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Vector-based SARS-CoV-2 vaccines have been associated with vaccine- induced thrombosis with thrombocytopenia syndrome (VITT/TTS), but the causative factors are still unresolved. We comprehensively analyzed the ChAdOx1 nCoV-19 (AstraZeneca) and Ad26.COV2.S (Johnson and Johnson) vaccines. ChAdOx1 nCoV-19 contains significant amounts of host cell protein impurities, including functionally active proteasomes, and adenoviral proteins. A much smaller amount of impurities was found in Ad26.COV2.S. Platelet factor 4 formed complexes with ChAdOx1 nCoV-19 constituents, but not with purified virions from ChAdOx1 nCoV-19 or with Ad26.COV2.S. Vascular hyperpermeability was induced by ChAdOx nCoV-19 but not by Ad26.COV2.S. These differences in impurities together with EDTAinduced capillary leakage might contribute to the higher incidence rate of VITT associated with ChAdOx1 nCoV-19 compared to Ad26.COV2.S.
Platelet adhesion and spreading at the sites of vascular injury is vital to hemostasis. As an integral part of the innate immune system, platelets interact with opsonized bacterial pathogens through FcγRIIA and contribute to host defense. As mechanoscavangers, platelets actively migrate and capture bacteria via cytoskeleton-rich, dynamic structures, such as filopodia and lamellipodia. However, the role of human platelet FcγRIIA in cytoskeleton-dependent interaction with opsonized bacteria is not well understood. To decipher this, we used a reductionist approach with well-defined micropatterns functionalized with immunoglobulins mimicking immune complexes at planar interfaces and bacteriamimetic microbeads. By specifically blocking of FcγRIIA and selective disruption of the platelet cytoskeleton, we show that both functional FcγRIIA and cytoskeleton are necessary for human platelet adhesion and haptotaxis. The direct link between FcγRIIA and the cytoskeleton is further explored by single-particle tracking. We then demonstrate the relevance of cytoskeleton-dependent differential mobilities of FcγRIIA on bacteria opsonized with the chemokine platelet factor 4 (PF4) and patient-derived anti-PF4/polyanion IgG. Our data suggest that efficient capture of opsonized bacteria during host-defense is governed by mobility dynamics of FcγRIIA on filopodia and lamellipodia, and the cytoskeleton plays an essential role in platelet morphodynamics at biological interfaces that display immune complexes.
Divalent magnesium restores cytoskeletal storage lesions in cold-stored platelet concentrates
(2022)
Cold storage of platelet concentrates (PC) has become attractive due to the reduced risk of bacterial proliferation, but in vivo circulation time of cold-stored platelets is reduced. Ca2+ release from storage organelles and higher activity of Ca2+ pumps at temperatures < 15 °C triggers cytoskeleton changes. This is suppressed by Mg2+ addition, avoiding a shift in Ca2+ hemostasis and cytoskeletal alterations. We report on the impact of 2–10 mM Mg2+ on cytoskeleton alterations of platelets from PC stored at room temperature (RT) or 4 °C in additive solution (PAS), 30% plasma. Deformation of platelets was assessed by real-time deformability cytometry (RT-DC), a method for biomechanical cell characterization. Deformation was strongly affected by storage at 4 °C and preserved by Mg2+ addition ≥ 4 mM Mg2+ (mean ± SD of median deformation 4 °C vs. 4 °C + 10 mM Mg2+ 0.073 ± 0.021 vs. 0.118 ± 0.023, p < 0.01; n = 6, day 7). These results were confirmed by immunofluorescence microscopy, showing that Mg2+ ≥ 4 mM prevents 4 °C storage induced cytoskeletal structure lesion. Standard in vitro platelet function tests showed minor differences between RT and cold-stored platelets. Hypotonic shock response was not significantly different between RT stored (56.38 ± 29.36%) and cold-stored platelets with (55.22 ± 11.16%) or without magnesium (45.65 ± 11.59%; p = 0.042, all n = 6, day 1). CD62P expression and platelet aggregation response were similar between RT and 4 °C stored platelets, with minor changes in the presence of higher Mg2+ concentrations. In conclusion, increasing Mg2+ up to 10 mM in PAS counteracts 4 °C storage lesions in platelets, maintains platelet cytoskeletal integrity and biomechanical properties comparable to RT stored platelets.
Inherited platelet disorders affecting the human platelet cytoskeleton result in increased bleeding risk. However, deciphering their impact on cytoskeleton-dependent intrinsic biomechanics of platelets remains challenging and represents an unmet need from a diagnostic and prognostic perspective. It is currently unclear whether ex vivo anticoagulants used during collection of peripheral blood impact the mechanophenotype of cellular components of blood. Using unbiased, high-throughput functional mechanophenotyping of single human platelets by real-time deformability cytometry, we found that ex vivo anticoagulants are a critical pre-analytical variable that differentially influences platelet deformation, their size, and functional response to agonists by altering the cytoskeleton. We applied our findings to characterize the functional mechanophenotype of platelets from a patient with Myosin Heavy Chain 9 (MYH9) related macrothrombocytopenia. Our data suggest that platelets from MYH9 p.E1841K mutation in humans affecting platelet non-muscle myosin heavy chain IIa (NMMHC-IIA) are biomechanically less deformable in comparison to platelets from healthy individuals.
Platelets within one individual display heterogeneity in reactivity, size, age, and expression of surface receptors. To investigate the combined intraindividual contribution of platelet size, platelet age, and receptor expression levels on the reactivity of platelets, we studied fractions of large and small platelets from healthy donors separated by using differential centrifugation. Size-separated platelet fractions were perfused over a collagen-coated surface to assess thrombus formation. Multicolor flow cytometry was used to characterize resting and stimulated platelet subpopulations, and platelet age was determined based on RNA and HLA-I labeling. Signal transduction was analyzed by measuring consecutive phosphorylation of serine/threonine-protein kinase Akt. Compared with small platelets, large platelets adhered faster to collagen under flow and formed larger thrombi. Among the large platelets, a highly reactive juvenile platelet subpopulation was identified with high glycoprotein VI (GPVI) expression. Elevated GPVI expression correlated with high HLA-I expression, RNA content, and increased platelet reactivity. There was a stronger difference in Akt phosphorylation and activation upon collagen stimulation between juvenile and older platelets than between large and small platelets. GPVI expression and platelet reactivity decreased throughout platelet storage at 22°C and was better maintained throughout cold storage at 4°C. We further detected higher GPVI expression in platelets of patients with immune thrombocytopenia. Our findings show that high GPVI expression is a feature of highly reactive juvenile platelets, which are predominantly found among the large platelet population, explaining the better performance of large platelets during thrombus formation. These data are important for studies of thrombus formation, platelet storage, and immune thrombocytopenia.
Thrombozyten haben neben ihrer Funktion in der Hämostase eine wichtige Rolle in der Immunabwehr. Sie interagieren hierbei mit Komponenten des angeborenen und des adaptiven Immunsystems und sind in der Lage, direkte anti-mikrobielle Einflüsse zu vermitteln. Die Interaktion von Thrombozyten mit Gram-positiven Bakterien unterscheidet sich von jener mit Gram-negativen Erregern. Bei beiden Gruppen von Bakterien scheint die Aktivierung von Thrombozyten und Freisetzung anti-mikrobieller Peptiden aus den Granula ein wichtiger Bestandteil der direkten Pathogenabwehr durch Thrombozyten zu sein. Hierbei führt die Interaktion mit S. aureus direkt zu einer starken pathogen-induzierten Thrombozytenaktivierung, während bei Gram-negativen Organismen wie E. coli eine Verstärkung durch die Opsonierung mit PF4 und anti-PF4/H IgG notwendig scheint. Vermutlich ist die Bindung von PF4 und anti-PF4/H IgG an Gram-positive Bakterien von größerer Bedeutung für die Opsonierung für andere Immunzellen als für den direkten bakteriziden Effekt der Thrombozyten.
Der Gram-positive S. pneumoniae führt durch Funktionsstörung und Exposition von Phosphatidylserin zu einer Schädigung der Thrombozyten. Dieser schädigende Effekt auf Thrombozyten durch S. pneumoniae wird unter anderem durch Pneumolysin, ein porenbildendes Toxin der Pneumokokken, vermittelt. Dieses induziert bereits in geringen Konzentrationen die Porenbildung in der Thrombozytenmembran und führt zur Induktion von Apoptose.
In der Arbeit konnten die initialen Fragestellungen folgendermaßen beantwortet werden:
1.Thrombozyten können einen direkten schädigenden Effekt auf Gram-positive Bakterien vermitteln.
2.PF4 und anti-PF4/Polyanion IgG spielen in der direkten Thrombozyten-vermittelten Pathogenabwehr bei Gram-positiven Erregern, trotz der Bindung an Gram-positive Bakterien, eine untergeordnete Rolle. Sie verstärken weder die Thrombozyten-aktivierung noch den anti-bakteriellen Effekt.
3.Die Auswirkung der Co-Inkubation mit Bakterien auf die Thrombozyten ist heterogen und abhängig vom untersuchten Bakterienstamm. Es kommt zur Aktivierung der Thrombozyten durch S. aureus und zur Schädigung der Thrombozyten durch S. pneumoniae.
Im Falle einer neuen, sich rasch ausbreitenden viralen Erkrankung empfiehlt die WHO die Verwendung von Blutplasma von bereits Genesenen (Rekonvaleszenten-plasma) zur ersten Therapie bevor Impfstoffe entwickelt werden können, da dieses Plasma bereits Immunglobuline gegen den Erreger enthält. In dieser zeitkritischen Situation kann die von der deutschen Richtlinie für Hämotherapie geforderte Quarantänelagerung für Plasmen für mindestens vier Monate nicht immer ein-gehalten werden. Eine Alternative zur Quarantänelagerung stellen Verfahren zur Pathogeninaktivierung des Plasmas dar. Es muss jedoch sichergestellt werden, dass die enthaltenen Immunglobuline durch die Pathogeninaktivierung nicht beeinträchtigt werden. In dieser Arbeit konnte gezeigt werden, dass die Pathogeninaktivierung von Plasma mittels Methylenblau-Behandlung keinen signifikanten Einfluss auf Immun-globuline M und G hat. Die Ergebnisse können die Sicherheit der Verwendung von Rekonvaleszentenplasma zu Beginn einer Ausbreitung einer viralen Erkrankung deutlich erhöhen.
Blutplasma muss generell blutgruppenkompatibel transfundiert werden, um Blutgruppeninkompatibilitäten durch enthaltene gegen die Blutgruppenantigene A und B gerichetete Antikörper (Isoagglutinine) zu vermeiden. Plasma von Spendern der Blutgruppe AB enthält keine Isoagglutinine und kann somit Patienten aller Blutgruppen transfundiert werden. Der Anteil der Spender in der mitteleuropäischen Bevölkerung mit Blutgruppe AB ist mit 4 % jedoch sehr gering. Daher besteht der Bedarf Isoagglutinine aus Plasma der Blutguppe A, B und 0 zu entfernen, um dieses „universell“ transfundierbar zu machen. Das so hergestellte Isoagglutinin-depletierte Plasma kann auch für Notfalltransfusionen bei Patienten mit unbekannter Blutgruppe angewendet werden. Kleinere Krankenhäuser können ihre Logistik effizienter gestalten und lediglich diese Art von Plasma bevorraten.
Im Rahmen dieser Arbeit wurden ein geschlossenes Beutelsystem und ein Prozess entwickelt, welcher die Depletion von Isoagglutininen aus Plasma mit Mitteln ermöglicht, die in jeder transfusionsmedizinischen Einrichtung vorhanden sind. Es konnte gezeigt werden, dass die Qualität des Plasmas der des bereits zugelassenen humanen gefrorenen Frischplasmas entspricht. Durch Poolen im Herstellungs-prozess können mehrere Plasmen gleichzeitig bereitgestellt werden. Das Verfahren hat das Potenzial, die klinische Praxis der Plasmatherapie zu beeinflussen.
Background
Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a prothrombotic, heparin-induced thrombocytopenia (HIT)-mimicking, adverse reaction caused by platelet-activating anti-platelet factor 4 (PF4) antibodies that occurs rarely after adenovirus vector-based COVID-19 vaccination. Strength of PF4-dependent enzyme immunoassay (EIA) reactivity—judged by optical density (OD) measurements—strongly predicts platelet-activating properties of HIT antibodies in a functional test. Whether a similar relationship holds for VITT antibodies is unknown.
Objectives
To evaluate probability for positive platelet activation testing for VITT antibodies based upon EIA OD reactivity; and to investigate simple approaches to minimize false-negative platelet activation testing for VITT.
Methods
All samples referred for VITT testing were systematically evaluated by semiquantitative in-house PF4/heparin-EIA (OD readings) and PF4-induced platelet activation (PIPA) testing within a cohort study. EIA-positive sera testing PIPA-negative were retested following 1/4 to 1/10 dilution. Logistic regression was performed to predict the probability of a positive PIPA per magnitude of EIA reactivity.
Results
Greater EIA ODs in sera from patients with suspected VITT correlated strongly with greater likelihood of PIPA reactivity. Of 61 sera (with OD values >1.0) testing negative in the PIPA, a high proportion (27/61, 44.3%) became PIPA positive when tested at 1/4 to 1/10 dilution.
Conclusions
VITT serology resembles HIT in that greater EIA OD reactivity predicts higher probability of positive testing for platelet-activating antibodies. Unlike the situation with HIT antibodies, however, diluting putative VITT serum increases probability of a positive platelet activation assay, suggesting that optimal complex formation depends on the stoichiometric ratio of PF4 and anti-PF4 VITT antibodies.