Immune-mediated hemolytic anemia (IMHA) is a common, and often life-threatening, blood disorder in dogs. In this condition, the dog's immune system attacks its own red blood cells, leading to a severe anemia that is treated with immunosuppressive drugs. However, affected dogs suffer from more than just anemia. They also have over-active clotting systems that lead to abnormal blood clot formation. These blood clots can be fatal if they block off the blood supply and delivery of nutrients and oxygen to vital tissues, causing serious organ damage and failure. We now treat dogs with blood thinners to try and prevent these clots from forming, but dogs with IMHA continue to suffer from lethal blood clots, indicating that we need to identify more effective therapy. When clots form in the body, they are gradually broken down by enzymes - this normal process is called fibrinolysis. If clots are not broken down properly, they will persist in the blood vessels, causing tissue damage. We suspect that clot breakdown is defective in dogs with IMHA, leading to persistence of blood clots. We believe the decreased fibrinolysis is caused by too much of a blood protein, called PAI-1. PAI-1 protein is the main inhibitor of clot breakdown and if it is too high, clots remain in blood vessels and prevent normal blood flow. Our theory that high PAI-1 levels prevent normal clot breakdown is based on recently published data on dogs with IMHA. These dogs had high levels of the P AI-1 precursor (mRNA) in the circulation - in fact, the mRNA was up to 17-times higher than in healthy dogs. Our preliminary studies have also shown decreased clot breakdown in blood samples from dogs with IMHA.  In this proposal, we plan to determine whether dogs with IMHA have high levels of active PAI-1 protein (not just mRNA levels) as a major cause of reduced clot breakdown. In this project, we will collect blood samples from 40 dogs with IMHA and 40 healthy control dogs and measure PAl-1 protein activity levels and mRNA levels, and perform laboratory tests of clot breakdown. We will also test whether a drug that blocks PAI-1 activity can improve fibrinolysis in these test samples. Importantly, the blood thinning drugs currently given to dogs with IMHA to prevent clot formation do not affect clot breakdown at all. If we find that high P Al-1 levels result in reduced clot breakdown in dogs with IMHA, then PAI-1 inhibitor drugs will open up new possibilities for more effective treatment. Our goal is to improve on current IMHA treatment so that abnormal blood clot formation no longer limits dogs' survival.