S-Nitrosoglutathione Alters Fibrin Architecture Figure 2. GSNO alters human fibrin fiber density and fiber diameter. Platelet-poor plasma was incubated with GSNO as described in methods. Images of native fibrin clots were acquired using multiphoton microscopy and analyzed using custom designed computer software. Panels A,B,C,G show fibrin clot ‘‘read out’’ images at 0,1,2.5,3.75 mM GSNO, respectively. GSNO decreased fibrin fiber density, panel D, but increased fiber diameter to a maximum at 1 mM GSNO, panel E. Panel F plots fibrin density against fiber diameter. Panel C contained abnormal fibrin clusters with numerous thin diameter fibers protruding from a high intensity core. They decreased the average fiber diameter and shifted the expected density- diameter relationship panel F, point C (2.5 mM GSNO). Fibrin agglomerates were detected at the highest GSNO concentration tested, panel G. Fibrin clot parameters displayed on clot ‘‘read out’’ images: n (number of fibers), fiber density (fibers/100 um), fiber intensity (au, mean and CV), fiber diameter (nm, mean or median and CV), fiber thin-thick ratio vs control (diam-TTR (ctrl)), void area (%), crossing fibers (xfibers/fiber), trifunctional 2 2 2 junctions (xbranching/fiber), fibrinogen clusters (FbgClusters/mm ) and fibrin agglomerates (AG/mm ). *p,0.05 vs control. Area is 59659 um . Colour bars are image intensity. Scale bars are 8.5 um. doi:10.1371/journal.pone.0043660.g002 inspection revealed considerably more empty space in the GSNO treated clot than control. A plot of void volume against GSNO concentration showed that clot void volume remained relatively insensitive to GSNO below 1.75 mM (approx. 70%), indicating that spatial losses due to decreases in fiber density were offset by spatial increases in fiber diameter. However, as GSNO concen- tration increased beyond this point, there was a dramatic increase in void volume (from 72.7 to 85.2 to 100%) and projected void area (from 38.7 to 63.7 to 100%), Figure 3D, as both fiber density and fiber diameter decreased. These imaging results show that GSNO dramatically altered the porosity of human fibrin clots above a concentration threshold. GSNO Decreases Trifunctional Junctions, but has No Effect on the Fiber Density/Branch Point Ratio During fibrin network formation, protofibrils are known to elongate and form trifunctional junctions. These branch points are characterized predominantly by three fibers intersecting at a point, Figure 3. GSNO increases human fibrin clot void volume. Human fibrin clots were prepared and imaged as described in methods. 2D multiphoton images contain spatial information that can be used to quantify clot void volume and projected clot void area from a stack of three images. The calculation uses binary images of clots acquired at 40,50,60 microns above the clot surface, where white pixels are fibers and black pixels are empty space. Panel A shows 3 image stacks for control (C1,C2,C3) and 2.5 mM GSNO (B1,B2,B3), resulting in projected image C and B, respectively. Both clot void volume and projected clot area were insensitive to GSNO at low concentrations, but increased once GSNO exceeded 1.7 mM, panel D. At 3.75 mM GSNO, only fibrin agglomerates were present in plasma, rendering clot void volume effectively 100%, panel D. *p,0.05, compared to control.Scale bars are 8.5 um. doi:10.1371/journal.pone.0043660.g003 PLOS ONE | www.plosone.org 6 August 2012 | Volume 7 | Issue 8 | e43660