Context: Inferior vena cava (IVC) anomalies have a 0.5% incidence rate and could be associated with other congenital abnormalities. In later stage of the disease, trophic ulcers with or without deep vein thrombosis (DVT) is consistent finding.Case Report: A 29-year-old male patient presented with recurrent lower extremity ulcers. Further workup revealed an absent infrahepatic inferior vena cava, prominently dilated azygos and hemiazygos veins with enlarged retroperitoneal collaterals without DVT.Conclusion: IVC anomaly should be suspected in a young patient presenting with unexplained venous thrombosis and recurrent ulcers of a lower extremity. IVC anomaly would inherently lead to blood flow stasis and endothelial injury. Thus per Virchow's triad, other risk factors for hypercoagulability such as physical inactivity, smoking tobacco, oral contraceptive pills should be avoided and when hereditary thrombophilias or other irreversible risk factors are present, lifelong anticoagulation should be considered.

Introduction: Cancer associated thrombosis is a well-recognized phenomenon that results in considerable patient morbidity and mortality. Malignancy conveys an increased risk for thrombosis and chemotherapy further elevates this risk. The pathophysiological mechanisms underlying this process remain poorly defined. Materials and Methods: A human acute monocytic leukemia cell line (THP-1) was treated with commonly used anthracycline chemotherapeutics at concentrations similar to those found in the plasma of cancer patients. Cells were analyzed for tissue factor (TF) mRNA, protein, and activity. Microparticle (MP) TF activity was also measured. Phosphatidylserine (PS) exposure on cells and MPs was analyzed by flow cytometry. PS levels on MPs was also evaluated in an annexin V capture assay. Results: Anthracycline treatment of THP-1 cells resulted in a concentration-dependent increase in cellular TF activity without a change in TF protein, which was associated with increased PS exposure on the cell surface and apoptosis. The increase in TF activity was abolished by annexin V or lactadherin indicating that PS exposure was required. Anthracycline treatment of THP-1 cells also increased the number of TF-positive MPs. Conclusion: Treatment of THP-1 cells with anthracyclines induces apoptosis and increases cellular TF activity. The increased activity required an increase in exposure of PS. Additionally, anthracyclines increase the release of TF-positive MPs from THP-1 cells. We propose that the increase in cellular TF activity in circulating leukemic cells, combined with increased numbers of TF-positive MPs, may contribute to thrombosis in cancer patients receiving chemotherapy.

ADAMTS13 is a metalloprotease that regulates the length, and thus, the platelet-capturing capacity of von Willebrand factor. The regulation of ADAMTS13 activity remains poorly understood. Numerous circulating proteases cleave ADAMTS13 in vitro, impairing its activity, but the physiological significance of this mechanism remains unknown. Two commonly cleaved regions within ADAMTS13 were identified and mutants were developed: two with one of each region mutated (T4L and T8L mutants), one with both regions mutated (T4L/T8L or “double” mutant), and one with an additional elastase site mutated (T4L/T8L + I380G). This work characterizes the mutants’ resistance to proteolysis and compares the activity of the double mutant to wild-type ADAMTS13 (WT). Each mutant and WT was incubated with purified coagulation and neutrophil proteases, activated neutrophils, or added to plasma before initiating coagulation with or without tissue plasminogen activator. Cleavage patterns were visualized with western blot. FRETS-VWF73 and microfluidic flow assays were used to compare WT and mutant activity. Coagulation proteases cleave both predicted sites within WT, and the double mutant exhibits near complete resistance to cleavage over 3 hours. Resistance to degradation by neutrophil proteases is prolonged in the double mutant, but additional cleavage sites are present. Elastase cleavage is prevented in the T4L/T8L + I380G mutant. In plasma, WT is degraded upon initiating coagulation and subsequent fibrinolysis, which is prevented in the double mutant. WT is also degraded in the presence of activated neutrophils, and the double and T4L/T8L + I380G mutants exhibit improved but incomplete resistance. Finally, the mutants exhibit similar activity to WT using FRETS-VWF73 and the microfluidic assay. This work validates the location of two protease-sensitive regions within ADAMTS13 and confirms the resistance of the double mutant to coagulation proteases in vitro. Future work will complete the activity analysis, and compare the mutants’ therapeutic efficacy to WT in vivo. / Thesis / Master of Science (MSc) / Current drugs used to dissolve blood clots can cause major bleeding. Therefore, safer treatments need to be developed. An important step in the clotting pathway is platelet accumulation in the injured vessel. Platelets stick to string-like protein, von Willebrand Factor (VWF), and ADAMTS13 is a protein that regulates this by cutting VWF strings. ADAMTS13 shows promise as a treatment for clots without causing bleeding, but it is unclear how its activity is controlled. ADAMTS13 can be degraded by other proteins, however the importance of this process in the body is unknown. This work characterizes a degradation-resistant ADAMTS13 mutant, which may be used to study whether ADAMTS13 degradation reduces its therapeutic effectiveness. The mutant has normal VWF-cutting activity, is resistant to degradation by clotting proteins, and is partially resistant to proteins released by neutrophils, an important immune cell in clotting. Future studies will investigate its effectiveness at treating clots in animals.

Recent studies suggest that factor (F) XII, which is dispensable for hemostasis, is important for thrombus stabilization and growth. Therefore, FXIIa inhibition may attenuate thrombosis without disrupting hemostasis. FXII activation is stimulated by polyanions such as polyphosphates released from activated platelets, and nucleic acids released by cells. Previously, we showed that histidine-rich glycoprotein (HRG) binds FXIIa with high affinity, inhibits FXII autoactivation and FXIIa-mediated activation of FXI, and attenuates ferric chloride-induced arterial thrombosis in mice. Thus, HRG has the capacity to downregulate the contact pathway in vitro and in vivo. This thesis aimed to identify the complementary binding domains of HRG and FXIIa, and to further explore the anticoagulants effects of HRG on FXIIa-mediated contact activation. We hypothesized that FXIIa binds to the zinc-binding histidine-rich region (HRR) of HRG and that HRG binds to the non-catalytic heavy chain of FXIIa to exert its anticoagulant activities on FXIIa-mediated contact activation. We have localized the complementary binding sites of HRG and FXIIa to be within the HRR domain of HRG and NH2-FNII-EGF1 (NFE) domains of FXIIa. Moreover, we show that the HRR binds to short chain polyphosphate with high affinity, suggesting a dynamic complex between HRG, FXIIa, and polyphosphate (polyP) on activated platelets. We provide evidence for two potential mechanisms through which HRG modulates the contact system. These include by 1) inhibiting FXIIa activity and 2) attenuating the procoagulant effect of polyanions, such as polyP on FXIIa-mediated reactions. Indeed, we show that the interaction of HRG with FXIIa and polyphosphate is predominantly mediated by the HRR domain and that HRR analogs have the capacity to recapitulate the anticoagulant effects of HRG in purified and plasma systems. Therefore, by modulating FXIIa-mediated contact pathway reactions, like HRG, HRR analogs may attenuate thrombosis without disrupting hemostasis. / Thesis / Doctor of Philosophy (Medical Science)

Abstract available through the Journal of Investigative Medicine.

This research will investigate techniques to create a sensor that is responsive to methane at 23°C. The approach will use the integration of a very thin film, which changes its resistive properties when methane gas is applied, deposited atop the surface of a piezoelectric substrate. An aluminum thin film interdigital transducer will launch a surface acoustic wave (SAW) that travels under the sensor’s gas-sensitive resistive thin film. The SAW/resistive film interaction changes the SAW amplitude, phase and delay. For this work, three films, tin dioxide (SnO2), zinc oxide (ZnO) and palladium (Pd) [1, 2] will be studied. Gas detection will be shown when combining ZnO and Pd, and, observable change in SAW propagation loss is measured when methane gas is present at the film.

Venous thromboembolism (VTE) is a chronic illness that affects nearly 10 million people every year worldwide. Anticoagulant therapy with direct oral anticoagulants is the mainstay of treatment for patients with VTE, and should be continued for at least 3-6 months. Thereafter, a decision should be made to discontinue anticoagulation or continue it indefinitely. This decision is most challenging for patients with a first unprovoked VTE because of uncertainty in estimates for the long-term benefits (e.g., reduction in recurrent VTE) and harms (e.g., increase in major bleeding) of extended anticoagulation, and the trade-offs between them. The overarching aim of this doctoral thesis was to address these key evidence gaps that are pertinent to making decisions regarding the duration of anticoagulation for patients with a first unprovoked VTE. The first three studies of this thesis synthesized contemporary and reliable estimates for the long-term risks and consequences of recurrent VTE and major bleeding, with and without extended anticoagulation (parameters that can influence the clinical and cost-effectiveness of discontinuing versus continuing anticoagulation indefinitely). Broadly, these systematic reviews and meta-analyses found that: 1) the long-term risks and consequences of major bleeding during extended anticoagulation are considerable, particularly with vitamin K antagonists as well as in older patients, patients using antiplatelet therapy, and in patients with kidney disease, a history of bleeding, or anemia; and 2) the long-term risks of recurrent VTE during extended anticoagulation and major bleeding after discontinuing anticoagulation are reassuringly low but not negligible. The fourth study incorporated the synthesized evidence to compare the lifetime clinical benefits, harms, and costs of discontinuing versus continuing anticoagulation indefinitely. This decision analytic modelling study showed that indefinite anticoagulation is unlikely to either result in a net clinical benefit or be cost-effective in all (i.e., unselected) patients with a first unprovoked VTE. Findings from this thesis can serve to impact clinical practice and health policy by informing patient prognosis to guide shared decision-making regarding the duration of treatment for unprovoked VTE, and informing future research to ultimately identify which patients should receive anticoagulation indefinitely in order to maximize health benefits for the available healthcare resources.

The endothelial protein C receptor (EPCR) binds to protein C (PC) and increases the rate of activated protein C (APC) generation by the thrombin-thrombomodulin (TM) complex. APC exerts anticoagulant, anti-inflammatory, and cytoprotective effects, which are EPCR-dependent. The thrombin-TM complex is also a potent activator of thrombin activable fibrinolysis inhibitor (TAFI), leading to impaired clot lysis. Mutations and polymorphisms identified in the EPCR gene, which can affect the efficiency of PC activation, have been associated with an increased risk of thrombosis. In this thesis we investigate the impact of impaired PC binding to EPCR on coagulation, inflammation, and fibrinolysis using novel in vitro and in vivo models. Using a murine model that harbours a variant of EPCR that does not bind PC (R84A), we demonstrate that upon thrombotic challenge, there is an increase in thrombin generation and fibrin deposition in the lungs. Upon inflammatory challenge, impaired PC/EPCR interactions also result in increased thrombin generation and increased neutrophil infiltration into the lungs. Using cells that express TM and a human variant of EPCR that does not bind PC (R96C), we demonstrate that clot lysis is delayed in normal plasma independent of TAFI activation, suggesting PC and TAFI do not compete for activation by the thrombin-TM complex. In contrast, delayed clot lysis in plasma deficient of PC is a result of greater TAFI activation by the thrombin-TM complex. Taken together, impairment of the PC pathway contributes to thrombosis through pro-coagulant, pro-inflammatory and anti-fibrinolytic mechanisms. Interestingly, mice with EPCR variant R84A, develop bone marrow failure and splenomegaly, revealing a novel role for EPCR in the bone marrow. Taken together, PC/EPCR interactions regulate the coagulation, inflammation, and fibrinolytic pathways, which may have a significant impact on maintaining hematopoietic homeostasis. / Thesis / Doctor of Philosophy (PhD) / Under normal conditions, blood is maintained in a fluid state. Upon injury or infection, the blood begins to form a clot to prevent bleeding. Once bleeding has stopped the clot is dissolved and blood regains its fluid state. The formation of a blood clot is a serious and potentially life threatening disease. A blood clot formed inside a blood vessel can block the flow of blood through the circulation, leading to organ damage. Approximately 50% of blood clots are caused by known genetic or environmental factors, leaving 50% of blood clots caused by unknown factors. In this thesis we investigate the unknown factors that contribute to blood clotting. In patients who have experienced blood clots with no known cause, we have identified genetic mutations in a blood vessel wall protein, known as the endothelial protein C receptor (EPCR) that renders it non-functional. We demonstrate both in vitro and in vivo that non-functional EPCR not only leads to the formation of a blood clot but also delays the removal of the blood clot. Our in vivo studies have also revealed a previously unknown role for EPCR in the bone marrow, likely through its effects on blood coagulation. Taken together, loss of EPCR function contributes to the development of clot formation and likely impacts other organ systems.

Whether a patient presents with deep vein thrombosis (DVT) or pulmonary embolism (PE) varies based on clinical factors. Patients with factor V Leiden (FVL) typically present with DVT while cancer patients present with PE. The biological mechanisms that determine DVT stability in the progression of DVT to PE are not known. Thus, little is known about the mechanism of thrombus stability, the factors involved, or the effect of anticoagulants on embolization and PE burden. In order to answer these questions, we first need to (i) develop a mouse model to evaluate DVT stability and its relationship with PE burden when treated with anticoagulants, (ii) determine if anticoagulants, by inhibiting thrombin, require FXIII to decrease thrombus stability, (iii) determine the effects of attenuating fibrinolysis, using epsilon aminocaproic acid (ε-ACA or EACA), supplemental FXIII and α2-AP, on clot stability and (iv) utilize our model to explain the FVL paradox. For our thrombus stability model, the femoral vein of C57BL/6, FXIII deficient (FXIII-/-), FVL heterozygous, or FVL homozygous female mice was subjected to ferric chloride (FeCl3) injury to initiate a non-occlusive thrombus. Treatment with saline, dalteparin, dabigatran, EACA or FXIII was administered 12 minutes after thrombus formation. Intravital videomicroscopy recorded the thrombus sizes and embolic events leaving the thrombus for 2 hours. Lungs were harvested, sectioned and stained for the presence of PE. Total and large embolic events were highest after dabigatran treatment compared to saline or dalteparin in wild-type (WT) mice. Variations in amounts of embolic events were not attributed to variations in thrombus size since thrombus size was similar between the groups. The number of emboli per lung slice was higher in dabigatran-treated mice. Large embolic events correlated positively with the number of emboli per lung slice independent of treatment. Dabigatran treatment in FXIII-/- mice did not alter embolization patterns suggesting that FXIII is required for dabigatran to decrease thrombus stability. EACA increases thrombus size significantly and therefore would not be a feasible alternative to IVC filters, as it will increase DVT size. FXIII marginally increased thrombus size. Treatment with FXIII decreases total and large embolic events in saline-, dalteparin- or dabigatran-treated mice, similar to EACA-treated mice. The number of emboli per lung slice was reduced after treatment with FXIII and EACA compared to non-treated mice. PE burden was not significantly different between FXIII anticoagulated mice or EACA-treated mice. The large embolic events correlate positively with PE burden. FVL heterozygous and homozygous mice had significantly reduced embolization and thrombus size grew significantly over time, this contrasted with WT mice, where thrombus size remained similar to the initial injury. PE burden was significantly reduced in the FVL mice compared to WT. Collectively, these data shows that we have successfully developed a mouse model of acute venous thrombus stability that can quantify emboli and PE burden. Consistent with clinical data, dabigatran, a DTI, was shown to acutely decrease thrombus stability and increase PE burden compared to LMWH or saline; an effect that was FXIII-dependent. Also, attenuating fibrinolysis with EACA, but not FXIII, increases thrombus size; but both increase DVT stability and decrease PE burden. Supplementing α2-AP did not alter thrombus stability. This suggests that administration of FXIII may be a better treatment option for DVT patients who are bleeding than EACA, since EACA may increase DVT size. Lastly, our model can explain the FVL paradox. Those with FVL have stable thrombus formation leading to an increased incidence of symptomatic DVT and a decreased risk of PE. / Thesis / Doctor of Philosophy (PhD)

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