B-001 Preliminary evaluation of a canine PAI-1 kit for use as a biomarker of immunothrombosis

Abstract Background Immunothrombosis describes the pathologic formation of thrombi triggered by a dysregulated inflammatory response. This process is recognized to increase mortality from infectious diseases, such as COVID-19, and chronic inflammatory syndromes, such as obesity. Development of animal models and translational biomarkers to study immunothrombosis is challenging due to species differences in the inflammatory response and the lack of laboratory assays that define prothrombotic imbalance. Plasminogen activator inhibitor 1 (PAI-1) is a major plasma regulator of fibrinolysis conserved among species. Primarily synthesized in the liver, PAI-1 is also released from endothelial cells, adipocytes, and platelets. High plasma PAI-1 has been associated with impaired fibrinolysis and prothrombotic imbalance in response to inflammation with endothelial injury and platelet activation in human and rodent model studies. We undertook a preliminary evaluation of a commercial canine active PAI-1 kit (Canine Active PAI-1 ELISA, Innovative Research) to test its potential utility for characterizing fibrinolysis in dogs with inflammatory disease. Methods Performance of an active PAI-1 ELISA configured with urokinase-coated plates (PAI-1 capture) and an anti-canine PAI-1 sandwich antibody to measure active PAI-1 concentration in canine plasma was evaluated in the following samples: pooled canine plasma (n=20 healthy dogs), individual healthy dogs (n=5), spiked pool containing 10 ng/mL PAI-1 standard, spiked buffer containing 50 and 0.5 ng/mL canine PAI-1, and 2 dogs with sepsis and signs of a systemic inflammatory response. Results Dilutions of the assay’s canine PAI-1 standard demonstrated quantitative recovery through the range of 0 to 100 ng/mL PAI-1. The mean active PAI-1 concentration for all healthy dog samples was 0.33 ng/mL (median = 0.44, range 0 to 0.66), intra-assay coefficient of variation (CV) of pooled plasma = 9.8% (n=6 replicates), inter-assay CV of 50 ng/mL and 0.5 mL ng/mL samples = 8.9% and 12.3%, respectively (n=5 replicates). Observed recovery of 10 ng/mL PAI-1 spiked into pooled plasma was 80%. PAI-1 concentrations in dogs with sepsis were markedly higher than controls, at 12.8 ng/mL and 6.5 ng/mL. Conclusion The canine active PAI-1 assay revealed acceptable performance characteristics to warrant further verification and investigation for its diagnostic utility. In this preliminary evaluation, healthy dogs had PAI-1 concentrations below 1 ng/mL and dogs with sepsis demonstrated an approximately 10-fold increase. Measurement of plasma active PAI-1 concentration may be useful in characterizing immunothrombosis in canine models of inflammatory disease.