R. A. Jacobson1,2, O. Zaborina1, J. C. Alverdy1 1The University Of Chicago,Surgery,Chicago, IL, USA 2Rush University/Cook County,Surgery,Chicago, IL, USA
Introduction: Recent work in our lab has demonstrated that Enterococcus faecalis (E. faecalis) alone can cause anastomotic leak (AL) in rodents by activating tissue associated pro-matrix metalloprotease 9 (pro-MMP9) leading to pathologic degradation of submucosal collagen in the anastomotic wound. However the fibrinolytic protease plasminogen (PLG) 1) is concentrated and cleaved to active plasmin at sites of tissue injury 2) activates pro-MMP9 and 3) directly cleaves collagen, therefore its role in AL pathogenesis needs to be clarified. Here we hypothesized that PLG is also activated by E. faecalis and acts synergistically with MMP9 leading to further enhancement of collagen degradation. Therefore, the aim of this study was to define the relative contribution of PLG in the molecular pathogenesis of AL via its effect on collagen degradation.
Methods: A collagenolytic E. faecalis strain (E44), previously identified to play a putative role in AL was used in these experiments. Plasmin(ogen) activation was assessed with a fluorogenic substrate specific to plasmin. Fluorescein-tagged gelatin, type 1 collagen and type 4 collagen assays were employed to assess E. faecalis-mediated collagenolylsis in the presence of PLG, the known plasminogen activator urokinase (uPA), and pro-MMP9. All purified enzymes were purified recombinant human forms Tranexamic acid (TXA) was used to inhibit plasmin(ogen) activation.
Results:
Plasmin(ogen) activation: E. faecalis alone activated PLG in a concentration-dependent fashion (reaction velocity increased by 0.32 RFU/s per E. faecalis colony forming unit, R2 = 0.99 ). The effect was amplified in the presence of uPA (1.57 RFU/s per CFU, R2 = 0.96). This process was inhibited by TXA in a concentration-dependent fashion.
Collagenolysis/gelatinolysis: E. faecalis-mediated cleavage of type 4 collagen was unchanged by the addition of pro-MMP9, but was significantly increased in the presence of PLG with and without its activator uPA. In the presence of PLG and uPA, addition of pro-MMP9 appeared to synergistically increase collagenolytic activity (Panel A). TXA diminished overall gelatinolytic activity in a dose dependent manner (1.16 AU 10mM TXA vs 3.50 AU E.faecalis + PLG/uPA/proMMP9, p<0.05, panel B). Results were redemonstrated in gelatin and type 1 collagen cleavage assays.
Conclusion: Our data suggest that the molecular pathogenesis of anastomotic leak may involve synergistic activation of plasmin(ogen) and pro-MMP9 in a manner that drives collagenolytic activity to supraphysiologic levels. That this process can be suppressed by the use of TXA, offers a novel therapeutic role to apply this FDA approved agent to high risk anastomotic surgery.
