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Title:Polyphosphate and tissue factor/factor VIIA as initiators of coagulation
Author(s):Gajsiewicz, Joshua M
Director of Research:Morrissey, James H
Doctoral Committee Chair(s):Morrissey, James H
Doctoral Committee Member(s):Rienstra, Chad; Tajkhorshid, Emad; Fratti, Rutilio
Department / Program:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Blood coagulation
Protein-membrane interactions
Tissue factor (TF)
Abstract:Protein-membrane interactions are a critical component of the coagulation cascade. For many coagulation factors, these interactions are mediated via γ-carboxyglutamate rich regions, or GLA domains, in a Ca2+-dependent manner. Nearly all studies of GLA domain-containing coagulation factors, and coagulation in general, have utilized supraphyisologic Ca2+ concentrations in order to fully occupy the 7 GLA domain metal binding sites (and thus maximize activity). Recent work has indicated that under physiologic conditions, 2-3 of the GLA domain metal binding sites are actually occupied by Mg2+; furthermore, the concentration of Ca2+ required to maximize coagulation factor activity is reduced to near-physiologic levels in the presence of physiologic concentrations of Mg2+. Together, these results suggest an important role for Mg2+ in coagulation. To investigate the role of Mg2+ further, I first demonstrated that tissue factor is required for the Mg2+-dependent rate enhancements and that tissue factor itself does not bind Mg2+. To determine which residues of TF mediate the effect of Mg2+, I performed alanine scanning mutagenesis and substrate activation experiments, identifying several key residues mediating these effects. Importantly, these residues reside in or near the putative substrate binding exosite, suggesting that Mg2+ occupation of GLA domain metal binding sites may mediate both protein-membrane and protein-protein interactions. Furthermore, in collaboration with the lab of Dr. Ryan Bailey in the department of analytical chemistry at UIUC, we have probed the protein-membrane interactions of coagulation factors in a high-throughput manner utilizing photonic microring resonators. Additionally, we have investigated the binding of factor X to the tissue factor/factor VIIa complex assembled on Nanodiscs, leading to a deeper understanding of the effects of membrane composition and complex formation dynamics. The roles of anionic polymers such as inorganic polyphosphate and nucleic acids in both coagulation and inflammation have recently come to light. To better understand and delineate their effects, I performed a head-to-head comparison of the abilities of polyphosphate, DNA, and RNA to activate the contact pathway of coagulation, as well as to abrogate the anticoagulant activity of tissue factor pathway inhibitor (TFPI). Polyphosphate was a superior contact pathway activator on both a weight and molar basis, and was significantly more effective in abrogating TFPI activity than DNA or RNA purified from biologic samples. Both polyphosphate and nucleic acids are linear, anionic polymers; thus, it is possible that utilizing nucleic acid purification techniques on samples containing both nucleic acids and polyphosphate may result in co-purification of these polymers. To assess the purity of DNA or RNA samples purified from biological sources, I examined the co-purification characteristics of polyphosphate with DNA or RNA using numerous commercially-available nucleic acid purification techniques. Polyphosphate readily co-purified with both DNA and RNA, though to a significantly greater extent with the former. In addition, I demonstrated that purified cell-derived DNA contained contaminant polyP using PAGE and staining with DAPI. Because polyphosphate can co-purify with nucleic acids, investigations into the effects of DNA and RNA on coagulation might be indirectly probing the effects of polyphosphate contamination within the purified nucleic acid sample. To investigate, I treated purified cellular DNA with nucleases and polyphosphatases then assessed their ability to activate the contact pathway. Interestingly, treatment with either enzyme abrogated the procoagulant effect of purified DNA. Though physiologic anionic polymers have recently been identified as activators of the contact pathway, their precise mode of action has not been elucidated. The contact pathway consists of 3 proteolytically active enzymes, each of which have been reported to bind –and active –in the presence of anionic polymers. Furthermore, an important cofactor, high molecular weight kininogen, also binds to anionic surfaces and has been demonstrated as important for mediating the activities of contact pathway proteins. Nonetheless, it is often overlooked when studying the contact pathway in vitro. To investigate the reaction-by-reaction effects of physiologic polyanions, I performed a series of activity assays with purified proteins of the contact pathway. These assays were run in the presence of polyanions, and in some experiments HK. The results demonstrate the disparate effects of polyanions and HK, and highlight the importance of understanding contact pathway activation on a per-reaction level in addition to the pathway as a whole.
Issue Date:2016-10-11
Rights Information:Copyright 2016 Joshua Gajsiewicz
Date Available in IDEALS:2017-03-01
Date Deposited:2016-12

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