Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Pharmacology and Toxicology

Supervisor

Karel Tyml

Abstract

Sepsis, a systemic inflammatory response to an infection, is a significant cause of morbidity and mortality worldwide. The microcirculation during sepsis fails, in part, due to microthrombosis and the resulting plugging of capillaries, precipitating organ failure. Intravenous injection of ascorbate has been shown to reduce capillary plugging, however the mechanism of this protective effect is unclear. We hypothesized that ascorbate-mediated destabilization of the microthrombi through promoting fibrinolysis could contribute to this protection.

We showed that streptokinase, a pro-fibrinolytic agent, reduced the capillary plugging to a similar degree as ascorbate. This similarity provided the impetus for studying the effect of ascorbate on fibrinolysis. Sepsis increased the urokinase plasminogen activator (u-PA) and tissue plasminogen activator (t-PA) mRNA expression in the skeletal muscle and liver in mice. No effect of ascorbate was observed on u-PA or t-PA expression levels. Sepsis also increased the plasminogen activator inhibitor 1 (PAI-1) mRNA and protein expression and activity in these tissues, but ascorbate did not affect these increases.

The local PAI-1 release by both platelets and endothelial cells may play a critical role in microthrombus formation in capillaries. We observed that PAI-1 released by isolated endothelial cells was not affected by ascorbate. However, thrombin-induced PAI-1 release from platelets was inhibited by ascorbate pH-dependently. We have also discovered that the PAI-1 release from platelets was nitric oxide independent.

It has been shown that PAI-1 has a protective role in sepsis, namely that PAI-1 knockout leads to increased bacterial content, increased neutrophil apoptosis and increased mortality. Therefore, the lack of effect of ascorbate on PAI-1 in the tissue may maintain PAI-1’s beneficial role in sepsis. Consistently, we observed that sepsis-induced increases in bacterial count, PAI-1 expression and myeloperoxidase content in various organs were not affected by ascorbate.

Overall, the lack of effect of ascorbate indicates that the protection by ascorbate through reduced capillary plugging is not through a fibrinolytic mechanism. Other mechanisms such as platelet-endothelial cell adhesion and changes in red blood cell deformability in the capillaries should be explored as possible mechanisms of protection by ascorbate.