Plasma free fatty acid levels influence Zn²⁺-dependent histidine-rich glycoprotein-heparin interactions via an allosteric switch on serum albumin

Background: Histidine-rich glycoprotein (HRG) regulates coagulation, through its ability to bind and neutralize heparins. HRG associates with zinc ions (Zn²⁺) to stimulate HRG-heparin complex formation. Under normal conditions the majority of plasma Zn²⁺ associates with human serum albumin (HSA). However, free fatty acids (FFA) allosterically disrupt Zn²⁺ binding to HSA. Thus high levels of circulatory FFA, as are associated with diabetes, obesity and cancer, may increase the proportion of plasma Zn²⁺ associated with HRG contributing to an increased risk of thrombotic disease.

Objectives: The aims were to characterize Zn²⁺ binding by HRG, examine the influence FFA have on Zn²⁺ binding by HSA and establish whether FFA-mediated displacement of Zn²⁺ from HSA may influence HRG-heparin complex formation.

Methods: Zn²⁺ binding to HRG and to HSA in the presence of different FFA (myristate) concentrations were examined by isothermal titration calorimetry (ITC) and the formation of HRG-heparin complexes in the presence of different Zn²⁺ concentrations by both ITC and an ELISA-based assay.

Results and conclusions: We reveal that HRG possesses 10 Zn²⁺ sites (K´=1.63 x 10⁵) and that cumulative binding of FFA to HSA perturbed its ability to bind Zn²⁺. Also Zn²⁺ binding was shown to increase the affinity by which HRG interacts with unfractionated heparins but has no effect upon its interaction with low molecular weight heparin (ca. 6850 kDa). Speciation modeling of plasma Zn²⁺ based upon the data obtained suggests that FFA-mediated displacement of Zn²⁺ from serum albumin would be likely to contribute to the development of thrombotic complications in individuals with high plasma FFA levels.