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Optimizing human apyrase to treat arterial thrombosis and limit reperfusion injury without increasing bleeding risk.

ABSTRACT: In patients with acute myocardial infarction undergoing reperfusion therapy to restore blood flow through blocked arteries, simultaneous inhibition of platelet P2Y12 receptors with the current standard of care neither completely prevents recurrent thrombosis nor provides satisfactory protection against reperfusion injury. Additionally, these antiplatelet drugs increase the risk of bleeding. To devise a different strategy, we engineered and optimized the apyrase activity of human nucleoside triphosphate diphosphohydrolase-3 (CD39L3) to enhance scavenging of extracellular adenosine diphosphate, a predominant ligand of P2Y12 receptors. The resulting recombinant protein, APT102, exhibited greater than four times higher adenosine diphosphatase activity and a 50 times longer plasma half-life than did native apyrase. Treatment with APT102 before coronary fibrinolysis with intravenous recombinant human tissue-type plasminogen activator in conscious dogs completely prevented thrombotic reocclusion and significantly decreased infarction size by 81% without increasing bleeding time. In contrast, clopidogrel did not prevent coronary reocclusion and increased bleeding time. In a murine model of myocardial reperfusion injury caused by transient coronary artery occlusion, APT102 also decreased infarct size by 51%, whereas clopidogrel was not effective. These preclinical data suggest that APT102 should be tested for its ability to safely and effectively maximize the benefits of myocardial reperfusion therapy in patients with arterial thrombosis.

SUBMITTER: Moeckel D

PROVIDER: S-EPMC4291186 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Optimizing human apyrase to treat arterial thrombosis and limit reperfusion injury without increasing bleeding risk.

Moeckel Douglas D Jeong Soon Soeg SS Sun Xiaofeng X Broekman M Johan MJ Nguyen Annie A Drosopoulos Joan H F JH Marcus Aaron J AJ Robson Simon C SC Chen Ridong R Abendschein Dana D

Science translational medicine 20140801 248

In patients with acute myocardial infarction undergoing reperfusion therapy to restore blood flow through blocked arteries, simultaneous inhibition of platelet P2Y12 receptors with the current standard of care neither completely prevents recurrent thrombosis nor provides satisfactory protection against reperfusion injury. Additionally, these antiplatelet drugs increase the risk of bleeding. To devise a different strategy, we engineered and optimized the apyrase activity of human nucleoside triph ...[more]

PMID: 25100739

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Project description:Background Deep vein thrombosis (DVT) is the primary cause of pulmonary embolism and the third most life-threatening cardiovascular disease in North America. Post-DVT anticoagulants, such as warfarin, heparin, and direct oral anticoagulants, reduce the incidence of subsequent venous thrombi. However, all currently used anticoagulants affect bleeding time at various degrees, and there is therefore a need for improved therapeutic regimens in DVT. It has recently been shown that mast cells play a crucial role in a DVT murine model. The underlying mechanism involved in the prothrombotic properties of mast cells, however, has yet to be identified. Methods and Results C57BL/6 mice and mouse mast cell protease-4 (mMCP-4) genetically depleted mice (mMCP-4 knockout) were used in 2 mouse models of DVT, partial ligation (stenosis) and ferric chloride-endothelial injury model of the inferior vena cava. Thrombus formation and impact of genetically repressed or pharmacologically (specific inhibitor TY-51469) inhibited mMCP-4 were evaluated by morphometric measurements of thrombi immunochemistry (mouse and human DVT), color Doppler ultrasound, bleeding times, and enzymatic activity assays ex vivo. Recombinant chymases, mMCP-4 (mouse) and CMA-1 (human), were used to characterize the interaction with murine and human plasmin, respectively, by mass spectrometry and enzymatic activity assays. Inhibiting mast cell-generated mMCP-4, genetically or pharmacologically, resolves and prevents venous thrombus formation in both DVT models. Inferior vena cava blood flow obstruction was observed in the stenosis model after 6 hours of ligation, in control- but not in TY-51469-treated mice. In addition, chymase inhibition had no impact on bleeding times of healthy or DVT mice. Furthermore, endogenous chymase limits plasmin activity in thrombi ex vivo. Recombinant mouse or human chymase degrades/inactivates purified plasmin in vitro. Finally, mast cell-containing immunoreactive chymase was identified in human DVT. Conclusions This study identified a major role for mMCP-4, a granule-localized protease of chymase type, in DVT formation. These findings support a novel pharmacological strategy to resolve or prevent DVT without affecting the coagulation cascade through the inhibition of chymase activity.

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Optimizing human apyrase to treat arterial thrombosis and limit reperfusion injury without increasing bleeding risk.

Publications

Optimizing human apyrase to treat arterial thrombosis and limit reperfusion injury without increasing bleeding risk.

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