The Metal Dependency of Coagulation Factor IX as it relates to its Structure and Function

Amanda Sutton, University of Nebraska at Lincoln

A DISSERTATION Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Doctor of Philosophy, Major: Interdepartmental Area of Engineering (Chemical & Biomolecular Engineering), Under the Supervision of Professor William H. Velander. Lincoln, Nebraska: May 2010
Copyright (c) 2010 Amanda Sutton

Abstract

Hemophilia B is a bleeding disorder caused by deficiency in blood clotting factor IX (FIX). FIX circulates as a 55-57 kDa inactive precursor of the serine protease, FIXa. Activation results in the release of the activation peptide to generate an amino terminal light chain of Mr~18,000 and a carboxyl terminal heavy chain of Mr~28,000 linked by a disulfide bridge. The light chain consists of the gamma-carboxyglutamic acid rich gla domain, followed by two epidermal growth factor-like domains. The gla domain contains several low affinity divalent metal ion binding sites and primarily gives FIX its metal-dependent conformation. The heavy chain contains the catalytic domain, conferring proteolytic activity to FIXa. The biosynthesis of recombinant human FIX in the milk of transgenic pigs (tg-rhFIX) at >100 units coagulation activity per ml creates several major subpopulations that differ primarily in the content of gamma-carboxyglutamic acid. Using a final purification step of size exclusion chromatography with saturating [Ca2+], we generated three distinctive subpopulation pools of tg-rhFIX based on gla content: low gla (< 6), > 6 gla and high gla (> 9 gla). Each pool exhibited different biological and structural characteristics. Biological activity and FIXa content increased for higher gla pools. Binding to metal-dependent monoclonal antibodies and phospholipid also increased for higher gla pools.

Recent pharmacokinetic studies with tg-rhFIX show differences in the biological recoveries of the three pools while antigen recoveries are similar. We hypothesize that the three pools undergo proteolytic degradation differently. In vitro treatment by plasmin of tg-rhFIX with varied gla exhibited contrasting degradation in the presence of calcium or EDTA. Higher gla content species showed greater plasmin mediated proteolysis to form Factor IXalpha in the presence of Ca2+ and the lower gla content species showed increased production of Factor IXgamma. While Factor IXalpha retains procoagulation activity, Factor IXgamma has its putative exosite for FX binding disrupted thereby rendering it inactive. This is the first report of gla-dependant conformational effects upon plasmin cleavage of FIX.

Advisor: William H. Velander