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

 

 Buy Article Permissions and Reprints

Summary

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.

^* Presented in part at the 94th Annual Clinical Congress of the American College of Surgeons, Surgical Research Forum, October 14, 2008.

• References

• 1 Wakefield TW. et al. Mechanisms of venous thrombosis and resolution. Arterioscler Thromb Vasc Biol 2008; 28: 387-391.
  CrossrefPubMedGoogle Scholar
• 2 Meissner MH. et al. Determinants of chronic venous disease after acute deep venous thrombosis. J Vasc Surg 1998; 28: 826-833.
  CrossrefPubMedGoogle Scholar
• 3 Williams FM. et al. Identification of quantitative trait loci for fibrin clot pheno-types: the EuroCLOT study. Arterioscler Thromb Vasc Biol 2009; 29: 600-605.
  CrossrefPubMedGoogle Scholar
• 4 Kahn SR. et al. Determinants and time course of the postthrombotic syndrome after acute deep venous thrombosis. Ann Intern Med 2008; 149: 698-707.
  CrossrefPubMedGoogle Scholar
• 5 Lijnen HR. et al. Function of the plasminogen/plasmin and matrix metalloproteinase systems after vascular injury in mice with targeted inactivation of fibrinolytic system genes. Arterioscler Thromb Vasc Biol 1998; 18: 1035-1045.
  CrossrefPubMedGoogle Scholar
• 6 Bugge TH. et al. Urokinase-type plasminogen activator is effective in fibrin clearance in the absence of its receptor or tissue-type plasminogen activator. Proc Natl Acad Sci USA 1996; 93: 5899-5904.
  CrossrefPubMedGoogle Scholar
• 7 Singh I. et al. Failure of thrombus to resolve in urokinase-type plasminogen activator gene-knockout mice: rescue by normal bone marrow-derived cells. Circulation 2003; 107: 869-875.
  CrossrefPubMedGoogle Scholar
• 8 Schuster V. et al. Plasminogen deficiency. J Thromb Haemost 2007; 5: 2315-2322.
  CrossrefPubMedGoogle Scholar
• 9 Meltzer ME. et al. Fibrinolysis and the risk of venous and arterial thrombosis. Curr Opin Hematol 2007; 14: 242-248.
  CrossrefPubMedGoogle Scholar
• 10 Galis ZS, Khatri JJ. Matrix metalloproteinases in vascular remodeling and athero-genesis: the good, the bad, and the ugly. Circ Res 2002; 90: 251-262.
  PubMedGoogle Scholar
• 11 Visse R, Nagase H. Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry. Circ Res 2003; 92: 827-839.
  CrossrefPubMedGoogle Scholar
• 12 Eeckhout Y, Vaes G. Further studies on the activation of procollagenase, the latent precursor of bone collagenase. Effects of lysosomal cathepsin B, plasmin and kallikrein, and spontaneous activation. Biochem J 1977; 166: 21-31.
  CrossrefPubMedGoogle Scholar
• 13 Keski-Oja J. et al. Proteolytic processing of the 72,000-Da type IV collagenase by urokinase plasminogen activator. Exp Cell Res 1992; 202: 471-476.
  CrossrefPubMedGoogle Scholar
• 14 Coats Jr WD. et al. Collagen content is significantly lower in restenotic versus nonrestenotic vessels after balloon angioplasty in the atherosclerotic rabbit model. Circulation 1997; 95: 1293-1300.
  CrossrefPubMedGoogle Scholar
• 15 Deatrick KB. et al. Vein wall remodeling after deep vein thrombosis involves matrix metalloproteinases and late fibrosis in a mouse model. J Vasc Surg 2005; 42: 140-148.
  CrossrefPubMedGoogle Scholar
• 16 Henke PK. et al. Fibrotic injury after experimental deep vein thrombosis is determined by the mechanism of thrombogenesis. Thromb Haemost 2007; 98: 1045-1055.
  Article in Thieme ConnectPubMedGoogle Scholar
• 17 Lijnen HR. Matrix metalloproteinases and cellular fibrinolytic activity. Biochemistry (Mosc). 2002; 67: 92-98.
  PubMedGoogle Scholar
• 18 Dewyer NA. et al. Plasmin inhibition increases mmp-9 activity and decreases vein wall stiffness during venous thrombosis resolution. J Surg Res 2007; 142: 357-363.
  CrossrefPubMedGoogle Scholar
• 19 Henke PK. et al. Targeted Deletion of CCR2 Impairs Deep Vein Thombosis Resolution in a Mouse Model. J Immunol 2006; 177: 3388-3397.
  CrossrefPubMedGoogle Scholar
• 20 Henke PK. et al. Deep vein thrombosis resolution is modulated by monocyte CXCR2-mediated activity in a mouse model. Arterioscler Thromb Vasc Biol 2004; 24: 1130-1137.
  CrossrefPubMedGoogle Scholar
• 21 Siu MK, Cheng CY. Interactions of proteases, protease inhibitors, and the beta1 integrin/laminin gamma3 protein complex in the regulation of ectoplasmic specialization dynamics in the rat testis. Biol Reprod 2004; 70: 945-964.
  CrossrefPubMedGoogle Scholar
• 22 Gallimore MJ. et al. Urokinase induced fibrinolysis in thromboelastography: a model for studying fibrinolysis and coagulation in whole blood. J Thromb Haemost 2005; 3: 2506-2513.
  CrossrefPubMedGoogle Scholar
• 23 Henke PK. et al. Viral IL-10 gene transfer decreases inflammation and cell adhesion molecule expression in a rat model of venous thrombosis. J Immunol 2000; 164: 2131-2141.
  CrossrefPubMedGoogle Scholar
• 24 Henke PK. et al. Neutrophils modulate post-thrombotic vein wall remodeling but not thrombus neovascularization. Thromb Haemost 2006; 95: 272-281.
  Article in Thieme ConnectPubMedGoogle Scholar
• 25 Ginzinger DG. Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. Exp Hematol 2002; 30: 503-512.
  CrossrefPubMedGoogle Scholar
• 26 Sevitt S. The vascularisation of deep-vein thrombi and their fibrous residue: a post mortem angio-graphic study. J Pathol 1973; 111: 1-11.
  CrossrefPubMedGoogle Scholar
• 27 Matrisian LM. The matrix-degrading metalloproteinases. Bioessays 1992; 14: 455-463.
  CrossrefPubMedGoogle Scholar
• 28 Moaveni DK. et al. Vein wall re-endothelialization after deep vein thrombosis is improved with low-molecular-weight heparin. J Vasc Surg 2008; 47: 616-624.
  CrossrefPubMedGoogle Scholar
• 29 Schafer K. et al. Different mechanisms of increased luminal stenosis after arterial injury in mice deficient for urokinase- or tissue-type plasminogen activator. Circ 2002; 106: 1847-1852.
  CrossrefPubMedGoogle Scholar
• 30 Humphries J. et al. Monocyte chemotactic protein-1 (MCP-1) accelerates the organization and resolution of venous thrombi. J Vasc Surg 1999; 30: 894-899.
  CrossrefPubMedGoogle Scholar
• 31 Henke PK, Wakefield TW. Thrombus resolution and vein wall injury: dependence on chemokines and leukocytes. Thromb Res 2009; 123 (Suppl) S73-S79.
  PubMedGoogle Scholar
• 32 Humphries J. et al. Monocyte urokinase-type plasminogen activator up-regulation reduces thrombus size in a model of venous thrombosis. J Vasc Surg 2009; 50: 1127-1134.
  CrossrefPubMedGoogle Scholar
• 33 Kolev K, Machovich R. Molecular and cellular modulation of fibrinolysis. Thromb Haemost 2003; 89: 610-621.
  Article in Thieme ConnectPubMedGoogle Scholar
• 34 Day SM. et al. Macrovascular thrombosis is driven by tissue factor derived primarily from the blood vessel wall. Blood 2005; 105: 192-198.
  CrossrefPubMedGoogle Scholar
• 35 Johnson C, Galis ZS. Matrix metalloproteinase-2 and –9 differentially regulate smooth muscle cell migration and cell-mediated collagen organization. Arterioscler Thromb Vasc Biol 2004; 24: 54-60.
  CrossrefPubMedGoogle Scholar
• 36 Momi S. et al. Loss of matrix metalloproteinase 2 in platelets reduces arterial thrombosis in vivo. J Exp Med 2009; 206: 2365-2379.
  CrossrefPubMedGoogle Scholar
• 37 Aimes RT, Quigley JP. Matrix metalloproteinase-2 is an interstitial collagenase. Inhibitor-free enzyme catalyzes the cleavage of collagen fibrils and soluble native type I collagen generating the specific 3/4– and 1/4-length fragments. J Biol Chem 1995; 270: 5872-5876.
  CrossrefPubMedGoogle Scholar
• 38 Reichel CA. et al. Gelatinases mediate neutrophil recruitment in vivo: evidence for stimulus specificity and a critical role in collagen IV remodeling. J Leukoc Biol 2008; 83: 864-874.
  CrossrefPubMedGoogle Scholar
• 39 Greenlee KJ. et al. Proteomic identification of in vivo substrates for matrix metal-loproteinases 2 and 9 reveals a mechanism for resolution of inflammation. J Immunol 2006; 177: 7312-7321.
  CrossrefPubMedGoogle Scholar
• 40 Campbell RA. et al. Contributions of extravascular and intravascular cells to fibrin network formation, structure, and stability. Blood 2009; 114: 4886-4896.
  CrossrefPubMedGoogle Scholar
• 41 Meissner MH. et al. Deep venous insufficiency: the relationship between lysis and subsequent reflux. J Vasc Surg 1993; 18: 596-606.
  CrossrefPubMedGoogle Scholar
• 42 Merli G. et al. Use of emerging oral anticoagulants in clinical practice: translating results from clinical trials to orthopedic and general surgical patient populations. Ann Surg 2009; 250: 219-228.
  CrossrefPubMedGoogle Scholar