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

Abstract

Summary: 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.