Plasminogen activation in cancer

The subject of this thesis focusses on the role of the plasminogen activation system in angiogenesis and cancer. The plasminogen activation system regulates fibrinolysis and controls cell migration and invasion by plasmin-mediated matrix proteolysis. Plasmin is formed upon cleavage of the zymogen plasminogen by plasminogen activators, urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). In contrast to uPA, plasminogen activation by tPA requires a cofactor, such as fibrin. Plasmin cleaves its substrates behind a basic amino-acid (lysine or arginine). Free carboxy-terminal lysines or arginines provide high affinity binding sites for plasminogen and facilitate efficient activation by plasminogen activators. Plasminogen activation can be inhibited by lysine analogues such as epsilon aminocaproic acid (?ACA) or tranexamic acid. Studies with these lysine analogues have indicated the importance of carboxy-terminal lysine residues in cancer and angiogenesis. In chapter 1 we give a literature overview of the plasminogen activation system in cancer. In addition we summarize possible ways to interfere with tumor growth by targeting this system. Thrombin-activatable fibrinolysis inhibitor (TAFI) is a circulating carboxypeptidase B-type enzyme that specifically cleaves carboxy-terminal lysine or arginine residues from proteins. Removal of carboxy-terminal lysines and arginines from fibrin abrogates efficient plasminogen binding and consequently leads to decreased plasmin formation. Given the importance of the plasminogen activation system in angiogenesis, tumor growth and metastasis, we have studied the role of TAFI in corneal angiogenesis (chapter 2) and subcutaneous tumor growth and experimental- and spontaneous metastasis formation in TAFI-deficient mice (chapter 3). Furthermore, we have determined TAFI antigen and TAFI activity levels in patients with primary and metastasized prostate cancer (chapter 4). We further investigated the mechanism of action of endostatin (chapter 5). Endostatin is a carboxy-terminal fragment of collagen XIII, and has originally been described as one of the most potent inhibitors of angiogenesis. In animal experiments different forms of endostatin are used. Although the soluble form of endostatin inhibits tumor growth, complete tumor regression has only been observed when using insoluble endostatin. We found that insoluble endostatin, in contrast to soluble endostatin, stimulates plasminogen activation by tPA, induces plasminogen-mediated endothelial cell detachment and matrix degradation and is toxic to neuronal cells (chapter 5,6). These effects were inhibited by carboxypeptidase B treatment indicating an essential role for carboxy-terminal lysines. Insoluble proteins may occur as amorphous aggregates or as highly ordered amyloid deposits. Structure analyses revealed that insoluble endostatin forms amyloid fibers (chapter 6) which was critical for the effects on plasminogen activation and endothelial and neuronal cells (chapter 5,6,7). We further established that the presence of amyloid fibers is a common feature in other tPA-ligands, including fibrin peptides, islet amyloid polypeptide (IAPP, associated with pancreatic ß cell toxicity in type II diabetes) and amyloid ß (associated with Alzheimers disease), and responsible for their ability to stimulate tPA-mediated plasminogen activation (chapter 7). To what extent may our observations be of value for the use of endostatin and other antiangiogenic fragments? In chapter 9 we discuss the possible implications of our finding