Hemostasis is maintained by a well-coordinated series of vascular and chemical reactions that regulate the balance between coagulation and fibrinolysis. Under certain pathological conditions an exaggerated or insufficiently haemostatic response may lead to a situation in which coagulation/fibrinolysis contribute to disease. In the first part of this work, we undertook a study of the function of fibrinogen (Fg) in a murine model of acute inflammation, viz., lipopolysaccharide (LPS)-induced lethal endotoxemia. This study demonstrated that mice with a total deficiency of fibrinogen (Fg-/-) presented with reduced mortality, coagulation, and inflammatory responses when compared to their wild-type (WT) counterpart. The attenuated inflammatory responses in Fg-/- mice correlated with a lack of fibrin deposition in organs. Inflammatory cells appeared early in the tissues of challenged WT mice, but occurred at later times in Fg-/- mice. This delayed response in Fg-/- mice was confirmed by studies that showed a strong dependence on Fg for binding of neutrophils to endothelial cells in the presence of LPS. Inflammatory cytokines were elevated in both genotypes, however their levels were generally lower at early times in Fg-/-. Therefore, a Fg deficiency enhances survival from lethal endotoxemia through attenuation of inflammatory responses that result from reduced leukocyte infiltration to the inflammatory foci, and, from downregulation of chemokine/cytokine expression. Our results suggest that fibrin(ogen) plays an important role as an early mediator in the cross-talk between coagulation and inflammation. In the second part of this dissertation, we utilized a biochemical approach to study the role of plasminogen (Pg) in the pathogenic mechanisms of Group A streptreptococcus (GAS). GAS is the etiologic agent responsible for a number of human diseases that range from common pharyngitis to severe infections. Streptokinase (SK) is a 414 amino acid protein secreted by several streptococcal species, and an efficient activator of Pg. Interestingly, SK is not an enzyme; it activates Pg indirectly by the formation of a 1:1 complex with Pg. Furthermore, Pg activation by SK is highly species specific with activity towards human Pg (hPg), but exhibiting no activity against mouse plasminogen (mPg). In an attempt to define which amino acid regions within Pg may account for the species specificity of SK, several mutants, and chimeric mouse-human Pg constructs were generated. The hPg light chain (hL) was identified as the region responsible for SK sensitivity, specifically the amino acid sequences encoded by exons 16 and 18. In addition, surface plasmon resonance (SPR) experiments demonstrated high affinity binding between all Pg variants and SK, including mPg, indicating that SK has the ability to form “catalytic complexes” in a non-species specific manner. However, no active site is formed within the moiety of the activator complex. In summary, we identified the loci within hPg that productively interacts with SK. The data generated herein presents novel insights for the understanding of the activation mechanism of hPg by SK.
|Advisor||Francis J. Castellino|
|Contributor||Francis J. Castellino, Committee Member|
|Contributor||Jeffrey S. Schorey, Committee Member|
|Contributor||Patricia L. Clark, Committee Member|
|Degree Level||Doctoral Dissertation|
|Degree Discipline||Chemistry and Biochemistry|
|Departments and Units|