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

Abstract

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.