Targeting CD39 Toward Activated Platelets Reduces Systemic Inflammation and Improves Survival in Sepsis: A Preclinical Pilot Study

Objectives: Sepsis is associated with a systemic inflammatory reaction, which can result in a life-endangering organ dysfunction. Pro-inflammatory responses during sepsis are characterized by increased activation of leukocytes and platelets, formation of platelet-neutrophil aggregates, and cytokine production. Sequestration of platelet-neutrophil aggregates in the microvasculature contributes to tissue damage during sepsis. At present no effective therapeutic strategy to ameliorate these events is available. In this preclinical pilot study, a novel anti-inflammatory approach was evaluated, which targets nucleoside triphosphate hydrolase activity toward activated platelets via a recombinant fusion protein combining a single-chain antibody against activated glycoprotein IIb/IIIa and the extracellular domain of CD39 (targ-CD39). Design: Experimental animal study and cell culture study. Setting: University-based experimental laboratory. Subjects: Human dermal microvascular endothelial cells 1, human platelets and neutrophils, and C57BL/6NCrl mice. Interventions: Platelet-leukocyte-endothelium interactions were evaluated under inflammatory conditions in vitro and in a murine lipopolysaccharide-induced sepsis model in vivo. The outcome of polymicrobial sepsis was evaluated in a murine cecal ligation and puncture model. To evaluate the anti-inflammatory potential of activated platelet targeted nucleoside triphosphate hydrolase activity, we employed a potato apyrase in vitro and in vivo, as well as targ-CD39 and as a control, nontarg-CD39 in vivo. Measurements and Main Results: Under conditions of sepsis, agents with nucleoside triphosphate hydrolase activity decreased platelet-leukocyte-endothelium interaction, transcription of pro-inflammatory cytokines, microvascular platelet-neutrophil aggregate sequestration, activation marker expression on platelets and neutrophils contained in these aggregates, leukocyte extravasation, and organ damage. Targ-CD39 had the strongest effect on these variables and retained hemostasis in contrast to nontarg-CD39 and potato apyrase. Most importantly, targ-CD39 improved survival in the cecal ligation and puncture model to a stronger extent then nontarg-CD39 and potato apyrase. Conclusions: Targeting nucleoside triphosphate hydrolase activity (CD39) toward activated platelets is a promising new treatment concept to decrease systemic inflammation and mortality of sepsis. This innovative therapeutic approach warrants further development toward clinical application. Prof. Peter and Dr. Straub contributed equally and share senior authorship. Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's website (http://bit.ly/29S62lw). Supported, in part, by grants from the German Research Foundation (Deutsche Forschungsgemeinschaft [DFG], Bonn, Germany) to Dr. Straub (grant number STR-687/4-1); Prof. Rosenberger (grant number DFG-RO 3672/6-2); and Prof. Mirakaj (MI-1506/4-1) and the Interdisciplinary Center for Clinical Research (IZKF) program of the University of Tübingen, Germany (grant number IZKF-No. PK 2015-1-05; granted to Prof. Rosenberger and Mr. Glück); by a grant of the Dr. Karl Kuhn Foundation to Dr. Straub; and Dr. Xang was supported by a future leader fellowship of the National Heart Foundation of Australia). Mr. Glück received scholarship support for 1 year and travel support for visits at scientific meetings (German Anaesthesiology Congress and German Intensive Care and Emergency Medicine Congress) by the "IZKF—Promotionskolleg" University of Tübingen (Germany). Mr. Glück and Dr. Straub disclosed targ-CD39 as an experimental agent. Prof. Peter was supported by a principal research fellowship from the National Health and Medical Research Council of Australia. Dr. Straub's institution received funding from Deutsche Forschungsgemeinschaft (German Research Foundation) and Interdisciplinary Center for Clinical Research (IZKF) program of the University of Tübingen, Germany, and he received speaker honoraria from CSL Behring GmbH Munich, Germany and Schöchl Medical Education GmbH Mattsee, Austria. The remaining authors have disclosed that they do not have any potential conflicts of interest. Address requests for reprints to: Andreas Straub, MD, Department of Anesthesiology and Intensive Care Medicine, Eberhard-Karls University Tübingen, Waldhörnlestr. 22, 72072 Tübingen, Germany. E-mail: andreas.straub@uni-tuebingen.de Copyright © by 2019 by the Society of Critical Care Medicine and Wolters Kluwer Health, Inc. All Rights Reserved.

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