Systemic inflammatory diseases, such as immune-mediated hemolytic anemia (IMHA) and sepsis, predispose dogs to thrombosis, which increases morbidity and mortality of the primary disease. Mechanisms involving thrombus formation in these disease states are not well-understood. We now know that dogs with such diseases are not only hypercoagulable but are also hypofibrinolytic. Both hypercoagulability and hypofibrinolysis promote thrombosis, but the role of fibrinolysis in thrombotic disorders has been largely unexplored in dogs. This oversight is mostly due to the lack of suitable assays to evaluate hypofibrinolysis and causes thereof. Only one study has been published showing that dogs with prothrombotic disorders are hypofibrinolytic. Our global hypothesis is that increased activity of inhibitors of fibrinolysis, particularly plasminogen activator inhibitor type 1 (PAI-1), contributes to thrombosis in dogs with predisposing diseases such as IMHA. Support for this hypothesis comes from a recent whole blood transcriptome sequencing study in dogs with IMHA, which showed marked upregulation of the PAI-1 gene (SERPINE1). Dogs with IMHA are also hypofibrinolytic (Dr. Goggs, unpublished data), which we suspect is due to PAI-1. However, we currently lack suitable assays that can measure canine PAI-1 activity or PAI-1’s role in fibrinolysis. Our first aim is to evaluate a commercial ELISA-based assay for measurement of PAI-1 activity. Use of the assay for clinical studies in dogs has not been previously verified. We will determine if the assay can detect canine PAI-1, via spiking with a constitutively active recombinant canine PAI-1 mutant. We will also evaluate two specific PAI-1 inhibitors, tiplaxtinin and TM5275, for their ability to inhibit PAI-1 activity. Our second aim is to develop a viscoelastic-based global hemostatic assay to measure PAI-1’s inhibitory activity in canine plasma. We selected this testing platform because it is used in emergency and referral veterinary clinics to assess hemostasis in ill patients at point-of-care. We will perform recombinant PAI-1 spike-in experiments through a concentration range of PAI-1, and assess the ability of the PAI-1 inhibitors to restore fibrinolysis in the presence of PAI-1. In this viscoelastic thromboelastographic (TEG) assay, clot formation and lysis are monitored over time, with a read out of percentage lysis after 30 and 60 minutes. Initial experiments will be performed using a single lot of canine plasma to minimize inter-individual variation in coagulation factors and inhibitors. We will then measure PAI-1 activity and its action as an inhibitor of fibrinolysis in the plasma of 20 healthy dogs with the ELISA and TEG assay, respectively. After verification of one or both assay’s ability to measure PAI-1’s activity and its contribution to fibrinolysis, we will then use these assays in future studies to measure PAI-1 in dogs at risk of thrombosis, such as those with IMHA. If we find PAI-1 activity is increased in dogs with thrombosis and suppresses fibrinolysis, this will provide solid evidence that PAI-1 inhibitors may be useful therapeutic agents for helping to treat or prevent thrombosis in dogs.