Abstract 9: Two Distinct Roles of Plasminogen Activator Inhibitor-2 (PAI) and PAI-1 in Deep Vein Thrombosis

Abstract

Objectives: Urokinase-plasminogen activator (uPA) has a vital role in deep vein thrombosis (DVT). While PAI-1 is the main inhibitor of uPA in vivo , PAI-2, found mostly intracellularly, has proven to have more anti-inflammatory and anti-apoptotic than anti-fibrinolytic activities. We explored the hypothesis that PAI-1 and PAI-2 play roles in DVT through either fibrinolytic or inflammatory pathways. Methods: Wild type C57Bl/6 (WT), PAI-2 -/- , and PAI-1 -/- mice underwent surgical caval ligation. Subsequent thrombus was harvested at various time points and measured for thrombus weight (TW) normalized to body weight. Tissue was analyzed by western blot, ELISAs, zymography and flow cytometry. Results: PAI-2 deficiency resulted in unchanged thrombus formation (TW day 4: WT = 1.037 ± .043 mg/g n=17; PAI-2 -/- = 1.028 ± .067 mg/g n=17, p=.91), while it enhanced thrombus resolution (TW day 12: WT = .43 ± .03 mg/g n=13; PAI-2 -/- = .319 ± .021 mg/g, n=16, p=.004). PAI-1 deficiency caused decreased formation (TW day 12: PAI-1 -/- = .901 ± .043 mg/g n=11, p=.045) and enhanced resolution (TW day 12: PAI-1 -/- = .248 ± .011 mg/g n=10, p=.00004). Though total uPA levels in thrombi were similar between groups, active uPA by ELISA was increased by 5 fold in PAI-1 -/- mice and 40 fold in PAI-2 -/- mice compared to WT mice at day 12, indicating increased uPA-mediated fibrinolysis. Exploring other mediators of resolution, we found that PAI-1 and PAI-2 deficiencies lead to contrasting results. By zymography, PAI-1 deficiency caused an early increase in matrix metalloproteinase (MMP) activity that decreased over time. Conversely, PAI-2 deficiency trended towards less early MMP activity that then increased. Using flow cytometry to assess clot cell populations, on day 4, PAI-2 -/- mice had an increase in neutrophils with a decrease in macrophages, whereas PAI-1 -/- mice had an increase in macrophages. Conclusions: This identifies PAI-2 as a novel regulator of venous thrombus resolution and suggests the first context in vivo in which PAI-2 has an anti-fibrinolytic role. Both PAI-1 and PAI-2 deficiencies enhance thrombus resolution, but through diverging mechanisms. Further examination of these pathways may lead to potential therapeutic prospects in accelerating thrombus resolution.