Abstract
Zn2+ is an essential regulator of coagulation and is released from activated platelets. In plasma, the free Zn2+ concentration is fine-tuned through buffering by human serum albumin (HSA). Importantly, the ability of HSA to bind/buffer Zn2+ is compromised by co-transported non-esterified fatty acids (NEFAs). Given the role of Zn2+ in blood clot formation, we hypothesise that Zn2+ displacement from HSA by NEFAs in certain conditions (such as type 2 diabetes mellitus, T2DM) impacts on the cellular and protein arms of coagulation. To test this hypothesis, we assessed the extent to which increasing concentrations of a range of medium- and long-chain NEFAs reduced Zn2+-binding ability of HSA. Amongst the NEFAs tested, palmitate (16 : 0) and stearate (18 : 0) were the most effective at suppressing zinc-binding, whilst the mono-unsaturated palmitoleate (16 : 1c9) was markedly less effective. Assessment of platelet aggregation and fibrin clotting parameters in purified systems and in pooled plasma suggested that the HSA-mediated impact of the model NEFA myristate on zinc speciation intensified the effects of Zn2+ alone. The effects of elevated Zn2+ alone on fibrin clot density and fibre thickness in a purified protein system were mirrored in samples from T2DM patients, who have derranged NEFA metabolism. Crucially, T2DM individuals had increased total plasma NEFAs compared to controls, with the concentrations of key saturated (myristate, palmitate, stearate) and mono-unsaturated (oleate, cis-vaccenate) NEFAs positively correlating with clot density. Collectively, these data strongly support the concept that elevated NEFA levels contribute to altered coagulation in T2DM through dysregulation of plasma zinc speciation.
Highlights
Zinc is an essential modulator of coagulation controlling multiple aspects.[1]
When a non-esterified fatty acids (NEFAs) molecule binds to FA2, the protein conformation changes to create a linear tunnel for the NEFA molecule that stretches across domains I and II; concomitantly, zinc site A is disrupted (the Zn2+-coordinating nitrogen of His[67] moves $8 Arelative to His[247] and Asp249),[22] dramatically reducing the Zn2+ affinity of human serum albumin (HSA).[10,17,18,19]
Using isothermal titration calorimetry (ITC), we previously demonstrated that myristate (14 : 0) impacts upon Zn2+ binding to HSA at the highest affinity Zn2+ site,[20] whilst mutagenesis and X-ray crystallography studies con rmed this to be site A.22
Summary
Zinc is an essential modulator of coagulation controlling multiple aspects.[1]. Molecular regulation of coagulation by zinc is complex, as it binds numerous plasma proteins to in uence their activities.[1]. In addition to binding/buffering Zn2+, HSA transports non-esteri ed fatty acids (NEFAs) at 5 medium- to high-affinity binding sites (FA1-5), one of which (FA2) is located at the interface between domains I and II, close to the main Zn2+ binding site (site A).[17,18,19] A secondary Zn2+ site with weaker affinity is present on HSA, but is unlikely to contribute greatly to Zn2+ binding under normal conditions.[20] Fig. 1A shows the structure of HSA with myristate bound.[21] When a NEFA molecule binds to FA2, the protein conformation changes to create a linear tunnel for the NEFA molecule that stretches across domains I and II; concomitantly, zinc site A is disrupted (the Zn2+-coordinating nitrogen of His[67] (domain I) moves $8 Arelative to His[247] and Asp[249] (domain II; Fig. 1B)),[22] dramatically reducing the Zn2+ affinity of HSA.[10,17,18,19] when plasma NEFA levels are elevated, the Zn2+ buffering ability of HSA is reduced and plasma Zn2+ speciation is altered
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