S-Nitrosoglutathione Alters Fibrin Architecture Figure 6. High concentrations of GSNO induced formation of abnormal fibrin clusters and fibrin agglomerates. Human fibrin clots were prepared and imaged as described in methods. When platelet-poor plasma was incubated with 2.5 mM GSNO, image analysis revealed the presence of abnormal fibrin clusters, characterized by high intensity cores with numerous protruding fibers, panels A and B. An unexpected finding was that 3.75 mM GSNO resulted in the formation of fibrin agglomerates, panel C. Panel D is a histogram of agglomerate long axis diameter. A projected binary image from 30,50,70 microns within the plasma sample, revealed the heterogeneous nature of the agglomerates, panel E. See data supplement figure 3 for additional images of agglomerates. Based on the GSNO dose-response curves, panel F, fibrin cluster formation preceded fibrin agglomerate formation. *p,0.05, compared to control. Scale bars for panels A,E are 8.5 um. Scale bars for panels B,C are 4.25 um. doi:10.1371/journal.pone.0043660.g006 Abnormal Fibrinogen Cluster Network Structural Elements At low concentrations, GSNO decreased both fibrin fiber density and branch point density, while increasing fiber diameter. However, as GSNO concentration increased above 1.75 mM, we detected abnormal structural elements, characterized by a dense core of fibrin with numerous thin fibers protruding from the periphery. These abnormal fibrin clusters were incorporated into PLOS ONE | www.plosone.org 10 August 2012 | Volume 7 | Issue 8 | e43660