Vol. 124, No. 3, 1984 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS November 14, 1984 Pages 745-751 EVIDENCE FOR ESSENTIAL LYSINRS IN HEPARIN COFACTOR II Frank C. Church' and Michael J. Griffith Departments of Pathology and Medicine The Center for Thrombosis and Hemostasis University of North Carolina, Chapel Hill, NC 27514 Received September 25, 1984 Covalent modification with pyridoxal 5'-phosphate was used to study the function of lysyl residues in heparin cofactor II, a heparin-dependent plasma protease inhibitor. Reduction of the Schiff base with sodium borohydride resulted in modification of 3-4 lysyl residues of heparin cofactor II at high concentrations of pyridoxal 5'-phosphate, one of which was protected in the presence of heparin. The antithrombin activity of modified heparin cofactor II was enhanced compared to the native protein. However, the heparin cofactor activity for thrombin inhibition was reduced significantly or completely elimi- nated in the modified protease inhibitor depending on the extent of phosphopyri- doxylation. In contrast to native heparin cofactor II, the modified protease inhibitor did not bind to a heparin-agarose column. The results suggest that lysyl residues are essential for heparin cofactor activity during thrombin inhibition. 0 1984 Academic Press. Inc. Antithrombin III (l-3) and heparin cofactor II (4-9) are two heparin- dependent thrombin inhibitors isolated from human plasma. There are significant differences in the structural properties of heparin cofactor II compared to antithrombin III, these include: molecular weights (7,9), protease specificity (4,5,7-g), heparin affinity (4-lo), NH2-terminal amino acid sequences (11,12), HPLC-tryptic peptide maps (12), immunological reactivity (6-9) and glycosamino- glycan activation specificity (10,13,14). However, there is evidence to suggest that the two protease inhibitors possess a general similarity with regard to the kinetic mechanism of heparin-enhanced thrombin inhibition (lO)and selected regions of limited sequence homology (reactive-site peptide) (12). Heparin binding to antithrombin III is dependent on ionic interactions between the net negative charge of the sulfated mucopolysaccharide and the positively charged 'To whom correspondence should be addressed at, Division of Hematology, Department of Medicine, 416 Burnett-Womack Bldg. 2298, University of North Carolina, Chapel Hill, NC 27514. This investigation was supported in part by NIH grants HL-07255, HL-06350 and HL-32656. 0006-291X/84 $1.50 145 Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.