Abstract 513: Localised Anticoagulation Via Gene Therapy: Towards Prevention Of Cardioembolic Stroke

Abstract

Introduction: The coagulation cascade thrives as the haematological gatekeeper ensuring equilibrium between blood flow and thrombosis. Under pathological conditions, the positive-feedback loops compound upon one another, disrupting this equilibrium and increasing the risk of thrombosis and embolic events. Cardiac thromboembolism persists as the culprit behind most ischemic strokes; a majority arising from the left atrial appendage. In order to combat this disease and overcome the limitations of systemic oral anticoagulants, we developed a novel anticoagulant viral vector to focally target cardiac regions of increased thrombotic potential. Hypothesis: Our novel recombinant lentiviral and adeno-associated viral vectors expressing thrombomodulin will induce a significant cell autonomous anticoagulant effect when expressed by endothelial cells of the left atrial appendage. Methods: The anticoagulant phenotype of these vectors was functionally assessed through in vitro and ex vivo platforms. For in vitro testing, a novel, cell-based version of the calibrated automated thrombogram assay, coupled with the overall haemostatic potential assay were utilised to determine thrombokinetics in cell culture models. For ex vivo testing, freshly isolated and characterized porcine left atrial appendage endothelial cells were used. These cells were used to endothelialise microfluidic devices, which were stimulated with TNF-α prior to perfusion with human whole blood. Changes in fibrin, platelets and neutrophils were investigated in the presence and absence of our novel anticoagulant viral vectors. Results: Experimental findings from our in vitro and ex vivo work showed a significant decrease in the endogenous thrombin potential of vector-transduced cells, coupled with a significant decrease in the velocity of thrombogenesis. Furthermore, transduced cells had a marked decrease in the peak rate of thrombin generation and a significant increase in the time taken to achieve peak thrombin generation. Conclusion: These findings show the impact of our anticoagulant vectors through inhibiting thrombosis and guide the development of focal gene therapy strategies to target thrombosis at its point of origin in the prevention of cardioembolic-stroke.