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 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 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 drive thrombosis in VITT. The data support a two-step mechanism underlying VITT that resembles the pathogenesis of (autoimmune) heparin-induced thrombocytopenia.

Epidemiological studies found an association between increased platelet size and the risk for thrombotic complications, but functional differences of large and small platelets remain poorly understood due to a lack of standardized protocols separating platelets with different size. We designed a protocol to separate large and small platelets from 15 mL whole blood. Separated large and small platelet fractions differed in mean platelet volume: 12.1 fl (10.3–13.8 fl) versus 7.7 fl (6.8–9.5 fl, p < 0.01), and forward scatter mean fluorescence intensity: 24.75 (19.9–30.9) versus 16.85 (11.3–20.6; p < 0.01). Similar fold differences were observed in cell diameter and plateletcrit. Large platelets express 30 to 50% more glycoprotein (GP) Ia, GPIb, GPIIIa, GPVI and P2Y12 on their membranes compared with small ones. Single large platelets covered a 50% larger area on a collagen surface. Adhesion to collagen was faster in large platelets compared with small ones indicating enhanced outside-in signal transduction in large platelets via collagen receptors. In contrast, integrin activation was more pronounced in small platelets after ADP stimulation. Proteome analysis revealed that 80 of the 894 proteins quantified differed in abundance: ADP-ribosylation factor 1/3, guanosine triphosphate-binding protein SAR1a, Voltage-dependent anion-selective channel protein 3 and guanylate cyclase soluble sub-unit α-3 were higher abundant in large, whereas immunoglobulins, haptoglobin, hemopexin, α-1-antitrypsin, serotransferrin and vitronectin were more abundant in small platelets. We conclude that some functions and the protein composition of large and small platelets differ, which cannot only be explained by the size difference. Our data suggest different functional roles of large and small platelets.

Community-acquired pneumonia by primary or superinfections with Streptococcus pneumoniae can lead to acute respiratory distress requiring mechanical ventilation. The pore-forming toxin pneumolysin alters the alveolar-capillary barrier and causes extravasation of protein-rich fluid into the interstitial pulmonary tissue, which impairs gas exchange. Platelets usually prevent endothelial leakage in inflamed pulmonary tissue by sealing inflammation-induced endothelial gaps. We not only confirm that S pneumoniae induces CD62P expression in platelets, but we also show that, in the presence of pneumolysin, CD62P expression is not associated with platelet activation. Pneumolysin induces pores in the platelet membrane, which allow anti-CD62P antibodies to stain the intracellular CD62P without platelet activation. Pneumolysin treatment also results in calcium efflux, increase in light transmission by platelet lysis (not aggregation), loss of platelet thrombus formation in the flow chamber, and loss of pore-sealing capacity of platelets in the Boyden chamber. Specific anti-pneumolysin monoclonal and polyclonal antibodies inhibit these effects of pneumolysin on platelets as do polyvalent human immunoglobulins. In a post hoc analysis of the prospective randomized phase 2 CIGMA trial, we show that administration of a polyvalent immunoglobulin preparation was associated with a nominally higher platelet count and nominally improved survival in patients with severe S pneumoniae–related community-acquired pneumonia. Although, due to the low number of patients, no definitive conclusion can be made, our findings provide a rationale for investigation of pharmacologic immunoglobulin preparations to target pneumolysin by polyvalent immunoglobulin preparations in severe community-acquired pneumococcal pneumonia, to counteract the risk of these patients becoming ventilation dependent. This trial was registered at www.clinicaltrials.gov as #NCT01420744.

Platelets are most important in providing cellular hemostasis but also take part in inflammation and immune processes. Increased platelet size has been regarded as a feature describing a young and more reactive subpopulation until studies were published which questioned this concept. Moreover, changes of platelet size given by the mean platelet volume (MPV) were described for immune thrombocytopenia, cardiovascular disease, atherosclerosis, venous thromboembolism, chronic lung disease, sepsis, cancer‐associated thrombosis, autoimmune disorders, and others. This review summarizes the literature on what is known about platelets with different size and describes controversies of studies with large and small platelets putting a focus on their thrombogenicity, age, and on the association of MPV with the mentioned diseases.

BackgroundSARS-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.MethodsWe investigated vaccine, PF4, and VITT patient-derived anti-PF4 antibody interactions using dynamic light scattering, 3D-super-resolution microscopy, and electron microscopy. Mass spectrometry was used to analyze vaccine composition. We investigated the mechanism for early post-vaccine inflammatory reactions as potential co-stimulant for anti-PF4 immune response. Finally, we evaluated VITT antibodies for inducing release of procoagulant DNA-containing neutrophil extracellular traps (NETs), and measured DNase activity in VITT patient serum.ResultsBiophysical analyses showed formation of complexes between PF4 and vaccine constituents, including virus proteins that were recognized by VITT antibodies. EDTA, a vaccine constituent, increased microvascular leakage in mice allowing for circulation of virus- and virus-producing cell culture-derived proteins. Antibodies in normal sera cross-reacted with human proteins in the vaccine and likely contribute to commonly observed acute ChAdOx1 nCov-19 post-vaccination inflammatory reactions. Polyphosphates and DNA enhanced PF4-dependent platelet activation by VITT antibodies. In the presence of platelets, PF4 enhanced VITT antibody-driven procoagulant NETs formation, while DNase activity was reduced in VITT sera, with granulocyte-rich cerebral vein thrombosis observed in a VITT patient.ConclusionsChAdOx1 nCoV-19 vaccine constituents (i) form antigenic complexes with PF4, (ii) EDTA increases microvascular permeability, and (iii) vaccine components cause acute inflammatory reactions. Antigen formation in a proinflammatory milieu offers an explanation for anti-PF4 antibody production. High-titer anti-PF4 antibodies activate platelets and induce neutrophil activation and NETs formation, fueling the VITT prothrombotic response.

Vaccination is crucial in combatting the SARS-CoV-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. We present a widely applicable whole blood standard flow cytometric assay to identify the pathogenic antibodies associated with vaccine-induced immune thrombotic thrombocytopenia (VITT) after ChAdOx1 nCov-19 vaccination. This assay will enable rapid diagnosis by many laboratories.

Circulating platelets consist of subpopulations with different age, maturation state and size. In this review, we address the association between platelet size and platelet function and summarize the current knowledge on platelet subpopulations including reticulated platelets, procoagulant platelets and platelets exposing signals to mediate their clearance. Thereby, we emphasize the impact of platelet turnover as an important condition for platelet production in vivo. Understanding of the features that characterize platelet subpopulations is very relevant for the methods of platelet concentrate production, which may enrich or deplete particular platelet subpopulations. Moreover, the concept of platelet size being associated with platelet function may be attractive for transfusion medicine as it holds the perspective to separate platelet subpopulations with specific functional capabilities.

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