Protein disulfide isomerase (PDI) is an oxidoreductase essential for folding proteins in the endoplasmic reticulum. The domain structure of PDI is a–b–b′–x–a′, wherein the thioredoxin-like a and a′ domains mediate disulfide bond shuffling and b and b′ domains are substrate binding. The b′ and a′ domains are connected via the x-linker, a 19-amino-acid flexible peptide. Here we identify a class of compounds, termed bepristats, that target the substrate-binding pocket of b′. Bepristats reversibly block substrate binding and inhibit platelet aggregation and thrombus formation in vivo. Ligation of the substrate-binding pocket by bepristats paradoxically enhances catalytic activity of a and a′ by displacing the x-linker, which acts as an allosteric switch to augment reductase activity in the catalytic domains. This substrate-driven allosteric switch is also activated by peptides and proteins and is present in other thiol isomerases. Our results demonstrate a mechanism whereby binding of a substrate to thiol isomerases enhances catalytic activity of remote domains.

N itric oxide is a secondary messenger involved in the cGMP cascade, vasodilation, and a known inhibitor of platelet aggregation. S-nitrosothiols (RSNOs) are formed via nitrosation of thiols under aerobic conditions (1). Apart from being cellular sources of NO, they also prolong its half-life (2, 3). RSNOs are involved in signaling pathways, immune responses, and the actions of nitrovasodilating compounds (4-7). Therefore, a study involving the effects and transport of S-nitroso-BSA (BSA-NO) into live cells is of utmost physiological importance and the center of pharmacological interest (8).Many indirect, discontinuous fluorescent, electrochemical, and colorometric assays have been developed for RSNOs (8, 9). However, none of these are conducive to measuring the transport of RSNO-bound NO into live cells, in vitro. The probe for NO influx presented here is N-dansylhomocysteine (DnsHCys). The fluorescence of this compound is completely quenched on its S-nitrosation, yielding N-dansyl-S-nitrosohomocysteine (DnsHCysNO). Here we show that DnsHCysNO is a direct fluorogenic substrate for protein disulfide isomerase (PDI).PDI acts as a chaperone molecule in the endoplasmic reticulum where it catalyses protein thiol exchange reactions. Hotchkiss et al. (10) have reported that PDI is also secreted by endothelial cells as well as deposited on the cell surface. Recent studies have presented indirect evidence for the involvement of cell-surface PDI (csPDI) in the influx of 12). Stamler and coworkers (13) also have shown that the export of intracellular NO from red blood cells to be facilitated by S-nitrosation of the cysteine residues in the hemoglobin-binding cytoplasmic domain of the anion exchanger AE1. Membranebound PDI may catalyze the formation of AE1-SNO and the subsequent export of cytosolic NO.Here, the extracellular RSNO-dependent quenching of the DnsHCys fluorescence was shown to be a csPDI-dependent process with the aid of antisense-mediated underexpression of PDI and the sense-mediated overexpression of PDI in HT1080 fibrosarcoma cells as well as with a cell-impermeant inhibitor that reacts with vicinal dithiols. In addition, N-dansylhomocystine (DnsHCys 2 ) is shown to be a sensitive intracellular probe for the kinetic characterization of csPDI in human umbilical vein endothelial cells (HUVECs), hamster lung fibroblasts, and HT1080 fibrosarcoma cells. Based on this data, a mechanism for csPDI-meditated intracellular S-nitrosation, by extracellular RSNOs, has been proposed. Materials and Methods Synthesis of S-Nitrosoglutathione (GSNO).Glutathione (GSH, Sigma) was dissolved in ice-cold 0.5 M HCl. Equimolar sodium nitrite was added and the reaction was carried out in the dark at 4°C for 40 min. The pH of the reaction mixture was adjusted to 7.0 and crystallized by the slow addition of cold acetone. BSA-NO was synthesized by using the above-mentioned protocol (3). Synthesis of DnsHCysNO.HCysNO was prepared by treating HCys (Sigma) with acidified nitrite. Dansylation was carried out in 0.1 M phosphate buffer (pH ...

The generation of an 'angiogenic switch' is essential for tumor growth, yet its regulation is poorly understood. In this investigation, we explored the linkage between metastasis and angiogenesis through CXCL12/CXCR4 signaling. We found that CXCR4 regulates the expression and secretion of the glycolytic enzyme phosphoglycerate kinase 1 (PGK1). Overexpression of PGK1 reduced the secretion of vascular endothelial growth factor and interleukin-8 and increased the generation of angiostatin. At metastatic sites, however, high levels of CXCL12 signaling through CXCR4 reduced PGK1 expression, releasing the angiogenic response for metastastic growth. These data suggest that PGK1 is a critical downstream target of the chemokine axis and an important regulator of an 'angiogenic switch' that is essential for tumor and metastatic growth.

Key Points• The major subpopulation of platelets involved in thrombus development form via regulated necrosis involving cyclophilin D.• Necrotic platelets may be targeted independent of platelet activation.A subpopulation of platelets fulfills a procoagulant role in hemostasis and thrombosis by enabling the thrombin burst required for fibrin formation and clot stability at the site of vascular injury. Excess procoagulant activity is linked with pathological thrombosis. The identity of the procoagulant platelet has been elusive. The cell death marker 4-[N-(S-glutathionylacetyl)amino]phenylarsonous acid (GSAO) rapidly enters a subpopulation of agonist-stimulated platelets via an organic anion-transporting polypeptide and is retained in the cytosol through covalent reaction with protein dithiols. Labeling with GSAO, together with exposure of P-selectin, distinguishes necrotic from apoptotic platelets and correlates with procoagulant potential. GSAO 1 platelets form in occluding murine thrombi after ferric chloride injury and are attenuated with megakaryocyte-directed deletion of the cyclophilin D gene. These platelets form a procoagulant surface, supporting fibrin formation, and reduction in GSAO 1 platelets is associated with reduction in platelet thrombus size and fibrin formation. Analysis of platelets from human subjects receiving aspirin therapy indicates that these procoagulant platelets form despite aspirin therapy, but are attenuated by inhibition of the necrosis pathway. These findings indicate that the major subpopulation of platelets involved in fibrin formation are formed via regulated necrosis involving cyclophilin D, and that they may be targeted independent of platelet activation. (Blood. 2015;126(26):2852-2862

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Protein action in nature is largely controlled by the level of expression and by post-translational modifications. Post-translational modifications result in a proteome that is at least two orders of magnitude more diverse than the genome. There are three basic types of post-translational modifications: covalent modification of an amino acid side chain, hydrolytic cleavage or isomerization of a peptide bond, and reductive cleavage of a disulfide bond. This review addresses the modification of disulfide bonds. Protein disulfide bonds perform either a structural or a functional role, and there are two types of functional disulfide: the catalytic and allosteric bonds. The allosteric disulfide bonds control the function of the mature protein in which they reside by triggering a change when they are cleaved. The change can be in ligand binding, substrate hydrolysis, proteolysis, or oligomer formation. The allosteric disulfides are cleaved by oxidoreductases or by thiol/disulfide exchange, and the configurations of the disulfides and the secondary structures that they link share some recurring features. How these bonds are being identified using bioinformatics and experimental screens and what the future holds for this field of research are also discussed.

Most proteins in nature are chemically modified after they are made to control how, when, and where they function. The 3 core features of proteins are posttranslationally modified: amino acid side chains can be modified, peptide bonds can be cleaved or isomerized, and disulfide bonds can be cleaved. Cleavage of peptide bonds is a major mechanism of protein control in the circulation, as exemplified by activation of the blood coagulation and complement zymogens. Cleavage of disulfide bonds is emerging as another important mechanism of protein control in the circulation. Recent advances in our understanding of control of soluble blood proteins and blood cell receptors by functional disulfide bonds is discussed as is how these bonds are being identified and studied.

We demonstrate mechanochemical regulation of platelet adhesion to von Willebrand factor in thrombosis and hemostasis.