Author ORCID Identifier
Date Available
11-20-2023
Year of Publication
2023
Degree Name
Doctor of Philosophy (PhD)
Document Type
Doctoral Dissertation
College
Medicine
Department/School/Program
Molecular and Cellular Biochemistry
First Advisor
Dr. Jeremy Paul Wood
Second Advisor
Dr. Sidney Waldo Whiteheart
Abstract
Protein S (PS) is a key regulator, which links inflammation and coagulation and performs multiple proposed functions in both processes. PS exists in the blood as a free soluble form (~40%), bound to complement component 4b-binding protein/ C4BP (~60%), and packaged in platelet α-granules (~2.5%). Subendothelial tissue factor (TF), upon exposure to blood, initiates coagulation, a proteolytic cascade which results in the activation of thrombin, the enzyme responsible for formation of a fibrin clot. PS is a critical anticoagulant that inhibits multiple steps of this process. Only the free fraction of PS has full anticoagulant properties, as C4BP blocks this activity. PS also negatively regulates complement system activation via its association with C4BP, and is a ligand for the anti-inflammatory TAM (Tyro3, Axl, and Mer) tyrosine kinase receptors expressed by monocytes, macrophages and other cell types. Congenital deficiency of PS is rare in the general population and is associated with increased thrombotic risk, whereas acquired PS deficiency commonly occurs secondary to several viral infections, including HIV-1 and SARS-CoV-2, both of which are associated with an increased risk of life-threatening thrombotic events. The mechanisms of this deficiency and its association with thrombosis are unclear. HIV-1-infected individuals have an ~10-fold increased risk for thromboembolic diseases, and antiretroviral therapy does not reduce this risk. While HIV-1 infection is associated with various hematological changes, acquired PS deficiency is the most common coagulation abnormality, occurring in up to 76% of infected individuals. PS deficiency correlates with disease progression, and both total and free plasma PS are decreased. However, despite this high prevalence, its pathologic consequences were unclear because PS concentration does not correlate with plasma thrombin generation ex vivo. We developed a PS-sensitive plasma thrombin generation assay, utilized it to measure thrombotic potential in HIV-1 patient plasmas, and showed that plasma PS negatively correlates with and contributes to the thrombotic risk in this population. SARS-CoV-2 infection (COVID-19) is associated with a much higher thrombotic risk than HIV-1, occurring in up to 30% of hospitalized patients. We identified a specific deficiency of free PS in COVID-19 patients, which was not explained by known PS-binding proteins, such as C4BP. We next determined that this free PS deficiency is caused by a shear-dependent interaction with von Willebrand Factor (VWF), a previously unrecognized PS-binding protein. Using mass spectrometric and immunoblotting analyses we showed that PS binding to VWF is increased > 10,000,000-fold by shear-induced unfolding of VWF, and that sheared VWF dose-dependently inhibits free PS, but not total PS, antigen measurements and interferes with PS anticoagulant activity. Thus, we identified a novel mechanism of acquired PS deficiency, which occurs in COVID-19 patients, reducing the available anticoagulant pool and shifting the hemostatic balance. This mechanism also likely contributes to PS deficiency in other inflammatory conditions where vascular shear flow is elevated, including viral infections and autoimmune diseases. The goal of this thesis is to identify the molecular and cellular mechanisms of anticoagulant PS deficiency and its contribution to virus-associated thrombotic risk, using HIV-1 and SARS-CoV-2 as models. In addition, we also evaluated the contribution of procoagulant Tissue Factor (TF), which is the initiator of coagulation often elevated during inflammation, to thrombotic risk, not only in viral infections but also in traumatic brain injury. This study, which utilizes novel assays including PS-sensitive thrombin generation, provides new insights into the molecular mechanisms of thrombosis in viral infections in general and in HIV-1 and SARS-CoV-2 specifically, and informs on the potential of PS as a biomarker, and on potential therapeutic approaches. We provided the first description of VWF-dependent PS deficiency and are now working to fully understand its physiological and pathological implications. Based on our results, it is likely that this interaction contributes to the thrombotic events that are among the leading causes of death in this pandemic, and may contribute to thrombosis in many other inflammatory conditions that share similar features to COVID-19. Finally, the implications extend beyond pathological conditions. VWF is exposed to shear and unfolds at injury sites as part of the normal hemostatic response, so the VWF-PS interaction may represent a regulatory step in normal clot formation, which we are only now beginning to recognize.
Digital Object Identifier (DOI)
https://doi.org/10.13023/etd.2023.269
Funding Information
This study was supported by University of Kentucky Center for Clinical and Translational Science in 2020, National Heart, Lung, and Blood Institute (K99 HL129193 and R00 HL129193) in 2015-2021, Pfizer grant in 2021-2023, and University of Kentucky Alliance Research Initiative in 2020-2023.
Recommended Citation
Sim, Martha Mega Silvia, "PROTEIN S IN COAGULATION AND INFLAMMATION" (2023). Theses and Dissertations--Molecular and Cellular Biochemistry. 66.
https://uknowledge.uky.edu/biochem_etds/66
