Susannah J. Bauman, Ph.D.

Dissertation research performed under the direction of Frank C. Church

ABSTRACT
    Heparin cofactor II (HCII) is a serine protease inhibitor (serpin) found in plasma.  Thrombin inhibition by HCII is greatly accelerated in the presence of glycosaminoglycan such as heparin and dermatan sulfate.  The localization of dermatan sulfate containing proteoglycans has led some to speculate the HCII is an extravascular inhibitor of thrombin.  We serendipitously discovered an HCII mutant with enhanced antithrombin activity, especially in the presence of heparin, a component of the majority of intravascular proteoglycans.  This mutant, carboxyl-terminal hexahistidine tagged HCII (rHCII-CHis6) was expressed in a baculoviral system and its activities were characterized and its mechanism of increased activity was explored.
    Our results indicate that rHCII-CHis6 is a potent inhibitor of thrombin in the presence of subnormal concentrations of heparin that rival the requirements of antithrombin III (ATIII)-glycosaminoglycan enhanced thrombin inhibition.  The data indicate rHCII-CHis6 functions through a similar mechanism to wild-type HCII (rHCII).  The effect of the hexahistidine tag is specific for HCII, since a tagged ATIII does not exhibit enhanced thrombin inhibition despite the high degree of homology of these two serpins.  We have investigated the character of the hexahistidine tag by comparing rHCII-CHis6 to hexaalanine- and hexalysine-tagged HCII.  The data indicate that neither a carboxyl-terminal extension nor a positively charged tag is responsible for the increased activity.  The activity seems to be specific to the histidine tag.  In addition, we have probed the contribution of the unique amino-terminal region of HCII to the gain of activity with rHCII-CHis6. These studies, which involved sequential deletions of the amino terminal region provided invaluable information about the mechanism of rHCII-CHis6 inhibition of thrombin.  In addition, we have acquired knowledge about the structure of HCII, in particular the amino terminal region.
    The role of the active site region of thrombin in its interaction with macromolecular inhibitors was also examined.  Specific mutations of the 60-insertion loop as well as amino acid Glu192 were studied to further define the requirements for macromolecular substrate (or pseudo-substrate) access to the catalytic triad.  From these studies, we deduced that the interaction of thrombin with its substrates is highly dependent on the particular substrate being investigated.