Oxidation shuts down an auto-inhibitory mechanism of von Willebrand factor.

The blood protein von Willebrand factor (VWF) is a key link between inflammation and pathological thrombus formation. In particular, oxidation of methionine residues in specific domains of VWF due to the release of oxidants in inflammatory conditions has been linked to an increased platelet-binding activity. However, the atomistic details of how methionine oxidation activates VWF have not been elucidated to date. Yet understanding the activation mechanism of VWF under oxidizing conditions can lead to the development of novel therapeutics that target VWF selectively under inflammatory conditions in order to reduce its thrombotic activity while maintaining its haemostatic function. In this manuscript, we used a combination of a dynamic flow assay and molecular dynamics (MD) simulations to investigate how methionine oxidation removes an auto-inhibitory mechanism of VWF. Results from the dynamic flow assay revealed that oxidation does not directly activate the A1 domain, which is the domain in VWF that contains the binding site to the platelet surface receptor glycoprotein Ibα (GpIbα), but rather removes the inhibitory function of the neighboring A2 and A3 domains. Furthermore, the MD simulations combined with free energy perturbation calculations suggested that methionine oxidation may destabilize the binding interface between the A1 and A2 domains leading to unmasking of the GpIbα-binding site in the A1 domain.

14-3-3ß protein expression in eosinophilic meningitis caused by Angiostrongylus cantonensis infection.

BACKGROUND: Angiostrongylus cantonensis is a parasite endemic in the Southeast Asian and Pacific regions. Humans are incidentally infected either by eating uncooked intermediate hosts or by consuming vegetables containing the living third-stage larvae. The 14-3-3ß protein is a cerebrospinal fluid (CSF) marker of neuronal damage during the development of Creutzfeldt-Jakob disease. In addition, increased 14-3-3ß protein is also found in CSF from patients with a variety of neurological disorders. The goal of this study is to determine the roles of serum/CSF14-3-3ß protein in patients with eosinophilic meningitis. METHODS: In a cohort study among nine Thai laborers with eosinophilic meningitis due to eating raw snails (Pomacea canaliculata), we examined the CSF weekly while patients were still hospitalized and followed up the serum for 6 months. The levels of 14-3-3ß protein in CSF were analyzed by western blot and an in-house 14-3-3ß enzyme-linked immunosorbent assay (ELISA) measurement was established and tested in an animal model of eosinophilic meningitis. RESULTS: The elevated 14-3-3ß level was detected in the CSF from eight out of nine (81%) patients After 2 weeks of treatment, all patients showed a declined level or cleared of 14-3-3ß protein in the CSF. By developing an in-house ELISA for measurement of 14-3-3ß protein, it was found that the serum 14-3-3ß level was significantly increased in patients during initial visit. . This finding was consistent to the animal experiment result in which there was severe blood brain barrier damage three weeks after infection and increased 14-3-3ß protein expression in the CSF and serum by western blot and in house ELISA. After treatment, the serum 14-3-3ß level in meningitis patients was rapidly returned to normal threshold. There was a correlation between initial CSF 14-3-3ß level with severity of headache (r = 0.692, p = 0.039), CSF pleocytosis (r = 0.807, p = 0.009) and eosinophilia (r = 0.798, p = 0.01) in the CSF of patients with eosinophilic meningitis (Spearman's correlation test). CONCLUSIONS: The serum 14-3-3ß concentrations may constitute a useful marker for blood brain barrier damage severity and follow up in patients with eosinophilic meningitis caused by A. cantonensis.