Modulation of purinergic signaling is critical to myocardial homeostasis. Ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD-1; CD39), which converts the proinflammatory molecules ATP or ADP to AMP is a key regulator of purinergic modulation. However, the salutary effects of transgenic over expression of ENTPD1 on myocardial response to ischemic injury have not been tested to date. Therefore we hypothesized that ENTPD1 over-expression affords myocardial protection from ischemia-reperfusion injury via specific cell signaling pathways. ENTPD-1 transgenic mice, which over-express human ENTPDase-1, and wild-type (WT) littermates were subjected to either ex vivo or in vivo ischemia-reperfusion injury. Infarct size, inflammatory cell infiltrate and intracellular signaling molecule activation were evaluated. Infarct size was significantly reduced in ENTPD-1 versus WT hearts in both ex vivo and in vivo studies. Following ischemia-reperfusion injury, ENTPD-1 cardiac tissues demonstrated an increase in the phosphorylation of the cellular signaling molecule extracellular signal-regulated kinases 1/2 (ERK 1/2) and glycogen synthase kinase-3β (GSK-3β). Resistance to myocardial injury was abrogated by treatment with a non-selective adenosine receptor antagonist, 8-SPT or the more selective A2B adenosine receptor antagonist, MRS 1754, but not the A1 selective antagonists, DPCPX. Additionally, treatment with the ERK 1/2 inhibitor PD98059 or the mitochondrial permeability transition pore opener, atractyloside, abrogated the cardiac protection provided by ENTPDase-1 expression. These results suggest that transgenic ENTPDase-1 expression preferentially conveys myocardial protection from ischemic injury via adenosine A2B receptor engagement and associated phosphorylation of the cellular protective signaling molecules, Akt, ERK 1/2 and GSK-β that prevents detrimental opening of the mitochondrial permeability transition pore.

Ventilator-associated pneumonia and catheter-related bacteremia are the most common and severe infections caused by Acinetobacter baumannii. Besides the capsule, lipopolysaccharides, and the outer membrane porin OmpA, little is known about the contribution of secreted proteins to A. baumannii survival in vivo. Here we focus on CpaA, a potentially recently acquired virulence factor that inhibits blood coagulation in vitro. We identify coagulation factor XII as a target of CpaA, map the cleavage sites, and show that glycosylation is a prerequisite for CpaA-mediated inactivation of factor XII. We propose adding CpaA to a small, but growing list of bacterial proteases that are specific for highly glycosylated components of the host defense system.

The serine protease inhibitor (SERPIN) plasminogen activator inhibitor-1 (PAI-1) is a key regulator of the fibrinolytic system, inhibiting the serine proteases tissue- and urokinase-type plasminogen activator (tPA and uPA, respectively). Missense variants render PAI-1 non-functional through misfolding, leading to its turnover as a protease substrate, or to a more rapid transition to the latent/inactive state. Deep mutational scanning was performed to evaluate the impact of amino acid sequence variation on PAI-1 inhibition of uPA using an M13 filamentous phage display system. Error prone PCR was used to construct a mutagenized PAI-1 library encompassing ~ 70% of potential single amino acid substitutions. The relative effects of 27% of all possible missense variants on PAI-1 inhibition of uPA were determined using high-throughput DNA sequencing. 826 missense variants demonstrated conserved inhibitory activity while 1137 resulted in loss of PAI-1 inhibitory function. The least evolutionarily conserved regions of PAI-1 were also identified as being the most tolerant of missense mutations. The results of this screen confirm previous low-throughput mutational studies, including those of the reactive center loop. These data provide a powerful resource for explaining structure–function relationships for PAI-1 and for the interpretation of human genomic sequence variants.

Objective Circulating blood cells and endothelial cells express Ectonucleoside triphosphate diphosphohydrolase-1 (CD39) and ecto-5’-nucleotidase (CD73). CD39 hydrolyzes extracellular ATP or ADP to AMP. CD73 hydrolyzes AMP to adenosine. The goal of this study was to examine the interplay between CD39 and CD73 cascade in arterial thrombosis. Approach and Results To determine how CD73 activity influences in vivo thrombosis, the time to FeCl3-induced arterial thrombosis was measured in CD73-null mice. In response to 5% FeCl3, but not to 10% FeCl3, there was a significant decrease in the time to thrombosis in CD73-null mice compared to wild-type (WT) mice. In mice overexpressing CD39, ablation of CD73 did not inhibit the prolongation in the time to thrombosis conveyed by CD39 overexpression. However, the CD73 inhibitor α-β-methylene-ADP nullified the prolongation in the time to thrombosis in hC39-Tg/CD73-null mice. To determine if hematopoietic-derived cells or endothelial cell CD39 activity regulates in vivo arterial thrombus, bone marrow transplant studies were conducted. FeCl3-induced arterial thrombosis in chimeric mice revealed a significant prolongation in the time to thrombosis in hCD39-Tg reconstituted WT mice, but not on WT reconstituted hCD39-Tg mice. Monocyte depletion with clodronate-loaded liposomes normalized the time to thrombosis in hCD39-Tg mice compared to hCD39-Tg mice treated with control liposomes, demonstrating that increased CD39 expression on monocytes protects against thrombosis. Conclusions These data demonstrate that ablation of CD73 minimally effects in vivo thrombosis, but increased CD39 expression on hematopoietic-derived cells, especially monocytes, attenuates in vivo arterial thrombosis.

Modulation of purinergic signaling, which is critical for vascular homeostasis and the response to vascular injury, is regulated by hydrolysis of proinflammatory ATP and/or ADP by ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD-1; CD39) to AMP, which then is hydrolyzed by ecto-5'-nucleotidase (CD73) to adenosine. We report here that compared with littermate controls (wild type), transgenic mice expressing human ENTPDase-1 were resistant to the formation of an occlusive thrombus after FeCl(3)-induced carotid artery injury. Treatment of mice with the nonhydrolyzable ADP analog, adenosine-5'-0-(2-thiodiphosphate) trilithium salt, Ado-5'-PP[S], negated the protection from thrombosis, consistent with a role for ADP in platelet recruitment and thrombus formation. ENTPD-1 expression decreased whole-blood aggregation after stimulation by ADP, an effect negated by adenosine-5'-0-(2-thiodiphosphate) trilithium salt, Ado-5'-PP[S] stimulation, and limited the ability to maintain the platelet fibrinogen receptor, glycoprotein α(IIb)/β(3), in a fully activated state, which is critical for thrombus formation. In vivo treatment with a CD73 antagonist, a nonselective adenosine-receptor antagonist, or a selective A(2A) or A(2B) adenosine-receptor antagonist, negated the resistance to thrombosis in transgenic mice expressing human ENTPD-1, suggesting a role for adenosine generation and engagement of adenosine receptors in conferring in vivo resistance to occlusive thrombosis in this model. In summary, our findings identify ENTPDase-1 modulation of purinergic signaling as a key determinant of the formation of an occlusive thrombus after vascular injury.

Antioxidant enzymatic pathways form a critical network that detoxifies ROS in response to myocardial stress or injury. Genetic alteration of the expression levels of individual enzymes has yielded mixed results with regard to attenuating in vivo myocardial ischemia-reperfusion injury, an extreme oxidative stress. We hypothesized that overexpression of an antioxidant network (AON) composed of SOD1, SOD3, and glutathione peroxidase (GSHPx)-1 would reduce myocardial ischemia-reperfusion injury by limiting ROS-mediated lipid peroxidation and oxidative posttranslational modification (OPTM) of proteins. Both ex vivo and in vivo myocardial ischemia models were used to evaluate the effect of AON expression. After ischemia-reperfusion injury, infarct size was significantly reduced both ex vivo and in vivo, ROS formation, measured by dihydroethidium staining, was markedly decreased, ROS-mediated lipid peroxidation, measured by malondialdehyde production, was significantly limited, and OPTM of total myocardial proteins, including fatty acid-binding protein and sarco(endo)plasmic reticulum Ca(²+)-ATPase (SERCA)2a, was markedly reduced in AON mice, which overexpress SOD1, SOD3, and GSHPx-1, compared with wild-type mice. These data demonstrate that concomitant SOD1, SOD3, and GSHPX-1 expression confers marked protection against myocardial ischemia-reperfusion injury, reducing ROS, ROS-mediated lipid peroxidation, and OPTM of critical cardiac proteins, including cardiac fatty acid-binding protein and SERCA2a.

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Objective: To determine the effect of increased CD39 expression on circulating blood components on in vivo arterial thrombosis in mice. Approach and Results: We have previously demonstrated that increased global expression of hCD39 conveys an anti-thrombotic phenotype in mice suggesting a pivotal role for CD39 in modulating the balance between a pro-thrombotic and anti-thrombotic milieu. We hypothesized the expression of CD39 on circulating blood components can regulate occlusive thrombus formation. Bone marrow transplantations were performed (WT to WT; WT to hCD39-Tg; hCD39-Tg to hCD39-Tg; hCD39-Tg to WT) to determine the contribution of CD39 expressed on circulating blood cells and platelets on occlusive arterial thrombus formation in mice. Complete blood counts and fluorescence-activated cell sorting (FACS) analyses were performed on whole blood obtained from mice 60 days after transplantation of bone marrow. No significant differences in the circulating levels of white blood cells, red blood cells, and platelets were observed between the groups. FACS analysis confirmed that the circulating blood components were reconstituted from the transplanted bone marrow. Carotid artery thrombosis was induced by ferric chloride (FeCl 3 ) application and the time to thrombotic occlusion (blood flow of 0 ml/min) was measured. The operator was blinded to the animal genotype and treatment during all experiments. Transplantation of CD39-Tg bone marrow into WT mice resulted in a marked resistance to thrombosis (CD39-Tg to WT: 315.7.00 ± 54.6 min; WT to WT: 7.58 ± 0.89 min; P <0.001). CD39-Tg mice that received WT bone marrow demonstrated a significantly lower time to vessel occlusion than CD39-Tg mice that received CD39-Tg bone marrow (WT to CD39-Tg: 17.15 ± 6 min; CD39-Tg to CD39-Tg 414.3 ± 61.95 min; P <0.001). Conclusion: Increased expression of CD39 on circulating bone-marrow-derived blood components markedly prolongs the time to carotid artery occlusion following ferric-chloride-induced injury in mice.