Rebecca A. Shirk, Ph.D.

Dissertation research performed under the guidence of Dr. Frank C. Church

ABSTRACT
    Protein C inhibitor (PCI), antithrombin (AT), and heparin cofactor II (HC II) are serine proteinase inhibitors (serpins) involved in the regulation of hemostasis. Inhibition of target proteinases is accelerated by heparin and involves binding of both inhibitor and proteinase to heparin. These studies were undertaken (i) to identify the heparin binding site in PCI, and (ii) to compare the mechanism by which AT and HC II demonstrate heparin-accelerated thrombin inhibition. Inactive truncated recombinant PCI (rPCI) containing two putative heparin binding sites (the A+ and H helices) was expressed in E. coli as a fusion protein. The PCI moiety of the fusion bound to heparin-Sepharose and blocked heparin-catalyzed proteinase inhibition. Deletion of the H but not the A+ helix markedly reduced heparin binding compared to the 'parent' fusion protein. Deletion of the A+ helix from full-length active rPCI (obtained by collaboration) did not reduce heparin-accelerated thrombin and activated protein C inhibition compared to wild-type rPCI. Next, two H helix substitution mutants (Arg269,Lys270 to Ala and Lys276,Lys277 to Ala) expressed in a Baculovirus system were compared to wild-type rPCI. Both mutants bound to heparin-Sepharose with reduced affinity. However, only Arg269,Lys270 to Ala rPCI showed reduced heparin-accelerated proteinase inhibition, a 50-60% reduction in the rate increase, compared to wild-type rPCI at optimum heparin concentrations. Collectively, these results demonstrate that the A+ helix is not essential for heparin binding to PCI but that, unlike AT and HC II, the H helix is a primary heparin binding site. They also indicate that H helix residues Arg269 and/or Lys270 are critical for maximal heparin-enhanced proteinase inhibition. Dysfunctional thrombin mutant Quick I (Arg67 to Cys, in anion binding exosite-I) was used to compare HC II and AT interactions. Quick I was inhibited similarly to a-thrombin by AT in the absence or presence of heparin and by HC II in the absence of glycosaminoglycan. However, glycosaminoglycan-accelerated Quick I inhibition by HC II was greatly reduced, indicating that anion binding exosite-I is critical for glycosaminoglycan-accelerated inhibition by HC II.