Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Pharmacology and Toxicology

Supervisor

Dr. Karel Tyml

Abstract

Sepsis is a systemic inflammatory response to an infection. The overwhelming inflammation has many deleterious effects, including cessation of capillary blood flow. This cessation may lead to organ failure and subsequent death, but the cause of cessation during sepsis is not fully understood. Ascorbate (reduced vitamin C) has been shown to restore capillary blood flow by an unknown mechanism. I hypothesized that activation of both platelets and the coagulation pathway in sepsis contributes to the cessation of capillary blood flow and that ascorbate protects against cessation by reducing platelet activation.

Using intravital microscopy in the mouse hindlimb skeletal muscle in vivo, I observed that sepsis impairs capillary blood flow and increases both adhesion of platelets/platelet aggregates to the capillary wall and deposition of fibrin plaques in the same capillaries. Platelet depletion, blocking of P-selectin (a key adhesion molecule), antithrombin, and eptifibatide (anti-aggregatory agent) all reduced the capillary blood flow impairment and platelet adhesion. Intravenous bolus injection of ascorbate reduced platelet adhesion in capillaries, via the endothelial nitric oxide synthase (eNOS) system.

To study any direct effects of ascorbate on platelet function, I used an ex vivo model (isolated mouse platelets) examining platelet aggregation under septic conditions. Here, thrombin, ADP, and thromboxane (agents released into the blood during sepsis), but not lipopolysaccharide (LPS), tumor necrosis factor (TNFa) or septic plasma, increased platelet aggregation and surface P-selectin protein expression. Ascorbate inhibited the increased aggregation and P-selectin expression.

Next, an in vitro mouse microvascular endothelial cells model was used to study the effect of ascorbate on platelet-endothelial cell adhesion. LPS and TNFa increased platelet adhesion and P-selectin mRNA expression in endothelial cells. LPS also increased P-selectin-containing endothelial granule secretion. Ascorbate prevented the increased adhesion and granule secretion but did not affect mRNA expression.

Thus, I conclude that impairment of blood flow in the septic microvasculature requires platelets and is reduced by anti-coagulant/anti-aggregatory agents. Ascorbate prevents platelet-endothelial adhesion and platelet aggregation, partly through reducing P-selectin protein expression at the platelet/endothelial cell surface. Thus ascorbate reduces adhering platelets in septic capillaries leading to restoration of blood flow.


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