SUMMARY/SCOPE OF WORK BACKGROUND AND SIGNIFICANCE: Thrombotic diseases are the leading cause of death and disability worldwide. Genetic, environmental, and pharmacological factors, as well as acute and chronic inflammatory diseases, are linked to exacerbated thrombosis. Despite this heterogeneity of etiology and risk factors, thrombosis pathogenesis converges on a common pathway culminating in the conversion of soluble fibrinogen to an insoluble fibrin matrix that serves as the primary structural component of blood clots. However, there are currently no therapeutic approaches that directly modulate fibrinogen. RATIONALE: Thrombosis risk and severity are increased substantially with elevated circulating fibrinogen levels and other perturbations that promote the formation of a dense fibrin network that is resistant to degradation. This proposal is based on the concept that fibrin(ogen) can be directly targeted to produce porous fibrin clot structures with poor red blood cell retention and enhanced susceptibility to fibrinolysis with the ultimate goal of reducing thrombosis across diverse settings regardless of other confounding sequelae. The central hypothesis is that specific and selective reduction of circulating fibrinogen levels or directed suppression of fibrin matrix formation significantly protects against venous and arterial thrombosis while maintaining hemostatic potential. SPECIFIC AIMS: Innovative studies will be performed using (i) a newly developed lipid nanoparticle delivery system of a small interfering RNA (siRNA) that knocks down fibrinogen to any desired target level and (ii) genetic and pharmacological strategies (mRNA delivery) utilizing a customized fibrinogen variant (fibrinogenLOCK) that suppresses fibrin polymerization. These approaches will be used to uncover the contributions of fibrinogen and fibrin matrix formation in the development of venous and arterial thrombosis as well as to hemostasis and platelet plug formation. The specific aims are to: (1) Determine the changes in venous and arterial thrombus formation following targeted reduction in circulating fibrinogen or selective suppression of fibrin matrix formation; (2) Determine qualitative and quantitative changes in bleeding and platelet plug formation during hemostatic challenges following fibrinogen reduction or suppression of fibrin matrix formation; (3) Determine the efficacy of fibrinogen reduction or introduction of fibrinogenLOCK in mitigating thrombosis during endotoxemia (acute inflammation) and sickle cell disease (chronic inflammatory disease). EXPECTED OUTCOMES AND DELIVERABLES: This work will provide novel mechanistic insight into how quantitative and qualitative changes in circulating fibrinogen or fibrin matrix formation alters thrombogenesis. Moreover, the proposed studies will provide essential preclinical data on a novel management strategy for thrombotic disease across diverse etiologies. We expect to demonstrate that knocking down fibrinogen or i...