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Background: Lipoprotein apheresis is an important therapeutic option in homozygous familial hypercholesterolemia, progressive atherosclerosis, or when depletion of lipoprotein(a) is indicated. It is generally regarded as safe, but drops in platelet counts as well as sporadic episodes of thrombocytopenia have been reported. We assessed the influence of platelet desialylation, which may be induced by endogenous or pathogen-derived neuraminidases, on platelet adhesion to polyacrylate-based adsorbents for whole blood lipoprotein apheresis. Methods: Medical grade platelet concentrates were incubated with neuraminidase in vitro and were circulated over adsorbent columns downscaled from clinical application. Results: Cleavage of terminal sialic residues resulted in platelet activation with significantly elevated expression of platelet factor 4 (PF4) and in enhanced platelet adhesion to the adsorbent, accompanied by a pronounced drop in platelet counts in the column flow-through. Conclusion: Alterations in endogenous neuraminidase activity or exogenous (pathogen-derived) neuraminidase may trigger enhanced platelet adhesion in whole blood lipoprotein apheresis.

Inflammation and thrombosis are closely intertwined in numerous disorders, including ischemic events and sepsis, as well as coronavirus disease 2019 (COVID-19). Thrombotic complications are markers of disease severity in both sepsis and COVID-19 and are associated with multiorgan failure and increased mortality. Immunothrombosis is driven by the complement/tissue factor/neutrophil axis, as well as by activated platelets, which can trigger the release of neutrophil extracellular traps (NETs) and release further effectors of immunothrombosis, including platelet factor 4 (PF4/CXCL4) and high-mobility box 1 protein (HMGB1). Many of the central effectors of deregulated immunothrombosis, including activated platelets and platelet-derived extracellular vesicles (pEVs) expressing PF4, soluble PF4, HMGB1, histones, as well as histone-decorated NETs, are positively charged and thus bind to heparin. Here, we provide evidence that adsorbents functionalized with endpoint-attached heparin efficiently deplete activated platelets, pEVs, PF4, HMGB1 and histones/nucleosomes. We propose that this elimination of central effectors of immunothrombosis, rather than direct binding of pathogens, could be of clinical relevance for mitigating thrombotic complications in sepsis or COVID-19 using heparin-functionalized adsorbents.

Graphical abstract The emerging role of extracellular vesicles (EVs) as biomarkers and their envisioned therapeutic use require advanced techniques for their detailed characterization. In this context, we investigated gas-phase electrophoresis on a nano electrospray gas-phase electrophoretic mobility molecular analyzer (nES GEMMA, aka nES differential mobility analyzer, nES DMA) as an alternative to standard analytical techniques. In gas-phase electrophoresis, single-charged, surface-dry, native, polydisperse, and aerosolized analytes, e.g., proteins or bio-nanoparticles, are separated according to their electrophoretic mobility diameter, i.e., globular size. Subsequently, monodisperse particles are counted after a nucleation step in a supersaturated atmosphere as they pass a focused laser beam. Hence, particle number concentrations are obtained in accordance with recommendations of the European Commission for nanoparticle characterization (2011/696/EU from October 18th, 2011). Smaller sample constituents (e.g., co-purified proteins) can be detected next to larger ones (e.g., vesicles). Focusing on platelet-derived EVs, we compared different vesicle isolation techniques. In all cases, nanoparticle tracking analysis (NTA) confirmed the presence of vesicles. However, nES GEMMA often revealed a significant co-purification of proteins from the sample matrix, precluding gas-phase electrophoresis of less-diluted samples containing higher vesicle concentrations. Therefore, mainly peaks in the protein size range were detected. Mass spectrometry revealed that these main contaminants belonged to the group of globulins and coagulation-related components. An additional size exclusion chromatography (SEC) step enabled the depletion of co-purified, proteinaceous matrix components, while a label-free quantitative proteomics approach revealed no significant differences in the detected EV core proteome. Hence, the future in-depth analysis of EVs via gas-phase electrophoresis appears feasible. Platelet-derived extracellular vesicles (EVs)with/without additional size exclusion chromatographic (SEC) purification were subjected to nanoparticle tracking analysis (NTA) and gas-phase electrophoresis (nES GEMMA). The latter revealed presence of co-purified proteins, targetable via mass spectrometry (MS). MS also revealed that SEC did not influence EV protein content. To conclude, nES GEMMA is a valuable tool for quality control of EV-containing samples under native conditions allowing for detection of co-purified proteins from complex matrices. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03692-y.

There is increasing evidence that C-reactive protein (CRP) can mediate inflammatory reactions following the transformation of functionally inert pentameric CRP (pCRP) into its structural isoform pCRP* and into monomeric CRP (mCRP). This conversion can occur on the membranes of apoptotic or activated cells or on extracellular vesicles (EVs) shed from the cell surface. Here, we characterized the association of CRP with EVs in plasma from sepsis patients using flow cytometry, and found highly elevated levels of total EV counts and CRP+ EVs as compared to healthy individuals. We further assessed the ability of PentraSorb CRP, an extracorporeal device for the adsorption of CRP, to deplete free CRP and CRP+ EVs. Treatment of septic plasma with the adsorbent in vitro resulted in almost complete removal of both, free CRP and CRP+ EVs, while total EV counts remained largely unaffected, indicating the detachment of CRP from the EV surface. EVs from septic plasma elicited a release of interleukin-8 from cultured human monocytes, which was significantly reduced by adsorbent treatment prior to EV isolation. Our findings provide evidence that CRP+ EVs exhibit pro-inflammatory characteristics and can contribute to the spreading of inflammation throughout the circulation on top of their pro-coagulant activity.

The timely recognition of sepsis and the prediction of its clinical course are challenging due to the complex molecular mechanisms leading to organ failure and to the heterogeneity of sepsis patients. Treatment strategies relying on a “one-fits-all” approach have failed to reduce mortality, suggesting that therapeutic targets differ between patient subgroups and highlighting the need for accurate analysis of the molecular cascades to assess the highly variable host response. Here, we characterized a panel of 44 inflammatory mediators, including cytokines, chemokines, damage-associated molecular patterns, and coagulation-related factors, as well as markers of endothelial activation in 30 patients suffering from renal failure in the course of sepsis. All patients received continuous veno-venous hemodialysis with either high cut-off filters or with standard filters, and mediators were quantified for all patients at the initiation of dialysis and after 24 h and 48 h. Mediator concentrations in individual patients ranged widely, demonstrating the heterogeneity of sepsis patients. None of the mediators correlated with SAPS III or TISS scores. The overall in-hospital mortality of the study population was 56.7% (57.1% vs. 56.3% for high cut-off vs. standard filter). The two filter groups differed regarding most of the mediator levels at baseline, prohibiting conclusions regarding the effect of standard filters versus high cut-off filters on mediator depletion. The elevation and correlation of damage-associated molecular patterns and markers of endothelial activation gave evidence of severe tissue damage. In particular, extracellular histones were strongly increased and were almost 30-fold higher in nonsurvivors as compared to survivors, indicating their diagnostic and prognostic potential.

Secretion and exchange of biomolecules via extracellular vesicles (EVs) are crucial mechanisms in intercellular communication, and the roles of EVs in infection, inflammation, or thrombosis have been increasingly recognized. EVs have emerged as central players in immune regulation and can enhance or suppress the immune response, depending on the state of donor and recipient cells. We investigated the interaction of blood cell-derived EVs with leukocyte subpopulations (monocytes and their subsets, granulocytes, B cells, T cells, and NK cells) directly in whole blood using a combination of flow cytometry, imaging flow cytometry, cell sorting, and high resolution confocal microscopy. Platelet-derived EVs constituted the majority of circulating EVs and were preferentially associated with granulocytes and monocytes, while they scarcely interacted with lymphocytes. Further flow cytometric differentiation of monocyte subsets provided clear indications for a preferential association of platelet-derived EVs with intermediate (CD14++CD16+) monocytes in whole blood.

Lipoprotein apheresis is applied to deplete low density lipoprotein and other apolipoprotein B containing lipoproteins in patients with severe familial hypercholesterolemia, hypertriglyceridemia associated pancreatitis, or lipoprotein (a)-hyperlipoproteinemia. Anticoagulation of the extracorporeal circuit may influence cellular activation, as evidenced by a reduction of inflammatory parameters during regional citrate anticoagulation with acid citrate dextrose A (ACD-A) commonly used in whole blood lipid apheresis. While the citrate concentration in the extracorporeal circuit has to ensure efficient anticoagulation, citrate infusion into the patient should be limited to avoid citrate overload. We assessed the influence of citrate concentration on cellular activation during in vitro circulation of whole blood containing 2.8 mM citrate (ACD-A 1:40), 5.6 mM citrate (ACD-A 1:20), or 13 mM citrate over polyacrylate-based adsorbents for lipoprotein apheresis. We found increased platelet adhesion for anticoagulation with 2.8 mM citrate as compared to 5.6 or 13 mM citrate, as shown by cell counting and confirmed by scanning electron microscopy of adsorbent beads as well as by elevated levels of platelet activation markers and of platelet-derived microvesicles. Leukocytes showed an equivalent adhesion pattern, while red blood cells remained unaffected at all citrate concentrations. Passage of blood over two consecutive columns resulted in enhanced platelet adhesion to the second column, presumably due to upstream preactivation. In conclusion, citrate influences activation and adhesion of platelets and leukocytes in a concentration-dependent manner, and ACD-A 1:20, equivalent to a citrate concentration of 5.6 mM in whole blood, ensures minimal cellular activation during passage of whole blood over polyacrylate-based adsorbents.

Human monocytes include CD14++CD16- (classical), CD14++CD16+ (intermediate), and CD14+CD16++ (non-classical) subsets with divergent roles in immune regulation and inflammation. Since the functional characterization of monocyte subsets is most commonly performed using isolated monocytes, we investigated the influence of different monocyte isolation protocols on the relative abundance of monocyte subsets. Using flow cytometric subset characterization directly in whole blood as a reference, we found that monocyte isolation by enrichment of peripheral blood mononuclear cells and subsequent depletion of non-monocytes by magnetic labeling did not alter the distribution of monocyte subsets. Particularly, we failed to detect a loss of CD16+ subsets upon monocyte isolation, although one of the negative depletion protocols used contained an anti-CD16 antibody to label granulocytes. Overnight storage of isolated monocytes induced a significant repartition of monocyte subsets towards CD14++CD16+ intermediate monocytes, which was barely seen in stored whole blood. We identified intermediate monocytes as main binding partners of platelet-derived extracellular vesicles (EVs) and propose that residual platelets contained in isolated monocyte preparations release EVs that induce the expression of the IgG receptor FcγRIII (CD16) on monocytes.

Secretion and exchange of biomolecules by extracellular vesicles (EVs) are crucial in intercellular communication and enable cells to adapt to alterations in their microenvironment. EVs are involved in a variety of cellular processes under physiological conditions as well as in pathological settings. In particular, they exert profound effects on the innate immune system, and thereby are also capable of modulating adaptive immunity. The mechanisms underlying their interaction with their recipient cells, particularly their preferential association with monocytes and granulocytes in the circulation, however, remain to be further clarified. Surface molecules exposed on EVs are likely to mediate immune recognition and EV uptake by their recipient cells. Here, we investigated the involvement of Tyro3, Axl, and Mer (TAM) tyrosine kinase receptors and of integrin CD11b in the binding of platelet-derived EVs, constituting the large majority of circulating EVs, to immune cells in the circulation. Flow cytometry and Western Blotting demonstrated a differential expression of TAM receptors and CD11b on monocytes, granulocytes, and lymphocytes, as well as on monocyte subsets. Of the TAM receptors, only Axl and Mer were detected at low levels on monocytes and granulocytes, but not on lymphocytes. Likewise, CD11b was present on circulating monocytes and granulocytes, but remained undetectable on lymphocytes. Differentiation of monocytes into classical, intermediate, and non-classical monocyte subsets revealed distinct expression patterns of Mer and activated CD11b. Co-incubation of isolated monocytes and granulocytes with platelet-derived EVs showed that the binding of EVs to immune cells was dependent on Ca++. Our data do not support a particular role for TAM receptors or for activated CD11b in the association of platelet-derived EVs with monocytes and granulocytes in the circulation, as anti-TAM antibodies did not interfere with EV binding to isolated immune cells, as binding was not dependent on the presence of TIM4 acting synergistically with TAM receptors, and as neither low levels of Gas6, required as a linker between phosphatidylserine (PS) on the EV surface and TAM receptors on immune cells, nor masking of PS on the EV surface did interfere with EV binding.

Extracellular vesicles are central players in intercellular communication and are released from the plasma membrane under tightly regulated conditions, depending on the physiological and pathophysiological state of the producing cell. Their heterogeneity requires a spectrum of methods for isolation and characterization, where pre-analytical parameters have profound impact on vesicle analysis, particularly in blood, since sampling, addition of anticoagulants, as well as post-sampling vesicle generation may influence the outcome. Here, we characterized microvesicles directly in whole blood using a combination of flow cytometry and imaging flow cytometry. We assessed the influence of sample agitation, anticoagulation, and temperature on post-sampling vesicle generation, and show that vesicle counts remained stable over time in samples stored without agitation. Storage with gentle rolling mimicking agitation, in contrast, resulted in strong release of platelet-derived vesicles in blood anticoagulated with citrate or heparin, whereas vesicle counts remained stable upon anticoagulation with EDTA. Using imaging flow cytometry, we could visualize microvesicles adhering to blood cells and revealed an anticoagulant-dependent increase in vesicle-cell aggregates over time. We demonstrate that vesicles adhere preferentially to monocytes and granulocytes in whole blood, while no microvesicles could be visualized on lymphocytes. Our data underscore the relevance of pre-analytical parameters in vesicle analysis and demonstrate that imaging flow cytometry is a suitable tool to study the interaction of extracellular vesicles with their target cells.

New anti-viral agents and strategies are urgently needed to fight rapidly mutating viruses, as vaccine programs cannot react fast enough to prevent pandemics. Recently, we have shown that interleukin-24 (IL-24) sensitizes tumor cells to toll-like receptor 3 (TLR3) mediated apoptosis. As influenza A virus stimulates the TLR3 receptor, we hypothesized that IL-24 might also exert an anti-viral effect. This study demonstrates that IL-24 reduces the titer of different influenza A virus subtypes independently of type I interferon in an apoptosis dependent manner. The anti-viral effect of IL-24 correlated with caspase-3 activation and could be blocked by a pan-caspase inhibitor and by small interfering RNA (siRNA) directed towards TLR3. Surprisingly, caspase-3 activation in influenza A virus/IL-24-stimulated cells correlated with the down-regulation of the B-cell lymphoma 2 (Bcl-2) family member myeloid cell leukemia 1 (Mcl-1). Correspondingly, knockdown of Mcl-1 by siRNA enhanced caspase activation in influenza A virus infected cells and was furthermore linked to a reduction of viral titers. We conclude that IL-24 exerts an anti-viral role selectively purging virally infected cells by leading to a down-regulation of Mcl-1. Our findings might therefore represent the first step towards a new rational concept in the development of anti-viral strategies based on the induction of apoptosis.

Background Regional citrate anticoagulation has been associated with enhanced biocompatibility in hemodialysis, but the optimal dose of citrate remains to be established. Here, we compared parameters related to cellular activation during in vitro dialysis, using two doses of citrate. Methods Human whole blood, anticoagulated with either 3 mM or 4 mM of citrate, was recirculated in an in vitro miniaturized dialysis setup. Complement (C3a-desArg), soluble platelet factor 4 (PF4), thromboxane B2 (TXB2), myeloperoxidase (MPO), as well as platelet- and red blood cell-derived extracellular vesicles (EV) were quantified during recirculation. Dialyzer fibers were examined by scanning electron microscopy after recirculation to assess the activation of clotting and the deposition of blood cells. Results Increases in markers of platelet and leukocyte activation, PF4, TXB2, and MPO were comparable between both citrate groups. Complement activation tended to be lower at higher citrate concentration, but the difference between the two citrate groups did not reach significance. A strong increase in EVs, particularly platelet-derived EVs, was observed during in vitro dialysis for both citrate groups, which was significantly less pronounced in the high citrate group at the end of the experiment. Assessment of dialyzer clotting scores after analysis of individual fibers by scanning electron microscopy revealed significantly lower scores in the high citrate group. Conclusions Our data indicate that an increase in the citrate concentration from 3 mM to 4 mM further dampens cellular activation, thereby improving biocompatibility. A concentration of 4 mM citrate might therefore be optimal for use in clinical practice.

Cells release diverse types of vesicles constitutively or in response to proliferation, injury, inflammation, or stress. Extracellular vesicles (EVs) are crucial in intercellular communication, and there is emerging evidence for their roles in inflammation, cancer, and thrombosis. We investigated the thrombogenicity of platelet-derived EVs, which constitute the majority of circulating EVs in human blood, and assessed the contributions of phosphatidylserine and tissue factor exposure on thrombin generation. Addition of platelet EVs to vesicle-free human plasma induced thrombin generation in a dose-dependent manner, which was efficiently inhibited by annexin V, but not by anti-tissue factor antibodies, indicating that it was primarily due to the exposure of phosphatidylserine on platelet EVs. Platelet EVs exhibited higher thrombogenicity than EVs from unstimulated monocytic THP-1 cells, but blockade of contact activation significantly reduced thrombin generation by platelet EVs. Stimulation of monocytic cells with lipopolysaccharide enhanced their thrombogenicity both in the presence and in the absence of contact activation, and thrombin generation was efficiently blocked by anti-tissue factor antibodies. Our study provides evidence that irrespective of their cellular origin, EVs support the propagation of coagulation via the exposure of phosphatidylserine, while the expression of functional tissue factor on EVs appears to be limited to pathological conditions.

Whole blood lipid apheresis is clinically applied to reduce low density lipoprotein cholesterol in patients with homozygous familial hypercholesterolemia. Here, we studied the correlation between physicochemical parameters, in particular, surface roughness and blood compatibility, of two polyacrylate-based and a dextran sulfate-based polymer for lipid apheresis. The adsorbent surface roughness was assessed by atomic force microscopy. Freshly isolated human thrombocytes were circulated over adsorbent columns downscaled equivalent to clinical use to study thrombocyte adhesion and microvesicle generation. Quantification of thrombocytes and microvesicles in the flow-through of the columns revealed that both thrombocyte adhesion and microvesicle generation increased with increasing adsorbent surface roughness. Activation of thrombocytes with thrombin receptor-activating peptide-6 favored their adhesion to the adsorbents, as demonstrated by preferential depletion of CD62(+) and PAC-1(+) thrombocytes. Taken together, enhanced polymer surface roughness fostered cell adhesion and microvesicle release, underscoring the role of extracellular microvesicles as markers of cellular activation and of blood compatibility.

Whole blood lipid apheresis is clinically applied in patients with familial hypercholesterolemia to reduce low density lipoprotein and other apolipoprotein B 100 containing lipoproteins. Here, the hemocompatibility of two polyacrylate-coated polyacrylamide-based polymers for lipid apheresis by evaluating the adhesion of blood cells to the adsorbent polymers, their respective activation, as well as the release of microvesicles during circulation of whole blood over the polymers was studied. Characterization of the adsorbents by scanning electron microscopy, atomic force microscopy, and X-ray photoelectron spectroscopy revealed differences with respect to their surface morphology and their surface chemical composition. Despite these differences, equivalent amounts of leukocytes and platelets adhered to both polymers during circulation of whole blood over the adsorbent columns. The release of phosphatidylserine-exposing microvesicles, in contrast, increased significantly with increasing surface roughness and with the amount of polyacrylate groups at the adsorbent surface. The majority of microvesicles generated during blood-material contact were platelet-derived, and their release was associated with enhanced thrombin generation. Microvesicles were present in free and in cell-bound form, and 75% of all monocytes, but only 0.2% and 2.3% of red blood cells and platelets, respectively, were associated with microvesicles, pointing to a role of monocytes in the clearance of released microvesicles. Taken together, microvesicles are sensitive indicators for biomaterial-induced activation of blood cells in apheresis. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 636-646, 2017.