Anti-PF4 immunothrombosis without proximate heparin or adenovirus vector vaccine exposure.

ABSTRACT: Platelet-activating anti-platelet factor 4 (PF4)/heparin antibodies and anti-PF4 antibodies cause heparin-induced thrombocytopenia (HIT) and vaccine-induced immune thrombocytopenia and thrombosis (VITT), respectively. Diagnostic and treatment considerations differ somewhat between HIT and VITT. We identified patients with thrombocytopenia and thrombosis without proximate heparin exposure or adenovirus-based vaccination who tested strongly positive by PF4/polyanion enzyme-immunoassays and negative/weakly positive by heparin-induced platelet activation (HIPA) test but strongly positive by PF4-induced platelet activation (PIPA) test (ie, VITT-like profile). We tested these patients by a standard chemiluminescence assay that detects anti-PF4/heparin antibodies found in HIT (HemosIL AcuStar HIT-IgG(PF4-H)) as well as a novel chemiluminescence assay for anti-PF4 antibodies found in VITT. Representative control sera included an exploratory anti-PF4 antibody-positive but HIPA-negative/weak cohort obtained before 2020 (n = 188). We identified 9 patients with a clinical-pathological profile of a VITT-like disorder in the absence of proximate heparin or vaccination, with a high frequency of stroke (arterial, n = 3; cerebral venous sinus thrombosis, n = 4), thrombocytopenia (median platelet count nadir, 49 × 109/L), and hypercoagulability (greatly elevated D-dimer levels). VITT-like serological features included strong reactivity by PIPA (aggregation <10 minutes in 9/9 sera) and positive testing in the novel anti-PF4 chemiluminescence assay (3/9 also tested positive in the anti-PF4/heparin chemiluminescence assay). Our exploratory cohort identified 13 additional patient sera obtained before 2020 with VITT-like anti-PF4 antibodies. Platelet-activating VITT-like anti-PF4 antibodies should be considered in patients with thrombocytopenia, thrombosis, and very high D-dimer levels, even without a proximate exposure to heparin or adenovirus vector vaccines.

FcγRIIA-specific DARPins as novel tools in blood cell analysis and platelet aggregation.

Fc receptors are involved in a variety of physiologically and disease-relevant responses. Among them, FcγRIIA (CD32a) is known for its activating functions in pathogen recognition and platelet biology, and, as potential marker of T lymphocytes latently infected with HIV-1. The latter has not been without controversy due to technical challenges complicated by T-B cell conjugates and trogocytosis as well as a lack of antibodies distinguishing between the closely related isoforms of FcγRII. To generate high-affinity binders specific for FcγRIIA, libraries of designed ankyrin repeat proteins (DARPins) were screened for binding to its extracellular domains by ribosomal display. Counterselection against FcγRIIB eliminated binders cross-reacting with both isoforms. The identified DARPins bound FcγRIIA with no detectable binding for FcγRIIB. Their affinities for FcγRIIA were in the low nanomolar range and could be enhanced by cleavage of the His-tag and dimerization. Interestingly, complex formation between DARPin and FcγRIIA followed a two-state reaction model, and discrimination from FcγRIIB was based on a single amino acid residue. In flow cytometry, DARPin F11 detected FcγRIIA+ cells even when they made up less than 1% of the cell population. Image stream analysis of primary human blood cells confirmed that F11 caused dim but reliable cell surface staining of a small subpopulation of T lymphocytes. When incubated with platelets, F11 inhibited their aggregation equally efficient as antibodies unable to discriminate between both FcγRII isoforms. The selected DARPins are unique novel tools for platelet aggregation studies as well as the role of FcγRIIA for the latent HIV-1 reservoir.

Indocyanine Green-Labeled Platelets for Survival and Recovery Studies.

Introduction: Before being implemented in daily clinical routine, new production strategies for platelet concentrates (PCs) must be validated for their efficacy. Besides in vitro testing, the establishment of new methods requires the labeling of platelets for in vivo studies of platelets' survival and recovery. Indocyanine green (ICG) is a Food and Drug Administration-approved near-infrared (NIR) fluorescent dye for diagnostic use in vivo, suitable for non-radioactive direct cell labeling of platelets. Methods: Platelets from PCs in storage solutions with different plasma concentrations were labeled with ICG up to concentrations of 200 µm. Whole blood (WB) was used as an ex vivo matrix to monitor the labeling stability of ICG-labeled platelets. The impact of labeling processes was assessed by the quantification of CD62P expression and PAC-1 binding as platelet function markers. Platelet aggregation was analyzed by light transmission aggregometry. ICG-labeling efficiency and stability of platelets were determined by flow cytometry. Results: Platelets from PCs could be successfully labeled with 10 µm ICG after 1 and 4 days of storage. The best labeling efficiency of 99.8% ± 0.1% (immediately after labeling) and 81% ± 6.2% (after 24 h incubation with WB) was achieved by plasma replacement by 100% platelet additive solution for the labeling process. Since the washing process slightly impaired platelet function, ICG labeling itself did not affect platelets. Immediately after the ICG-labeling process, plasma was re-added, resulting in a recovered platelet function. Conclusion: We developed a Good Manufacturing Practice compatible protocol for ICG fluorescent platelet labeling suitable for survival and recovery studies in vivo as a non-radioactive labeling alternative.

We developed a simple, reproducible method according to Good Manufacturing Practice guidelines for labeling of platelets from platelet concentrates (PCs) with indocyanine green (ICG) as a non-radioactive alternative. PCs are medicinal drugs that are transfused to prevent or treat bleeding. They consist of the blood cells' platelets which are responsible for clotting processes in the body. Manufacturing procedures of PCs are continuously refined, and for in vivo testing, these platelets have to be labeled to track and to distinguish them from proband's or patient's own cells. Radioactive labeling, for a long time the gold standard for cell labeling, is no longer accepted. ICG is a fluorescent dye approved by the drug authorities and already used for diagnostic purposes in humans. We used ICG to label platelets from PCs. With our method, we achieved a labeling efficiency of 99.8%. We used whole blood (WB) as an ex vivo matrix to monitor the labeling stability of ICG-labeled platelets. After the addition of ICG-labeled platelets to WB, the labeling efficiency decreased to 81% after 24 h. However, we were still able to distinguish the ICG-labeled platelets from the WB platelets. We could also show that platelet function was not impaired by the labeling processes. The good tolerance of ICG indicates a short path to clinical application in healthy volunteers and investigations of novel PC-manufacturing procedures. As a read-out system, flow cytometry systems equipped with NIR lasers and filters could offer the possibility of rapid visualization, cell tracking, re-isolation, and ex vivo studies.

A flow cytometric assay to detect platelet-activating antibodies in VITT after ChAdOx1 nCov-19 vaccination.

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.

Insights in ChAdOx1 nCoV-19 vaccine-induced immune thrombotic thrombocytopenia.

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.

Anti-platelet factor 4 antibodies causing VITT do not cross-react with SARS-CoV-2 spike protein.

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.

Comparative analysis of ChAdOx1 nCoV-19 and Ad26.COV2.S SARS-CoV-2 vector vaccines.

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.

The platelet proteasome and immunoproteasome are stable in buffy-coat derived platelet concentrates for up to 7 days.

OBJECTIVES: Characterization of the proteasome and its stability in buffy-coat derived platelet concentrates (PCs) during storage. BACKGROUND: The proteasome plays a key role in cell homeostasis by processing misfolded or abnormal proteins and regulating the levels and activities of a high number of proteins contributing to cell cycle, survival, and proliferation. Controversial data exist, whether inhibition of the proteasome affects platelet function. Little is known about function, expression, and stability of the proteasome in PCs during storage, and the potential role of the platelet proteasome in storage lesions. STUDY DESIGN AND METHODS: PCs were produced by the buffy-coat method in additive solution and stored at room temperature under agitation. Platelet aggregation was monitored by light transmission aggregometry. Proteasome complexes were assessed by immunoprecipitation and immunoblotting, and proteasome activity was measured using fluorogenic substrates specific for the three different proteolytic activities over 7 days of storage. RESULTS: Proteasome inhibition led to a decreased platelet aggregation response after activation with collagen, ADP, TRAP-6, and thrombin. There were no changes in the expression of the catalytic active subunits as well as the proteasome activity during storage of PCs, comparing baseline and day 7. DISCUSSION: Platelet proteasome function is relevant for platelet aggregation in response to various agonists. The constitutive and stable expression of the active standard- and immunoproteasome in platelets makes it unlikely that loss of proteasome function is a relevant cause of storage lesions.

Effect of Methylene Blue Pathogen Inactivation on the Integrity of Immunoglobulin M and G.

INTRODUCTION: In the light of the ongoing SARS-CoV-2 pandemic, convalescent plasma is a treatment option for CO-VID-19. In contrast to usual therapeutic plasma, the therapeutic agents of convalescent plasma do not represent clotting factor activities, but immunoglobulins. Quarantine storage of convalescent plasma as a measure to reduce the risk of pathogen transmission is not feasible. Therefore, pathogen inactivation (e.g., Theraflex®-MB, Macopharma, Mouvaux, France) is an attractive option. Data on the impact of pathogen inactivation by methylene blue (MB) treatment on antibody integrity are sparse. METHODS: Antigen-specific binding capacity was tested before and after MB treatment of plasma (n = 10). IgG and IgM isoagglutinin titers were tested by agglutination in increasing dilutions. Furthermore, the binding of anti-EBV and anti-tetanus toxin IgG to their specific antigens was assessed by ELISA, and IgG binding to Fc receptors was assessed by flow cytometry using THP-1 cells expressing FcRI and FcRII. RESULTS: There was no significant difference in the isoagglutinin titers, the antigen binding capacity of anti-EBV and anti-tetanus toxin IgG, as well as the Fc receptor binding capacity before and after MB treatment of plasma. CONCLUSION: MB treatment of plasma does not inhibit the binding capacity of IgM and IgG to their epitopes, or the Fc receptor interaction of IgG. Based on these results, MB treatment of convalescent plasma is appropriate to reduce the risk of pathogen transmission if quarantine storage is omitted.

Interaction between the Staphylococcus aureus extracellular adherence protein Eap and its subdomains with platelets.

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 D3D4 specifically interacts and rapidly activates human platelets. Furthermore, we validate our finding by using site directed directional immobilization of Eap D3D4 through N-terminal His6 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.

Flucloxacillin-induced immune thrombocytopenia.

BACKGROUND: Drug-induced immune thrombocytopenia (DITP) is an adverse drug reaction associated with platelet (PLT) destruction by drug-dependent antibodies. Demonstration of drug-dependent PLT antibodies is often difficult and can only be rendered by extensive laboratory testing. In this report, we present the first serologically confirmed case of DITP caused by the antibiotic flucloxacillin. CASE REPORT: A 68-year-old man developed severe thrombocytopenia that was first suspected to be related to heparin-induced thrombocytopenia. The absence of PLT-activating heparin-dependent antibodies and the abrupt decrease in PLT count to fewer than 20 × 10(9) /L raised the suspicion of DITP. DISCUSSION: Flucloxacillin-dependent antibodies were detected in patient serum using whole PLT enzyme immunoassay and flow cytometry. The glycoprotein (GP) specificity was identified to be against GP IIb/IIIa complexes using the monoclonal antibody immobilization of PLT antigens assay. Interestingly, antibody binding was abolished when EDTA plasma was used and restored after adding calcium. CONCLUSION: In summary, DITP should be considered in cases of acute thrombocytopenia during treatment with flucloxacillin.

Assessment of human platelet survival in the NOD/SCID mouse model: technical considerations.

BACKGROUND: The NOD/SCID mouse model is a unique and sophisticated method to study the survival of human platelets (PLTs) in vivo. Meanwhile, several research groups adopted this model to analyze a wide range of PLT antibodies. Differences exist between the research groups regarding the method of PLT injection, the amount and route of antibody injection, and the preparation of blood samples collected from the animal, making it difficult to compare results between studies. STUDY DESIGN AND METHODS: We compared the survival of human PLTs infused into NOD/SCID mice via the tail vein or the retro-orbital plexus. The percentage of circulating human PLTs in the mouse circulation was determined by flow cytometry. Murine blood samples were prepared using two different methods: 1) direct fixation of whole blood samples and 2) isolation of PLTs by density gradient centrifugation. RESULTS: Recovery of human PLTs after tail vein injection was comparable to retro-orbital injection (13% vs. 11% of all circulating PLTs, p = 0.401). However, the survival rate of tail vein-infused PLTs was higher than that of retro-orbitally injected PLTs (median PLT survival after 5 hr 84% vs. 56%, p = 0.025). Moreover, we observed that determination of circulating human PLTs in directly fixed murine whole blood samples shows better reproducibility compared to the density gradient centrifugation method. CONCLUSIONS: Tail vein injection of human PLTs into the NOD/SCID mice is superior to retro-orbital injection in terms of human PLT survival. Direct fixation of whole blood samples allows better reproducibility of results compared to the density gradient centrifugation method.

Impact of priming on the response of neutrophils to human neutrophil alloantigen-3a antibodies.

BACKGROUND: Human neutrophil alloantigen-3a (HNA-3a) antibodies can induce transfusion-related acute lung injury (TRALI). The severity of TRALI varies largely among the affected patients. Severe comorbidity seems to increase the susceptibility for TRALI, potentially by priming of neutrophils. Thus, the impact of neutrophil priming on HNA-3a antibody-mediated neutrophil aggregation and CD11b surface expression was investigated. STUDY DESIGN AND METHODS: Neutrophils were primed using formyl-methionyl-leucyl-phenylalanine (fMLP) or bacterial lipopolysaccharide (LPS). Granulocyte aggregation and CD11b surface expression were evaluated by the granulocyte agglutination test and by flow cytometry (FC), respectively. Priming-induced changes in the surface expression of choline transporter-like protein 2 (CTL2) and the CTL2 mRNA expression were assessed by FC and quantitative real-time polymerase chain reaction, respectively. RESULTS: Priming of neutrophils lowered the amount of HNA-3a antibodies required for inducing granulocyte aggregation in a dose-dependent manner by 50% to 75%. The priming agent concentration necessary for this response differed between donors. Priming slightly enhanced binding of HNA-3a antibodies to neutrophils. However, CTL2 de novo synthesis was not induced after priming with LPS, indicating that increased HNA-3a antibody binding was likely caused by translocation of intracellular CTL2 to the surface or by increased affinity of HNA-3a antibodies to CTL2. HNA-3a antibodies influenced CD11b surface expression on neutrophils only marginally, which was also not potentiated by priming with fMLP or LPS. CONCLUSION: This study provides experimental evidence supporting the "threshold model" of TRALI. Priming of neutrophils with fMLP or LPS increases their aggregation response to HNA-3a antibodies by lowering the required antibody amount.

Aggregates of nonmuscular myosin IIA in erythrocytes associate with GATA1- and GFI1B-related thrombocytopenia.

BACKGROUND: The transcription factor GATA1 is an essential regulator of erythroid cell gene expression and maturation and is also relevant for platelet biogenesis. GATA1-related thrombocytopenia (GATA1-RT) is a rare X-linked inherited platelet disorder (IPD) characterized by macrothrombocytopenia and dyserythropoiesis. Enlarged platelet size, reduced platelet granularity, and noticeable red blood cell anisopoikilocytosis are characteristic but unspecific morphological findings in GATA1-RT. OBJECTIVES: To expand the investigation of platelet phenotype of patients with GATA1-RT by light- and immunofluorescence microscopy on a blood smear. METHODS: We assessed blood smears by light- and immunofluorescence microscopy after May-Grünwald Giemsa staining using a set of 13 primary antibodies against markers belonging to different platelet structures. Antibody binding was visualized by fluorescently labeled secondary antibodies. RESULTS: We investigated 12 individuals with genetically confirmed GATA1-RT from 8 unrelated families. While confirming the already known characteristic of platelet morphology (platelet macrocytosis and reduced expression of markers for α-granules), we also found aggregates of nonmuscular myosin heavy chain II A (NMMIIA) in the erythrocytes in all individuals (1-3 aggregates/cell, 1-3 µm diameter). By systematically reanalyzing blood smears from a cohort of patients with 19 different forms of IPD, we found similar NMMIIA aggregates in the red blood cells only in subjects with GFI1B-related thrombocytopenia (GFI1B-RT), the other major IPD featured by dyserythropoiesis. CONCLUSION: Aggregates of NMMIIA in the erythrocytes associate with GATA1-RT and GFI1B-RT and can facilitate their diagnosis on blood smears. This previously unreported finding might represent a novel marker of dyserythropoiesis assessable in peripheral blood.

Expression of the CTL2 transcript variants in human peripheral blood cells and human tissues.

BACKGROUND: The HNA-3a antigen is an important antibody target in the pathophysiology of transfusion-related acute lung injury (TRALI). It is encoded by the choline transporter-like protein 2 (CTL2) gene, which exists in the two transcript variants TV1 and TV2, differing in the upstream promoter and coding region. Only TV1 has been demonstrated to enable choline transport across the cell membrane. STUDY DESIGN AND METHODS: The aim of this study was to determine the CTL2 transcript pattern in human peripheral blood cells and tissues and its capacity to bind HNA-3a antibodies. RNA was isolated from human whole blood, isolated neutrophils, mononuclear blood cells, leukoreduced platelets, human lung, liver, and colon. After reverse transcription, the single-stranded cDNA was amplified using primer combinations specific for the respective transcript. Plasmids containing the entire CTL2 coding cDNA of the transcript variant TV1 or TV2 served as controls. HEK293T cells expressing both variants were used to determine the binding of HNA-3a antibodies. RESULTS: The shorter TV2 transcript was demonstrated in each RNA sample derived from human peripheral blood tested so far, as well as in human lung and liver, whereas the longer TV1 transcript was only detected in human lung and colon. TV1 and TV2 had the same binding capacity to HNA-3a antibodies. CONCLUSION: The expression of TV1 and TV2 is tissue and cell specific, with peripheral blood cells expressing only TV2. This does not affect binding of HNA-3a antibodies. Whether the unequal expression might be relevant in the pathogenesis of TRALI remains to be investigated.

Gas plasma-induced platelet activation corresponds to reactive species profiles and lipid oxidation.

Surgical-induced hemostasis is a critical step in the closure of incisions, which is frequently achieved via electrocauterization and subsequent tissue necrotization. The latter is associated with postoperative complications. Recent in vivo work suggested reactive species-producing gas plasma technology as a pro-homeostatic agent acting via platelet activation. However, it remained elusive how platelet activation is linked to lipid and protein oxidation and the reactive species compositions. A direct relation between the reactive species composition and platelet activation was revealed by assessing the production of several reactive species and by using antioxidants. In addition, platelet lipidome and proteome analysis identified significantly regulated key lipids in the platelet activation pathway, such as diacylglycerols and phosphatidylinositol as well as oxylipins like thromboxanes. Lipid oxidation products mainly derived from phosphatidylethanolamine and phosphatidylserine species were observed at modest levels. In addition, oxidative post-translational modifications were identified on key proteins of the hemostasis machinery. This study provides new insights into oxidation-induced platelet activation in general and suggests a potential role of those processes in gas plasma-mediated hemostasis in particular.

Cytoskeleton Dependent Mobility Dynamics of FcγRIIA Facilitates Platelet Haptotaxis and Capture of Opsonized Bacteria.

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.

Ex vivo anticoagulants affect human blood platelet biomechanics with implications for high-throughput functional mechanophenotyping.

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.

Divalent magnesium restores cytoskeletal storage lesions in cold-stored platelet concentrates.

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.