The role of the plasminogen activating system in the proteolytic and phagocytic clearance of non-viable cells

The plasminogen activator (PA) system is an enzymatic cascade involved in the breakdown of fibrin, the structural component of a blood clot. tPA co-localises with plasminogen on the fibrin surface via C-terminal lysine residues, leading to the generation of plasmin and subsequent fibrin removal. In this thesis, both tPA and plasminogen were shown to interact with proteins in non-viable cells. Analogous to fibrinolysis, plasminogen binding, but not tPA binding was lysine dependent. Hence, tPA-mediated plasmin formation results in the proteolytic degradation of dead cells. This finding also led to the hypothesis that plasmin would also engage the innate immune system, given the known association of innate immunity in dead cell clearance. Dendritic cells (DCs) are antigen presenting phagocytes that link the innate and adaptive immune systems. tPA-mediated plasmin formation was shown to induce profound morphological changes in human monocyte-derived DCs (MoDCs) with the development of extended cell projections, yet this did not result in cell maturation. This effect of plasmin on cell morphology was dependent upon active plasmin and the binding to a lysine containing cell surface protein, coincided with the activation of MMP9 and release of TGFβ1. Although the identity of this cell surface protein was not determined, a number of known plasminogen receptors were excluded including annexin A2 fragment and the plg-RKT receptor. tPA-mediated plasmin formation on dead cells also significantly affected functional attributes of MoDCs. Indeed, plasmin formation significantly increases the capacity by MoDCs to uptake dead cells. Plasmin was also shown to selectively increase the ability of MoDCs to uptake microparticles (500nm) but not nanoparticles (40nm). This effect of plasmin on microparticle uptake was also seen using mouse-derived DCs. Moreover, the ability of plasmin to increase phagocytic capacity of mouse DCs was cell type specific, as CD11cpositive DCs and Gr-1intermediate myeloid cells were both sensitive to plasmin mediated microparticle uptake, whereas Gr-1high granulocytic-like cells were not responsive. DCs are crucial for the presentation of antigens to naïve T-cells to mount an adaptive immune response. A key finding of this thesis was that plasmin blocked the ability of MoDCs to invoke an allogeneic response raising the speculation that plasminogen activation on DCs influences the adaptive immune response. Intradermal co-injection of tPA/plasminogen and microparticles was also shown to cause selective expansion of CD3posCD8negCD4neg population. As this cell population is involved in immunosuppression, we hypothesis that plasmin may induce proliferation of this cell type in the presence of large amounts of phagocytic material which in turn results in a reduction in an adaptive immune response. The findings presented within this thesis show that plasmin influences dead cell removal by direct proteolysis of dead cell proteins and by increasing phagocytosis by DCs. Furthermore, plasmin increases phagocytosis without inducing an adverse stimulation of the adaptive immune system. The broader significance of this may be apparent in situations where large amounts of self-protein need to be cleared in a manner that avoids activation of the adaptive immune response, thereby minimising an autoimmune reaction.