Andreas Straub

Using reagent-supported thromboelastometry (ROTEM®) to monitor haemostatic changes in congenital heart surgery employing deep hypothermic circulatory arrest

OBJECTIVE Cardiac surgery employing cardiopulmonary bypass (CPB) and deep hypothermic circulatory arrest (DHCA) can induce coagulation disturbances and bleeding complications that may be especially severe in infants. A better understanding of the coagulopathy and a quick method for its evaluation would be helpful in the management of patients exposed to CPB and DHCA. This study aimed to monitor coagulation defects in congenital heart surgery using rotational thromboelastometry (ROTEM), standard coagulation tests and platelet flow cytometry. METHODS The study comprised 10 infants undergoing surgery for congenital heart disease on CPB and DHCA. Blood was sampled at skin incision, after heparinisation during CPB (directly pre- and directly post-DHCA) and after protamine administration post-CPB. ROTEM using different reagents including a heparinase-containing assay to evaluate coagulation during heparinisation, APTT and INR, and flow cytometry to evaluate platelet activation were performed. RESULTS During CPB, the ROTEM indicated CPB-induced clotting factor depletion and platelet dysfunction that persisted after CPB and heparin neutralisation. ROTEM results were available within 15 min and therefore much faster than standard tests. ROTEM-guided specific blood product treatment resulted in satisfactory coagulatory function. The highest degree of platelet activation was found directly after DHCA. CONCLUSIONS A major benefit of ROTEM is the quick detection of a developing coagulopathy already during CPB. ROTEM guides quick and specific blood product treatment after CPB, which may decrease bleeding complications in cardiac surgery. The finding of maximal platelet activation directly after DHCA suggests that not only CPB but also hypothermia activates platelets in vivo, thereby contributing to platelet dysfunction.

The neuroimmune guidance cue netrin-1 controls resolution programs and promotes liver regeneration.

Hepatic ischemia/reperfusion (I/R) is a major adverse reaction to liver transplantation, hemorrhagic shock, or resection. Recently, the anti‐inflammatory properties of the axonal guidance cue netrin‐1 were reported. Here, we demonstrate that netrin‐1 also impacts the resolution of inflammation and promotes hepatic repair and regeneration during liver I/R injury. In initial studies, we investigated the induction of netrin‐1 and its receptors in murine liver tissues after I/R injury. Hepatic I/R injury was performed in mice with a partial genetic netrin‐1 deficiency (Ntn1+/−) or wild‐type C57BL/6 treated with exogenous netrin‐1 to examine the endogenous and therapeutically administered impact of netrin‐1. These investigations were corroborated by studies determining the characteristics of intravascular leukocyte flow, clearance of apoptotic neutrophils (polymorphonuclear cells [PMNs]), production of specialized proresolving lipid mediators (SPMs), generation of specific growth factors contributing to the resolution of inflammation, and liver repair. Hepatic I/R was associated with a significant reduction of netrin‐1 transcript and protein in murine liver tissue. Subsequent studies in netrin‐1‐deficient mice revealed lower efficacies in reducing PMN infiltration, proinflammatory cytokine levels, and hepatic‐specific injury enzymes. Conversely, mice treated with exogenous netrin‐1 exhibited increased liver protection and repair, reducing neutrophil influx into the injury site, decreasing proinflammatory mediators, increasing efferocytosis of apoptotic PMNs, and stimulating local endogenous biosynthesis of SPMs and the generation of specific growth factors. Finally, genetic studies implicated the A2B adenosine receptor in netrin‐1‐mediated protection during hepatic I/R injury. Conclusion: The present study indicates a previously unrecognized role for netrin‐1 in liver protection and its contribution to tissue homeostasis and regeneration. (Hepatology 2016;63:1689‐1705)

Combined Blockade of ADP Receptors and PI3-Kinase p110β Fully Prevents Platelet and Leukocyte Activation during Hypothermic Extracorporeal Circulation

Extracorporeal circulation (ECC) and hypothermia are used to maintain stable circulatory parameters and improve the ischemia tolerance of patients in cardiac surgery. However, ECC and hypothermia induce activation mechanisms in platelets and leukocytes, which are mediated by the platelet agonist ADP and the phosphoinositide-3-kinase (PI3K) p110β. Under clinical conditions these processes are associated with life-threatening complications including thromboembolism and inflammation. This study analyzes effects of ADP receptor P2Y12 and P2Y1 blockade and PI3K p110β inhibition on platelets and granulocytes during hypothermic ECC. Human blood was treated with the P2Y12 antagonist 2-MeSAMP, the P2Y1 antagonist MRS2179, the PI3K p110β inhibitor TGX-221, combinations thereof, or PBS and propylene glycol (controls). Under static in vitro conditions a concentration-dependent effect regarding the inhibition of ADP-induced platelet activation was found using 2-MeSAMP or TGX-221. Further inhibition of ADP-mediated effects was achieved with MRS2179. Next, blood was circulated in an ex vivo ECC model at 28°C for 30 minutes and various platelet and granulocyte markers were investigated using flow cytometry, ELISA and platelet count analysis. GPIIb/IIIa activation induced by hypothermic ECC was inhibited using TGX-221 alone or in combination with P2Y blockers (p<0.05), while no effect of hypothermic ECC or antiplatelet agents on GPIIb/IIIa and GPIbα expression and von Willebrand factor binding was observed. Sole P2Y and PI3K blockade or a combination thereof inhibited P-selectin expression on platelets and platelet-derived microparticles during hypothermic ECC (p<0.05). P2Y blockade alone or combined with TGX-221 prevented ECC-induced platelet-granulocyte aggregate formation (p<0.05). Platelet adhesion to the ECC surface, platelet loss and Mac-1 expression on granulocytes were inhibited by combined P2Y and PI3K blockade (p<0.05). Combined blockade of P2Y12, P2Y1 and PI3K p110β completely inhibits hypothermic ECC-induced activation processes. This novel finding warrants further studies and the development of suitable pharmacological agents to decrease ECC- and hypothermia-associated complications in clinical applications.

Inhibition of neogenin dampens hepatic ischemia-reperfusion injury.

Objective:Liver ischemia and reperfusion injury is a common source of significant morbidity and mortality following liver transplantation, hemorrhagic shock, or major hepatic surgery. Based on studies showing a critical role for the neuronal guidance receptor neogenin (Neo1) outside the nervous system in mediating tissue adaption during acute inflammation, we hypothesized that Neo1 enhances hepatic ischemia and reperfusion injury. Design:Animal study. Setting:University-based experimental laboratory. Subjects:Wid-type, neogenin deficient and chimeric mice. Interventions:Neogenin expression was evaluated during inflammatory stimulation in vitro and during ischemia and reperfusion injury in vivo, intravital microscopy performed to study intravascular flow characteristics. The extent of liver injury was evaluated using histology, serum levels of lactate dehydrogenase, aspartate, and alanine aminotransferase. The functional role of Neo1 during liver IR was evaluated in mice with gene targeted repression of neogenin (Neo1-/-), bone marrow chimeric animals and controls. In addition, functional inhibition of neogenin was performed using antibody injection. Measurements and Main Results:We observed an induction of Neo1 during inflammation in vitro and ischemia and reperfusion in vivo. Intravital microscopy demonstrated a decreased ability of Neo1−/− leukocytes to attach to endothelial vascular wall during inflammation. Subsequent studies in Neo1−/− mice showed attenuated serum levels of lactate dehydrogenase, aspartate, alanine, and proinflammatory cytokines during hepatic ischemia and reperfusion injury. This was associated with improved hepatic histology scores. Studies in chimeric animals demonstrated that the hematopoietic Neo1 expression to be crucial for the observed results. Treatment with an anti-Neo1 antibody resulted in a significant reduction of experimental hepatic ischemia and reperfusion injury, involving attenuated variable of lactate dehydrogenase, alanine, aspartate, and cytokine levels. Conclusions:These data provide a unique role for Neo1 in the development of hepatic ischemia and reperfusion injury and identified Neo1 as a potential target to prevent liver dysfunction in the future.

The volatile anesthetic sevoflurane inhibits activation of neutrophil granulocytes during simulated extracorporeal circulation.

Extracorporeal circulation (ECC) is an essential tool for the execution of cardiac operations. However, ECC is also associated with undesirable side effects. These include the induction of a systemic inflammatory response associated with leukocyte activation and cytokine release as well as potentially life-threatening complications. The volatile anesthetic sevoflurane has been reported to exert anti-ischemic and anti-inflammatory effects. We therefore investigated whether sevoflurane modulates the ECC-triggered inflammatory response. Heparinized human blood was circulated for 90 min in a normothermic (37°C) ex vivo ECC circuit. An air-oxygen mixture was administered via an oxygenator in controls (n=5). Sevoflurane (2 vol.%) was added to the gas mixture in a second group (n=5). At baseline and after 30, 60 and 90 min of ECC, blood samples were taken. In each sample whole blood counts were determined. Expression of the activation-indicating Mac-1 receptor on granulocytes and monocytes as well as leukocyte-platelet aggregate formation was measured in flow cytometry. Levels of the granulocyte activation marker PMN-elastase and of the cytokines IL-1β, IL-8 and TNF-α were analyzed using ELISA. ECC induced significant increases in Mac-1 expression on granulocytes (p<0.001) and PMN-elastase release (p<0.001). Sevoflurane decreased granulocyte Mac-1 expression during ECC (p<0.05) and inhibited the ECC-induced PMN-elastase release (p<0.05). Sevoflurane had no effect on whole blood cell counts, leukocyte-platelet aggregate formation and cytokine release during ECC. Sevoflurane inhibits granulocyte activation during ex vivo ECC and therefore has the potential to decrease the ECC-triggered inflammatory response. This promising finding warrants further investigation under clinical conditions.

Periprocedural platelet inhibition with cangrelor in P2Y12-inhibitor naïve patients with acute coronary syndromes — A matched-control pharmacodynamic comparison in real-world patients

BACKGROUND Effective inhibition of platelet aggregation during PCI in high risk patients with ACS is of utmost importance. The new intravenous short acting P2Y12-receptor inhibitor cangrelor is available for use in PCI-treated patients. We aimed to study platelet inhibition during treatment with cangrelor and transition phase with oral P2Y12-receptor inhibitors in patients with acute coronary syndromes (ACS). METHODS Cangrelor was administered during PCI to 21 P2Y12-inhibitor naïve patients with ACS. Patients received a loading dose of ticagrelor at the time of procedure or prasugrel 30min before end of the cangrelor infusion. Platelet inhibition was measured by multiple electrode aggregometry (MEA) and thromboelastography (TEG), before and after PCI, immediately and 2h after stopping the infusion. Platelet inhibition after PCI was compared to a matched cohort of patients treated with oral P2Y12-inibitors only. RESULTS There was a significant reduction of platelet reactivity measured by MEA-ADP from 46.7U to 17.9U and by TEG MA ADP from 43.1mm to 22.0mm before infusion and after PCI respectively (p<0.001). There was also sustained platelet inhibition after stopping of cangrelor infusion and 2h later. Significant higher platelet inhibition was observed at the end of PCI in comparison to control cohort without cangrelor (MEA 17.9U vs. 54.2U, p=0.001). CONCLUSION We demonstrate significantly improved platelet inhibition during PCI in ACS patients treated with cangrelor in comparison to early treatment with potent oral P2Y12-inhibitors. Cangrelor should be considered for periprocedural treatment of high risk patients with acute coronary syndrome.

Using six-colour flow cytometry to analyse the activation and interaction of platelets and leukocytes--A new assay suitable for bench and bedside conditions.

INTRODUCTION Platelets are main effector cells in haemostasis and also promote inflammation. Platelet-leukocyte complexes are key mediators in a variety of thromboinflammatory disorders and consecutive organ failure. Cell-specific epitopes and activation markers on platelets and leukocytes can be measured using flow cytometry. However, until recently a major restriction has been a paucity in antibody combinations and lack of detection strategies. We aimed to develop a six-colour flow cytometry method which depicts multiple aspects of platelet and leukocyte interactions in human whole blood. MATERIALS AND METHODS Platelets, including microparticles and aggregates, were detected in flow cytometry using a platelet-specific anti-CD41-FITC antibody and size-defined regions. The morphology of platelet-leukocyte complexes (including granulocyte and monocyte content) were depicted using anti-CD45-PerCP, anti-CD66b-PE-Cy7, and anti-CD14-APC antibodies in a single sample. Expression of platelet and leukocyte activation markers P-selectin and CD11b were detected using anti-CD62P-PE and anti-CD11b-BV421 antibodies, respectively. RESULTS The sensitivity of this assay to detect the effects of various agonists (TRAP-6, ADP, collagen, epinephrine, TNF-α and LPS) is demonstrated. Furthermore, the assay is shown to detect platelet and leukocyte activation induced by extracorporeal circulation in vitro. The suitability of this assay for bedside analysis is demonstrated exemplarily in a patient treated with mechanical circulatory life support. CONCLUSIONS Using the concurrent assessment of multiple parameters, this method gives detailed insights into the complexity and dynamics of platelet-leukocyte interactions. This assay carries the potential to increase our understanding of the mechanisms and pathophysiology of platelet-leukocyte interaction in the research laboratory and clinical setting.

Inhibition of Adenosine Kinase Attenuates Acute Lung Injury.

Objectives:Extracellular adenosine has tissue-protective potential in several conditions. Adenosine levels are regulated by a close interplay between nucleoside transporters and adenosine kinase. On the basis of the evidence of the role of adenosine kinase in regulating adenosine levels during hypoxia, we evaluated the effect of adenosine kinase on lung injury. Furthermore, we tested the influence of a pharmacologic approach to blocking adenosine kinase on the extent of lung injury. Design:Prospective experimental animal study. Setting:University-based research laboratory. Subjects:In vitro cell lines, wild-type and adenosine kinase+/– mice. Interventions:We tested the expression of adenosine kinase during inflammatory stimulation in vitro and in a model of lipopolysaccharide inhalation in vivo. Studies using the adenosine kinase promoter were performed in vitro. Wild-type and adenosine kinase+/– mice were subjected to lipopolysaccharide inhalation. Pharmacologic inhibition of adenosine kinase was performed in vitro, and its effect on adenosine uptake was evaluated. The pharmacologic inhibition was also performed in vivo, and the effect on lung injury was assessed. Measurements and Main Results:We observed the repression of adenosine kinase by proinflammatory cytokines and found a significant influence of nuclear factor kappa-light-chain-enhancer of activated B-cells on regulation of the adenosine kinase promoter. Mice with endogenous adenosine kinase repression (adenosine kinase+/–) showed reduced infiltration of leukocytes into the alveolar space, decreased total protein and myeloperoxidase levels, and lower cytokine levels in the alveolar lavage fluid. The inhibition of adenosine kinase by 5-iodotubercidin increased the extracellular adenosine levels in vitro, diminished the transmigration of neutrophils, and improved the epithelial barrier function. The inhibition of adenosine kinase in vivo showed protective properties, reducing the extent of pulmonary inflammation during lung injury. Conclusions:Taken together, these data show that adenosine kinase is a valuable target for reducing the inflammatory changes associated with lung injury and should be pursued as a therapeutic option.

Phosphorylation of vasodilator-stimulated phosphoprotein contributes to myocardial ischemic preconditioning

Ischemic preconditioning (IP) is a well-known strategy to protect organs against cell death following ischemia. The previous work has shown that vasodilator-stimulated phosphoprotein (VASP) is involved in cytoskeletal reorganization and that it holds significant importance for the extent of myocardial ischemia reperfusion injury. Yet, the role of VASP during myocardial IP is, to date, not known. We report here that VASP phosphorylation at serine157 and serine239 is induced during hypoxia in vitro and during IP in vivo. The preconditioning-induced VASP phosphorylation inactivates the GP IIb/IIIa integrin receptor on platelets, which results in the reduced formation of organ compromising platelet neutrophil complexes. Experiments in chimeric mice confirmed the importance of VASP phosphorylation during myocardial IP. When studying this in VASP−/− animals and in an isolated heart model, we were able to confirm the important role of VASP on myocardial IP. In conclusion, we were able to show that IP-induced VASP phosphorylation in platelets is a protective mechanism against the deleterious effects of ischemia.