Doctor of Philosophy in Biochemistry and Molecular Biology (PhD)
Using Lipid Nanoparticles to Modulate Platelet Function and Create Designer Platelets
Platelets are small, anucleate cells that circulate in the blood stream and mediate hemostasis, inflammation, and angiogenesis. Platelet transfusions are used to treat active bleeding as well as to prevent bleeding during thrombocytopenia or prior to surgery. Yet there are situations where transfusions do not adequately stop bleeding, such as during trauma, which is associated with platelet dysfunction. A method for genetically modifying platelets might enhance their efficacy and lead to new therapeutic uses for platelets. As platelets are anucleate, directly modifying platelets requires messenger RNA (mRNA). Attempts to transfect platelets with mRNA have not been successful, and it is unknown whether lipid-based materials could be used as mRNA transfection agents for platelets.Lipid nanoparticles (LNPs) have been used for nucleic acid delivery in vitro and in vivo. In this thesis, the ability of four different classes of LNPs to deliver mRNA to platelets was compared. These classes consisted of LNPs containing cationic lipids (cLNPs) that are highly effective in vitro, LNPs containing ionizable cationic lipids (icLNPs) developed for in vivo use, LNPs without a cationic lipid commonly used to encapsulate proteins or small molecules, and a commercially available agent previously used for short interfering RNA delivery to platelets. To identify ideal conditions for transfection with mRNA, uptake under various storage conditions and the ability of the LNPs to alter platelet activation was quantified. Finally, the ability of platelets to translate and release the mRNA was assessed. Two approaches were taken for mRNA delivery. In one approach, mRNA was synthesized inside of liposomes, indicating proteins, DNA, and small molecules can be delivered to platelets using LNPs. In the second approach, in vitro transcribed mRNA was directly delivered to platelets using icLNPs and cLNPs, and mRNA delivered to platelets using cLNPs was released in microparticles. These were the first examples of direct delivery of mRNA to platelets, and the first step towards creating genetically modified platelets. While protein synthesis in LNP-treated platelets was not detected, optimizing the LNP formulations used here may lead to a transfection agent for platelets that allows for de novo synthesis of exogenous proteins in the future.
No abstract available.
Coagulation factor XIII (FXIII) is a protransglutaminase enzyme that is activated at the end of the coagulation cascade. Activated FXIII (FXIIIa) stabilizes the blood clot from premature lysis by covalently crosslinking fibrin molecules to itself and to other anti-fibrinolytic proteins. Although the role of FXIIIa as an antifibrinolytic protein has been well characterized, the role of fibrinolysis in regulating FXIII and FXIIIa has not been characterized. FXIIIa lies in close proximity to fibrin during the hemostatic processes; therefore, the effect of plasmin on FXIIIa activity was examined. This thesis shows that plasmin preferentially cleaves the active enzyme FXIIIa, but not the zymogen FXIII. The primary cleavage site identified by mass spectrometry was between K468-Q469. Inactivation of FXIIIa occurred during clot lysis, but not during clot formation. These results indicate FXIIIa activity can be modulated by fibrinolytic enzymes, and suggest that changes in fibrinolytic activity may influence cross-linking of blood proteins.Thrombosis patients who are treated with thrombolytic therapy receive high doses of fibrinolytic enzymes. Since FXIIIa is inactivated by plasmin, the stability of FXIII and FXIIIa during thrombolytic therapy was examined using purified proteins and blood collected from nine DVT patients undergoing CDT. During CDT, FXIII levels were decreased by more than 40% in 5 of 9 patients and FXIIIa levels were decreased by more than 85% in 2 patients when it was activated. FXIII and FXIIIa can decrease during CDT in some patients, warranting further research into the role of FXIIIa in bleeding from thrombolysis.Amyloid beta (Aβ) peptide inhibits fibrinolysis and can form complexes with FXIIIa. Although Aβ can be crosslinked by tissue transglutaminase, the ability of FXIIIa to crosslink Aβ has not been demonstrated. The thesis shows that FXIIIa covalently crosslinked Aβ40 into dimers and oligomers, as well as to fibrin, and to blood clots under flow in vitro. Aβ40 also increased the stiffness of platelet-rich plasma clots in the presence of FXIIIa. These results suggest that crosslinking of Aβ40 by FXIIIa may contribute to the formation of vascular deposits in cerebral amyloid angiopathy.
Uncontrollable hemorrhage remains a leading cause of mortality in many situations, including during surgery and following trauma. Many hemostatic interventions have been developed, but mortality and morbidity remain high because they are ineffective or difficult to use in cases of severe bleeding. Blood flow can rapidly wash away topically applied hemostatic agents and prevents them from reaching leaking vessels and forming robust clots. We hypothesized that increasing the transport of hemostatic agents into wounds could improve their ability to manage bleeding. To test this hypothesis, I developed self-propelling particles that can transport through flowing blood and into wounds. These particles, consisting of calcium carbonate and an organic acid, delivered two hemostatic agents, thrombin and tranexamic acid (TXA), and improved their ability to stop bleeding in mice (Chapter 3). Next, I showed that self-propelling particles loaded with thrombin applied with gauze (PTG) significantly improved survival in a swine model of massive traumatic bleeding without compression, compared to those agents on gauze without propulsion (Chapter 4). This demonstrates that increasing transport of hemostatic agents could increase their utility for managing clinically relevant hemorrhage, such as from battlefield trauma. In two sheep models of surgical hemorrhage, PTG’s ability to stop bleeding was compared to two clinical standard interventions (Chapter 5). In a model of endoscopic surgical bleeding, PTG significantly reduced bleeding time compared to control gauze. In a model of massive open surgical bleeding, PTG achieved hemostasis in more cases than a standard thrombin-containing hemostatic agent. These demonstrate that increased transport of hemostatic agents could enable improved management of surgical bleeding. Finally, formulating TXA with self-propelling particles increased its ability to inhibit fibrinolysis in vitro and reduce bleeding in mice in vivo, demonstrating that these particles could transport a variety of hemostatic agents and increase their efficacy too (Chapter 6). The findings of this thesis suggest that improving the transport of hemostatic agents into wounds, such as by using self-propelling particles, is a promising approach for increasing the efficacy of those agents, and may present an opportunity to reduce mortality, morbidity, and the clinical burden associated with bleeding.
Platelets are small, anucleate blood cells that are important mediators of many physiological and pathological processes. These include hemostasis, thrombosis, wound healing, inflammation, immunity, and malignancy. There are currently several uses for platelet therapy in the clinic, such as to increase platelet counts for the prevention of spontaneous bleeding, and to stop uncontrolled bleeding during trauma and surgery. Although platelet transfusions are an efficacious component in preventing and stopping bleeding in most cases, they are still insufficient to stop the most severe cases of surgical and traumatic bleeding. Traumatic bleeding is further complicated by trauma-induced coagulopathy, which often presents with platelet dysfunction and is not corrected by transfusions of normal platelets. Strategies to enhance the endogenous function of platelets to increase the efficacy of platelet transfusions has not been rigorously explored, especially during active bleeding in trauma-induced coagulopathy.When activated by specific stimuli, platelets locally secrete a variety of biologically active molecules in order to contribute to many physiological and pathophysiological processes. For example, platelets can recognize areas of vascular damage and respond by locally adhering, aggregating, and activating to initiate primary hemostasis. Platelets also release procoagulant molecules and mediate the formation of active coagulation factor complexes to ultimately form an insoluble fibrin clot and seal the wound. Taken together, developing strategies to modify the endogenous function of platelets may be a first step towards a platform system that could target many diseases. Moreover, strategies to load platelets with biomolecules could allow for the local delivery of therapeutics to disease sites using endogenous platelet machinery. This provides significant motivation to test our overarching hypothesis, that the endogenous function of platelets can be modified ex vivo through the delivery of liposome-encapsulated enzymes.The objectives of this thesis were to: i) develop a platform approach to deliver biomolecules to platelets, ii) engineer anucleate platelets to transcribe RNA, and iii) increase the coagulability of transfusable platelets. The results shown here demonstrate proof-of-concept that endogenous platelet function can be extended through the delivery of lipid-encapsulated enzymes, and provides new approaches to potentially enhancing current platelet transfusions.