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Fibrinogen plays a crucial role in hemostasis, the regulation and maintenance of blood flow, and mediates surface-induced thrombosis at blood-contacting materials (e.g., implantable medical devices). Fibrinogen binds platelets and polymerizes to form fibrin, the structural scaffold of blood clots. The mechanisms by which fibrin formation is influenced by the surface properties of a material or therapeutic agents used to prevent platelet aggregation (e.g., S-nitrosothiols) are unclear. In addition, the exact mechanism of fibrin formation in solution remains controversial. Herein, the mechanism of fibrin formation in solution and at surfaces was investigated using surface-based analytical methods (surface plasmon resonance and atomic force microscopy). Additionally, the influence of Snitrosoglutathione (a potent anti-platelet therapeutic) and its decomposition products on the mechanism of fibrin formation were examined by turbidity measurements. The affinities of two fibrin fragments (desA-NDSK and desAB-NDSK) binding to fibrinogen were compared by SPR. The affinities of desA- and desAB-NDSK (5.8 plus/minus 1.1 and 3.7 plus/minus 0.7 ìM, respectively) were not statistically different from one another. Peptide inhibition studies showed “B-b” interactions occurred between desAB-NDSK but not desANDSK and fibrinogen, indicating B-b interactions may occur simultaneously to A-a interactions and between the same to interacting molecules. An atomic force microscopy method was developed to study the ruptures that occur upon the forced dissociation of desANDSK and desAB-NDSK from fibrinogen. The protein immobilization strategy and data collection procedures were varied to minimize multiple interactions and non-specific forces. Unlike the SPR experiments, B-b interactions were not detected in the forced dissociation of desAB-NDSK from fibrinogen. The influence of S-nitrosoglutathione on the mechanism of fibrin formation was studied using turbidity measurements. S-nitrosogluathione (GSNO) and its decomposition products (reduced and oxidized glutathione, GSH and GSSG, respectively) each inhibited fibrin formation as evidenced by longer lag times, lower Vmax values and lower final optical densities. GSH, GSSG and GSNO have numerous roles in physiology. However, the work presented herein is the first report of the inhibition of fibrin formation by GSH and GSSG. Lastly, the role of surface properties on the mechanism of fibrin formation at surfaces was studied by SPR. Fibrinogen was adsorbed to a hydrophobic and negatively charged surface and the availability of fibrinopeptide A (FpA), a critical site for thrombin activation, were measured by antibody binding. Approximately 3 times more FpA was measured on fibrinogen adsorbed at the hydrophobic surface compared to the negatively charged surface, indicating surface properties strongly influence the availability of FpA on adsorbed fibrinogen. By better understanding the mechanism of fibrin formation in solution and at surfaces, researchers may be able design materials or therapies to improve the standard of care in hemostatic disorders and improve the blood-compatibility of currently available medical devices.