S-Nitrosoglutathione Acts as a Small Molecule Modulator of Human Fibrin Clot Architecture Ryon M. Bateman1,2*, Christopher G. Ellis3, Makoto Suematsu1, Keith R. Walley2 1 Department of Biochemistry, School of Medicine, Keio University, Tokyo, Japan, 2 Heart Lung Institute, University of British Columbia, Vancouver, Canada, 3 Department of Medical Biophysics, University of Western Ontario, London, Canada Abstract Background: Altered fibrin clot architecture is increasingly associated with cardiovascular diseases; yet, little is known about how fibrin networks are affected by small molecules that alter fibrinogen structure. Based on previous evidence that S- nitrosoglutathione (GSNO) alters fibrinogen secondary structure and fibrin polymerization kinetics, we hypothesized that GSNO would alter fibrin microstructure. Methodology/Principal Findings: Accordingly, we treated human platelet-poor plasma with GSNO (0.01–3.75 mM) and imaged thrombin induced fibrin networks using multiphoton microscopy. Using custom designed computer software, we analyzed fibrin microstructure for changes in structural features including fiber density, diameter, branch point density, crossing fibers and void area. We report for the first time that GSNO dose-dependently decreased fibrin density until complete network inhibition was achieved. At low dose GSNO, fiber diameter increased 25%, maintaining clot void volume at approximately 70%. However, at high dose GSNO, abnormal irregularly shaped fibrin clusters with high fluorescence intensity cores were detected and clot void volume increased dramatically. Notwithstanding fibrin clusters, the clot remained stable, as fiber branching was insensitive to GSNO and there was no evidence of fiber motion within the network. Moreover, at the highest GSNO dose tested, we observed for the first time, that GSNO induced formation of fibrin agglomerates. Conclusions/Significance: Taken together, low dose GSNO modulated fibrin microstructure generating coarse fibrin networks with thicker fibers; however, higher doses of GSNO induced abnormal fibrin structures and fibrin agglomerates. Since GSNO maintained clot void volume, while altering fiber diameter it suggests that GSNO may modulate the remodeling or inhibition of fibrin networks over an optimal concentration range. Citation: Bateman RM, Ellis CG, Suematsu M, Walley KR (2012) S-Nitrosoglutathione Acts as a Small Molecule Modulator of Human Fibrin Clot Architecture. PLoS ONE 7(8): e43660. doi:10.1371/journal.pone.0043660 Editor: Pieter H. Reitsma, Leiden University Medical Center, The Netherlands Received April 19, 2012; Accepted July 23, 2012; Published August 20, 2012 Copyright: 2012 Bateman et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Heart and Stroke Foundation of Canada (http://www.heartandstroke.com/site/c.ikIQLcMWJtE/b.2796497/k.BF8B/ Home.htm) and Michael Smith Foundation for Health Research (http://www.msfhr.org/) Fellowships (R.M.B.), MSFHR Distinguished Scholar Award (K.R.W.), and the Ministry of Education, Culture, Sports, Science and Technology, Japan, Global COE Program for Human Metabolomic Systems Biology (M.S., M.B.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: rbateman@z6.keio.jp Introduction Changes in fibrin clot architecture are increasingly recognized as an important risk factor for cardiovascular disease and thrombotic complications [1–5]. Fibrin clots form when thrombin catalyzes the conversion of fibrinogen to fibrin monomers that form double stranded protofibrils that elongate, branch (diverge and converge to form predominantly trifunctional branch junctions [6–8]), and laterally associate to form a three-dimen- sional fibrin network, visible at the gel point. Concomitantly, thrombin activated FXIII stabilizes and strengthens the fibrin network by cross-linking protofibrils and fibers [9]. The compo- sition of a network’s structural elements, including fiber density, diameter and branch point density [8] are determined by the clotting conditions, including fibrinogen, thrombin and calcium concentrations, FXIIIa activity, pH, ionic strength, temperature, whether fibrin is formed in plasma and by changes in fibrinogen structure [8,10–16]. S-nitrosoglutathione (GSNO) is a low molecular weight member of a class of molecules referred to as S-nitrosothiols that have emerged as important biological signaling molecules and impor- tant mediators of liver damage and animal survival during bacterial infection [17]. In blood, S-nitrosothiols/GSNO play an essential role in vascular homeostasis [17–19] and have relative plasma concentrations of 7 uM/100–300 nM, respectively [18,20], which can increase 10–30 fold in pneumonia or the absence of GSNO reductase [17,18,21]. Additionally, GSNO modulates hemostasis by inhibiting platelet activation and aggregation, and decreasing fibrinogen binding to platelets [22– 25]. GSNO has been used clinically as an antithrombotic agent [26,27] and is also being incorporated into drug-eluting stents to prevent restenosis [28]. GSNO has been reported to bind to fibrinogen a-chain c- termini, altering fibrinogen secondary structure and fibrin polymerization kinetics [29,30] and inhibit factor XIIIa activity and fiber cross-linking in a dose-dependent manner [31]. PLOS ONE | www.plosone.org 1 August 2012 | Volume 7 | Issue 8 | e43660