Dabbagh, Karim; (1997) Effect of thrombin and alpha-1-antitrypsin on mesenchymal cell proliferation and procollagen production. Doctoral thesis (Ph.D.), University College London (United Kingdom).

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Abstract

The mechanisms of tissue repair following injury are partially understood. Disruption of blood vessels results in the extravasation of blood derived proteins with subsequent platelet aggregation and fibrin clot formation. The repair processes that follow involve acute phase and inflammatory reactions, accompanied by extracellular matrix synthesis and remodelling. Mesenchymal cells are responsible for the latter processes which are thought to be co-ordinated by cytokines and growth factors. Aberrations in the proliferation and connective tissue metabolism by these cells can lead to the development of fibroproliferative disorders. Proteases and antiproteases play key roles in blood coagulation, inflammatory reactions and the subsequent repair processes. Two such molecules are thrombin and α1-antitrypsin (AAT). Thrombin, a serine protease involved in blood coagulation, also promotes mesenchymal cell chemotaxis and proliferation. It is therefore believed to play a role in tissue repair processes and fibrotic disorders in organs such as the lung, kidney and blood vessels. AAT on the other hand, is an acute phase serine protease inhibitor which protects tissues from the effects of neutrophil elastase. Increased levels of AAT have been associated with liver fibrosis, and its absence in the inflamed lung results in destruction of the connective tissue and emphysema. At present, AAT has not been reported to directly influence mesenchymal cell behaviour, and the effect of thrombin on collagen metabolism by these cells remains unexplored. The aim of this thesis was therefore to address the hypothesis that thrombin and AAT can play roles in tissue repair processes by directly activating mesenchymal cells. More specifically, the effects of thrombin and AAT on mesenchymal cell proliferation and procollagen metabolism were investigated. The results showed that thrombin stimulated fibroblast and smooth muscle cell procollagen production in a dose- and time-dependent manner. This stimulation occurs via increases in ?1(I) procollagen mRNA levels and requires de novo protein synthesis. Further, the mechanisms which mediate these stimulatory effects involve the proteolytic activation of the PAR-1 thrombin receptor, and preliminary experiments investigating the intracellular signalling pathways showed that tyrosine kinase- and PKC-linked pathways play a role in mediating these effects. In addition, this thesis reports for the first time, that AAT stimulated fibroblast proliferation and procollagen production. AAT also induced rapid protein tyrosine phosphorylation and the stimulatory effects on fibroblast proliferation were mediated via tyrosine kinase- and MAP kinase-linked pathways, suggesting that AAT exerts these stimulatory effects via a receptor mediated mechanism. In summary, this thesis demonstrates for the first time that thrombin stimulates mesenchymal cell procollagen production, and that AAT promotes fibroblast proliferation and procollagen production. It also partially characterises the mechanisms which mediate these stimulatory effects. These data support the hypothesis that thrombin and AAT can play a role in tissue repair processes by influencing mesenchymal cell proliferation and procollagen metabolism. This supports the view that proteases and antiproteases can exert similar effects to cytokines and growth factors and may form an additional mechanism by which connective tissue repair can be regulated.

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