the fibrin network, as there was no evidence of fibrin movement within the image. Similar to our finding with GSNO, nitrated fibrinogen in the blood of cigarette smokers was found to generate fibrin networks with ‘‘clusters of fibrin’’ [16]. These ‘‘cluster’’ structures had .8 fibers crossing at a single point, and were related to the extent of fibrinogen nitration in the carboxy terminus of the fibrinogen b-chain (proximal region of the D domain). However, contrary to GSNO, nitrated fibrinogen increased the rate of fibrin polymerization, without affecting fibrin diameter. In another study, Marchi et al [38] reported that fibrin clots generated from human fibrinogen with a point deletion in the a-helical coiled-coil region (fibrinogen Caracus VI, deletion of Aa- Asn 80) resulted in fibrin networks with ‘‘some regions highly dense’’. Consistent with GSNO, this dysfibrinogenemia decreased the rate of fibrin polymerization, but unlike GSNO there was no change in fiber diameter. Taken together, different structural modifications of fibrinogen (GSNO binding or thiolation within the aC region, nitration of tyrosine in the bC-terminal region or ASN80 deletion in the a-helical coiled-coil region), result in both increased and decreased rates of fibrin polymerization; yet, seemingly produce similarly abnormal fibrinogen cluster struc- tures, suggesting that fibrinogen cluster structures result from a common, yet unknown, defect in the polymerization process. Fibrin Agglomerates We report for the first time, that high concentrations of GSNO completely inhibit fibrin network formation, while generating a heterogeneous population of fibrin agglomerates. These agglomer- ates were heterogeneous in both size (0.5–8 mm) and shape (spherical to irregular). Image analysis revealed that some of the agglomerates shared structural features with fibrin clusters (blunted protruding fibers form a central core), suggesting the latter may be intermediate forms of the former. It is reasonable to assume fibrin agglomerates consist of short protofibrils and oligomers that have failed to elongate and branch, but have retained the ability to laterally aggregate, or agglomerate. The role these agglomerates may play in vivo is unknown; however, if not quickly recycled into platelets or cleared from the circulation, they may increase the risk of microvascular complications given that a large number of agglom- erates exceed the luminal diameter of capillary beds (,5–6 um) and could thereby block capillary blood flow. In contrast to fibrin agglomerates generated during fibrin polymerization in the presence of GSNO, which remained in suspension, Sakharov et al. [32] reported that fibrin agglomerates (2–4 um) formed within a 5–8 micron superficial layer during fibrinolysis, eventually dissolved. Accordingly, fibrin agglomerates seemingly play a beneficial role in clearing fibrin fragments during fibrinolysis, or a potentially deleterious role if generated under coagulation conditions and not removed from the circulation. This may be of significance in the design of drug-eluting stents to prevent restenosis, where GSNO is combined with a polymeric coating, and has the property of inhibiting platelet aggregation with an EC50 of 5.0 mM [28]. Our results suggest this concentration of GSNO could induce local formation of fibrin agglomerates that may enter the circulation and potentially plug the downstream microcirculation. RBC-GSNO Theoretical Mechanism for Thrombus Remodeling While nitrosative stress can lead to increased circulating levels of S-nitrosothiols [17] the relatively high in vitro concentrations of GSNO required to first alter fibrin network architecture and then inhibit fibrin formation suggests that local sources of GSNO would likely be involved in vivo. Possible local sources of GSNO within S-Nitrosoglutathione Alters Fibrin Architecture blood clots are red blood cells, which release S-nitrosothiols in response to hypoxic gradients [19,40,41] platelet derived nitric oxide [42] and S-nitrosylated-albumin [43] which could both transnitrosate [44] plasma glutathione forming GSNO. Under the appropriate hypoxic conditions, which may exist for example in an intraluminal thrombus [45], it is possible that trapped erythrocytes within a fibrin network modulate on-going fibrin polymerization and remodel clot architecture [46] by releasing GSNO into the local clot environment, where it is free to diffuse within the clot and interact with fibrinogen or S-nitrosylate the free sulfhyryl group in factor XIIIa [31]. While the theory that RBC GSNO modulates fibrin architecture is intriguing, and somewhat com- plementary to the finding that RBCs carrying plasminogen activators modulate clot lysis [47], more research is required to determine if such a mechanism occurs in vivo. Conclusion The picture that emerges from this study is that GSNO dramatically alters human fibrin clot architecture in a dose- dependent manner, acting as a small molecule modulator of fibrin clot architecture. While many structural modifications of fibrin- ogen are associated with fine fibrin clot structures that are stiffer and resistant to clot lysis, GSNO produces coarse clot networks with decreased fibrin density and increased fiber diameter. Such clots are known to be more susceptible to clot lysis [36]; though, further research is required to determine the effects of GSNO on fibrinolysis. Our image analysis clearly shows that during fibrin polymerization, fibrin network architecture is sensitive to elevated GSNO concentration, where excessive GSNO leads to abnormal fibrin networks and even the generation of fibrin agglomerates. We conclude that GSNO may attenuate the risk of thrombosis and facilitate thrombus remodeling at low levels, but induce fibrin polymerization disorders at high levels. Supporting Information Figure S1 Computer analysis of fibrin clot networks. Fibrin clot images were acquired using multiphoton microscopy and analyzed using in-house custom designed software. A series of horizontal test lines along the image y-axis (at LN171,LN341,LN512,LN682,LN853) were generated to sample fluorescence intensity and determine fiber density. The corre- sponding intensity profiles are shown in panel B, where the various intensity peaks correspond to fluorescent fibers. The software then numbered and mapped all peaks in the intensity profile back into the clot image and calculated fiber density as the number of fibers per 100 um test line. The software program is interactive and allowed the operator to verify the identity of each fiber and edit false positives or false negative fibers, if necessary, using the graphical user interface shown in panel C. The four panels on the left, from top to bottom, are two subimages of the test line being evaluated (LN512), showing the test line in red and the numbered fibers. The bottom two panels are the intensity profile and first derivative plot of the intensity profile, respectively. More than one fiber was found to intersect at a branch point, as shown in the dashed circle area. Next, the diameter of each fiber was determined automatically. The right side of panel C shows a subimage of a fiber and the position where the diameter measurement was made. The solid circle corresponds to the fiber measurement. Automatic diameter measurements were validated against manual diameter measurements, panel D, and found to be in good agreement, panel E, p,0.001. Scale bar is 8.5 um. (PDF) PLOS ONE | www.plosone.org 11 August 2012 | Volume 7 | Issue 8 | e43660