Mechanism of damage to fibronectin by myeloperoxidase derived oxidants

Atherosclerosis is characterised by lipid deposition in the arterial wall and chronic low-grade inflammation. Leukocytes migrate to the area of injury and release the heme enzyme myeloperoxidase (MPO) into the extracellular matrix (ECM). This enzyme converts H2O2 to hypohalous acids (e.g.HOCl from Cl-, HOSCN from SCN-). There is considerable evidence that these oxidants, particularly HOCl, damage cells and the ECM, and contribute to cardiovascular disease. HOCl is a highly damaging oxidant, whereas HOSCN is less reactive and generates reversible modifications. The ECM contains multiple proteins, including fibronectin (FN), which are critical to matrix assembly and cell function. FN possesses multiple functionally-important epitopes including a cell binding fragment (CBF) and a heparin binding fragment (HBF). The co-localisation of FN and MPO in the ECM, makes FN a likely target for MPO-derived oxidants. Therefore, this project aimed to elucidate the effects of HOCl and HOSCN on FN, and whether the increasing SCN- concentrations modulated ECM damage induced by HOCl. Exposure of human plasma FN to HOCl resulted in fragmentation and aggregation of the protein, formation of modified amino acids, and alterations to the CBF and HBF. Human coronary artery endothelial cells exposed to modified FN showed poor adhesion, impaired cell spreading, reduced metabolic activity and altered gene expression. In contrast, reagent HOSCN generated limited modifications. Studies using an enzymatic MPO/H2O2 system with either Cl- or SCN-, showed that MPO/H2O2/Cl- gave extensive FN modifications, whereas MPO/H2O2/SCN- induced only minor changes. When both Cl- and SCN- were present as competing substrates, increasing concentrations of SCN- decreased the extent of chemical and structural modifications detected on FN supporting the hypothesis that increasing the concentration of SCN- may mitigate damage generated by the MPO/Cl-/H2O2 system and potentially modulate disease development