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Tracking Oxidation-Induced Alterations in Fibrin Clot Formation by NMR

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Tracking Oxidation-Induced Alterations in Fibrin Clot Formation by NMR www.nature.com/scientificreports OPEN Tracking oxidation‑induced alterations in fbrin clot formation by NMR‑based methods Wai‑Hoe Lau1, Nathan J. White2, Tsin‑Wen Yeo1,4, Russell L. Gruen3* & Konstantin Pervushin5* Plasma fbrinogen is an important coagulation factor and susceptible to post‑translational modifcation by oxidants. We have reported impairment of fbrin polymerization after exposure to hypochlorous acid (HOCl) and increased methionine oxidation of fbrinogen in severely injured trauma patients. Molecular dynamics suggests that methionine oxidation poses a mechanistic link between oxidative stress and coagulation through protofbril lateral aggregation by disruption of AαC domain structures. However, experimental evidence explaining how HOCl oxidation impairs fbrinogen 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 fbrinogen conformation and T2 relaxation time of water protons in the fbrin gels. We have demonstrated that both HOCl oxidation of purifed fbrinogen and addition of HOCl‑ oxidized fbrinogen to plasma fbrinogen solution disrupted lateral aggregation of protofbrils similarly to competitive inhibition of fbrin polymerization using a recombinant AαC fragment (AαC 419–502). DOSY NMR measurement of fbrinogen protons demonstrated that the difusion coefcient of fbrinogen increased by 17.4%, suggesting the oxidized fbrinogen was more compact and fast motion in the prefbrillar state. 2D‑NMR analysis refected that water protons existed as bulk water (T2) and intermediate water (T2i) in the control plasma fbrin. Bulk water T2 relaxation time was increased twofold and correlated positively with the level of HOCl oxidation. However, T2 relaxation of the oxidized plasma fbrin gels was dominated by intermediate water. Oxidation induced thinner fbers, in which less water is released into the bulk and water fraction in the hydration shell was increased. We have confrmed that T2 relaxation is afected by the self‑assembly of fbers and stifness of the plasma fbrin gel. We propose that water protons can serve as an NMR signature to probe oxidative rearrangement of the fbrin clot. Fibrinogen is a 340 kDa glycoprotein, physiologically present in blood plasma at concentrations from 2 to 4 g/L, that self-associates to form the fbrin clot at wounds afer its activation by thrombin1. It is composed of two pairs of three non-identical chains Aα, Bβ, and γ connected with 29 disulfde bonds. Together, the chains comprise a symmetrical molecule consisting of one globular E region fanked on each side by globular D regions that are connected by three-stranded alpha-helical coiled coils2,3. Fibrin is formed by thrombin-mediated proteolytic cleavage and removal of N-terminal fbrinopeptides from the respective Aα and Bβ chains. Te exposed bind- ing sites of α- and β-“knobs” are complementary to a- and b-“holes” in the γ and β nodules in the D regions of adjoining monomers. Knob-hole associations each result in the formation of half-staggered, double-stranded protofbrils, which then associate laterally to form fbrin fbers, and ultimately the branching hydrogel network structure of the hemostatic clot 4. Lateral association is contributed to by interactions between αC regions of the fbrin monomers (αC-αC interactions) during polymerization, which is important for mechanical stifness, stability, and durability of fbrin clots5–7. Polymerization of fbrinogen lacking its αC regions is delayed but not completely inhibited7. 1Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore. 2Department of Emergency Medicine, University of Washington School of Medicine, Seattle, Washington, USA. 3ANU College of Health and Medicine, The Australian National University, Canberra, ACT , Australia. 4Communicable Diseases Centre, Institute of Infectious Disease and Epidemiology, Tan Tock Seng Hospital, Singapore, Singapore. 5School of Biological Sciences, Nanyang Technological University, Singapore, Singapore. *email: [email protected]; [email protected] Scientifc Reports | (2021) 11:15691 | https://doi.org/10.1038/s41598-021-94401-3 1 Vol.:(0123456789) www.nature.com/scientificreports/ a) 0.8 b) nm 0Mµ 10 µM ) -1 250 µ s A350 25 M 6 - t 0.6 50 µM a 10 nit) 75 µM 200 (x yu 100Mµ ding a 0.4 125Mµ * e trar 150 i 150Mµ rate er rb on (a *** 0.2 100 *** zati i *** r orbanc *** e s m 50 Ab 0.0 010203040506070 Poly 0 Time (minute) 010255075100 125150 c) HOCl concentration(μM) 40 Thrombin time d) Reptilasetime 300 ) 30 ) 250 (sec 200 (mg/dL time 20 150 10 100 Clotting Fibrinogen 50 0 0 25 50 75 100125 150 0 0255075100 125150 e) HOCl concentration(μM) 1.0 nm f) 0% ) -1 300 10% s 6 A350 0.8 - ) 20% at 10 250 30% unit 0.6 40% (x e ry t 200 a 50% ra eading r n bitr 0.4 150 o ar * ( zati i 100 ** 0.2 r 50 Absorbance 0.0 Polyme 0 010203040506070 01020304050 Time (minute) %HOCl-oxidized fibrinogen g) 0.8 h) nm 0% ) 350 -1 250 5% s 6 - tA 0.6 10% nit) ga 200 u 20% (x10 30% ** rary 0.4 40% rate 150 eadin t i b er r *** (a 100 0.2 *** banc r *** 50 Abso 0.0 Polymerization 0102030405060 0 0510 20 30 40 Time (minute) %AαC fragment Scientifc Reports | (2021) 11:15691 | https://doi.org/10.1038/s41598-021-94401-3 2 Vol:.(1234567890) www.nature.com/scientificreports/ ◂Figure 1. (a) Turbidity curves of non-oxidized fbrinogen (0 µM HOCl) and purifed fbrinogen solutions oxidized with an increasing HOCl concentrations (10, 25, 50, 75, 100, 125, 150 μM). Fibrinogen without HOCl oxidation (0 µM HOCl) was used as a control. (b) Polymerization rate for fbrinogen control and the fbrinogen solutions oxidized with increasing HOCl concentrations (10, 25, 50, 75, 100, 125, 150 μM). (c) Trombin and reptilase times for the fbrinogen control and fbrinogen solutions oxidized with increasing HOCl concentrations. Trombin and reptilase time measured the clotting time needed for fbrin clot formation afer the addition of thrombin and reptilase, receptively. Te normal value of thrombin time and reptilase time are < 21 s and < 24 s, respectively. Te red and blue dotted lines indicate the upper limit of the thrombin and reptilase times required for normal fbrin clot formation. (d) Te concentration of functional fbrinogen in fbrinogen solution afer HOCl oxidation. Clauss fbrinogen was used to measure the concentration of functional fbrinogen in the fbrinogen control and purifed fbrinogen solutions oxidized with an increasing HOCl. Te normal fbrinogen level is at the range of 2–4 mg/mL (200–400 mg/dL). Te black dotted line indicates the upper limit of normal fbrinogen concentration. (e) Turbidity curves of control plasma fbrinogen and plasma fbrinogen solution added with an increasing level of HOCl-oxidized fbrinogen (10, 20, 30, 40, and 50%). Plasma fbrinogen solution without HOCl-oxidized fbrinogen (0% HOCl-oxidized fbrinogen) was used as control. (f) Polymerization rate for control plasma fbrinogen and plasma fbrinogen solution added with increasing HOCl-oxidized fbrinogen (pre-oxidation of purifed fbrinogen by 150 µM HOCl). (g) Turbidity curves of fbrinogen control and the purifed fbrinogen solutions added with an increasing percentage of AαC fragment (5, 10, 20, 30, and 40%). Te copolymerization of the protein mixtures was activated by incubation with 0.16 NIH U/mL thrombin at 37 °C for 1 h. (h) Polymerization rate of fbrinogen control and the purifed fbrinogen added with an increasing percentage of AαC fragment. P values that were 0.05 and less were considered statistically signifcant, as assessed using ANOVA-Bonferroni test. * P < 0.05; ** P < 0.01; *** P < 0.001. Fibrinogen is also highly susceptible to many post-translational modifcations (PTMs), especially from oxi- dants and free radicals3. Oxidant agents are a potentially important source of oxidative fbrinogen PTMs that have been identifed in trauma patients having impaired clot formation 13. Neutrophils are recruited to the blood afer trauma and are activated to release histones and DNA as neutrophil extracellular traps (NETs) and myelop- 8 eroxidase (MPO), which synthesizes HOCl by the MPO/ hydrogen peroxide (H 2O2) oxidant system . HOCl is predominantly generated in the plasma, where it can oxidize methionine residues to methionine sulfoxide as a host-mediated bacterium killing mechanism9. Recent evidence supports an important role for fbrinogen in the regulation of these responses by sequestering histones as histone-fbrinogen complexes10. We have previously reported that exposure of fbrinogen to HOCl in vitro has profound efects upon fbrin polymerization, produc- ing weak, sof, and thin fbered fbrin clots that are resistant to enzymatic degradation by the protease plasmin 11. We found that methionine at positions AαM476, BβM367, and γM78 were oxidized to methionine sulfoxide by HOCl, though the AαC domain was preferentially oxidized 11. Molecular dynamics (MD) simulation revealed that oxidation within the AαC domain may promote the opening of key beta-hairpin structures making αC domain dimerization energetically unfavorable in comparison with native structures 12,13. Tese data suggest a relationship and potential mechanism by which PTMs by oxidants generated in the blood afer trauma injury can contribute directly to impaired clot formation. Tis is potentially signifcant because the risk of death for trauma patients presenting with impaired clot formation is increased up to sixfold14. NMR is an important and useful tool for assessing the structure of gels by examining the movement of water molecules which can be afected by fbrillar structures, cells, vessels, extracellular matrix, and other macromol- 15 ecules contained therein . Parameters including spin–lattice (T1) relaxation and spin–spin (T2) relaxation time and difusion coefcients can be used to identify important structural characteristics 15,16. Molecular fngerprints of complex biological fuids such as blood have been encoded using two-dimensional (2D) T 1/T2 correlations. NMR relaxometry has been used to investigate oxygenated (oxy-Hb), deoxygenated (deoxy-Hb), and oxidized 17 (oxidized Hb) hemoglobin (Hb) derivatives with respect to their altered T1/T2 relaxation states .
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