Early platelet dysfunction in patients receiving extracorporeal membrane oxygenation is associated with mortality.

Extracorporeal membrane oxygenation (ECMO) is used for patients with cardiopulmonary failure and is associated with severe bleeding and poor outcome. Platelet dysfunction may be a contributing factor. The aim of this prospective observational study was to characterize platelet dysfunction and its relation to outcome in ECMO patients. Blood was sampled from thirty ECMO patients at three timepoints. Expression of CD62P, CD63, activated GPIIb/IIIa, GPVI, GPIbα and formation platelet-leukocyte aggregates (PLA) were analyzed at rest and in response to stimulation. Delta granule storage-pool deficiency and secretion defects were also investigated. Fifteen healthy volunteers and ten patients with coronary artery disease served as controls. Results were also compared between survivors and non-survivors. Compared to controls, expression of platelet surface markers, delta granule secretion and formation of PLA was reduced, particularly in response to stimulation. Baseline CD63 expression was higher and activated GPIIb/IIIa expression in response to stimulation was lower in non-survivors on day 1 of ECMO. Logistic regression analysis revealed that these markers were associated with mortality. In conclusion, platelets from ECMO patients are severely dysfunctional predisposing patients to bleeding complications and poor outcome. Platelet dysfunction on day 1 of ECMO detected by the platelet surface markers CD63 and activated GPIIb/IIIa is associated with mortality. CD63 and activated GPIIb/IIIa may therefore serve as novel prognostic biomarkers, but future studies are required to determine their true potential.

Cross-TCR Antagonism Revealed by Optogenetically Tuning the Half-Life of the TCR Ligand Binding.

Activation of T cells by agonistic peptide-MHC can be inhibited by antagonistic ones. However, the exact mechanism remains elusive. We used Jurkat cells expressing two different TCRs and tested whether stimulation of the endogenous TCR by agonistic anti-Vß8 antibodies can be modulated by ligand-binding to the second, optogenetic TCR. The latter TCR uses phytochrome B tetramers (PhyBt) as ligand, the binding half-life of which can be altered by light. We show that this half-life determined whether the PhyBt acted as a second agonist (long half-life), an antagonist (short half-life) or did not have any influence (very short half-life) on calcium influx. A mathematical model of this cross-antagonism shows that a mechanism based on an inhibitory signal generated by early recruitment of a phosphatase and an activating signal by later recruitment of a kinase explains the data.

Extracellular Vesicles Are Associated With Outcome in Veno-Arterial Extracorporeal Membrane Oxygenation and Myocardial Infarction.

Background: Veno-arterial extracorporeal membrane oxygenation (VA-ECMO) is being increasingly applied in patients with circulatory failure, but mortality remains high. An inflammatory response syndrome initiated by activation of blood components in the extracorporeal circuit may be an important contributing factor. Patients with ST-elevation myocardial infarction (STEMI) may also experience a systemic inflammatory response syndrome and are at risk of developing cardiogenic shock and cardiac arrest, both indications for VA-ECMO. Extracellular vesicles (EV) are released by activated cells as mediators of intercellular communication and may serve as prognostic biomarkers. Cardiomyocyte EV, released upon myocardial ischemia, hold strong potential for this purpose. The aim of this study was to assess the EV-profile in VA-ECMO and STEMI patients and the association with outcome. Methods: In this prospective observational study, blood was sampled on day 1 after VA-ECMO initiation or myocardial reperfusion (STEMI patients). EV were isolated by differential centrifugation. Leukocyte, platelet, endothelial, erythrocyte and cardiomyocyte (caveolin-3+) Annexin V+ EV were identified by flow cytometry. EV were assessed in survivors vs. non-survivors of VA-ECMO and in STEMI patients with normal-lightly vs. moderately-severely reduced left ventricular function. Logistic regression was conducted to determine the predictive accuracy of EV. Pearson correlation analysis of EV with clinical parameters was performed. Results: Eighteen VA-ECMO and 19 STEMI patients were recruited. Total Annexin V+, cardiomyocyte and erythrocyte EV concentrations were lower (p ≤ 0.005) while the percentage of platelet EV was increased in VA-ECMO compared to STEMI patients (p = 0.002). Total Annexin V+ EV were increased in non-survivors of VA-ECMO (p = 0.01), and higher levels were predictive of mortality (AUC = 0.79, p = 0.05). Cardiomyocyte EV were increased in STEMI patients with moderately-severely reduced left ventricular function (p = 0.03), correlated with CK-MBmax (r = 0.57, p = 0.02) and time from reperfusion to blood sampling (r = 0.58, p = 0.01). Leukocyte EV correlated with the number of coronary stents placed (r = 0.60, p = 0.02). Conclusions: Elevated total Annexin V+ EV on day 1 of VA-ECMO are predictive of mortality. Increased cardiomyocyte EV on day 1 after STEMI correlate with infarct size and are associated with poor outcome. These EV may aid in the early identification of patients at risk of poor outcome, helping to guide clinical management.

Optogenetic control shows that kinetic proofreading regulates the activity of the T cell receptor.

The immune system distinguishes between self and foreign antigens. The kinetic proofreading (KPR) model proposes that T cells discriminate self from foreign ligands by the different ligand binding half-lives to the T cell receptor (TCR). It is challenging to test KPR as the available experimental systems fall short of only altering the binding half-lives and keeping other parameters of the interaction unchanged. We engineered an optogenetic system using the plant photoreceptor phytochrome B (PhyB) as a ligand to selectively control the dynamics of ligand binding to the TCR by light. This opto-ligand-TCR system was combined with the unique property of PhyB to continuously cycle between the binding and non-binding states under red light, with the light intensity determining the cycling rate and thus the binding duration. Mathematical modeling of our experimental datasets showed that indeed the ligand-TCR interaction half-life is the decisive factor for activating downstream TCR signaling, substantiating KPR.