Role of lysine 173 in heparin binding to heparin cofactor II

Heparin cofactor II (HC) is a plasma serine proteinase inhibitor (serpin) that inhibits α-thrombin in a reaction that is dramatically enhanced by heparin and other glycosaminoglycans/polyanions. We investigated the glycosaminoglycan binding site in HC by: (i) chemical modification with pyridoxal 5′-phosphate (PLP) in the absence and presence of heparin and dermatan sulfate; (ii) molecular modeling; and (iii) site-directed oligonucleotide mutagenesis. Four lysyl residues (173, 252, 343, and 348) were protected from modification by heparin and to a lesser extent by dermatan sulfate. Heparin-protected PLPHC retained both heparin cofactor and dermatan sulfate cofactor activity while dermatan sulfate-protected PLPHC retained some dermatan sulfate cofactor activity and little heparin cofactor activity. Molecular modeling studies revealed that Lys¹⁷³ and Lys²⁵² are within a region previously shown to contain residues involved in glycosaminoglycan binding. Lys³⁴³ and Lys³⁴⁸ are distant from this region, but protection by heparin and dermatan sulfate might result from a conformational change following glycosaminoglycan binding to the inhibitor. Site-directed mutagenesis of Lys¹⁷³ and Lys³⁴³ was performed to further dissect the role of these two regions during HC-heparin and HC-dermatan sulfate interactions. The Lys³⁴³ → Asn or Thr mutants had normal or only slightly reduced heparin or dermatan sulfate cofactor activity and eluted from heparin-Sepharose at the same ionic strength as native recombinant HC. However, the Lys¹⁷³ → Gln or Leu mutants had greatly reduced heparin cofactor activity and eluted from heparin-Sepharose at a significantly lower ionic strength than native recombinant HC but retained normal dermatan sulfate cofactor activity. Our results demonstrate that Lys¹⁷³ is involved in the interaction of HC with heparin but not with dermatan sulfate, whereas Lys³⁴³ is not critical for HC binding to either glycosaminoglycan. These data provide further evidence for the determinants required for glycosaminoglycan binding to HC.