Search

SARS-CoV-2 vaccine ChAdOx1 nCoV-19 (AstraZeneca) causes a thromboembolic complication termed vaccine-induced immune thrombotic thrombocytopenia (VITT). Using biophysical techniques, mouse models, and analysis of VITT patient samples, we identified determinants of this vaccine-induced adverse reaction. Super-resolution microscopy visualized vaccine components forming antigenic complexes with platelet factor 4 (PF4) on platelet surfaces to which anti-PF4 antibodies obtained from VITT patients bound. PF4/vaccine complex formation was charge-driven and increased by addition of DNA. Proteomics identified substantial amounts of virus production-derived T-REx HEK293 proteins in the ethylenediaminetetraacetic acid (EDTA)-containing vaccine. Injected vaccine increased vascular leakage in mice, leading to systemic dissemination of vaccine components known to stimulate immune responses. Together, PF4/vaccine complex formation and the vaccine-stimulated proinflammatory milieu trigger a pronounced B-cell response that results in the formation of high-avidity anti-PF4 antibodies in VITT patients. The resulting high-titer anti-PF4 antibodies potently activated platelets in the presence of PF4 or DNA and polyphosphate polyanions. Anti-PF4 VITT patient antibodies also stimulated neutrophils to release neutrophil extracellular traps (NETs) in a platelet PF4-dependent manner. Biomarkers of procoagulant NETs were elevated in VITT patient serum, and NETs were visualized in abundance by immunohistochemistry in cerebral vein thrombi obtained from VITT patients. Together, vaccine-induced PF4/adenovirus aggregates and proinflammatory reactions stimulate pathologic anti-PF4 antibody production that drives thrombosis in VITT. The data support a 2-step mechanism underlying VITT that resembles the pathogenesis of (autoimmune) heparin-induced thrombocytopenia.

Vaccination is crucial in combatting the severe acute respiratory syndrome coronavirus 2 pandemic. The rare complication of thrombocytopenia and thrombotic complications at unusual sites after ChAdOx1 nCov-19 vaccination is caused by platelet-activating antibodies directed against platelet factor 4 (PF4). We present a widely applicable whole-blood standard flow cytometric assay to identify the pathogenic antibodies associated with vaccine-induced immune-mediated thrombotic thrombocytopenia (VITT) after ChAdOx1 nCov-19 vaccination. This assay will enable rapid diagnosis by many laboratories. This trial was registered at www.clinicaltrials.gov as #NCT04370119.

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 2+ addition, avoiding a shift in Ca2+ hemostasis and cytoskeletal alterations. We report on the impact of 2–10 mM Mg2+ addition 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 + 10mM Mg2+ 0.073 ± 0.021 vs. 0.118 ± 0.023, p 2+ ≥ 4mM 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 reduced in cold-stored platelets (45.65 ± 11.59% vs. RT stored platelets 56.38 ± 29.36; p = 0.042) but normal at 4°C + 10 mM Mg2+ (55.22 ± 11.16%, 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.

Without cellular blood products such as platelet concentrates (PC), red blood cell concentrates (RCC), and hematopoietic stem cells (HPSC) modern treatments in medicine would not be possible. An unresolved challenge is the assessment of their quality with minimal cell manipulation. Minor changes in production, storage conditions, or blood bag composition may impact cell function, which can have important consequences on product integrity. This is especially relevant for personalized medicine, such as autologous T-cell therapy. Today a robust methodology that globally determines cell status directly before transfusion or transplantation is lacking. We demonstrate that measuring viscoelastic characteristics of peripheral blood cells using real-time deformability cytometry (RT-DC) provides comprehensive information on product quality, which is not accessible using conventional quality control tests. In addition, RT-DC requires few cells, a minimal sample volume and has a rapid turnaround time. We compared RT-DC to standard in vitro quality assays assessing: i) PC after storage at 4 °C and room temperature; ii) magnetic nanoparticle labeled platelets; iii) RCC stored in blood bags with different plasticizers; iv) RCC after gamma irradiation; and v) HPSC after cryopreservation with 5% or 10% dimethyl sulfoxide, respectively. Additionally, we evaluated the engraftment time of patients' platelets and leukocytes after transplantation of HPSC products. Our results demonstrate that label-free mechano-phenotyping can be used as a potential biomarker for quality assessment of cell-based pharmaceutical products.

Background: SARS-CoV-2 vaccine ChAdOx1 nCov-19 rarely causes vaccine-induced immune thrombotic thrombocytopenia (VITT) that-like autoimmune heparin-induced thrombocytopenia-is mediated by platelet-activating anti-platelet factor 4 (PF4) antibodies. Aims: To understand how the SARS-CoV-2 vaccine ChAdOx1 nCov-19 can induce anti-PF4 antibodies and how these antibodies induce thrombosis Methods: We investigated vaccine, PF4, and VITT patient-derived anti-PF4 antibody interactions using 3D-super-resolution microscopy, dynamic light scattering, and transmission electron microscopy. Vaccine composition was analyzed by mass spectrometry. Experimental vascular leakage models assessed early post-vaccine reactions. We evaluated VITT antibody-mediated platelet activation and formation of procoagulant DNA-containing neutrophil extracellular traps (NETs), including within VITT patient cerebral venous thrombi. Results: Biophysical analyses showed HEK cell line proteins and free virus proteins in the vaccine, formation of complexes between PF4 and vaccine constituents (including viral proteins) that were recognized by VITT antibodies. In a murine model, EDTA (vaccine constituent) increased microvascular leakage with dissemination of virus-and cell culture-derived human proteins. Free viral proteins and preexisting antibodies in normal human sera reacting with vaccine-containing constituents, likely contribute to commonly observed acute ChAdOx1 nCov-19 post-vaccination inflammatory reactions. PF4-binding polyanions (polyphosphates, DNA) enhanced PF4-dependent platelet activation by VITT antibodies. In the presence of platelets, PF4 enhanced VITT antibody-driven NETs formation;further evidence for NETosis included elevated NETs biomarkers and low DNase activity in VITT sera, and NETs/ neutrophil-rich cerebral vein thrombi extracted from VITT patients. Conclusions: ChAdOx1 nCoV-19 vaccine constituents form antigenic complexes with PF4, and EDTA increases microvascular permeability, enhancing risk of early post-vaccination acute inflammatory reactions;PF4/polyanion antigen formation in a proinflammatory milieu offers an explanation for anti-PF4 antibody production. Resulting high-titer anti-PF4 antibodies activate platelets and induce neutrophil activation with NETosis, further fueling the VITT prothrombotic response.

Magnetic nanoparticles have recently shown great potential in nonradioactive labeling of platelets. Platelet labeling efficiency is enhanced when particles are conjugated with proteins like human serum albumin (HSA). However, the optimal HSA density coated on particles and the uptake mechanism of single particles in platelets remain unclear. Here, we utilized single-molecule force spectroscopy (SMFS) and other complementary methods to characterize the interaction of particles when interacting with platelets and to determine the optimal HSA amount required to coat particles. An HSA concentration of 0.5-1.0 mg/mL for coating particles is most efficient for platelet labeling. Binding pathways could be elucidated by linking a single HSA particle to SMFS tips via polyethylene glycol (PEG) linkers of different lengths and allowing them to interact with immobilized platelets on the substrate. Depending on the PEG length (i.e., short ∼2 nm, medium ∼30 nm, and long ∼100 nm), particles interact differently with platelets as shown by one, two, or three force distributions, which correspond up to three different binding pathways, respectively. We propose a model that the short PEG linker allows the particle to interact only with the platelet membrane, whereas the medium and long PEG linkers promote the particle to transfer from open canalicular system to another target inside platelets. Our study optimizes magnetic platelet labeling and provides details of particle pathways in platelets.

Platelets are the smallest blood cells and important for hemostasis. Platelet concentrates (PC) are medicinal products transfused to prevent or treat bleeding. Typically, platelets in PCs are assessed by in vitro tests for their function. However, in vivo testing of these platelets is highly desirable. To distinguish transfused platelets from patients or probands own cells after PC transfusions within the scope of clinical studies, platelets need to be efficiently labeled with minimal preactivation prior to transfusion. Here we report on a method for improved cell uptake of ferucarbotran magnetic nanoparticles contained in Resovist, an FDA-approved MRI contrast agent, by modifying the nanoparticle shell with human serum albumin (HSA). Both HSA-ferucarbotran nanoparticles and magnetically labeled platelets were produced according to EU-GMP guidelines. Platelet function after labeling was evaluated by light transmission aggregometry and by determination of expression of CD62P as platelet activation marker. Magnetic labeling does not impair platelet function and platelets showed reasonable activation response to agonists. Platelet survival studies in NOD/SCID-mice resulted in comparable survival behavior of magnetically labeled and nonlabeled platelets. Additionally, labeled platelets can be recovered from whole blood by magnetic separation.

Platelet transfusion has become essential therapy in modern medicine. Although the clinical advantage of platelet transfusion has been well established, adverse reactions upon transfusion, especially transmission of bacterial infection, still represent a major challenge. While bacterial contamination is favored by the storage of platelets at room temperature, cold storage may represent a solution for this important clinical issue. In this study, we aimed to clarify whether plasma has protective or detrimental effects on cold-stored platelets. We investigated the impact of different residual plasma contents in apheresis-derived platelet concentrates, stored at 4°C or room temperature, on platelet function and survival. We found that platelets stored at 4°C have higher expression of apoptosis marker compared to platelets stored at room temperature, leading to accelerated clearance from the circulation in a humanized animal model. While cold-induced apoptosis was independent of the residual plasma concentration, cold storage was associated with better adhesive properties and higher response to activators. Interestingly, delta (δ) granule-related functions, such as ADP-mediated aggregation and CD63 release, were better preserved at 4°C, especially in 100% plasma. An extended study to assess cold-stored platelet concentrates produced under standard care Good Manufacturing Practice conditions showed that platelet function, metabolism and integrity were better compared to those stored at room temperature. Taken together, our results show that residual plasma concentration does not have a cardinal impact on the cold storage lesions of apheresis-derived platelet concentrates and indicate that the current generation of additive solutions represent suitable substitutes for plasma to store platelets at 4°C.

Background The THERAFLEX UV-Platelets pathogen reduction system for platelet concentrates (PCs) operates with ultraviolet C light (UVC; 254 nm) only without addition of photosensitizers. This phase I study evaluated safety and tolerability of autologous UVC-irradiated PCs in healthy volunteers. Methods Eleven volunteers underwent two single (series 1 and 2) and one double apheresis (series 3). PCs were treated with UVC, stored for 48 h and retransfused in a dose-escalation scheme: 12·5, 25% and 50% of a PC (series 1); one complete PC (series 2); two PCs (series 3). Platelet counts, fibrinogen, activated partial thromboplastin time, prothrombin time, D-dimer, standard haematology, temperature, heart rate, blood pressure and clinical chemistry parameters were measured. One- and 24-h corrected count increments were determined in series 2 and 3. Platelet-specific antibodies were assessed before and at the end of the study. Results Neither adverse reactions related to transfusions nor antibodies against UVC-treated platelets were observed. Corrected count increments did not differ between series 2 and 3. Conclusions Repeated transfusions of autologous UVC-treated PCs were well tolerated and did not induce antibody responses in all volunteers studied. EudraCT No. 2010-023404-26.

Platelets play a dominant role in the pathogenesis of bleeding disorders and cardiovascular pathology (e.g., myocardial infarction). Nonradioactive labeling of platelets may offer several clinical applications, ranging from survival studies of transfused platelet concentrates to studies on the pathogenesis of stroke. We used ferucarbotran superparamagnetic nanoparticles (NPs) for cell labeling. Platelets incorporated these NPs by endocytosis (without linkers or binding agents). Flow cytometry using FITC-conjugated magnetic NPs showed ex vivo labeling of about 98% of platelets; NPs were predominantly located inside the platelet granules as confirmed by fluorescence microscopy and transmission electron microscopy. Iron concentrations of 2 pg per platelet were reached as determined by atomic absorption spectroscopy. This will enable sensitive ex vivo determination of transfused labeled platelets, allowing survival studies. In vitro, labeled platelets gave a clear signal by 7 Tesla magnetic resonance tomography. Magnetic labeling of platelets may offer a new tool for diagnosis and research in transfusion medicine and cardiovascular medicine. From the Clinical Editor In this study a platelet labeling method is discussed and described for in vivo and ex vivo applications, using a binder or linker free fluorescent superparamagnetic iron nanoparticle system. Magnetic labeling of platelets may offer a new tool for diagnosis and research in transfusion medicine and cardiovascular medicine.