Abstract 196: Matrix Metalloproteinases Regulation in Thrombus Resolution Is Independent of TGF-β in a PAI-1 Ko Mouse Model of Venous Thrombosis

Abstract

Introduction: Plasminogen activator inhibitor (PAI-1) is the primary regulator of urokinase (u-PA) and tissue (t-PA) plasminogen activators. Observations in mice with impaired fibrinolysis have demonstrated an alternate pathway of thrombus resolution by matrix metalloproteinase (MMP)- 9 and 2. TGF-β is a potent inducer of TIMP-1 expression and is found in both thrombus and vein wall post-thrombosis. Hypothesis: PAI-1 deficiency and resulting hyperfibrinolytic state will improve thrombus resolution and result in reciprocal downregulation of MMP-2 and MMP-9 via TGF-beta induction of TIMP-1, resulting in decreased vein wall fibrosis. Methods: Inferior vena cava (IVC) ligation was performed in wild-type (WT) mice (N=194) and PAI-1 deficent mice (N=164). IVC, thrombus and blood samples were collect at 2, 6 and 14 days post procedure. The IVC and thrombus were assessed for thrombus weight and samples collected for zymography (MMP-2 and MMP-9), ELISA (TIMP-1, TGF-β) and histology. Results: PAI-1 deficient mice had similar thrombus weight (TW) versus WT at 2 days, but smaller at days 6 (p<0.0001) and 14 (p=0.0085). In thrombus and vein wall (VW), active MMP-9 was decreased in acute thrombosis (p<0.0001), and MMP-2 was decreased during late thrombus resolution (p<0.05). TIMP-1 levels in VW were elevated across all time points (p<0.05), whereas VW TGF-beta was not different compared to WT. Neutrophils were decreased at day 2 (p = 0.0309) and monocytes decreased at day 14 (p = 0.0028). Conclusions: (1) PAI-1 deficient mice exhibit a hyperfibrinolytic phenotype with normal thrombotic response to IVC ligation, but improved thrombus resolution when compared to WT mice. (2) MMP-9 and MMP-2 were suppressed, confirming a reciprocal relationship between plasmin and MMP activation as mediators of thrombus resolution. (3) TIMP-1 production was elevated in vein wall independent of TGF-beta. (4) Decreased neutrophils at day 2 correlated with decreased MMP-9 and decreased monocytes at day 14 correlated with decreased MMP-2, suggesting a plasmin-induced inhibition of leukocyte recruitment. (5) Final histology showed no difference in vein wall fibrosis or thickness at chronic time points, indicating that improved thrombus resolution does not lessen vein wall fibrosis.