S-Nitrosoglutathione Alters Fibrin Architecture Figure 5. Crossing fibers are more sensitive than fiber density to GSNO. Images of native human fibrin clots were acquired using multiphoton microscopy and analyzed using custom designed computer software, as described in methods. Panels A,B,E,F show surface plots of fibrin fibers observed in the corresponding multiphoton image and contour figure, panels C,D respectively. Arrows connect the regions of interest. When two fibers cross at a point in space, there is a .40+/215% increase in fluorescence intensity at the point of contact, that is proportional to fiber intensity. Panel A shows a trifunctional junction (F1 = F2+F3), panel B shows one fiber (F2) passing beneath another (F1), panel E shows two fibers (F1 and F2) crossing and panel F shows six fiber segments (or possibly three fibers) crossing at a point. As GSNO concentration increased, crossing fibers per area decreased, panel G. The ratio of fiber density to crossing fibers increased with increasing GSNO concentration, indicating the decrease in crossing fibers was greater than the decrease in fiber density, panel H. *p,0.05, compared to control. Area is 59659 um2. Colour bars are image intensity. Scale bars are 700 nm. doi:10.1371/journal.pone.0043660.g005 GSNO Decreased Crossing Fibers and Increased the Fiber Density/Crossing Fiber Ratio In addition to trifunctional junctions, we also observed crossing fibers at a point in space within the network. These network structural elements were readily identified in contour images and surface plots, as shown in Figure 5, by high regions of intensity at the point of contact between fibers. During computer analysis, identified fibers were sampled along their respective lengths. If a local increase in fiber intensity was detected, then a search was made for a putative crossing fiber, which was similarly sampled along its length. If both fibers had proportional increases in intensity .40%, they were considered to be in contact; otherwise, one fiber was simply passing above or below the other, as shown in Figure 5B. As GSNO concentration increased, crossing fibers per unit area decreased, Figure 5G. When compared to changes in fiber density (Figure 2D), however, the ratio of fiber density to crossing fibers was found to increase from 0.760.1 to 1.560.2, p,0.05 with increasing GSNO concentration, Figure 5H, sug- gesting that crossing fibers were more sensitive to GSNO than fiber density. (Note, we list crossing fiber statistics as xlinks in clot ‘‘read out’’ images, Figure 2, panels A,B,C,G.) Abnormal Fibrinogen Polymerization and Clot Structures As GSNO concentration increased to 2.5 mM, visual inspection of human fibrin clot images revealed the presence of abnormal network structures, referred to as fibrin clusters. Fibrin clusters were incorporated into the fibrin network and defined by an irregularly shaped high intensity core with .8–10 thin fibers protruding from the core periphery, Figure 6, panels A and B. And when plasma was treated with 3.75 mM GSNO, we discovered that fibrin fibers had been replaced by fibrin agglomerates. Fibrin agglomerates were suspended in plasma and had a range of irregular shapes and sizes (0.5–8 um) with some having thin blunted fibers protruding from the core, as seen in multiphoton and projected binary images, Figure 6, panel C and E, respectively, and Figure S3. Measurement along the agglomerate long axis found them to have a mean diameter of 2.8 um, with 11.5% having diameters greater than that of an average capillary (5–6 mm). Of note was that agglomerates were only detected at GSNO concentrations greater than that required to generate fibrin clusters, Figure 6, panel F. Discussion Summary In this study, we used multiphoton microscopy to investigate the effects of S-nitrosoglutathione (GSNO) on native human fibrin clot architecture. We report for the first time that a series of structural changes occurred as fibrin network formation was progressively inhibited by increasing GSNO concentration. GSNO decreased fibrin density to the point of completely inhibiting fibrin network formation. Concomitantly, fiber diameter increased by 25% to a maximum diameter of 835 nm at 1 mM GSNO, after which fibers became thinner as GSNO was further increased. Image analysis showed that clot void volume (,70%) was relatively insensitive to changes in GSNO over a lower concentration range (0.01– 1.75 mM), as decreases in fibrin density were spatially offset by increases in fiber diameter. However, clot void volume rapidly increased as GSNO increased above this lower limit, as both fibrin density and fiber diameter decreased. This significant increase in clot void area coincided with the appearance of abnormal network structural elements, referred to here as ‘‘fibrin clusters’’. These highly dense abnormal structures may signal the GSNO threshold for the onset of abnormal fibrin polymerization. Notwithstanding the obvious structural abnormalities, image analysis found no evidence of fiber movement, indicating the clot network remained stable [9]. This inherent stability was likely due, in part, to the finding that fiber branching was insensitive to GSNO concentra- tion. Over the same concentration range, crossing fibers decreased, suggesting that clot stiffness was likely affected. All clots reached their respective gel points within 5 minutes of thrombin activation, except for human plasma treated with the highest GSNO tested. In this case, fibrin networks were completely inhibited and replaced, unexpectedly, by fibrin agglomerates. Changes in Fibrinogen Structure and Altered Fibrin Polymerization are Associated with GSNO Induced Changes in Fibrin Clot Architecture Increasing evidence indicates that fibrinogen is susceptible to a variety of structural modifications [10,16,29,30,38,39] that impact on its ability to polymerize and form fibrin networks. The mechanism whereby GSNO alters fibrinogen secondary structure is controversial. Akheter et al [29] reported that GSNO interacted with fibrinogen S-nitrosothiol binding domains in the aromatic rich aC-terminal region, increasing a-helical content and altering fibrinogen secondary structure. The effect was reversible, suggesting GSNO was an allosteric inhibitor. In contrast, Geer et al [30] proposed that GSNO alters fibrin polymerization by thiolating an exposed disulfide in the aC-region. While the mechanism remains uncertain, both studies found GSNO decreased the initial rate of polymerization. In the Geer study [30], this was related to increased lag time and decreased final turbidity. Here, we extended these biochemical findings by imaging and quantifying the nature and extent of the GSNO induced changes in fibrin network structure. We found that GSNO decreased fibrin fiber density and fibrin branch point density while increasing fiber diameter, resulting in ‘‘coarse’’ fibrin networks, known to be more susceptible to fibrinolysis [36] and have weaker mechanical properties [8]. It is intriguing that GSNO maintained clot void volume, while altering fiber diameter, suggesting GSNO may modulate the remodeling or break down of fibrin networks over an optimal concentration range. Additionally, GSNO maintained the fiber density to branch point density ratio, an important feature of clot structure [37]; yet, it increased the fiber density to crossing fiber ratio, suggesting fiber interactions were more sensitive to GSNO than fiber branching. Taken together, GSNO functions as a small molecule modulator of fibrin network structure over a range of concentrations. PLOS ONE | www.plosone.org 9 August 2012 | Volume 7 | Issue 8 | e43660