Jennifer Wemhoff Mitchell, Ph.D.

Dissertation research performed under the direction of Frank C. Church

ABSTRACT
    The physiological process of blood coagulation is an essential part of our host defense mechanism.  An important component of coagulation is the protease thrombin, which cleaves fibrinogen to generate a hemostatic plug.  Thrombin also participates in inflammation and wound healing; therefore, thrombin activity must be carefully regulated.  One significant regulator of thrombin is heparin cofactor II (HCII), a serine protease inhibitor that forms a bimolecular complex with thrombin.  The rate of thrombin inhibition by HCII is significantly increased by glycosaminoglycans such as heparin or dermatan sulfate.
    Thrombin recognizes and cleaves the P1-P1' bond typically consisting of Arg-Ser residues in macromolecular substrates.  However, HCII has an unusual Leu-Ser sequence within its reactive site loop.  Since this is an atypical thrombin recognition sequence, HCII must have other structural elements that participate in thrombin inhibition, specifically, the unique N-terminal acidic domain of HCII.  The proposed mechanism implies that the negatively charged acidic region 2 (AR2) of HCII’s acidic domain interacts ionically with the positively charged D-helix region of HCII in the absence of glycosaminoglycans.  In the presence of glycosaminoglycans that bind to the D-helix, AR2 is displaced allowing acidic region 1 (AR1) to bind anion-binding exosite-1 (exosite-1) of thrombin, thus facilitating thrombin’s inhibition.  This dissertation provides evidence that specific amino acid residues in AR2 (Asp72, Tyr73 and Asp75) are important in HCII’s mechanism of inhibition.  Furthermore, by changing HCII’s reactive site loop from Leu444 to Arg444, the sequence of a typical thrombin substrate, HCII’s acidic domain is no longer necessary.  This supports the current theory that HCII’s acidic domain plays a significant role in wild-type HCII thrombin inhibition.
    Studies have suggested that HCII may play its greatest role in thrombin inhibition outside of the vasculature in contrast to antithrombin, the primary intravascular coagulation inhibitor.  We confirm that HCII thrombin inhibition increases in the presence of fibroblasts and smooth muscle cells.  In addition, inflammatory cytokines interleukin-1b and transforming growth factor-b cause changes in HCII inhibitory activity due to altered proteoglycan levels on these cells and their extracellular matrix.  This bolsters the evidence that HCII is a physiological thrombin inhibitor in the extravasculature.