Influence of zinc on glycosaminoglycan neutralisation during coagulation

Abstract

Heparan sulfate (HS), dermatan sulfate (DS) and heparin are glycosaminoglycans (GAGs) that serve as key natural and pharmacological anticoagulants. During normal clotting such agents require to be inactivated or neutralised. Several proteins have been reported to facilitate their neutralisation, which reside in platelet α-granules and are released following platelet activation. These include histidine-rich-glycoprotein (HRG), fibrinogen and high-molecular-weight kininogen (HMWK). Zinc ions (Zn2+) are also present in α-granules at a high concentration and participate in the propagation of coagulation by influencing the binding of neutralising proteins to GAGs. Zn2+ in many cases increases the affinity of these proteins to GAGs, and is thus an important regulator of GAG neutralisation and haemostasis. Binding of Zn2+ to HRG, HMWK and fibrinogen is mediated predominantly through coordination to histidine residues but the mechanisms by which Zn2+ increases the affinity of the proteins for GAGs are not yet completely clear. Here we will review current knowledge of how Zn2+ binds to and influences the neutralisation of GAGs and describe the importance of this process in both normal and pathogenic clotting.

Fig. 1. Coagulation control by glycosaminoglycans and Zn²⁺. (A) Anticoagulant glycosaminoglycans bind to antithrombin and enhance its neutralisation of thrombin (and/or factor Xa). (B) When platelets become activated, the Zn²⁺ released from the α-granules of platelets bind to the GAG neutralising proteins, increasing their affinity for GAGs and allowing them to neutralise them. Once neutralised, the GAG cannot promote the inhibition of thrombin and clotting occurs. (C) Sources of Zn²⁺ in plasma. During coagulation, Zn²⁺ is released by activated platelets. However, erythrocytes, lymphocytes and neutrophils contain Zn²⁺ which may be released under certain conditions. In some disease states, elevated levels of free fatty acids may also influence available Zn²⁺ levels through release from serum albumin. Atherosclerotic plaques contain up to six time more Zn²⁺ than healthy tissue and could potentially release Zn²⁺ when they rupture. The structure of human serum albumin (with stearate bound) was taken from PDB ; 1E7I.

Fig. 2. Structure of histidine-rich glycoprotein. N1 and N2 are N-terminal domain 1 and 2, they have a GAG binding activity; PRR1 and PRR2 are proline-rich regions; HRR is the histidine rich region that binds Zn²⁺ and GAG; C is the C-terminal domain.

Fig. 3. Structure of high-molecular-weight kininogen. Domains 1, 2 and 3 are cystatin-like domains, with 2 and 3 having a cysteine protease inhibitor activity; domain 4 is bradykinin and another peptide; domain 5 is the surface-binding domain containing the histidine-rich region that binds GAGs and Zn²⁺; domain 6 is the domain binding prekallikrein and activated coagulation factor XI.

Fig. 4. Structure of fibrinogen. The protein forms hexamer made of three different strands (Aα Bβ γ)₂. All of the N-terminals are in the E domain which is the heparin binding domain. The three strands then coil together until they reach the D domains where the C-terminal of the β and γ strands are located. This domain is a Zn²⁺-binding domain. The α strand goes back toward the E domain where its C-terminal forms the αC domain, another Zn²⁺ binding domain. Insert 1. Crystal structure of fibrinogen D domain (one of the Zn²⁺ binding domain) and part of the coil–coil region (PDB structure ; 3GHG). The histidine residues which have the potential to be involved in Zn²⁺ binding are represented in pink with the residues His217 and His234 known to be involved represented in yellow. Most of those residues are hidden beneath the surface of the protein. Insert 2. Crystal structure of fibrinogen E domain (the heparin binding domain) and part of the coil–coil regions, (PDB structure ; 3GHG). The positive charges are represented in blue and the negative charges in red. The Lys and Arg residues which usually constitutes the main binding partners of GAGs are represented in green. The first few residues of the Bβ chain are mobile and so they are not visible in the crystal structure, with β58 the first residue that can be observed (represented in yellow). This residue is a protruding Lys that is believed to be important for GAG binding. As the GAG binding affinity of fibrinogen is enhanced when the protein is converted into fibrin by cleavage of the A and B peptides, the absence in the crystal structure of the first few residues of the Bβ chain may be the reason for exposure of the β58 residue. The αC domain is attached to the E domain; binding of Zn²⁺ ions to its His α544 and His α545 is thought to change the conformation of the protein and thus facilitate GAG binding to the E domain.