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.

The uncoordinated-5 homolog B (UNC5B) receptor increases myocardial ischemia-reperfusion injury.

The UNC5 receptor family are chemorepulsive neuronal guidance receptors with additional functions outside the central nervous system. Previous studies have implicated that the UNC5B receptor influences the migration of leukocytes into sites of tissue inflammation. Given that this process is a critical step during the pathophysiology of myocardial ischemia followed by reperfusion (IR) we investigated the role of UNC5B during myocardial IR. In initial in-vitro experiments, the functional inhibition of UNC5B resulted in a significant reduction of chemotactic migration of neutrophils. In-vivo, using a model of acute myocardial ischemia in UNC5B(+/-) and wild type (WT) animals, we found a significant reduction of infarct sizes in UNC5B(+/-) animals. This was associated with significantly reduced levels of troponin-I and IL-6 in UNC5B(+/-) mice. The repression of UNC5B using siRNA and the functional inhibition of UNC5B significantly dampened the extent of myocardial IR injury. Following depletion of neutrophils, we were not able to observe any further reduction in infarct size through functional inhibition of UNC5B in WT and UNC5B(+/-) mice. In summary our studies demonstrate an important role for UNC5B during myocardial IR injury, and that UNC5B might be a potential therapeutic target to control reperfusion injury in the future.