Institut für Immunologie u. Transfusionsmedizin - Abteilung Transfusionsmedizin
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Microenvironment contains biophysical and biochemical elements to maintain survival, growth, proliferation, and differentiation of cells. Any change can lead to cell response to the mechanical forces, which can be described by elasticity. It is an indicator of a cell’s state since it plays an important role in many cellular processes. In many cases, cell elasticity is measured by using discontinuous manner, which may not allow elucidating real-time activity of individual live cells in physiological condition or cell response against microenvironmental changes. I argue that measuring cell elasticity using continuously repetitive nanoindentation technique is important that should be considered. As an example, I discuss mechanics of human embryonic kidney (HEK) cells in various conditions. In resting cells, there is an activity of the cytoskeleton whose oscillation amplitude is strongly affected by the intracellular calcium, and the collective activity of myosin motor proteins induces elasticity oscillation. Experimental results also reveal that actin cytoskeleton and cell membrane determine cell mechanics.
Background: Plasma transfusion is one of the basic treatments in patients with major blood loss. The anti-A and anti-B antibodies contained in the plasma demand ABO blood group compatibility. This is limiting the use of plasma in emergency situations and can cause a shortage in the supply of plasma of certain blood groups. We developed a method for anti-A and anti-B depletion by adsorbing plasma isoagglutinins using red blood cells.
Materials and Methods: Three units of fresh frozen plasma were thawed after quarantine storage, pooled, and an aliquot of red cell concentrate was added. After 2 h of incubation at room temperature antibody-red-cell complexes were removed by centrifugation, the isoagglutinin-depleted plasma was split into three units and deep frozen. Isoagglutinin titers, free hemoglobin, residual red cells, clotting factor activity, and sterility of plasma units were determined after isoagglutinin depletion and a double freeze-thawing procedure.
Results: Anti-B titers in group A plasma were reduced from values of 1:64 to 1:1 or lower, anti-A titers in group B plasma decreased from values of 1:128 to at least 1:16. Postprocedure clotting factor activities were preserved with 88.0 ± 7.3% (factor V), 106.9 ± 11.4% (factor VIII), and 84.0 ± 7.5% (factor XI) fulfilling the quality control requirements. No residual red cells were found, but free hemoglobin slightly increased to 53.7 ± 5.2 μmol/L. All units were sterile.
Discussion: We described a method for the production of anti-A- and anti-B-depleted plasma in a closed system that uses standard equipment. The resulting isoagglutinin-depleted plasma may allow for blood group independent plasma transfusion.
Acute respiratory distress syndrome (ARDS) ist eine gefürchtete Komplikation bei Patienten mit Pneumokokken-Pneumonien. Die Zerstörung von Thrombozyten durch Pneumolysin, das Haupttoxin der Pneumokokken, trägt vermutlich zur Entwicklung und dem Fortschreiten von ARDS bei. Eine Inhibition der Pneumolysin-induzierten Thrombozytenschädigung könnte daher eine unterstützende Therapie bei Patienten mit Pneumokokken-Pneumonien darstellen. Kandidaten für solch einen therapeutischen Ansatz sind polyvalente Immunglobulinpräparate. Eine experimentelle Studie zeigte kürzlich, dass ein IgG Immunglobulinpräparat (IVIG, 98 % IgG, CSL Behring) die Pneumolysin-induzierte Thrombozytenschädigung inhibieren kann. Eine klinische Studie wiederum zeigte kürzlich, dass ein mit IgM/IgA angereichertes Immunglobulinpräparat (trimodulin, 23 % IgM, 21 % IgA, 56 % IgG, Biotest AG) die Sterblichkeit bei Patienten mit schwerer Pneumonie senken kann.
Auf Grundlage dieser Ergebnisse wurde in der vorliegenden Studie die Inhibition der Pneumolysin-induzierten Thrombozytenschädigung durch trimodulin untersucht und dabei die Wirksamkeit mit derer von IVIG verglichen.
Mittels Bestimmung der CD62P-Freisetzung (Durchflusszytometrie), der intrazellulären Ca2+-Freisetzung und der Thrombozytenlyse (Aggregometrie) wurden das Ausmaß und die Inhibition der Thrombozytenschädigung ermittelt. Anschließend wurde die Aufrechterhaltung komplexerer Thrombozytenfunktionen untersucht. Hierbei wurde die Viabilität der Thrombozyten (Viability assay) und die Fähigkeit der Thrombozyten, Poren zu verschließen (Transwell assay), untersucht. Im Vollblut wurde die Fähigkeit zur Thrombusbildung nachgewiesen (Flow chamber assay). Um aufzuzeigen, dass die Pneumolysin-Antagonisierung vermutlich auf Pneumolysin-spezifisches IgG zurückzuführen ist, wurde dieses in trimodulin und IVIG detektiert (Western blot). Darüber hinaus wurden die relativen Anti-Pneumolysin-IgG-Spiegel in IVIG mit einer Kohorte von Pneumoniepatienten und einer gesunden Kohorte verglichen.
Insgesamt zeigten beide Immunglobulinpräparate eine wirksame Inhibition der Pneumolysin-induzierten Thrombozytenschädigung in vitro. Somit sind trimodulin und IVIG vielversprechende Kandidaten für eine mögliche unterstützende Therapie von Patienten mit schwerer Pneumonie. Allerdings zeigte sich im Vergleich der beiden Substanzen trimodulin weniger potent als IVIG. Nur bei Konzentrationen mit dem gleichen finalen IgG-Gehalt (trimodulin 1,79 mg/ml und IVIG 1 mg/ml mit je 1 mg/ml IgG) zeigten die Immunglobulinpräparate eine ähnliche Wirksamkeit in der Inhibition der Pneumolysin-induzierten Thrombozytenschädigung. Dies deutet darauf hin, dass in polyvalenten Immunglobulinpräparaten IgG die Hauptkomponente für die Inhibition der Pneumolysin-induzierten Thrombozytenschädigung in vitro ist.
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.
Platelet factor 4 (PF4, synonym: CXCL4) is an evolutionary old chemokine with proposed roles in hemostasis and antimicrobial defense. In addition, PF4 has attracted considerable attention as a crucial mediator of one of the most prothrombotic adverse drug effects affecting blood cells, heparin-induced thrombocytopenia (HIT). Interest in PF4 substantially increased in 2021 when it was identified as the target antigen in the life-threatening adverse effect, vaccine-induced immune thrombotic thrombocytopenia (VITT). We address the concept that a major biological function of PF4—a strongly cationic chemokine—is to bind to negatively-charged prokaryotic microorganisms, resulting in structural changes in PF4 that trigger a danger signal recognized by the adaptive immune system. Application of biophysical tools has provided substantial insights into the molecular mechanisms by which PF4 becomes immunogenic, providing insights into a new mechanism of autoimmunity. Binding of autoantibodies with high affinity induces conformational change(s) in the endogenous protein, which are then recognized as foreign antigen, as exemplified by the prothrombotic disorders, autoimmune HIT and VITT. The final part of our review summarizes current assays for HIT and VITT, explaining how structural aspects of anti-PF4 pathobiology relate to assay design and performance characteristics. Currently, functional (platelet activation) assays using washed platelets detect HIT antibodies when heparin is added, and VITT antibodies when PF4 is added. Solid-phase PF4-dependent immunoassays using microtiter plates are sensitive for both HIT and VITT antibodies, while rapid immunoassays, in which the PF4/heparin antigen is coated on beads, are sensitive and specific for HIT, but not for VITT antibodies.
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.
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.
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.