Deep vein thrombosis (DVT), caused by alterations in venous homeostasis, is the third most common cause of cardiovascular mortality, however, key molecular determinants in venous thrombosis have not been fully elucidated. Several lines of evidence indicate that DVT occurs at the intersection of dysregulated inflammation and coagulation. The enzyme ectonucleoside tri(di)phosphohydrolase (ENTPD1, also known as CD39) is a vascular ecto-apyrase on the surface of leukocytes and the endothelium that inhibits intravascular inflammation and thrombosis by hydrolysis of phosphodiester bonds from nucleotides released by activated cells. Here, we evaluated the contribution of CD39 to venous thrombosis in a restricted-flow model of murine inferior vena cava stenosis. CD39 deficiency conferred a greater than 2-fold increase in venous thrombogenesis, characterized by increased leukocyte engagement, neutrophil extracellular trap formation, fibrin, and local activation of tissue factor in the thrombotic milieu. This venous thrombogenesis was orchestrated by increased phosphorylation of the p65 subunit of NF-κB, activation of the NLR family pyrin domain–containing 3 (NLRP3) inflammasome, and IL-1β release in CD39-deficient mice. Substantiating these findings, an IL-1β–neutralizing antibody or the IL-1 receptor inhibitor anakinra attenuated the thrombosis risk in CD39-deficient mice. These data demonstrate that IL-1β is a key accelerant of venous thrombo-inflammation, which can be suppressed by CD39. CD39 inhibits in vivo crosstalk between inflammation and coagulation pathways and is a critical vascular checkpoint in venous thrombosis.

Substrate ambiguity and relaxed reaction specificity underlie the diversity of reactions catalyzed by the transsulfuration pathway enzymes, cystathionine β-synthase (CBS) and γ-cystathionase (CSE). These enzymes either commit sulfur metabolism to cysteine synthesis from homocysteine or utilize cysteine and/or homocysteine for synthesis of H₂S, a signaling molecule. We demonstrate that a kinetically controlled heme-dependent metabolite switch in CBS regulates these competing reactions where by cystathionine, the product of CBS, inhibits H₂S synthesis by the second enzyme, CSE. Under endoplasmic reticulum stress conditions, induction of CSE and up-regulation of the CBS inhibitor, CO, a product of heme oxygenase-1, flip the operating preference of CSE from cystathionine to cysteine, transiently stimulating H₂S production. In contrast, genetic deficiency of CBS leads to chronic stimulation of H₂S production. This metabolite switch from cystathionine to cysteine and/or homocysteine renders H₂S synthesis by CSE responsive to the known modulators of CBS: S-adenosylmethionine, NO, and CO. Used acutely, it regulates H₂S synthesis; used chronically, it might contribute to disease pathology.

Hydrogen sulfide (H₂S) regulates various physiological processes, including neuronal activity, vascular tone, inflammation, and energy metabolism. Moreover, H₂S elicits cytoprotective effects against stressors in various cellular models of injury. However, the mechanism of the signaling pathways mediating the cytoprotective functions of H₂S is not well understood. We previously uncovered a heme-dependent metabolic switch for transient induction of H₂S production in the trans-sulfuration pathway. Here, we demonstrate that increased endogenous H₂S production or its exogenous administration modulates major components of the integrated stress response promoting a metabolic state primed for stress response. We show that H₂S transiently increases phosphorylation of eukaryotic translation initiation factor 2 (eIF2α) resulting in inhibition of general protein synthesis. The H₂S-induced increase in eIF2α phosphorylation was mediated at least in part by inhibition of protein phosphatase-1 (PP1c) via persulfidation at Cys-127. Overexpression of a PP1c cysteine mutant (C127S-PP1c) abrogated the H₂S effect on eIF2α phosphorylation. Our data support a model in which H₂S exerts its cytoprotective effect on ISR signaling by inducing a transient adaptive reprogramming of global mRNA translation. Although a transient increase in endogenous H₂S production provides cytoprotection, its chronic increase such as in cystathionine β-synthase deficiency may pose a problem.

Objective—: Leukocyte flux contributes to thrombus formation in deep veins under pathological conditions, but mechanisms that inhibit venous thrombosis are incompletely understood. Ectonucleotide di(tri)phosphohydrolase 1 ( ENTPD1 or Cd39 ), an ectoenzyme that catabolizes extracellular adenine nucleotides, is embedded on the surface of endothelial cells and leukocytes. We hypothesized that under venous stasis conditions, CD39 regulates inflammation at the vein:blood interface in a murine model of deep vein thrombosis. Approach and Results—: CD39-null mice developed significantly larger venous thrombi under venous stasis, with more leukocyte recruitment compared with wild-type mice. Gene expression profiling of wild-type and Cd39 -null mice revealed 76 differentially expressed inflammatory genes that were significantly upregulated in Cd39 -deleted mice after venous thrombosis, and validation experiments confirmed high expression of several key inflammatory mediators. P-selectin, known to have proximal involvement in venous inflammatory and thrombotic events, was upregulated in Cd39 -null mice. Inferior vena caval ligation resulted in thrombosis and a corresponding increase in both P-selectin and VWF (von Willebrand Factor) levels which were strikingly higher in mice lacking the Cd39 gene. These mice also manifest an increase in circulating platelet-leukocyte heteroaggregates suggesting heterotypic crosstalk between coagulation and inflammatory systems, which is amplified in the absence of CD39. Conclusions—: These data suggest that CD39 mitigates the venous thromboinflammatory response to flow interruption.