Tracking oxidation-induced alterations in fibrin clot formation by NMR-based methods

Abstract

Plasma fibrinogen is an important coagulation factor and susceptible to post-translational modification by oxidants. We have reported impairment of fibrin polymerization after exposure to hypochlorous acid (HOCl) and increased methionine oxidation of fibrinogen in severely injured trauma patients. Molecular dynamics suggests that methionine oxidation poses a mechanistic link between oxidative stress and coagulation through protofibril lateral aggregation by disruption of AαC domain structures. However, experimental evidence explaining how HOCl oxidation impairs fibrinogen structure and function has not been demonstrated. We utilized polymerization studies and two dimensional-nuclear magnetic resonance spectrometry (2D-NMR) to investigate the hypothesis that HOCl oxidation alters fibrinogen conformation and T2 relaxation time of water protons in the fibrin gels. We have demonstrated that both HOCl oxidation of purified fibrinogen and addition of HOCl-oxidized fibrinogen to plasma fibrinogen solution disrupted lateral aggregation of protofibrils similarly to competitive inhibition of fibrin polymerization using a recombinant AαC fragment (AαC 419–502). DOSY NMR measurement of fibrinogen protons demonstrated that the diffusion coefficient of fibrinogen increased by 17.4%, suggesting the oxidized fibrinogen was more compact and fast motion in the prefibrillar state. 2D-NMR analysis reflected that water protons existed as bulk water (T2) and intermediate water (T2i) in the control plasma fibrin. Bulk water T2 relaxation time was increased twofold and correlated positively with the level of HOCl oxidation. However, T2 relaxation of the oxidized plasma fibrin gels was dominated by intermediate water. Oxidation induced thinner fibers, in which less water is released into the bulk and water fraction in the hydration shell was increased. We have confirmed that T2 relaxation is affected by the self-assembly of fibers and stiffness of the plasma fibrin gel. We propose that water protons can serve as an NMR signature to probe oxidative rearrangement of the fibrin clot.

Leukocyte activation primes fibrinogen for proteolysis by mitochondrial oxidative stress

“…Antifibrinolytics combined with aggressive fibrinogen replacement would likely be required to restore coagulation function in this setting. Other work examining the effects of fibrinogen oxidation by NMR demonstrated in vitro that adding oxidized fibrinogen to plasma changed the behavior of the bulk solution, altered fibrin clot structure, and induced coagulopathy [ 35 ]. Linear positive relationships were found between concentration of oxidized fibrinogen and fibrin polymerization in plasma, leading to significantly altered fibrin polymerization at concentrations above 30–40%.…”

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“…Peng et al introduce machine learning techniques to transform the nuclear magnetic resonance (NMR) correlational map into user-friendly information for point-of-care disease diagnostic and monitoring 11 . Lau et al utilize polymerization studies and two dimensional-nuclear magnetic resonance spectrometry (2D-NMR) to investigate the hypothesis that HOCl oxidation alters fibrinogen conformation and T2 relaxation time of water protons in the fibrin gels 12 . In the false alarm reduction problem, conventional machine learning based methods train a classification model using hand-crafted features as input to classify the alarms 13 .…”