The mechanism of activation of the contact system and fibrinolytic system

Abstract

The contact system and the fibrinolytic system are two enzymatic systems that are involved in haemostasis, as well as in a number of haemostasis-unrelated processes. We hypothesized that the enzymes factor XII (FXII) and tissue-type plasminogen activator (tPA), which initiate these systems, have functional roles in the recognition of misfolded proteins. We expected that this could provide insight into the role of the contact system in vivo, as well as a better understanding about the roles of the fibrinolytic system beyond haemostasis The contact system is responsible for the clotting of blood on a surface such as glass. This process is governed by Factor XII. However, this system does not have a clear role in vivo, since congenital absence of Factor XII does not lead to bleeding symptoms. We discovered that Factor XII, besides interacting with surfaces in vitro, also can interact with misfolded protein aggregates. However, this latter interaction does not lead to a clotting response, but rather to the formation of kallikrein, which mediates a number of proinflammatory and vasoactive processes. We therefore conclude that the role of Factor XII and the contact system may lie in the detection of misfolded protein aggregates, thereby aiding in their clearance. We also determined the binding site for misfolded proteins on FXII, tPA and their homologues Hepatocyte Growth Factor activator and Fibronectin, which is the Fibronectin type I (Finger) domain. Although the roles of these homologous proteins are different, our data suggests that these proteins can interact with misfolded proteins or ligands that adopt similar structural properties. Since tPA is an important activator of the fibrinolytic system and can interact with misfolded proteins, we investigated the generation of plasmin-alpha2-antiplasmin (PAP) complexes in the plasma of patients with systemic amyloidosis, a disease hallmarked by the presence of circulating amyloidogenic proteins. PAP-complex levels were elevated in systemic amyloidoses, as compared to age-and-sex matched controls. This indicates that the fibrinolytic system is activated, which may play a role in the bleeding tendency that can be observed in these patients. We also investigated whether fibrin, the natural ligand of tPA, has structural properties that are comparable to those of misfolded proteins. Protein misfolding is often accompanied by aggregation, enhanced affinity for certain dye compounds and, in some cases formation of beta-sheets. Structural analyses of fibrin indicated that these polymers contain amyloid cross-beta structure, a structural element only known to occur in protein misfolding diseases, such as Alzheimer’s disease. This suggests that the interaction of tPA with misfolded proteins may not be mechanistically different from its interaction with fibrin. Finally, we found that the misfolding of proteins is also corresponded by increased immunogenicity, which may provide new insights into the unwanted immune responses against biopharmaceutical products and “self” proteins. Our studies suggest that several well-known biological systems can identify misfolded proteins, most likely via a common structural denominator. Although not always beneficial, these responses are meant to clear toxic protein species and aid in protein homeostasis, which is a necessity for maintenance of mammalian life.