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General Session IV - Chronic Deep Venous Disease

General Session IV - Chronic Deep Venous Disease

Plasminogen Activator Inhibitor Protease Domain Drives Venous Fibrosis

Monday, March 4, 2024

2:07 PM – 2:17 PM EST

Location: Tampa Bay Ballroom Salons 1-4

Objective: Plasminogen activator inhibitor-1 (PAI-1) is a multifunctional enzyme that inhibits fibrinolysis and modulates tissue fibrosis. Two domains of PAI-1 drive these processes: an antiprotease (P) domain that inhibits plasmin generation and consequently matrix metalloprotease (MMP) activation, and a vitronectin (V) binding domain that interferes with inflammatory cell adhesion. To determine the relative importance of each PAI-1 domain in venous fibrosis, we induced venous thrombosis in CRISPR-Cas9 genetically modified mice with point mutations in each respective domain (V+P-, V-P+), as well as globally gene deleted (V-P-) and wild-type (WT) mice.

Methods: Venous thrombosis was induced by IVC ligation in each genotype mouse. Mice were harvested at 2, 7, and 14 days post thrombosis. IVC and surrounding tissue was harvested en bloc and underwent Masson’s Trichrome staining. Vein wall (VW) thickness, as a measure of vascular fibrosis, was measured on Image J and quantified. Monocytes were quantified by immunohistochemistry, from CCR2 (pro-inflammatory) and Arg-1 (anti-inflammatory) positively staining cells. Thrombus matrix metalloproteinase (MMP) activity from globally deficient PAI-1 mice was measured via zymography.

Results: Mice with loss of plasmin inhibitor domain function (V+P-) and globally deficient PAI-1 mice (V-P-) both demonstrated increased Vein Wall thickness at 7 days post thrombosis (Fig. 1A). In thrombi from mice globally deficient in PAI-1 we observed increased MMP2/9 activity, demonstrating the role of this enzyme in inhibition of MMP activation (Fig. 1B). In mice with loss of plasmin inhibitor domain function (V+P-) we observed accelerated thrombus resolution noted by smaller thrombi at day 7, consistent with loss of inhibition of fibrinolytic system (Fig. 1C). In thrombi from mice with loss of vitronectin binding domain (V-P+) we observed increased anti-inflammatory Arg-1 presence, with no observed differences in pro or anti-inflammatory intrathrombus monocyte infiltrate (Fig. 1D).

Conclusions:

The plasmin binding domain of PAI-1 is a key regulator of thrombus resolution. Loss of this domain potentiates vascular fibrosis, potentially via enhanced intrathrombus protease activity leading to tissue injury. Inflammatory cells infiltrate to the thrombus in unaffected by PAI-1-vitronectin interactions, suggesting that alternate ECM and cell-cell binding sites may predominate during post-thrombosis inflammatory cell influx.

Abstract Podium Presenter(s)

Kiran Kumar, BS

Research Associate
University of Michigan
Ann Arbor, Michigan, United States