Plasmin is a multifunctional protease: Roles include fibrinolysis, cleavage of extracellular matrix molecules, activation and liberation of growth factors, regulation of inflammation, angiogenesis and tumour cell and macrophage invasion.
Proteases and their inhibitors are interconnected in the “protease web” through activating and inactivating cleavages within and between protease classes e.g. the serine protease plasmin can activate several matrix metalloproteinases (MMPs). These interconnections can be important in therapies that target proteases since there may be unforeseen effects mediated through the protease web. For instance, the current treatment for ischaemic stroke is tissue plasminogen activator (tPA) administration: tPA activates plasminogen to plasmin which degrades fibrin clots to restore blood flow. The major side-effect of tPA therapy is intracerebral haemorrhage, involving upregulation and activation of MMP2 and MMP9 which degrade endothelial basement membranes as well as a myriad of bioactive mediators.
Our Terminal Amino Isotopic Labelling of Substrates (TAILS) strategy to identify the N-terminome has identified many novel matrix metalloproteinase (MMP) substrates in vitro and more recently in vivo. Here we apply TAILS to proteomes from cell lines (EAhy926 endothelial and MDA MB 231 breast cancer) and use dimethylation to differentially label the N-termini of plasmin vs. control secretomes to elucidate the degradome of plasmin. Neo N-terminal peptides generated by plasmin were identified by mass spectrometry (Bruker Impact mass spectrometer) and bona fide plasmin substrates and their cleavage sites discriminated with high confidence to allow the prediction of the substrate repertoire of plasmin. Predictions will be tested in cell culture models and in vitro assays.
Determining the degradome of plasmin will help us to understand the interdependence of the plasminogen system and MMPs that is key to developing safe new therapies for ischaemic stroke.