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 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 (FFAs) allosterically disrupt Zn2+ binding to HSA. Thus, high levels of circulating FFAs, 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: To characterize Zn2+ binding by HRG, examine the influence that FFAs 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 ELISA. Results and conclusions: We found that HRG possesses 10 Zn2+ sites (K′ = 1.63 × 105) and that cumulative binding of FFA to HSA perturbed its ability to bind Zn2+. Also Zn2+ binding was shown to increase the affinity with which HRG interacts with unfractionated heparins, but had no effect on its interaction with low molecular weight heparin (~ 6850 Da). [Correction added on 1 December 2014, after first online publication: In the preceding sentence, “6850 kDa” was corrected to “6850 Da”.] Speciation modeling of plasma Zn2+ based on 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.