Urokinase plasminogen activator independent early experimental thrombus resolution: MMP2 as an alternative mechanism

Abstract

Deep-vein thrombosis (DVT) resolution is thought to be primarily a urokinase plasminogen activator (uPA) -dependent mechanism, although observations suggest other non-fibrinolytic mechanisms may exist. We explored the role of matrix metalloproteinase (MMP) -2 and -9 in early DVT resolution in uPA-deficient mice. Male B6/SVEV (WT) and genetically matched uPA -/- mice underwent inferior vena cava (IVC) ligation to create stasis venous thrombi, with IVC and thrombus harvest. Thrombus size was similar between WT and uPA -/- mice at day 4, suggesting early non uPA-dependent resolution. Intrathrombus neutrophils and monocytes were reduced 3- and 3.5-fold in uPA -/- mice as compared with WT. By ELISA, tumour necrosis factor α and interleukin 1β were not altered, while interferon (IFN)γ was significantly elevated in uPA -/- mice. A compensatory increase in thrombus tPA was not observed, plasmin activity was reduced and PAI-1 was elevated 2.5-fold in uPA -/- mice. Active MMP2, but not MMP9, was elevated 3-fold in uPA -/- mice as compared with WT as well as MMP-14, an MMP2 activator. Collagen type IV and fibrinogen were reduced in uPA -/- mice thrombi as compared with WT. IFNγ induces MMP2, and blockade of IFNγ was associated with larger venous thrombi and reduced active MMP2, as compared with WT. Consistently, MMP2 -/- mice had larger VT as compared with WT controls, despite normal thrombus plasmin levels. Taken together, early experimental venous thrombus resolution is independent of uPA, and, in part, inflammatory cell influx. MMP2-dependent thrombolysis is an important compensatory mechanism of venous thrombus resolution, possibly by collagen type IV metabolism, and may represent an exploitable therapeutic avenue.

Intact Toll-like receptor 9 signaling in neutrophils modulates normal thrombogenesis in mice

“…25, 28 Primary antibodies were diluted in TBST, added to the membrane and incubated at 4°C overnight while gently shaking. For normalization of proteins on the western blots, the membranes were stripped and probed with anti-B-actin antibodies conjugated with HRP (Santa Cruz).…”

Matrix metalloproteinase-9 deletion is associated with decreased mid-term vein wall fibrosis in experimental stasis DVT

“…5, 13, 15 Activity was visualized as light staining bands on a dark background, and normalized to the total amount of protein present in each sample. Activity of the TIMPs (active and latent forms of TIMP-1, TIMP-2) was determined by reverse zymography on 10% SDS polyacrylamide gels, copolymerized with gelatin and human pro-MMP2, which degrades gelatin and is susceptible to inhibition by all TIMPs, as previously described.…”

Therapeutic effectiveness of bone marrow-derived mesenchymal stem cell administration against acute pulmonary thromboembolism in a mouse model

“…Therefore, we inferred that there was some other mechanism about the role of BMSCs on thrombus resolution. u-PA is an important naturally occurring fibrinolytic agent for early thrombus resolution [36,37] its deficiency is associated with fibrin deposition [38]. A recent study in ApoE-/-mice determined that undetectable u-PA correlated with a decrease in vein wall MMP-2, MMP-9, and MCP-1 expression and a decrease in monocyte recruitment, which greatly contribute to thrombus resolution [39].…”