Thiol Exchange Research Articles

Facile preparation of surface-exchangeable core@shell iron oxide@gold nanoparticles for magnetic solid-phase extraction: Use of gold shell as the intermediate platform for versatile adsorbents with varying self-assembled monolayers

The core@shell Fe3O4@Au nanoparticles (NPs) functionalized with exchangeable self-assembled monolayers have been developed for mode switching magnetic solid-phase extraction (MSPE) using high performance liquid chromatography with ultraviolet detection. The adsorbents were synthesized by chemical coprecipitation to prepare magnetic cores followed by sonolysis to produce gold shells. Functionalization of Fe3O4@Au NPs surface was realized through self-assembly of commercially available low molecular weight thiol-containing ligands using gold shells as intermediate platform and the dynamic nature of Au–S chemistry allowed substituent of one thiol-containing ligand with another simply by thiol exchange process. The resultant adsorbents were characterized by transmission electronic microscopy, Fourier transform infrared spectroscopy, elemental analysis, contact angle measurement, and vibrating sample magnetometry. To evaluate the versatile performance of the developed MSPE adsorbents, they were applied for normal-phase SPE followed by reversed-phase SPE. A few kinds of diphenols and polycyclic aromatic hydrocarbons (PAHs) were employed as model analytes, respectively. The predominant parameters affecting extraction efficiency were investigated and optimized. Under the optimum experimental conditions, wide dynamic linear range (6.25–1600μgL−1 for diphenols and 1.56–100μgL−1 for PAHs) with good linearity (r2≥0.989) and low detection limits (0.34–16.67μgL−1 for diphenols and 0.26–0.52μgL−1 for PAHs) were achieved. The advantage of the developed method is that the Fe3O4@Au NPs could be reutilized for preconcentrating diverse target analytes in different SPE modes sequentially simply through treatment with desired thiol-containing ligands.

The control of hyperhomocysteinemia through thiol exchange mechanisms by mesna

In hyperhomocysteinemic patients, after reaction with homocysteine-albumin mixed disulfides (HSS-ALB), mesna (MSH) forms the mixed disulfide with Hcy (HSSM) which can be removed by renal clearance, thus reducing the plasma concentration of total homocysteine (tHcy). In order to assess the HSS-ALB dethiolation via thiol exchange reactions, the distribution of redox species of cysteine, cysteinylglycine, homocysteine and glutathione was investigated in the plasma of healthy subjects: (i) in vitro, after addition of 35 μM reduced homocysteine (HSH) to plasma for 72 h, followed by MSH addition (at the concentration range 10-600 μM) for 25 min; (ii) in vivo, after oral treatment with methionine (methionine, 200 mg/kg body weight, observation time 2-6 h). In both experiments the distribution of redox species, but not the total amount of each thiol, was modified by thiol exchange reactions of albumin and cystine, with changes thermodynamically related to the pKa values of thiols in the corresponding mixed disulfides. MSH provoked a dose-response reversal of the redox state of aged plasma, and the thiol action was confirmed by in vivo experiments. Since it was observed that the dimesna production could be detrimental for the in vivo optimization of HSSM formation, we assume that the best plasma tHcy lowering can be obtained at MSH doses producing the minimum dimesna concentration in each individual.

Endo-PDI is required for TNFα-induced angiogenesis

Protein disulfide isomerase (PDI) and its homologs are oxidoreductases facilitating protein folding in the ER. Endo-PDI (also termed ERp46) is highly expressed in endothelial cells. It belongs to the PDI family but its physiological function is largely unknown. We studied the role of Endo-PDI in endothelial angiogenic responses. Stimulation of human umbilical vein endothelial cells (with TNFα (10ng/ml) increased ERK1/2 phosphorylation. This effect was largely attenuated by Endo-PDI siRNA, whereas JNK and p38 MAP kinase phosphorylation was Endo-PDI independent. Similarly, TNFα-stimulated NF-κB signaling determined by IκBα degradation as well as TNFα-induced ICAM expression was unaffected by Endo-PDI siRNA. The action of Endo-PDI was not mediated by extracellular thiol exchange or cell surface PDI as demonstrated by nonpermeative inhibitors and PDI-neutralizing antibody. Moreover, exogenously added PDI failed to restore ERK1/2 activation after Endo-PDI knockdown. This suggests that Endo-PDI acts intracellularly potentially by maintaining the Ras/Raf/MEK/ERK pathway. Indeed, knockdown of Endo-PDI attenuated Ras activation measured by G-LISA and Raf phosphorylation. ERK activation influences gene expression by the transcriptional factor AP-1, which controls MMP-9 and cathepsin B, two proteases required for angiogenesis. TNFα-stimulated MMP-9 and cathepsin B induction was reduced by silencing of Endo-PDI. Accordingly, inhibition of cathepsin B or Endo-PDI siRNA blocked the TNFα-stimulated angiogenic response in the spheroid outgrowth assays. Moreover ex vivo tube formation and in vivo Matrigel angiogenesis in response to TNFα were attenuated by Endo-PDI siRNA. In conclusion, our study establishes Endo-PDI as a novel, important mediator of AP-1-driven gene expression and endothelial angiogenic function.

Studies of Reversible Conjugate Additions

AbstractBenzalcyanoacetamides were designed and synthesized as reversible thiol conjugate addition acceptors. These thia‐conjugate additions can rapidly and reversibly achieve equilibrium under aqueous conditions at neutral pH. Kinetic studies show that electron‐withdrawing groups at the 4‐position of the phenyl ring of the benzalcyanoacetamides promote the conjugate addition at equilibrium. Dynamic thiol exchange of these conjugate acceptors is faster than singly activated α,β‐unsaturated carbonyl compounds. These thia‐conjugate additions can be assembled as potentially useful components in dynamic combinatorial chemistry.

Neutral polymeric micelles for RNA delivery.

RNA interference (RNAi) drugs have significant therapeutic potential, but delivery systems with appropriate efficacy and toxicity profiles are still needed. Here, we describe a neutral, ampholytic polymeric delivery system based on conjugatable diblock polymer micelles. The diblock copolymer contains a hydrophilic poly[N-(2-hydroxypropyl)methacrylamide-co-N-(2-(pyridin-2-yldisulfanyl)ethyl)methacrylamide) (poly[HPMA-co-PDSMA]) segment to promote aqueous stability and facilitate thiol-disulfide exchange reactions and a second ampholytic block composed of propylacrylic acid (PAA), dimethylaminoethyl methacrylate (DMAEMA), and butyl methacrylate (BMA). The poly[(HPMA-co-PDSMA)-b-(PAA-co-DMAEMA-co-BMA)] was synthesized using reversible addition-fragmentation chain transfer (RAFT) polymerization with an overall molecular weight of 22 000 g/mol and a PDI of 1.88. Dynamic light scattering and fluorescence measurements indicated that the diblock copolymers self-assemble under aqueous conditions to form polymeric micelles with a hydrodynamic radius and critical micelle concentration of 25 nm and 25 μg/mL, respectively. Red blood cell hemolysis experiments show that the neutral hydrophilic micelles have potent membrane destabilizing activity at endosomal pH values. Thiolated siRNA targeting glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was directly conjugated to the polymeric micelles via thiol exchange reactions with the pyridal disulfide groups present in the micelle corona. Maximum silencing activity in HeLa cells was observed at a 1:10 molar ratio of siRNA to polymer following a 48 h incubation period. Under these conditions 90% mRNA knockdown and 65% protein knockdown at 48 h was achieved with negligible toxicity. In contrast the polymeric micelles lacking a pH-responsive endosomalytic segment demonstrated negligible mRNA and protein knockdown under these conditions. The potent mRNA knockdown and excellent biocompatibility of the neutral siRNA conjugates demonstrate the potential utility of this carrier design for delivering therapeutic siRNA drugs.

“Isothermal” LCST transitions triggered by bioreduction of single polymer end-groups

The α-termini of RAFT-derived thermoresponsive polymers were functionalised via thiol exchange with pyridyl disulfide. Addition of hydrophilic end-groups dramatically increased the observed cloud point of the polymers, without changing the composition of the main chain. Selective cleavage of the disulfide-linked end-groups was observed under conditions intended to mimic intracellular glutathione concentration. This allowed the thermoresponsive behaviour to be 'switched on' without the need for a temperature stimulus - an 'isothermal' switch.

One pot room temperature synthesis of robust gold nanochains

We report a room temperature reduction and stabilization of gold nanoparticles (AuNPs) from auric chloride in the presence of 1.5 and 3 mM linear poly(amidoamine) (PAmAm), which leads to 1D assembly of AuNPs. At higher concentrations, isolated AuNPs are formed. Detailed investigations show that the morphology is governed primarily by pH rather than concentration. This is the first report to demonstrate the stability of nanochains against pH switching. The nanochains are robust as revealed by stability at higher temperature, salt concentration, thiol exchange, and would be useful in developing electronic devices for biological applications.

Quantification of Global Protein Disulfides and Thiol-Protein Mixed Disulfides to Study the Protein Dethiolation Mechanisms

The redox state of cellular thiols is widely studied because it was recently linked to many different diseases and pathologies. In this work we quantified the concentrations of protein disulfides (PSSP) and thiol-protein mixed disulfides (XSSP) in rat tissues (liver, kidney and heart) and cells (Raw 264.7) by an improved method of XSSP and PSSP determination after oxidative stress induced by diamide. Under native and denaturing conditions, a thiol block by N-ethymaleimide was introduced to avoid thiol exchange reaction activations by protein SH groups (PSH) (PSH + XSSP ←→ PSSP + XSH) and alterations of original XSSP/PSSP levels. Low molecular weight thiols (XSH) and PSH were respectively measured by HPLC on supernatants and on corresponding pellets by DTNB (Ellman’s reagent) after dithiothreitol reduction. PSSP concentrations of liver, heart and kidney were respectively 0.304, 0.605 and 0.785 μmoles/g and after diamide exposure they were significantly augmented of about 65%-70% in liver and heart, but not in the kidney. Normal XSSP, that were -20 times lower than normal PSSP were induced by diamide in liver and heart of about 40 times, but not in kidney. Thermodynamic criteria regarding the pKa values of thiols engaged as PSSP and GSSP were used to interpret dethiolation mechanisms via thiol exchange reactions.

Manipulating the Protein Energy Landscape

I will present new developments in our studies probing high-energy fluctuations on the energy landscape. using single molecule mechanical manipulation and thiol exchange techniques, we have been able to identify and monitor novel events on the conformational landscape. Our recent results on the differences in mechanical compliance between the molten globule and native states and our ability to monitor the conformational fluctuations leading to unfolding have important implications for mechanical processes in the cell.

Self-healing systems based on disulfide–thiol exchange reactions

New thermoset systems based on disulfide bonds were synthesized with self-healing capabilities. The self-healing mechanism is not related to disulfide–disulfide exchange reactions, but to thiol–disulfide exchange reactions that are pH-dependent. Stress relaxation experiments showed large relaxation for systems having PTM2 as a curing agent, which indicates that the system can rearrange its molecular structure as a mechanism to release stress. However, relaxation rates decreased for samples tested longer after production. This indicates the disappearance of thiol-groups probably caused by thiol–thiol oxidation.

Epimerization in peptide thioester condensation

Peptide segment couplings are now widely utilized in protein chemical synthesis. One of the key structures for the strategy is the peptide thioester. Peptide thioester condensation, in which a C-terminal peptide thioester is selectively activated by silver ions then condensed with an amino component, is a powerful tool. But the amino acid adjacent to the thioester is at risk of epimerization. During the preparation of peptide thioesters by the Boc solid-phase method, no substantial epimerization of the C-terminal amino acid was detected. Epimerization was, however, observed during a thioester-thiol exchange reaction and segment condensation in DMSO in the presence of a base. In contrast, thioester-thiol exchange reactions in aqueous solutions gave no epimerization. The epimerization during segment condensation was significantly suppressed with a less polar solvent that is applicable to segments in thioester peptide condensation. These results were applied to a longer peptide thioester condensation. The epimer content of the coupling product of 89 residues was reduced from 27% to 6% in a condensation between segments of 45 and 44 residues for the thioester and the amino component, respectively.

Tertiary amine-based glutathione peroxidase mimics: some insights into the role of steric and electronic effects on antioxidant activity

In this work, several tertiary amine-based diaryl diselenides were synthesized and evaluated for their glutathione peroxidase (GPx)-like antioxidant activities using hydrogen peroxide, tert-butyl hydroperoxide and cumene hydroperoxide as substrates and thiophenol (PhSH) and glutathione (GSH) as co-substrates. A comparison of the GPx-like activity of 4-methoxy-substituted N,N-dialkylbenzylamine-based diselenides with that of the corresponding 6-methoxy-substituted compounds indicates that the activity highly depends on the position of the methoxy substituent. Although the methoxy group at 4- and 6-position alters the electronic properties of selenium, the substitution at the 6-position provides the required steric protection for some of the key intermediates in the catalytic cycle. A detailed experimental and theoretical investigation reveals that the 6-methoxy substituent prevents the undesired thiol exchange reactions at the selenium centers in the selenenyl sulfide intermediates. The 6-methoxy substituent also prevents the formation of seleninic and selenonic acids. When PhSH is used as the thiol co-substrate, the 4-methoxy-substituted diselenides exhibit GPx-like activity similar to that of the parent compounds as the 4-methoxy substituent does not block the selenium center in the selenenyl sulfide intermediates from thiol exchange reactions. In contrast, the 4-methoxy substituent significantly enhances the GPx-like activity of the diselenides when glutathione (GSH) is used as the co-substrate.

Dynamic thiol exchange with β-sulfido-α,β-unsaturated carbonyl compounds and dithianes.

A reversible covalent bond exchange of thiols, β-sulfido-α,β-unsaturated carbonyls, and dithianes has been studied in DMSO and D(2)O/DMSO mixtures. The equilibrium between thiols and β-sulfido-α,β-unsaturated carbonyls is obtained within a few hours, while the equilibration starting with the β-dithiane carbonyls and thiols requires a few days. This time scale makes the system ideal for utilization in dynamic combinatorial chemistry.

RAFT Agent Design and Synthesis

This Perspective reviews the design and synthesis of RAFT agents. First, we briefly detail the basic design features that should be considered when selecting a RAFT agent or macro-RAFT agent for a given polymerization and set of reaction conditions. The RAFT agent should be chosen to have an optimal Ctr (in most circumstances higher is better) while at the same time it should exhibit minimal likelihood for retarding polymerization or undergoing side reactions. The RAFT agent should also have appropriate solubility in the reaction medium and possess the requisite end-group functionality for the intended application. In this light we critically evaluate the various methods that have been used for RAFT agent synthesis. These methods include reaction of a carbodithioate salt with an alkylating agent, various thioacylation procedures, thiation of a carboxylic acid or ester, the ketoform reaction, thiol exchange, radical substitution of a bis(thioacyl) disulfide, and radical-induced R-group exchange. We also co...

Perylene Monolayer Protected Gold Nanorods: Unique Optical, Electronic Properties and Self-Assemblies

Here we report the synthesis and characterization of organosoluble perylene monolayer protected gold nanorods. From 1H NMR, FT-IR, and differential scanning calorimetry experiments, the successful thiol exchange and stacking of perylene molecules on gold nanorods were confirmed. The resulting gold nanorods encapsulated with perylene thiol molecules via strong covalent Au–S linkages showed unique optical and electronic properties compared to the initial free perylene molecules and gold nanorods, indicating there were strong interactions between perylene chromophores and gold nanorods. When attached on gold nanorods, the perylene chromophores did not exhibit any typical UV–vis absorption or fluorescence emission signal, originating from the charge transfer from gold nanorods to perylene chromophores. However, the missing signals reappeared upon the addition of iodine, which detached the perylene molecules from gold nanorods. For the hybrid gold nanorods, particular electronic properties were also investigated by cyclic voltammetry and electron diffraction. Furthermore, with strong π–π intermolecular interactions, the perylene thiol monolayer protected gold nanorods were able to aggregate. When drying from highly diluted solution, gold nanorods formed well-organized side-by-side self-assembly arrays.

Role of thiol pathways in TF procoagulant regulation

The generation of procoagulant Tissue Factor (TF) is crucial for thrombosis. TF contains a surface exposed allosteric disulfide bond that stabilizes the carboxyl-terminal domain involved in ligand interactions with coagulation factors VIIa and X. TF procoagulant activation typically occurs following cellular perturbations that also cause the appearance of procoagulant phosphatidylserine in the outer leaflet of cell membranes. However, thiol modifying agents, without suppressing phosphatidylserine exposure, can prevent TF activation, implicating thiol-disulfide exchange reactions in the regulation of TF procoagulant activity of primary cells. Protein disulfide isomerase (PDI), a regulator of extracellular thiol exchange, is associated with cell surface TF and required for TF-dependent thrombosis in vivo. PDI regulates the thiol-dependent biogenesis of procoagulant microparticles that are released from myeloid cells and smooth muscle cells following activation of the purinergic P2X7 receptor. Genetic deletion of P2X7 signaling attenuates FeCl3-induced carotid artery thrombosis in mice, indicating that TF prothrombotic activity is regulated by specific cell signaling pathways in vivo.

Non-viral siRNA delivery vectors: dendritic molecular transporter and molecular transporter nanovectors for target gene silencing

Viable non-viral delivery vectors have been utilized for the intracellular delivery of siRNA duplexes to result in complete target gene silencing. Biologically active siRNA duplexes were attached to a dendritic molecular transporter (MT) or a molecular transporter-polymeric nanoparticle conjugate (MT-NP) via a thiol exchange reaction utilizing a dithiol pyridyl linker. The MT and the MT-NP delivery systems resulted in significant reduction of the target protein (lamin A/C) investigated for a range of conjugate concentrations. Both non-viral systems have proven effective for the delivery of siRNA into the cytosol of live cells and specifically the development of the MT-NP delivery system provides opportunities for targeted delivery of siRNA into selected cell types.

A structurally driven analysis of thiol reactivity in mammalian albumins

Understanding the structural basis of protein redox activity is still an open question. Hence, by using a structural genomics approach, different albumins have been chosen to correlate protein structural features with the corresponding reaction rates of thiol exchange between albumin and disulfide DTNB. Predicted structures of rat, porcine, and bovine albumins have been compared with the experimentally derived human albumin. High structural similarity among these four albumins can be observed, in spite of their markedly different reactivity with DTNB. Sequence alignments offered preliminary hints on the contributions of sequence-specific local environments modulating albumin reactivity. Molecular dynamics simulations performed on experimental and predicted albumin structures reveal that thiolation rates are influenced by hydrogen bonding pattern and stability of the acceptor C34 sulphur atom with donor groups of nearby residues. Atom depth evolution of albumin C34 thiol groups has been monitored during Molecular Dynamic trajectories. The most reactive albumins appeared also the ones presenting the C34 sulphur atom on the protein surface with the highest accessibility. High C34 sulphur atom reactivity in rat and porcine albumins seems to be determined by the presence of additional positively charged amino acid residues favoring both the C34 S⁻ form and the approach of DTNB.

Tissue Factor (TF) Activation by Antithymocyte Globulin Is Inhibited by a Novel Monoclonal Antibody Specific for Reduced TF Mutated at the Allosteric Cys186-Cys209 Disulfide.

AbstractAbstract 1132Increased TF expression on monocytes contributes to coagulation activation in atherothrombosis, sepsis and cancer. Although it is well established that TF is predominantly non-coagulant and cryptic on unperturbed cells, the molecular mechanisms underlying TF activation remain intensely debated. Antithymocyte globulin (ATG) is a polyclonal rabbit IgG used in transplantation medicine to prevent organ rejection and graft-versus-host disease, but it can cause thrombocytopenia and low-grade DIC. In this study, we investigated the effect of ATG on TF activation. ATG specifically activated TF on cells of the (myelo)monocytic lineage, including THP1, HL60 and U937 cells, as well as isolated peripheral blood monocytes, but not on myeloma, glioma and epithelial cancer cells. Membrane-bound ATG dose-dependently enhanced the TF-specific procoagulant activity (PCA) of THP1 cells 30 ± 15-fold compared to control IgG, as assessed by one-stage clotting, chromogenic Xa generation assay or thrombin generation measured by the Technothrombin™ TGA. ATG rapidly (within 2 min) activated THP1 cells dependent on the Fc portion, but independent of Fcγ receptors, and required heat-labile plasma components for TF induction. ATG did not further enhance TF activity after cell lysis and had no effect on clotting induced by recombinant TF, demonstrating specific effects on TF activation rather than the coagulation reaction itself. ATG-mediated TF activation on THP1 cells did not involve inhibition of surface-floated TFPI and was more potent than, and synergized with, calcium ionophore treatment. Although ATG variably induced exposure of low to moderate levels of phosphatidylserine (PS) on the plasma membrane, as measured by annexin V-FITC binding (38 ± 25 vs. 8 ± 5% positive cells, ATG vs. control IgG, n=22), robust TF activation was observed even in experiments with marginal PS upregulation. Consequently, there was no correlation between TF PCA and PS exposure on ATG-treated THP1 cells (r=-0.03), indicating that the primary effect of ATG was on the activation of TF itself. Previous studies have implicated protein disulfide isomerase (PDI) and the TF extracellular allosteric Cys186-Cys209 disulfide bond as determinants for TF activation. Consistently, PDI was detectable at low levels on the surface of unstimulated THP1 cells by flow cytometry and ATG-induced TF activation was inhibited by 70 ± 15% (n=9) with RL90, an inhibitory monoclonal antibody to PDI previously shown to have antithrombotic effects in vivo. RL90 did not decrease PS externalization on ATG-activated cells and had no effect when added to THP1 cells after TF activation had occurred, demonstrating that the antibody specifically blocked the TF activation process. In addition, blocking free thiols of THP1 cells with N-ethylmaleimide slightly increased TF activity, but completely abolished further TF activation by ATG, demonstrating that thiol exchange reactions were critically involved. In order to directly probe the involvement of the allosteric TF disulfide in ATG-mediated TF activation, we raised monoclonal antibodies to Cys186-Cys209 disulfide bond mutants of human TF. Panreactive and mutant TF-specific antibodies were identified. Monoclonal antibody 3D10 did not bind to oxidized recombinant soluble TF, but selectively recognized the Cys186Ala recombinant mutant that retained a free Cys209 prone to S-nitrosylation/glutathionation. Antibody 3D10 completely inhibited ATG-induced TF activation on THP1 cells, but was without effect when added to THP1 cells after TF activation had occurred. The inhibitory effect of 3D10 on TF activation was dose-dependent (5-50 μg/ml), did not require Fcγ receptor binding and did not involve competition with ATG binding to THP1 cells. Preincubation of THP1 cells with 3D10-derived Fab fragments also inhibited ATG-mediated TF activation by 68 ± 7% (n=8) without affecting PS exposure. Importantly, the combination of RL90 and 3D10 Fab had no additive effects, suggesting that both interfered with the same molecular activation pathway. In summary, these data provide novel evidence that TF cellular activation involves intermediates with an altered conformation of the allosteric Cys186-Cys209 disulfide in the TF extracellular domain and further substantiate the concept that TF decryption involves PDI-dependent pathways that are distinct from the exposure of procoagulant PS on cell surfaces. Disclosures:No relevant conflicts of interest to declare.

P2×7 Signaling Triggers Tissue Factor Activation and Protein Disulfide Isomerase-Dependent Release of TF+ Microparticles In Thrombosis

AbstractAbstract 344Thrombosis induced by the tissue factor (TF) pathway remains a major cause for morbidity and mortality in cardiovascular diseases. The cellular mechanisms of TF activation in primary cells implicated in pathological thrombosis are poorly understood and a controversy exists about the importance of thiol exchange involving protein disulfide isomerase (PDI) versus cell surface exposure of procoagulant anionic phosphatidylserine (PS). Myeloid cells are a source for TF positive (+) procoagulant microparticles (MP). We found that primed bone marrow-derived macrophages had undetectable TF activity. Upon stimulation of the purinergic P2X7 receptor with ATP, TF was readily decrypted. P2X7 signaling induced both the activation of TF cell surface procoagulant activity and thiol- and PDI-dependent generation of procoagulant MP in a pathway that was independent of cell surface PS exposure. Specifically, thiol blockade with dithio-bis-2-nitrobenzoic-acid (DTNB) prevented ATP-triggered release of procoagulant MP without effects on baseline or agonist-induced PS exposure. Similarly, anti-PDI antibody RL90, previously shown to have antithrombotic activities in vivo, attenuated the ATP-induced release of redox active TF+ MP without appreciable effects on cell surface TF activity or PS exposure. Importantly, P2X7 signaling also regulated TF activation and MP release from smooth muscle cells, implicated in TF thrombogenic pathways. Accordingly, P2X7 signaling-deficient mice displayed an attenuated TF-dependent thrombogenic response to an 8% FeCl3·6H2O carotid artery injury. In a more severe 10% injury, P2X7 deficient (-/-) mice eventually formed occluding thrombi that were inhibited by anti-FXI antibody given at a dose without effect in wild-type controls. In contrast, anti-TF did not prevent thrombosis, implying that P2X7-/- mice under these conditions rely on the intrinsic pathway for vascular occlusion and are generally impaired in TF-dependent thrombosis. Bone marrow transplantation experiments showed that P2X7 inactivation on both hematopoietic and vessel wall cells was required for protection from thrombosis. In further studies on this pathway, we identified another anti-PDI monoclonal antibody with unique activating effects that bypassed defective signaling of P2X7-/- cells, directly induced cell surface TF activity, and restored the thiol pathway leading to release of procoagulant MP from P2X7-/- smooth muscle cells and macrophages. The MP released from P2X7-/- macrophages by this antibody were remarkably similar to ATP-induced MP from wild-type mice and carried the thrombus targeting receptor P-selectin glycoprotein ligand-1 (PSGL-1), integrin b1, and PDI. In vivo, the activating anti-PDI antibody induced stable arterial occlusion in P2X7-/- mice challenged with 8% FeCl3·6H2O, and confirmed to be TF dependent since occlusion under these conditions was prevented by a function blocking anti-TF antibody. In conclusion, this study provides novel evidence that cell signaling is required for the activation of prothrombotic TF in vivo, thus challenging the current thinking that loss of vascular integrity or cell injury and PS exposure are the primary triggers for TF-dependent thrombosis. Our findings position cell surface PDI at a central node that controls P2X7-dependent TF cellular decryption and MP release. This novel activation pathway is critical for the generation of prothrombotic TF by vessel wall and blood cells and may be targeted as an alternative strategy to conventional anticoagulant therapy directed towards coagulation proteases. Disclosures:No relevant conflicts of interest to declare.