title: Development of a plasminogen activator inhibitor (PAI-1) assay and comparison of plasma PAI-1 activity in hyperlipidemic/dyslipidemic dogs with either hyperadrenocorticism or diabetes mellitus, and healthy dogs.
abstract: Thrombosis is a serious complication of many canine diseases and may be related to decreased fibrinolytic potential. Plasminogen activator inhibitor-1 (PAI-1) is the key regulator of fibrinolysis with increased levels demonstrated in states of pro-thrombosis and abnormal lipid metabolism. Our objective was to develop and validate a canine PAI-1 activity assay and test whether dogs with hyperadrenocorticism or diabetes mellitus that are hyperlipidemic/dyslipidemic have increased plasma PAI-1 activity. Functionally active PAI-1 in the plasma sample was incubated with recombinant tissue plasminogen activator (tPA), allowing the formation of a 1:1 stoichiometric inactive complex. Residual unbound tPA was then reacted with excess plasminogen in the presence of a colorimetric plasmin substrate. Plasmin production is quantified by computing the area under the curve of time (x) vs optical density (y) plot and converted to tPA IU/mL by comparison to a calibration curve of tPA standards. PAI-1 activity was determined by calculating the proportion of exogeneous tPA suppressed by PAI-1 in plasma. Assay verification included assessment of linearity, specificity, precision, sensitivity, and stability. PAI-1 activity was increased in hyperlipidemic compared to healthy dogs, but there was no significant difference between dogs with hyperadrenocorticism and diabetes mellitus. A near significant decrease in activity was detected in thawed plasma stored for 20h at 4°C. Our successfully validated assay offers a new tool for investigating fibrinolysis in dogs. Investigation of PAI-1 activity in dogs with other diseases associated with an increased risk of thrombosis would be valuable. Future studies of PAI-1 activity should consider its lability.
authors.affiliation: Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, 602 Tower Rd, Ithaca, NY 14853, United States. Electronic address: cjw228@cornell.edu.
authors.forename: Cheryl J
authors.initials: CJ
authors.lastname: Wong
authors.affiliation: Veterinary Specialists and Emergency Services, 825 White Spruce Blvd, Rochester, NY 14623, United States. Electronic address: kochm@att.net.
authors.forename: Michael
authors.initials: M
authors.lastname: Koch
authors.affiliation: Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, 602 Tower Rd, Ithaca, NY 14853, United States. Electronic address: eb58@cornell.edu.
authors.forename: Erica L
authors.initials: EL
authors.lastname: Behling-Kelly
mesh_terms: D000308:Adrenocortical Hyperfunction / Q000097:blood* / Q000150:complications / Q000378:metabolism; D000818:Animals; D003920:Diabetes Mellitus / Q000097:blood* / Q000378:metabolism; D004283:Dog Diseases / Q000097:blood / Q000378:metabolism*; D004285:Dogs; D005260:Female; D006801:Humans; D006949:Hyperlipidemias / Q000097:blood / Q000150:complications / Q000662:veterinary*; D008297:Male; D017395:Plasminogen Activator Inhibitor 1 / Q000097:blood / Q000235:genetics / Q000378:metabolism*; D012680:Sensitivity and Specificity; D012698:Serologic Tests
chemical_list: D017395:Plasminogen Activator Inhibitor 1
keywords: Cholesterol; Endocrinopathy; Fibrinolysis; Thrombosis; Triglycerides