Plasminogen Stimulates Formation Of Pathogenic Prion Clumps

Introduction

1.1 Background of the Study

Prion diseases are fatal neurodegenerative conditions, such as Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy and scrapie in animals. They are characterized by the accumulation in the brain of an abnormal isoform of the prion protein (PrPSc)1 in amyloid deposits (1). The normal prion protein (PrPC) is a glycoprotein expressed on the plasma membrane as a GPI-anchored protein and highly concentrated in synapses (2). PrPSc, which is rich in â-sheets, is generated from the largely R-helical PrPC by a conversion mechanism as yet unknown (3).

Prion diseases can all be experimentally transmitted to other animals, and PrPSc is inseparable from the infectious agent. The expression of PrPC is necessary for prion disease, as mice devoid of PrPC are resistant to infection (4). The generation of PrPSc from host PrPC implies that the mechanism by which PrPSc is generated is of fundamental importance in understanding the cause of these diseases and that understanding the difference between PrPC and PrPSc might lead to both diagnosis and possibly treatment of prion diseases. One significant difference between these proteins that has recently been described by Fischer et al. (5) is that some plasma proteins, notably plasminogen, bind to PrPSc but not to PrPC.

Several studies have demonstrated that PrPC is a copperbinding protein (6-9). However, recent studies have indicated that PrPSc does not bind copper. PrP isolated from mouse brains contains approximately 3 copper atoms per PrP molecule, and a further 2 atoms can be bound in vitro (10). In contrast, PrP isolated from brains of both scrapie-infected mice and humans with Creutzfeldt-Jakob disease contains less than 0.5 copper atoms per PrP molecule (11). Copper binding is thought to have both functional and structural effects on PrP. Copper both increases the rate of endocytosis of PrPC (12) and endows it with superoxide dismutase activity (9, 10). The N-terminal half of PrP contains four tandem copies of a highly conserved octapeptide repeat which is thought to bind copper by coordination to four histidine residues (13). This binding of copper imparts structure in this otherwise unstructured part of the molecule (14). Copper can be specifically incorporated into recombinant PrP (PrP23-231), but not PrP lacking the octapeptide repeat region (PrP∆51-90), during the refolding procedure and acquire properties resembling those of PrPC (9, 15).

Pericellular proteolytic activity plays an important role in many pathological as well as physiological situations in a variety of organs, including the brain. This proteolytic activity can act to degrade components of the extracellular matrix or activate bioactive molecules such as growth factors, making it a key regulator of cellular behavior (16). The broad specificity serine protease plasmin is one of the principal activities involved in these processes. Plasmin is generated from the abundant zymogen plasminogen by a single proteolytic cleavage catalyzed by either of the two plasminogen activators, uPA and tPA. At the functional level, the activity of the plasminogen activation system is largely regulated by mechanisms that enhance the generation of plasmin (17).

Thus uPA-catalyzed plasminogen activation is stimulated by the binding of uPA to its cell surface receptor uPAR (18) and tPA-catalyzed plasminogen activation by binding to fibrin (19) or cell surface binding sites (20). All of these situations involve the binding of plasminogen in juxtaposition to the plasminogen activator, either on the same cofactor molecule (as with fibrin) or to discrete cellular binding sites. Interactions with these molecules are mediated by “lysine binding sites” in the kringle modules of plasminogen, which can be antagonized by lysine and various aminocarboxylic acid analogues of lysine, such as ACA (21). These kringle modules preferentially bind C-terminal lysine residues (i.e., those with a free carboxylate group), which can either be present in the native proteins or generated by the proteolytic action of plasmin.

1.2 Statement Problem

The fibrinolytic system has been implicated in multiple biological functions such as wound healing, angiogenesis, tumor metastasis and brain development [1–3]. In addition to their biological functions, plasminogen activators are routinely used for therapeutic interventions in acute arterial thrombosis such as myocardial infarction [4] and stroke [5]. Several plasminogen activators with distinct biochemical properties are in clinical use. First generation fibrinolytic agents are strepto- kinase, anisoylated plasminogen-streptokinase activator complex (APSAC), urokinase, single chain urokinase and tissue-type plasminogen activator. Apart from tissue-type plasminogen activator (t-PA), none of the first generation fibrinolytic agents bind to fibrin and induce systemic plasmi- nogen activation with pronounced changes in the concentra- tions of hemostatic and fibrinolytic proteins.

By contrast, t-PA has a stringent cofactor requirement to protect the organism from the detrimental consequences of non-specific plasminogen activation. The formation of a ternary complex with fibrin and plasminogen is required to stimulate t-PA activity [6]. This fibrin specificity of t-PA is concentration dependent. At the concentrations used in fibrinolytic therapy, a systemic activation of the fibrinolytic system is still detectable as evidenced by consumption of hemostatic and fibrinolytic proteins. Nevertheless, this consumption is much less pro- nouncedcomparedwith the other first generation thrombolytic agents.

Here we test the hypothesis that, due to its reported ability to bind plasminogen, PrPSc is a regulator of plasminogen activation. We demonstrate that PrP can indeed regulate plasminogen activation but that, surprisingly, a critical determinant of this is an interaction between PrP and tPA. Furthermore, rather than being a result of the conversion of PrPC to PrPSc, the ability of PrP to interact with tPA and plasminogen, and to stimulate plasminogen activation, is related to the binding of copper to PrP. These data have implications for the diagnostic detection of the prion diseases and suggest that plasmin may have a role in prion-induced neurodegeneration.

1.3 Research Objective

The objective of this study was to evaluate the plasminogen stimulates formation of pathogenic prion clumps….