Herbert C. Whinna, M.D., Ph.D.

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

ABSTRACT
    Heparin cofactor II (HC) is a plasma serine proteinase inhibitor (serpin) that inhibits the blood coagulation proteinase thrombin in a reaction accelerated by glycosaminoglycans (GAGs) and other polyanions, such as fucoidan. HC-thrombin complex is rapidly formed in the presence of fucoidan when [125]I-labeled thrombin is incubated with human plasma. HC, not antithrombin III (AT, another plasma serpin that inhibits thrombin) is important for the anticoagulant activity of fucoidan. The characteristics of the reaction are consistent with formation of a ternary complex which implies binding of both inhibitor and proteinase to fucoidan. Modification of lysyl residues in HC by pyridoxal 5’-phosphate in the presence of GAG gave four lysyl residues (173, 252, 343, and 348) protected from modification by heparin and to a lesser extent by dermatan sulfate, compared to modification in the absence of GAG. Molecular modeling showed lysines 173 and 252 in one region of the inhibitor, lysines 343 and 348 in another. Site directed mutagenesis showed Lys173 to be important in heparin (but not dermatan sulfate) interactions with HC, while Lys343 was not found important for either GAG. The unique ability of HC to interact with dermatan sulfate (located in extracellular matrix as dermatan sulfate-containing proteoglycans), lead to a proposed role for HC as an extravascular thrombin inhibitor. Two such proteoglycans, biglycan and decorin, both in solution and bound to collagen accelerated thrombin inhibition by HC, but not by AT, further implying a physiological role for HC as an extravascular regulator of thrombin. Thrombin has multiple structural domains for interactions with macromolecules. Using the three-dimensional structure of thrombin to evaluate recent data on the interaction of thrombin with AT and HC, we determined that the active site and anion-binding exosites-I and -II are involved in the GAG accelerated inhibition by HC, but anion-binding exosite-II has little to do with GAG accelerated inhibition by AT. A synthetic peptide from the GAG-binding site of HC competed for heparin and dermatan sulfate in inhibition assays. GAGs increased the a-helical content of the peptide as follows: LMWH < heparin < dermatan sulfate, suggesting that a a-helical conformation is the important in binding heparin.