Circulatory zinc “speciation” and its relevance to cardiovascular disease

Abstract

Zn²⁺ ions are released from activated platelets and are important regulators of coagulation. Human serum albumin (HSA) controls the concentration of available Zn²⁺, but binding of free fatty acids (FFAs) to HSA disrupts Zn²⁺-binding. Elevated plasma FFAs concentrations are associated with disease states (e.g. diabetes, obesity and cancer) characterised by increased thrombotic risk. It is therefore important to understand this dynamic and the roles Zn²⁺ plays in coagulation. The aims of this project were to investigate the interplay between binding of Zn²⁺ and different FFAs to HSA using isothermal titration calorimetry. Increasing the FFA concentration (0-5 mol. eq.) or chain length (C8:0-C18:0) decreased Zn²⁺-binding to HSA. The effect of Zn²⁺ and FFAs upon fibrin clot formation and lysis was determined in a purified system, in pooled plasma and in plasma from subjects with type-1 and type-2 diabetes mellitus (T1DM and T2DM) using turbidimetric assays and scanning electron microscopy. Plasma concentrations of zinc, copper, magnesium and selenium in T1DM and T2DM were also measured. In both the purified system and pooled plasma, Zn²⁺ increased clot density, an effect further increased by the presence of FFAs. Clot density was found to be increased in T2DM subjects (compared to controls) and positively correlated with plasma FFA concentration. In T1DM subjects, clot lysis time was increased (compared to controls) and negatively correlated with magnesium concentration. Magnesium deficiency in T1DM likely contributes to increased thrombotic risk. Finally, the effects of Zn²⁺ on heparin neutralisation by histidine rich glycoprotein (HRG), fibrinogen and fibronectin was assessed using surface plasmon resonance and anti-factor Xa and anti-thrombin activity assays. The abilities of HRG and fibrinogen to neutralise heparins were found to increase in the presence of Zn²⁺, while neutralisation by fibronectin was unaffected. These results collectively support the hypothesis that FFAs impact on coagulation in vivo through mishandling of Zn²⁺.subjects (compared to controls) and positively correlated with plasma FFA concentration. In T1DM subjects, clot lysis time was increased (compared to controls) and negatively correlated with magnesium concentration. Magnesium deficiency in T1DM likely contributes to increased thrombotic risk. Finally, the effects of Zn²⁺ on heparin neutralisation by histidine rich glycoprotein (HRG), fibrinogen and fibronectin was assessed using surface plasmon resonance and anti-factor Xa and anti-thrombin activity assays. The abilities of HRG and fibrinogen to neutralise heparins were found to increase in the presence of Zn²⁺, while neutralisation by fibronectin was unaffected. These results collectively support the hypothesis that FFAs impact on coagulation in vivo through mishandling of Zn²⁺.