The Heparin Binding Properties of Heparin Cofactor II Suggest an Antithrombin-like Activation Mechanism

Abstract

The serpin heparin cofactor II (HCII) is a glycosaminoglycan-activated inhibitor of thrombin that circulates at a high concentration in the blood. The antithrombotic effect of heparin, however, is due primarily to the specific interaction of a fraction of heparin chains with the related serpin antithrombin (AT). What currently prevents selective therapeutic activation of HCII is the lack of knowledge of the determinants of glycosaminoglycan binding specificity. In this report we investigate the heparin binding properties of HCII and conclude that binding is nonspecific with a minimal heparin length of 13 monosaccharide units required and affinity critically dependent on ionic strength. Rapid kinetics of heparin binding indicate an induced fit mechanism that involves a conformational change in HCII. Thus, HCII binds to heparin in a manner analogous to the interaction of AT with low affinity heparin. A fully allosteric 2000-fold heparin activation of thrombin inhibition by HCII is demonstrated for heparin chains up to 26 monosaccharide units in length. We conclude that the heparin-binding mechanism of HCII is closely analogous to that of AT and that the induced fit mechanism suggests the potential design or discovery of specific HCII agonists.

A Hexasaccharide Containing Rare 2‐O‐Sulfate‐Glucuronic Acid Residues Selectively Activates Heparin Cofactor II

“…The k OBS of inhibition was found to increase linearly with the concentration of HX–serpin complex (Figure S17), which was used to derive the second‐order rate constant for the uncatalyzed inhibition ( k UNCAT ) from the intercept and HX‐catalyzed inhibition from the slope ( k HX ) (Table S2). The k UNCAT for HCII–TH system and AT–fXa system was found to be 1.2–1.6×10 3   m −1  s −1 and 2.2–2.4×10 3   m −1  s −1 , respectively, which compare favorably with the corresponding basal rates reported in the literature 8, 10. The k HX for HCII–TH reaction in the presence of HX1, HX3 and HX4 was measured to be (2.2–3.9)×10 5   m −1  s −1 .…”

“…All five hexasaccharides demonstrated a distinct and saturable change in fluorescence emission at pH 7.4 with both serpins from which the K D s were calculated (Figure S15). For HCII, the K D s were found to be in the range of 14 to 46 μ m (Table S2), which are in the range of affinities reported for the polymeric entities (20–40 μ m ) 10, 31. In comparison, the hexasaccharides bound AT with much more varied affinities (1 to 75 μ m , Table S2).…”

“…Yet, whereas AT binds specifically to fondaparinux sequence in heparin,7 HCII is considered to bind non‐specifically to heparin and HS. Further, although both serpins display a two‐step, induced‐fit, allosteric activation mechanism in inhibiting their target enzymes,8, 9, 10 no HS oligosaccharide has been discovered to induce robust activation of HCII.…”

Structure of Native Protein C Inhibitor Provides Insight into Its Multiple Functions

“…The bound protein was then eluted with a 10-column volume gradient from 50 mM to 1 M NaCl, and the conductivity of the peak was measured by the FPLC system (Amersham Biosciences). Accurate dissociation constants were obtained using TNS as an extrinsic fluorescence probe, essentially as described previously for HCII (27). Briefly, precisely size-fractionated heparin (41) was added to buffer (50 mM Tris-HCl, pH7.4, 50 mM NaCl, 0.1% PEG8000, and 1 mM EDTA) containing PCI (200 -400 nM) and TNS (10 M).…”

“…The crystal structures of native AT (24) and HCII (25) revealed the incorporation of the N terminus of the RCL (the hinge region) into ␤-sheet A. Heparin activation is effected, in part, by liberating the RCL from its contacts with the body of the serpin (26,27). Although PCI does not utilize helix D for heparin binding, a heparin-induced conformational change in PCI is possible, although it would presumably depend on the native structure of PCI resembling that of AT and HCII (i.e.…”