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A Microfluidic Approach for Evaluating Novel Antithrombotic Targets

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A Microfluidic Approach for Evaluating Novel Antithrombotic Targets University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2017 A Microfluidic Approach For Evaluating Novel Antithrombotic Targets Shu Zhu University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Chemical Engineering Commons Recommended Citation Zhu, Shu, "A Microfluidic Approach For Evaluating Novel Antithrombotic Targets" (2017). Publicly Accessible Penn Dissertations. 2670. https://repository.upenn.edu/edissertations/2670 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/2670 For more information, please contact [email protected]. A Microfluidic Approach For Evaluating Novel Antithrombotic Targets Abstract Microfluidic systems allow precise control of the anticoagulation/pharmacology protocols, defined reactive surfaces, hemodynamic flow and optical imaging outines,r and thus are ideal for studies of platelet function and coagulation response. This thesis describes the use of a microfluidic approach to investigate the role of the contact pathway factors XII and XI, platelet-derived polyphosphate, and thiol isomerases in thrombus growth and to evaluate their potential as safer antithrombotic drug targets. The use of low level of corn trypsin inhibitor allowed the study of the contact pathway on collagen/kaolin surfaces with minimally disturbed whole blood sample and we demonstrated the sensitivity of this assay to antithrombotic drugs. On collagen/tissue factor surfaces, we found the relative contributions of the extrinsic pathway, the contact pathway, and the thrombin feedback pathway vary with tissue factor surface concentration. Platelet-derived polyphosphate potentiated the thrombin feedback pathway at low tissue factor level but enhanced fibrin fiber structure regardless of tissue factor level. At locations with low tissue factor level, thrombosis may be druggable by contact pathway and polyphosphate inhibition, although thrombolytic susceptibility may benefit from polyphosphate antagonism regardless of tissue factor level. We developed a peptide-based platelet-targeting thiol reduction sensor to visualize thrombus- incorporated thiol reductase activity. Although distribution of thiol reductase activity was shown to be correlated with the level of platelet activation, protein disulfide isomerase inhibition showed a limited effect on platelet aggregation in microfluidic thrombosis assay. We also used the microfluidic system ot explore the injury patch size limit for triggering clotting. We observed a full clotting response of platelet deposition, thrombin generation and fibrin polymerization on one of the smallest biological units of a single collagen fiber presenting tissue factor and von Willebrand factor suggesting the lack of physiological injury patch size limit. Finally, we made the first estimation of thrombin flux from growing thrombus under flow using the microfluidic thrombosis assay in combination with enzyme-linked immunosorbent measurement of thrombin-antithrombin complex. We found thrombin is robustly generated within clots by the extrinsic pathway, followed by late-stage factor XIa contributions, with fibrin localizing thrombin via its antithrombin activity as a self-limiting hemostatic mechanism. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Chemical and Biomolecular Engineering First Advisor Scott L. Diamond Keywords Blood, Hemostasis, Microfluidics, Platelet, Thrombosis Subject Categories Chemical Engineering This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/2670 A MICROFLUIDIC APPROACH FOR EVALUATING NOVEL ANTITHROMBOTIC TARGETS Shu Zhu 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 2017 Supervisor of Dissertation _________________________ Scott L. Diamond Professor, Chemical and Biomolecular Engineering Graduate Group Chairperson __________________________ John C. Crocker, Professor, Chemical and Biomolecular Engineering Dissertation Committee Lawrence F. Brass, Professor, Department of Medicine Talid R. Sinno, Professor, Department of Chemical and Biomolecular Engineering Daeyeon Lee, Professor, Department of Chemical and Biomolecular Engineering A MICROFLUIDIC APPROACH FOR EVALUATING NOVEL ANTITHROMBOTIC TARGETS COPYRIGHT 2017 Shu Zhu ACKNOWLEDGMENT Firstly, I would like to express my deepest gratitude to my thesis advisor Dr. Scott Diamond for the guidance and continuous support for my PhD study. I feel so privileged to conduct research with a talented, enthusiastic and devoted scientist like Dr. Diamond. I would never be able to prepare this dissertation without the training provided by the Diamond lab. I would also like to extend my thanks to my other thesis committee members, Dr. Lawrence Brass, Dr. Talid Sinno and Dr. Daeyeon Lee for their challenging questions and insightful comments on my work and for the time and effort they spent serving on my committee. I owe equal appreciation to all past and current Diamond lab members, especially those who I have worked closely with, Dr. Thomas Colace, Dr. Ryan Muthard, Dr. John Welsh, Dr. Mei Yan Lee, Dr. Ruizhi Li, Dr. Jifu Tan, Bradley Herbig, Xinren Yu, and Huiyan Jing and to all the great scientists I have collaborated with, Dr. James Morrissey, Dr. Maurizio Tomaiuolo and Dr. Juan Jimenez. I want to thank all of you for offering me precious technical support and all the joyful and inspiring discussions. My special appreciation goes to my dearest parents for trusting my decision of studying and pursuing a doctoral degree abroad in the US. Thanks for the great internet, I can always feel the love from my family through daily conversations despite the 12-hour time difference between home and Philadelphia. Thank you dad for the encouragements whenever I am hesitant to move forward and thank you mom for all the comforting words to guide me through all the tough times. I want also to extend special thanks to my friends and other family members, especially Fay Wang, for always being curious about my research and being cheerful whenever I feel down. I feel lucky to always have this iii group of optimistic and caring people backing me up throughout my life away from home. Finally, I want to thank my husband and my best friend Yichen Lu for sharing this journey with me. I feel so fortunate to be in the same department and lab, which later led to both academic and life partners. Your intelligence, kindness, patience and devotion motivates me to be a better person. I feel truly blessed to be part of your life. Wherever the future leads us, I am excited to explore, to learn and to strive with you. iv ABSTRACT A MICROFLUIDIC APPROACH FOR EVALUATING NOVEL ANTITHROMBOTIC TARGETS Shu Zhu Scott L. Diamond Microfluidic systems allow precise control of the anticoagulation/pharmacology protocols, defined reactive surfaces, hemodynamic flow and optical imaging routines, and thus are ideal for studies of platelet function and coagulation response. This thesis describes the use of a microfluidic approach to investigate the role of the contact pathway factors XII and XI, platelet-derived polyphosphate, and thiol isomerases in thrombus growth and to evaluate their potential as safer antithrombotic drug targets. The use of low level of corn trypsin inhibitor allowed the study of the contact pathway on collagen/kaolin surfaces with minimally disturbed whole blood sample and we demonstrated the sensitivity of this assay to antithrombotic drugs. On collagen/tissue factor surfaces, we found the relative contributions of the extrinsic pathway, the contact pathway, and the thrombin feedback pathway vary with tissue factor surface concentration. Platelet-derived polyphosphate potentiated the thrombin feedback pathway at low tissue factor level but enhanced fibrin fiber structure regardless of tissue factor level. At locations with low tissue factor level, thrombosis may be druggable by contact pathway and polyphosphate inhibition, although thrombolytic susceptibility may benefit from polyphosphate antagonism regardless of tissue factor level. We developed a peptide-based platelet-targeting thiol reduction sensor to visualize thrombus-incorporated thiol reductase activity. Although distribution of thiol reductase activity was shown to be v correlated with the level of platelet activation, protein disulfide isomerase inhibition showed a limited effect on platelet aggregation in microfluidic thrombosis assay. We also used the microfluidic system to explore the injury patch size limit for triggering clotting. We observed a full clotting response of platelet deposition, thrombin generation and fibrin polymerization on one of the smallest biological units of a single collagen fiber presenting tissue factor and von Willebrand factor suggesting the lack of physiological injury patch size limit. Finally, we made the first estimation of thrombin flux from growing thrombus under flow using the microfluidic thrombosis assay in combination with enzyme-linked immunosorbent measurement of thrombin-antithrombin complex. We found thrombin is robustly generated within clots by the extrinsic pathway, followed by late-stage factor XIa contributions, with fibrin localizing thrombin via its antithrombin activity as a self-limiting hemostatic mechanism. vi TABLE OF CONTENTS ACKNOWLEDGMENT .........................................................................................................
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