserved physiological response that prevents excessive blood loss following vessel injury. It involves a sequence of plasma reactions leading to the formation of thromin (the coagulation cascade) as well as tightly controlled intracellular reactions mediating platelet activation. These two events are inextricably coupled, with the active platelet surface serving as a cofactor for coagulation factor assembly and thrombin serving as a potent platelet agonist. Using the technologies of automated liquid handling, high throughput experimental systems were developed that allowed individual exploration of these two components of the thrombotic response under diverse initial conditions. Based on this high dimensional experimental exploration, a “bottom-up” mechanism based Ordinary Differential Equation (ODE) description of thrombin generation kinetics and a “top-down” data driven Neural Network model of platelet activation were developed. In the first study, “contact activation” (and not “blood-borne TF” alone) despite the best available inhibitor to prevent it, was found build up enough autocatalytic strength to trigger coagulation in the absence of exogenous tissue factor, particularly upon activated platelets. Further, the “Platelet-Plasma model” successfully predicted the stability of blood under multiple perturbations with active enzymes at various physiologically realizable conditions. In the second study, “Pairwise Agonist Scanning” (PAS), a strategy that trains a Neural Network model based on measurements of cellular responses to individual and all pairwise combinations of input signals is described. PAS was used to predict calcium signaling responses of human platelets in EDTA-treated plasma to six different agonists (ADP, Convulxin, U46619, SFLLRN, AYPGKF and PGE2). The model predicted responses to sequentially added agonists, to ternary combinations of agonists and to 45 different combinations of four to six agonists (R=0.88). Furthermore, PAS was used to distinguish between the phenotypic responses of platelets from healthy human donors. Taken together, these two studies lay the groundwork for integration of coagulation reaction kinetics and donor specific descriptions of platelet function with models of convection and diffusion to simulate thrombosis under flow. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Chemical and Biomolecular Engineering First Advisor Scott L. Diamond Keywords Systems Biology, High Throughput Experimentation, Patient Specific Therapy, Combinatorial Experimentation, Thrombin, Platelet Activation Subject Categories Biochemical and Biomolecular Engineering This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/348 SYSTEMS BIOLOGY OF BLOOD COAGULATION AND PLATELET ACTIVATION Manash Shankar Chatterjee A DISSERTATION in Chemical and Biomolecular Engineering Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2011 __________________________________ Scott L. Diamond, Professor, Chemical and Biomolecular Engineering Supervisor of Dissertation __________________________________ Raymond J. Gorte, Professor, Chemical and Biomolecular Engineering Graduate Group Chairman Dissertation Committee Lawrence F. Brass, Professor, Department of Medicine Ravi Radhakrishnan, Associate Professor, Chemical and Biomolecular Engineering Matthew J. Lazzara, Assistant Professor, Chemical and Biomolecular Engineering SYSTEMS BIOLOGY OF BLOOD COAGULATION AND PLATELET ACTIVATION COPYRIGHT 2011 Manash Shankar Chatterjee ACKNOWLEDGMENTS First, I would like to thank my thesis supervisor Scott Diamond for scientific guidance throughout the past 4 years, and also for the freedom to explore independently and creatively. Thanks also to Skip Brass for his encouragement and advice, to Ravi Radhakrishnan and Matt Lazzara for helpful suggestions, and to all other faculty members at the University of Pennsylvania who have been involved in my education. I have worked with several talented past and present members of the Diamond and Brass labs, and have had the privilege of learning from all of them. Thanks in particular to Bill Denney for getting me started in the lab, Jeremy Purvis for the platelet model, and to Matthew Flamm, Sean Maloney, Dan Jaeger, Tom Colace, Roman Voronov and Andrew Dolan for several useful conversations. I would also like to thank all of my friends in the Chemical Engineering department for their interest in my work, cricket and Sourav Ganguly’s heroic exploits. I would also like to thank our phlebotomists Huyian Jing, Robert Bucki and Marie Guerraty and the numerous people who have volunteered to donate blood for my experiments. Finally I would like to thank my parents and sister. This work would not be possible without their unending support. iii ABSTRACT SYSTEMS BIOLOGY OF BLOOD COAGULATION AND PLATELET ACTIVATION Manash Shankar Chatterjee Supervisor: Scott L. Diamond Blood clotting is a highly conserved physiological response that prevents excessive blood loss following vessel injury. It involves a sequence of plasma reactions leading to the formation of thromin (the coagulation cascade) as well as tightly controlled intracellular reactions mediating platelet activation. These two events are inextricably coupled, with the active platelet surface serving as a cofactor for coagulation factor assembly and thrombin serving as a potent platelet agonist. Using the technologies of automated liquid handling, high throughput experimental systems were developed that allowed individual exploration of these two components of the thrombotic response under diverse initial conditions. Based on this high dimensional experimental exploration, a “bottom-up” mechanism based Ordinary Differential Equation (ODE) description of thrombin generation kinetics and a “top-down” data driven Neural Network model of platelet activation were developed. In the first study, “contact activation” (and not “blood-borne TF” alone) despite the best available inhibitor to prevent it, was found build up enough autocatalytic strength to trigger coagulation in the absence of exogenous tissue factor, particularly upon activated platelets. Further, the “Platelet-Plasma model” successfully predicted the stability of blood under multiple perturbations with active enzymes at various physiologically realizable conditions. In the second study, “Pairwise iv Agonist Scanning” (PAS), a strategy that trains a Neural Network model based on measurements of cellular responses to individual and all pairwise combinations of input signals is described. PAS was used to predict calcium signaling responses of human platelets in EDTA-treated plasma to six different agonists (ADP, Convulxin, U46619, SFLLRN, AYPGKF and PGE2). The model predicted responses to sequentially added agonists, to ternary combinations of agonists and to 45 different combinations of four to six agonists (R=0.88). Furthermore, PAS was used to distinguish between the phenotypic responses of platelets from healthy human donors. Taken together, these two studies lay the groundwork for integration of coagulation reaction kinetics and donor specific descriptions of platelet function with models of convection and diffusion to simulate thrombosis under flow. v Table of Contents ACKNOWLEDGMENTS ..............................................................................................III ABSTRACT……………………………………………….............................................IV CHAPTER 1 INTRODUCTION................................................................................. 1 1.1 THROMBUS FORMATION....................................................................................... 1 1.2 PLATELET ACTIVATION ........................................................................................ 3 1.2.1 Initiation of platelet activation on collagen surfaces ................................. 3 1.2.2 Growth of the platelet aggregate via autocatalytic signaling .................... 4 1.2.3 Stabilization of the platelet aggregate ........................................................ 8 1.3 THE BLOOD COAGULATION CASCADE................................................................. 10 1.3.1 The extrinsic pathway ............................................................................... 11 1.3.2 The intrinsic pathway................................................................................ 13 1.3.3 The common pathway ............................................................................... 14 1.3.4 Coagulation inhibition.............................................................................. 14 1.3.5 Fibrin formation.......................................................................................
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