The ability of nitric oxide (NO)-releasing xerogels to reduce fibrinogen-mediated adhesion of Staphylococcus aureus, Staphylococcus epidermidis, and Escherichia coli is described. A negative correlation was observed between NO surface flux and bacterial adhesion for each species tested. For S. aureus and E. coli, reduced adhesion correlated directly with NO flux from 0 to 30 pmol cm −2 s −1 . A similar dependence for S. epidermidis was evident from 18 to 30 pmol cm −2 s −1 . At a NO flux of 30 pmol cm −2 s −1 , surface coverage of S. aureus, S. epidermidis, and E. coli was reduced by 96, 48, and 88%, respectively, compared to non-NO-releasing controls. Polymeric NO release was thus demonstrated to be an effective approach for significantly reducing fibrinogen-mediated adhesion of both gram-positive and gram-negative bacteria in vitro, thereby illustrating the advantage of active NO release as a strategy for inhibiting bacterial adhesion in the presence of pre-adsorbed protein.

During blood vessel injury, fibrinogen is converted to fibrin, a polymer that serves as the structural scaffold of a blood clot. The primary function of fibrin is to withstand the large shear forces in blood and provide mechanical stability to the clot, protecting the wound. Understanding the biophysical forces involved in maintaining fibrin structure is of great interest to the biomedical community. Previous reports have identified the "A-a" knob-hole interaction as the dominant force responsible for fibrin's structural integrity. Herein, biochemical force spectroscopy is used to study knob-hole interactions between fibrin fragments and variant fibrinogen molecules to identify the forces occurring between individual fibrin molecules. The rupture of the "A-a" knob-hole interaction results in a characteristic profile previously unreported in fibrin force spectroscopy with two distinct populations of specific forces: 110 +/- 34 and 224 +/- 31 pN. In the absence of a functional "A" knob or hole "a", these forces cease to exist. We propose that the characteristic pattern represents the deformation of the D region of fibrinogen prior to the rupture of the "A-a" knob-hole bond.

Summary. Background: The formation of a fibrin clot is supported by multiple interactions, including those between polymerization knobs ÔAÕ and ÔBÕ exposed by thrombin cleavage and polymerization holes ÔaÕ and ÔbÕ present in fibrinogen and fibrin. Although structural studies have defined the ÔA-aÕ and ÔB-bÕ interactions in part, it has not been possible to measure the affinities of individual knob-hole interactions in the absence of the other interactions occurring in fibrin. Objectives: We designed experiments to determine the affinities of knob-hole interactions, either ÔA-aÕ alone or ÔA-aÕ and ÔB-bÕ together. Methods: We used surface plasmon resonance to measure binding between adsorbed fibrinogen and soluble fibrin fragments containing ÔAÕ knobs, desA-NDSK, or both ÔAÕ and ÔBÕ knobs, desAB-NDSK. Results: The desA-and desAB-NDSK fragments bound to fibrinogen with statistically similar K d Õs of 5.8 ± 1.1 lM and 3.7 ± 0.7 lM (P = 0.14), respectively. This binding was specific, as we saw no significant binding of NDSK, which has no exposed knobs. Moreover, the synthetic ÔAÕ knob peptide GPRP and synthetic ÔBÕ knob peptides GHRP and AHRPY, inhibited the binding of desA-and/or desAB-NDSK. Conclusions: The peptide inhibition findings show both ÔA-aÕ and ÔB-bÕ interactions participate in desAB-NDSK binding to fibrinogen, indicating ÔB-bÕ interactions can occur simultaneously with ÔA-aÕ. Furthermore, ÔA-aÕ interactions are much stronger than ÔB-bÕ because the affinity of desA-NDSK was not markedly different from desAB-NDSK.

A complex relationship exists between reduced, oxidized, and nitrosated glutathione (GSH, GSSG, and GSNO, respectively). Although previous studies have demonstrated S-nitrosoglutathione (GSNO) has potent antiplatelet efficacy, little work has examined the role of GSNO and related species on subsequent aspects of coagulation (e.g., fibrin polymerization). Herein, the effects of GSH, GSSG, and GSNO on the entire process of fibrin polymerization are described. Relative to normal fibrinogen, the addition of GSH, GSSG, or GSNO leads to prolonged lag times, slower rates of protofibril lateral aggregation and the formation of clots with lower final turbidities. Dose-dependent studies indicate the influence of GSH on fibrin formation is a function of both GSH and fibrinogen concentration. Studies with Aalpha251 recombinant fibrinogen (lacking alphaC regions) showed GSH had no influence on its polymerization, suggesting the glutathione species interact within the alphaC region of fibrinogen.

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