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)

<p>The effect of direction thrombin inhibition on acute deep vein thrombosis (DVT) stability has not been defined and could contribute to pulmonary embolism (PE) risk. Direct thrombin inhibitors (DTIs) effectively inhibit free and clot-bound thrombin, which could potentiate thrombus instability through disruption of platelet, fibrin, and FXIIIa stabilizing mechanisms. This could manifest as increased thrombus embolization. A clinically relevant mouse model of DVT stability could further our understanding of venous thrombosis pathophysiology and define the effect of direct thrombin inhibition on PE. We hypothesized that acute DTI administration would decrease acute DVT stability and potentially increase PE risk. Platelets were labeled <em>in vivo</em>, femoral vein thrombosis was induced using FeCl₃, and lepirudin (8U/g) was administered <em>after</em> clot formation. Using intravital videomicroscopy (IVM), real time embolization was quantified as a measurement of thrombus stability. Thrombus stability increased in the control group and decreased in the lepirudin-treated group over two hours. The decrease in α₂-antiplasmin (α₂-AP) content within lepirudin-treated thrombi, compared to control thrombi, could possibly contribute to the observed decrease in thrombus stability. Continued growth and embolization established the dynamic nature of formed thrombi. In both groups, emboli were detected in the pulmonary artery circulation. Therefore, we successfully developed a mouse model of venous thrombus stability, which imitated the clinical progression of DVT to PE. DTI administration in the acute DVT setting could decrease thrombus stability, demonstrated through increased embolization and PE. This model could be useful in examining the effect of other antithrombotics and risk factors settings on DVT stability.</p> / Master of Science (MSc)