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In order to improve the safety of COVID-19 vaccines, there is an urgent need to unravel the pathogenesis of vaccineinduced immune thrombotic thrombocytopenia (VITT), a severe complication of recombinant adenoviral vector vaccines used to prevent COVID-19, and likely due to anti-platelet factor 4 (PF4) IgG antibodies. In this study, we demonstrated that 1E12, a chimeric anti-PF4 antibody with a human Fc fragment, fully mimics the effects of human VITT antibodies, as it activates platelets to a similar level in the presence of platelet factor 4 (PF4). Incubated with neutrophils, platelets and PF4, 1E12 also strongly induces NETosis, and in a microfluidic model of whole blood thrombosis, it triggers the formation of large platelet/leukocyte thrombi containing fibrin(ogen). In addition, a deglycosylated form of 1E12 (DG-1E12), which still binds PF4 but no longer interacts with Fcγ receptors, inhibits platelet, granulocyte and clotting activation induced by human anti-PF4 VITT antibodies. This strongly supports that 1E12 and VITT antibodies recognize overlapping epitopes on PF4. In conclusion, 1E12 is a potentially important tool to study the pathophysiology of VITT, and for establishing mouse models. On the other hand, DG-1E12 may help the development of a new drug that specifically neutralizes the pathogenic effect of autoimmune anti-PF4 antibodies, such as those associated with VITT.

The multidrug resistance protein 4 (MRP4) is highly expressed in platelets and several lines of evidence point to an impact on platelet function. MRP4 represents a transporter for cyclic nucleotides as well as for certain lipid mediators. The aim of the present study was to comprehensively characterize the effect of a short-time specific pharmacological inhibition of MRP4 on signaling pathways in platelets. Transport assays in isolated membrane vesicles showed a concentrationdependent inhibition of MRP4-mediated transport of cyclic nucleotides, thromboxane (Tx)B2 and fluorescein (FITC)- labeled sphingosine-1-phosphate (S1P) by the selective MRP4 inhibitor Ceefourin-1. In ex vivo aggregometry studies in human platelets, Ceefourin-1 significantly inhibited platelet aggregation by about 30-50% when ADP or collagen was used as activating agents, respectively. Ceefourin-1 significantly lowered the ADP-induced activation of integrin aIIbb3, indicated by binding of FITC-fibrinogen (about 50% reduction at 50 mM Ceefourin-1), and reduced calcium influx. Furthermore, pre-incubation with Ceefourin-1 significantly increased PGE1- and cinaciguat-induced vasodilatorstimulated phosphoprotein (VASP) phosphorylation, indicating increased cytosolic cAMP as well as cGMP concentrations, respectively. The release of TxB2 from activated human platelets was also attenuated. Finally, selective MRP4 inhibition significantly reduced both the total area covered by thrombi and the average thrombus size by about 40% in a flow chamber model. In conclusion, selective MRP4 inhibition causes reduced platelet adhesion and thrombus formation under flow conditions. This finding is mechanistically supported by inhibition of integrin aIIbb3 activation, elevated VASP phosphorylation and reduced calcium influx, based on inhibited cyclic nucleotide and thromboxane transport as well as possible further mechanisms.

Platelets play an important role in the development and progression of respiratory distress. Functional platelets are known to seal inflammatory endothelial gaps and loss of platelet function has been shown to result in loss of integrity of pulmonary vessels. This leads to fluid accumulation in the pulmonary interstitium, eventually resulting in respiratory distress. Streptococcus pneumoniae is one of the major pathogens causing community-acquired pneumonia. Previously, we have shown that its major toxin pneumolysin forms pores in platelet membranes and renders them nonfunctional. In vitro, this process was inhibited by polyvalent intravenous immunoglobulins (IVIGs). In this study, we compared the efficacy of a standard IVIG preparation (IVIG, 98% immunoglobulin G [IgG]; Privigen, CSL Behring, United States) and an IgM/IgA-enriched immunoglobulin preparation (21% IgA, 23% IgM, 56% IgG; trimodulin, Biotest AG, Germany) to inhibit pneumolysin-induced platelet destruction. Platelet destruction and functionality were assessed by flow cytometry, intracellular calcium release, aggregometry, platelet viability, transwell, and flow chamber assays. Overall, both immunoglobulin preparations efficiently inhibited pneumolysin-induced platelet destruction. The capacity to antagonize pneumolysin mainly depended on the final IgG content. As both polyvalent immunoglobulin preparations efficiently prevent pneumolysin-induced platelet destruction and maintain platelet function in vitro, they represent promising candidates for clinical studies on supportive treatment of pneumococcal pneumonia to reduce progression of respiratory distress., Competing Interests: A.G. reports grants and nonfinancial support from Aspen, Boehringer Ingelheim, MSD, Bristol Myers Squibb (BMS), Bayer Healthcare, Instrumentation Laboratory; personal fees from Aspen, MSD, Macopharma, BMS, Chromatec, Instrumentation Laboratory, nonfinancial support from Portola, Ergomed, Biokit outside the submitted work. S.W. and J.S. are employees of Biotest AG. All other authors declare no conflict of interest., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/).)

Background: Toxins are key virulence determinants of pathogens and can impair the function of host immune cells, including platelets. Insights into pathogen toxin interference with platelets will be pivotal to improve treatment of patients with bacterial bloodstream infections., Materials and Methods: In this study, we deciphered the effects of Staphylococcus aureus toxins α-hemolysin, LukAB, LukDE, and LukSF on human platelets and compared the effects with the pore forming toxin pneumolysin of Streptococcus pneumoniae. Activation of platelets and loss of platelet function were investigated by flow cytometry, aggregometry, platelet viability, fluorescence microscopy, and intracellular calcium release. Thrombus formation was assessed in whole blood., Results: α-hemolysin (Hla) is known to be a pore-forming toxin. Hla-induced calcium influx initially activates platelets as indicated by CD62P and αIIbβ3 integrin activation, but also induces finally alterations in the phenotype of platelets. In contrast to Hla and pneumolysin, S. aureus bicomponent pore-forming leukocidins LukAB, LukED, and LukSF do not bind to platelets and had no significant effect on platelet activation and viability. The presence of small amounts of Hla (0.2 µg/ml) in whole blood abrogates thrombus formation indicating that in systemic infections with S. aureus the stability of formed thrombi is impaired. Damage of platelets by Hla was not neutralized by intravenous immune globulins., Conclusion: Our findings might be of clinical relevance for S. aureus induced endocarditis. Stabilizing the aortic-valve thrombi by inhibiting Hla-induced impairment of platelets might reduce the risk for septic (micro-)embolization., (© 2022 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

MYH9 -related disease patients with mutations in the contractile protein nonmuscle myosin heavy chain IIA display, among others, macrothrombocytopenia and a mild-to-moderate bleeding tendency. In this study, we used three mouse lines, each with one point mutation in the Myh9 gene at positions 702, 1424, or 1841, to investigate mechanisms underlying the increased bleeding risk. Agonist-induced activation of Myh9 mutant platelets was comparable to controls. However, myosin light chain phosphorylation after activation was reduced in mutant platelets, which displayed altered biophysical characteristics and generated lower adhesion, interaction, and traction forces. Treatment with tranexamic acid restored clot retraction in the presence of tPA and reduced bleeding. We verified our findings from the mutant mice with platelets from patients with the respective mutation. These data suggest that reduced platelet forces lead to an increased bleeding tendency in patients with MYH9 -related disease, and treatment with tranexamic acid can improve the hemostatic function.

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.

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. Ca 2+ release from storage organelles and higher activity of Ca 2+ pumps at temperatures < 15 °C triggers cytoskeleton changes. This is suppressed by Mg 2+ addition, avoiding a shift in Ca 2+ hemostasis and cytoskeletal alterations. We report on the impact of 2-10 mM Mg 2+ 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 Mg 2+ addition ≥ 4 mM Mg 2+ (mean ± SD of median deformation 4 °C vs. 4 °C + 10 mM Mg 2+ 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 Mg 2+  ≥ 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 Mg 2+ concentrations. In conclusion, increasing Mg 2+ 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., (© 2022. The Author(s).)

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.

Vaccine-induced immune thrombotic thrombocytopenia (VITT; synonym, thrombosis with thrombocytopenia syndrome, is associated with high-titer immunoglobulin G antibodies directed against platelet factor 4 (PF4). These antibodies activate platelets via platelet FcγIIa receptors, with platelet activation greatly enhanced by PF4. Here we summarize the current concepts in the pathogenesis of VITT. We first address parallels between heparin-induced thrombocytopenia and VITT, and provide recent findings on binding of PF4 to adenovirus particles and non-assembled adenovirus proteins in the 2 adenovirus vector-based COVID-19 vaccines, ChAdOx1 nCoV-19 and Ad26.COV2.S. Further, we discuss the potential role of vaccine constituents such as glycosaminoglycans, EDTA, polysorbate 80, human cell-line proteins and nucleotides as potential binding partners of PF4. The immune response towards PF4 in VITT is likely triggered by a proinflammatory milieu. Human cell-line proteins, non-assembled virus proteins, and potentially EDTA may contribute to the proinflammatory state. The transient nature of the immune response towards PF4 in VITT makes it likely that-as in heparin-induced thrombocytopenia -marginal zone B cells are key for antibody production. Once high-titer anti-PF4 antibodies have been formed 5 to 20 days after vaccination, they activate platelets and granulocytes. Activated granulocytes undergo NETosis and the released DNA also forms complexes with PF4, which fuels the Fcγ receptor-dependent cell activation process, ultimately leading to massive thrombin generation. Finally, we summarize our initial observations indicating that VITT-like antibodies might also be present in rare patients with recurrent venous and arterial thrombotic complications, independent of vaccination., Competing Interests: Conflicts of interest Dr Schönborn is the recipient of a young investigator grant of the medical faculty of the Universitätsmedizin Greifswald. Dr. Greinacher reports grants and non-financial support from Aspen, Boehringer Ingelheim, MSD, Bristol Myers Squibb (BMS), Paringenix, Bayer Healthcare, Gore Inc., Rovi, Sagent, Biomarin/Prosensa, personal fees from Aspen, Boehringer Ingelheim, MSD, Macopharma, BMS, Chromatec, Instrumentation Laboratory, nonfinancial support from Boehringer Ingelheim, Portola, Ergomed, GTH e.V. outside the submitted work. Dr. Thiele reports personal fees from Bristol Myers Squibb, Bayer, Daichii Sankyo, Pfizer, Novo Nordisk, Chugai Pharma, and Novartis, all of which are outside of the submitted manuscript. None of the other authors has to declare a conflict of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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., (© 2022. The Author(s).)

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., (© 2021 by The American Society of Hematology.)

Background: The classic Bernard-Soulier syndrome (BSS) is a rare inherited thrombocytopenia (IT) associated with severe thrombocytopenia, giant platelets, and bleeding tendency caused by homozygous or compound heterozygous variants in GP1BA, GP1BB, or GP9. Monoallelic BSS (mBSS) associated with mild asymptomatic macrothrombocytopenia caused by heterozygous variants in GP1BA or GP1BB may be a frequent cause of mild IT., Objective: We aimed to examine the frequency of mBSS in a consecutive cohort of patients with IT and to characterize the geno- and phenotype of mBSS probands and their family members. Additionally, we set out to examine if thrombopoietin (TPO) levels differ in mBSS patients., Patients/methods: We screened 106 patients suspected of IT using whole exome- or whole genome sequencing and performed co-segregation analyses of mBSS families. All probands and family members were phenotypically characterized. Founder mutation analysis was carried out by certifying that the probands were unrelated and the region around the variant was shared by all patients. TPO was measured by solid phase sandwich ELISA., Results: We diagnosed 14 patients (13%) with mBSS associated with heterozygous variants in GP1BA and GP1BB. Six unrelated probands carried a heterozygous variant in GP1BA (c.58T>G, p.Cys20Gly) and shared a 2.0 Mb region on chromosome 17, confirming that it is a founder variant. No discrepancy of TPO levels between mBSS patients and wild-type family members (P > .05) were identified., Conclusion: We conclude that the most frequent form of IT in Denmark is mBSS caused by the Copenhagen founder variant., (© 2021 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a severe adverse effect of ChAdOx1 nCoV-19 COVID-19 vaccine (Vaxzevria) and Janssen Ad26.COV2.S COVID-19 vaccine, and it is associated with unusual thrombosis. VITT is caused by anti-platelet factor 4 (PF4) antibodies activating platelets through their FcγRIIa receptors. Antibodies that activate platelets through FcγRIIa receptors have also been identified in patients with COVID-19. These findings raise concern that vaccination-induced antibodies against anti-SARS-CoV-2 spike protein cause thrombosis by cross-reacting with PF4. Immunogenic epitopes of PF4 and SARS-CoV-2 spike protein were compared using in silico prediction tools and 3D modeling. The SARS-CoV-2 spike protein and PF4 share at least 1 similar epitope. Reactivity of purified anti-PF4 antibodies from patients with VITT was tested against recombinant SARS-CoV-2 spike protein. However, none of the affinity-purified anti-PF4 antibodies from 14 patients with VITT cross-reacted with SARS-CoV-2 spike protein. Sera from 222 polymerase chain reaction-confirmed patients with COVID-19 from 5 European centers were tested by PF4-heparin enzyme-linked immunosorbent assays and PF4-dependent platelet activation assays. We found anti-PF4 antibodies in sera from 19 (8.6%) of 222 patients with COVID-19. However, only 4 showed weak to moderate platelet activation in the presence of PF4, and none of those patients developed thrombotic complications. Among 10 (4.5%) of 222 patients who had COVID-19 with thrombosis, none showed PF4-dependent platelet-activating antibodies. In conclusion, antibodies against PF4 induced by vaccination do not cross-react with the SARS-CoV-2 spike protein, indicating that the intended vaccine-induced immune response against SARS-CoV-2 spike protein is not the trigger of VITT. PF4-reactive antibodies found in patients with COVID-19 in this study were not associated with thrombotic complications., (© 2021 by The American Society of Hematology.)

Platelet activation and aggregation are essential to limit blood loss at sites of vascular injury but may also lead to occlusion of diseased vessels. The platelet cytoskeleton is a critical component for proper hemostatic function. Platelets change their shape after activation and their contractile machinery mediates thrombus stabilization and clot retraction. In vitro studies have shown that platelets, which come into contact with proteins such as fibrinogen, spread and first form filopodia and then lamellipodia, the latter being plate-like protrusions with branched actin filaments. However, the role of platelet lamellipodia in hemostasis and thrombus formation has been unclear until recently. This short review will briefly summarize the recent findings on the contribution of the actin cytoskeleton and lamellipodial structures to platelet function., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

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., (© 2020 by The American Society of Hematology.)

Cytoskeleton is composed of more than 100 proteins and represents a dynamic network of the cellular cytoplasm. Cytoskeletal functions include spatial organization of cellular components, structural connection of the cell with external environment, and biomechanical force generation. Cytoskeleton takes part, at different levels, in all phases of platelet biogenesis: megakaryocyte (MK) differentiation, MK maturation, and platelet formation. In addition, it also plays a major role in each stage of platelet function. Inherited platelet disorders (IPDs) are a group of rare diseases featured by low platelet count and/or impaired platelet function. Over the past decade, the investigation of platelet biomechanics has become a major and highly relevant theme of research due to its implications at every stage of development of human life. The initial use of diverse biophysical techniques (e.g., micropipette aspiration, atomic force and scanning ion conductance microscopy, real-time deformability cytometry) started unraveling biomechanical features of platelets that are expected to provide new explanations for physiological and pathological mechanisms. Although the impact of cytoskeletal alterations has been largely elucidated in various IPDs' pathogenesis, the understanding of their impact on biomechanical properties of platelets represents an unmet need. Regarding IPDs, improving biomechanical studies seems promising for diagnostic and prognostic implications. Potentially, these characteristics of platelets may also be used for the prediction of bleeding risk. This review addresses the current available methods for biophysical investigations of platelets and the possible implementations in the field of IPDs., Competing Interests: The authors declare that they have no conflict of interest., (Georg Thieme Verlag KG Stuttgart · New York.)

It is widely anticipated that large platelets are more reactive than small platelets. This was mainly shown in Ca 2+ -poor media albeit extracellular Ca 2+ is utilized by platelets for activation. We determined the impact of extracellular Ca 2+ on functional differences between large and small platelets in response to thrombin receptor activating peptide 6 (TRAP-6), adenosine diphosphate (ADP), and epinephrine. In Ca 2+ -poor buffer, large platelets responded stronger to TRAP-6 which equalized in Ca 2+ containing buffer. Large platelets contained and mobilized more Ca 2+ from their intracellular stores upon TRAP-6 stimulation explaining their better reactivity in Ca 2+ -poor media. Stronger aggregation of large platelets in response to ADP also equalized in presence of Ca 2+ , whereas large platelets responded weaker to ADP in flow cytometry (CD62P-expression: 9.7 mean fluorescence intensity [MFI] [4.4-17.9] vs. 17.5 MFI [6.1-45.6], p  = 0.0234) and PAC-1 binding (11.1 MFI [5.7-19.6] vs. 20.5 MFI [14.4-35.0], p  = 0.0078). Epinephrine response was stronger in large platelets (CD62P-expression: 11.8 MFI [6.8-33.0] vs. 6.8 MFI [2.5-15.2], p  = 0.0078; PAC-1 binding 18.9 MFI [13.6-38.4] vs. 13.0 MFI [6.8-22.4], p  = 0.0234; max. aggregation 82.9% [58.7-94.8] vs. 77.2% [19.8-88.8], p  = 0.0313), which expressed more α2A receptors. Epinephrine further increased phosphatidylserine (PS) exposure especially in large platelets. PS-positive platelets progressively divided into two subpopulations with high or basic intracellular Ca 2+ dependent on extracellular Ca 2+ . Thrombin generation was faster with small, but accelerated by PS exposure and epinephrine-coactivated large platelets. We show that responses of large and small platelets differ depending on extracellular Ca 2+ availability and the inductor. Careful control of extracellular Ca 2+ is necessary in functional studies with large and small platelets., Competing Interests: None of the authors has a conflict of interest to declare with regard to this manuscript. A.G. reports grants from Deutsche Forschungsgemeinschaft, during the conduct of the study; grants and nonfinancial support from Aspen, Boehringer Ingelheim, MSD, Bristol Myers Squibb (BMS), Paringenix, Bayer Healthcare, Gore Inc., Rovi, Sagent, Biomarin/Prosensa, personal fees from Aspen, Boehringer Ingelheim, MSD, Macopharma, Bristol Myers Squibb, Chromatec, Instrumentation Laboratory, nonfinancial support from Boehringer Ingelheim, Portola, and Ergomed, outside the submitted work. T.T. reports personal fees and other from Bristol Myers Squibb, Pfizer, and Chugai Pharma; personal fees from Bayer and Novartis; other from Novo Nordisk and Daichii Sankyo, outside the submitted work., (Georg Thieme Verlag KG Stuttgart · New York.)

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. The phenotypes of patients with the recently discovered, dominant, ETV6 -linked leukaemia predisposition and familial thrombocytopenia syndrome are variable, and the exact mechanism of leukaemogenesis remains unclear. Patients and Methods. Here, we present novel clinical and laboratory phenotypes of seven individuals from three families with ETV6 germline mutations and a refined genetic analysis of one child with additional high-hyperdiploid acute lymphoblastic leukaemia (HD-ALL), aiming to elucidate second oncogenic hits. Results. Four individuals from two pedigrees harboured one novel or one previously described variant in the central domain of ETV6 (c.592C>T, p.Gln198* or c.641C>T, p.Pro241Leu, respectively). Neutropenia was an accompanying feature in one of these families that also harboured a variant in RUNX1 (c.1098&#95;1103dup, p.Ile366&#95;Gly367dup), while in the other, an autism-spectrum disorder was observed. In the third family, the index patient suffered from HD-ALL and life-threatening pulmonary mucor mycosis, and had a positive family history of 'immune' thrombocytopenia. Genetic analyses revealed a novel heterozygous mutation in the ETS domain of ETV6 (c.1136T>C, p.Leu379Pro) along with absence of heterozygosity of chromosome (10)(q21.2q21.3), yielding a biallelic leukaemia risk allele in ARID5B (rs7090445-C). The neutrophil function was normal in all individuals tested, and the platelet immune histochemistry of all three pedigrees showed delta-storage-pool defect-like features and cytoskeletal defects. Conclusions. Our clinical observations and results of high-resolution genetic analyses extend the spectrum of possible phenotypes cosegregating with ETV6 germline mutations. Further, we propose ARID5B as potential leukaemogenic cofactor in patients with ETV6 -linked leukaemia predisposition and familial thrombocytopenia syndrome., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Heparin induced thrombocytopenia (HIT) is likely a misdirected bacterial host defense mechanism. Platelet factor 4 (PF4) binds to polyanions on bacterial surfaces exposing neo-epitopes to which HIT antibodies bind. Platelets are activated by the resulting immune complexes via FcγRIIA, release bactericidal substances, and kill Gram-negative Escherichia coli., Objectives: To assess the role of PF4, anti-PF4/H antibodies and FcγRIIa in killing of Gram-positive bacteria by platelets., Methods: Binding of PF4 to protein-A deficient Staphylococcus aureus (SA113Δspa) and non-encapsulated Streptococcus pneumoniae (D39Δcps) and its conformational change were assessed by flow cytometry using monoclonal (KKO,5B9) and patient derived anti-PF4/H antibodies. Killing of bacteria was quantified by counting colony forming units (cfu) after incubation with platelets or platelet releasate. Using flow cytometry, platelet activation (CD62P-expression, PAC-1 binding) and phosphatidylserine (PS)-exposure were analyzed., Results: Monoclonal and patient-derived anti-PF4/H antibodies bound in the presence of PF4 to both S. aureus and S. pneumoniae (1.6-fold increased fluorescence signal for human anti-PF4/H antibodies to 24.0-fold increase for KKO). Staphylococcus aureus (5.5 × 10 4 cfu/mL) was efficiently killed by platelets (2.7 × 10 4 cfu/mL) or their releasate (2.9 × 10 4 cfu/mL). Killing was not further enhanced by PF4 or anti-PF4/H antibodies. Blocking FcγRIIa had no impact on killing of S. aureus by platelets. In contrast, S. pneumoniae was not killed by platelets or releasate. Instead, after incubation with pneumococci platelets were unresponsive to TRAP-6 stimulation and exposed high levels of PS., Conclusions: Anti-PF4/H antibodies seem to have only a minor role for direct killing of Gram-positive bacteria by platelets. Staphylococcus aureus is killed by platelets or platelet releasate. In contrast, S. pneumoniae affects platelet viability., (© 2020 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

Platelets are the key cellular components of blood primarily contributing to formation of stable hemostatic plugs at the site of vascular injury, thus preventing excessive blood loss. On the other hand, excessive platelet activation can contribute to thrombosis. Platelets respond to many stimuli that can be of biochemical, cellular, or physical origin. This drives platelet activation kinetics and plays a vital role in physiological and pathological situations. Currently used bulk assays are inadequate for comprehensive biomechanical assessment of single platelets. Individual platelets interact and respond differentially while modulating their biomechanical behavior depending on dynamic changes that occur in surrounding microenvironments. Quantitative description of such a phenomenon at single-platelet regime and up to nanometer resolution requires methodological approaches that can manipulate individual platelets at submicron scales. This review focusses on principles, specific examples, and limitations of several relevant biophysical methods applied to single-platelet analysis such as micropipette aspiration, atomic force microscopy, scanning ion conductance microscopy and traction force microscopy. Additionally, we are introducing a promising single-cell approach, real-time deformability cytometry, as an emerging biophysical method for high-throughput biomechanical characterization of single platelets. This review serves as an introductory guide for clinician scientists and beginners interested in exploring one or more of the above-mentioned biophysical methods to address outstanding questions in single-platelet biomechanics., (© 2020 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals, Inc on behalf of International Society on Thrombosis and Haemostasis.)

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., Competing Interests: None of the authors has a conflict of interest to declare with regard to this manuscript. T.T. declares a conflict outside of the submitted work: personal fees from Bristol Myers Squibb, personal fees and other from Bayer, other from Novo Nordisk, personal fees and other from Pfizer, personal fees from Novartis. A.G. declares a conflict outside of the submitted work: personal fees and non-financial support from Maco Pharma, Boehringer Ingelheim, personal fees from ASPEN, personal fees from Bristol Myers Squibb, other from Bayer Healthcare, grants and other from Instrumentation laboratories., (Georg Thieme Verlag KG Stuttgart · New York.)

Competing Interests: Conflict-of-interest disclosure: The authors declare no competing financial interests.

The adaption of endothelial cells to local flow conditions is a multifunctional process which leads to distinct alterations in cell shape, the subcellular distribution of structural proteins, and cellular function. G-protein-coupled receptors (GPCRs) have been identified to be fundamentally involved in such processes. Recently, we and others have shown that the expression of the endothelial GPCR apelin receptor (APJ) is regulated by fluid flow and that activation of APJ participates in signaling pathways which are related to processes of mechanotransduction. The present study aims to illuminate these findings by further visualization of APJ function. We show that APJ is located to the cellular junctions and might thus be associated with platelet endothelial cell adhesion molecule-1 (PECAM-1) in human umbilical vein endothelial cells (HUVEC). Furthermore, siRNA-mediated silencing of APJ expression influences the shear-induced adaption of HUVEC in terms of cytoskeletal remodeling, cellular elasticity, cellular motility, attachment, and distribution of adhesion complexes. Taken together, our results demonstrate that APJ is crucial for complemented endothelial adaption to local flow conditions., (© 2018 Wiley Periodicals, Inc.)

S. aureus associated bacteremia can lead to severe infections with high risk of mortality (e.g. sepsis, infective endocarditis). Many virulence factors and adhesins of S. aureus are known to directly interact with platelets. Extracellular adherence protein, Eap, one of the most important virulence factors in S. aureus mediated infections is a multi-tandem domain protein and has been shown to interact with almost all cell types in the human circulatory system. By using amine reactive fluorescent N-hydroxysuccinimidyl (NHS)-ester dyes and by direct detection with primary fluorescently conjugated anti-histidine (His-tag) antibodies against detect N-terminal His6, we show Eap subdomain Eap D 3 D 4 specifically interacts and rapidly activates human platelets. Furthermore, we validate our finding by using site directed directional immobilization of Eap D 3 D 4 through N-terminal His 6 on nickel (II)-nitrilotriacetic acid (Ni-NTA) functionalized bacteriomimetic microbead arrays to visualize real-time platelet activation through calcium release assay. These methods offer an easily adoptable protocols for screening of S.aureus derived virulence factors and adhesins with platelets., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Staphylococcus aureus can cause bloodstream infections associated with infective endocarditis (IE) and disseminated intravascular coagulopathy (DIC). Both complications involve platelets. In view of an increasing number of antibiotic-resistant strains, new approaches to control systemic S. aureus infection are gaining importance. Using a repertoire of 52 recombinant S. aureus proteins in flow cytometry-based platelet activation and aggregation assays, we identified, in addition to the extracellular adherence protein Eap, three secreted staphylococcal proteins as novel platelet activating proteins. Eap and the chemotaxis inhibitory protein of S. aureus (CHIPS), the formyl peptide receptor-like 1 inhibitory protein (FLIPr) and the major autolysin Atl induced P-selectin expression in washed platelets and platelet-rich plasma. Similarly, AtlA, CHIPS and Eap induced platelet aggregation in whole blood. Fluorescence microscopy illustrated that P-selectin expression is associated with calcium mobilization and re-organization of the platelet actin cytoskeleton. Characterization of the functionally active domains of the major autolysin AtlA and Eap indicates that the amidase domain of Atl and the tandem repeats 3 and 4 of Eap are crucial for platelet activation. These results provide new insights in S. aureus protein interactions with platelets and identify secreted proteins as potential treatment targets in case of antibiotic-resistant S. aureus infection., Competing Interests: None., (Schattauer GmbH Stuttgart.)

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.

Binding assays with fluorescently labeled ligands and recombinant receptor proteins are commonly performed in 2D arrays. But many cell surface receptors only function in their native membrane environment and/or in a specific conformation, such as they appear on the surface of live cells. Thus, receptors on live cells should be used for ligand binding assays. Here, it is shown that antibodies preprinted on a glass surface can be used to specifically array a peptide receptor of the immune system, i.e., the major histocompatibility complex class I molecule H-2K b , into a defined pattern on the surface of live cells. Monoclonal antibodies make it feasible to capture a distinct subpopulation of H-2K b and hold it at the cell surface. This patterned receptor enables a novel peptide-binding assay, in which the specific binding of a fluorescently labeled index peptide is visualized by microscopy. Measurements of ligand binding to captured cell surface receptors in defined confirmations apply to many problems in cell biology and thus represent a promising tool in the field of biosensors., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objective: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies and is projected to be the second leading cause of cancer-related death by 2030. Despite extensive knowledge and insights into biological properties and genetic aberrations of PDAC, therapeutic options remain temporary and ineffective. One plausible explanation for the futile response to therapy is an insufficient and non-specific delivery of anticancer drugs to the tumour site., Design: Superparamagnetic iron oxide nanoparticles (SPIONs) coupled with siRNA directed against the cell cycle-specific serine-threonine-kinase, Polo-like kinase-1 (siPLK1-StAv-SPIONs), could serve a dual purpose for delivery of siPLK1 to the tumour and for non-invasive assessment of efficiency of delivery in vivo by imaging the tumour response. siPLK1-StAv-SPIONs were designed and synthesised as theranostics to function via a membrane translocation peptide with added advantage of driving endosomal escape for mediating transportation to the cytoplasm (myristoylated polyarginine peptides) as well as a tumour-selective peptide (EPPT1) to increase intracellular delivery and tumour specificity, respectively., Results: A syngeneic orthotopic as well as an endogenous cancer model was treated biweekly with siPLK1-StAv-SPIONs and tumour growth was monitored by small animal MRI. In vitro and in vivo experiments using a syngeneic orthotopic PDAC model as well as the endogenous LSL-Kras G12D , LSL-Trp53 R172H , Pdx-1-Cre model revealed significant accumulation of siPLK1-StAv-SPIONs in PDAC, resulting in efficient PLK1 silencing. Tumour-specific silencing of PLK1 halted tumour growth, marked by a decrease in tumour cell proliferation and an increase in apoptosis., Conclusions: Our data suggest siPLK1-StAv-SPIONs with dual specificity residues for tumour targeting and membrane translocation to represent an exciting opportunity for targeted therapy in patients with PDAC., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)