IntroductionEndothelium-derived relaxing factor is important for vascular homeostasis and possesses qualities that may modulate vascular injury, including vasodilation, platelet inhibition, and inhibition of smooth muscle proliferation. S-nitrososerum albumin is a naturally occurring adduct of nitric oxide (NO) with a prolonged biologic half-life and is a potent vasodilator and platelet inhibitor. Given the avidity of serum albumin for subendothelial matrix and the antiproliferative effects of NO, we investigated the effects of locally delivered S-nitroso-bovine serum albumin (S-NO-BSA) and a polythiolated form of bovine serum albumin (pS-BSA) modified to carry several S-nitrosothiol groups (pS-NO-BSA) on neointimal responses in an animal model of vascular injury.Locally delivered S-NO-BSA bound preferentially to denuded rabbit femoral vessels producing a 26-fold increase in local concentration compared with uninjured vessels (P = 0.029). pS-NO-BSA significantly reduced the intimal/medial ratio (P = 0.038) and did so in conjunction with elevations in platelet (P < 0.001) and vascular cGMP content (P < 0.001). pS-NO-BSA treatment also inhibited platelet deposition (P = 0.031) after denuding injury. Comparison of BSA, S-NO-BSA, pS-NO-BSA, and control revealed a dose-response relationship between the amount of displaceable NO delivered and the extent of inhibition of neointimal proliferation at 2 wk (P < 0.001).Local administration of a stable protein S-nitrosothiol inhibits intimal proliferation and platelet deposition after vascular arterial balloon injury. This strategy for the local delivery of a long-lived NO adduct has potential for preventing restenosis after angioplasty. (J. Clin. Invest. 1995Invest. . 96:2630Invest. -2638

High-throughput experimental techniques have made possible the systematic sampling of the single mutation landscape for many proteins, defined as the change in protein fitness as the result of point mutation sequence changes. In a more limited number of cases, and for small proteins only, we also have nearly full coverage of all possible double mutants. By comparing the phenotypic effect of two simultaneous mutations with that of the individual amino acid changes, we can evaluate epistatic effects that reflect non-additive cooperative processes. The observation that epistatic residue pairs often are in contact in the 3D structure led to the hypothesis that a systematic epistatic screen contains sufficient information to identify the 3D fold of a protein.To test this hypothesis, we examined experimental double mutants for evidence of epistasis and identified residue contacts at 86% accuracy, including secondary structure elements and evidence for an alternative all--helical conformation. Positively epistatic contactscorresponding to compensatory mutations, restoring fitnesswere the most informative. Folded models generated from top-ranked epistatic pairs, when compared with the known structure, were accurate within 2.4 Å over 53 residues, indicating the possibility that 3D protein folds can be determined experimentally with good accuracy from functional assays of mutant libraries, at least for small proteins. These results suggest a new experimental approach for determining protein structure.