Platelets bind plasminogen facilitating surface-bound plasmin generation. We previously reported that the plasminogen receptor, Plg-RKT, retains plasminogen on activated platelets. Here, we investigate the significance of the interaction of Plg-RKT on thrombus formation, growth and stability. Whole blood from Plg-RKT−/− or littermate Plg-RKT+/+ mice was flowed over collagen/tissue factor-coated microfluidic biochips at 250 or 1000 s−1 to reflect venous and arterial shear rates. AlexaFluor488-fibrinogen and Dylight633-labelled-plasminogen accumulation was monitored in real-time by fluorescence microscopy in the presence or absence of tissue plasminogen activator (tPA). At 1000 s−1, plasminogen accumulation was reduced in thrombi formed from Plg-RKT−/− mice compared to Plg-RKT+/+ mice. Fibrin(ogen) accumulation in Plg-RKT−/− mice persisted for the duration of the experiment, indicating impaired fibrinolysis compared to Plg-RKT+/+ mice. Mice were subjected to FeCl3 carotid artery model of thrombosis followed by tPA infusion. Initial platelet deposition was faster in Plg-RKT−/− mice compared to Plg-RKT+/+ mice. Fibrin(ogen) accumulation and persistence was enhanced in Plg-RKT−/− mice indicating impaired fibrinolysis. We demonstrate for the first time that under arterial shear, Plg-RKT facilitates plasminogen incorporation and limits both platelet recruitment to the forming thrombus and fibrin accumulation. These data highlight that Plg-RKT and potentially plasmin on the platelet surface regulate arterial thrombus growth. Plg-RKT regulates initial platelet deposition and fibrin(ogen) accumulation into thrombi thereby down-regulating thrombolysis. Plg-RKT functions to restrain thrombus growth and modify the evolving architecture.

The platelet immunoreceptor tyrosine-based activation motif receptors, glycoprotein VI (GPVI) and C-type lectin-like receptor 2 (CLEC-2), are activated by a diversity of ligands that bind to distinct epitopes, indicating that competitive antagonists will not block activation by all stimuli. In this study, we used 2-color single-particle tracking to investigate the dynamic nanoscale organization of the 2 receptors in the cell membrane to identify new strategies for inhibition. The studies were performed in CHO-K1 cells that lack the tyrosine kinase Syk. The results show that, when expressed at low level, GPVI and CLEC-2 diffuse over the cell surface as monomers with the presence of dimers due to random collisions. The addition of divalent and trivalent nanobody ligands induces homodimerization and cessation of movement of both receptors proportionate to ligand valency. The dimers of CLEC-2 are longer-lived than those of GPVI, despite a lower affinity of the monomeric nanobody that forms the ligand backbone. The dimerization of recombinant monomeric CLEC-2 but not GPVI was detected by surface plasmon resonance with an affinity constant of 18.5μM. The results suggest that the prolonged lifetime of the CLEC-2 interactions is due to synergy between ligand-induced cross-linking and receptor homodimerization. Blocking dimerization may be an effective way to inhibit the activation of CLEC-2 by its diverse range of ligands.

The platelet ITAM receptors, GPVI and CLEC-2, are activated by a diversity of ligands which bind to distinct epitopes indicating that competitive antagonists will not block activation by all stimuli. The present study used two-colour single-particle tracking to investigate the dynamic nanoscale organisation of the two receptors in the cell membrane to identify new strategies for inhibition. The studies were performed in CHO-K1 cells which lack the tyrosine kinase Syk. The results show that when expressed at low level, GPVI and CLEC-2 diffuse over the cell surface as monomers with the presence of dimers due to random collisions. The addition of divalent and trivalent nanobody ligands induces homodimerisation and cessation of movement of both receptors proportionate to ligand valency. Dimers of CLEC-2 are longer-lived than those of GPVI despite a lower affinity of the monomeric nanobody that forms the ligand backbone. Dimerisation of recombinant monomeric CLEC-2 but not GPVI was detected by surface plasmon resonance with a KD of 18.5 μM. The results suggest that the prolonged lifetime of the CLEC-2 interactions is due to synergy between ligand-induced crosslinking and receptor homodimerisation. Blocking dimerisation may be an effective way to inhibit activation of CLEC-2 by its diverse range of ligands.

Inflammation-induced thrombosis is a common consequence of bacterial infections, such as those caused by Salmonella Typhimurium (STm). The presentation of multi-organ thrombosis post-infection that develops and resolves with organ-specific kinetics raises significant challenges for its therapeutic control. Here, we identify specific inflammatory events driving thrombosis in the spleens and livers of STm-infected mice. IFN-γ or platelet expression of C-type lectin-like receptor CLEC-2, key drivers of thrombosis in liver, are dispensable for thrombosis in the spleen. Platelets, monocytes, and neutrophils are identified as core constituents of thrombi in both organs. Depleting either neutrophils or monocytic cells abrogates thrombus formation. Neutrophils and monocytes secrete TNF and blocking TNF diminishes both thrombosis and inflammation, which correlates with reduced endothelial expression of E-selectin and leukocyte infiltration. Moreover, inhibiting tissue factor and P-selectin glycoprotein ligand-1 pathways impairs thrombosis in both spleen and liver. Therefore, we identify organ-specific, and shared mechanisms driving thrombosis within a single infection. This may inform on tailoring treatments towards infection-induced inflammation, and single- or multi-organ thrombosis, based on the clinical need.

This series of illustrated capsules summarizes the presentations made by the speakers at the first International Advanced Course in Platelet Research held in Murcia (Spain) from 27 to 28 September, 2024. This is the first course to receive a Fundamental Research Workshop Grant from the International Society on Thrombosis and Haemostasis (ISTH) and was also supported administratively and scientifically by the Spanish Society of Thrombosis and Haemostasis (SETH). This unique course focused on new methodologies applied in platelet research and how these are increasing our understanding of platelet formation, their multifunctionality in different physiological and pathological contexts, and contributing to the development of new platelet-targeted therapies to improve the management of hemostatic/thrombotic pathologies. It aligns with the objectives of several Scientific and Standardization Committees of the ISTH, including Platelet Physiology and Genomics in Thrombosis and Haemostasis, as well as with the academic objectives of the ISTH and SETH. The program was designed by the coordinator (J. Rivera), and the scientific advisory board (SAB: S.P. Watson, K. Freson, A. Balduini, and J. Di Paola) and comprised 9 scientific sessions with 25 presentations, each with time for extensive open discussion. Additionally, 33 abstract posters were presented, with the 3 highest scoring selected as oral presentations. The course was held in a single location and with an informal atmosphere to facilitate networking among participants. The course received very positive feedback from the 140 attendees. The course was supported by the ISTH, SETH, University of Murcia, CIBERER-ISCIII, Fundación Séneca (22426/OC/24), the United Kingdom Platelet Society and various pharmaceutical companies. We believe that the extraordinary scientific and human experience of this course may act as a stimulus for future courses.

Phosphoinositides are a group of interconvertible lipids that are located in the membrane of eukaryotic cells. They turnover via complex network of reactions (called the phosphoinositide pathway) that respond rapidly to regulate many aspects of a cell's response to their environment. Given their low-abundance they are difficult to characterise experimentally. Here we utilise a new experimental method to generate an unusually large dataset that characterises the time-dependent changes in five membrane bound phospoinositides and a soluble inositide in platelet, downstream of its GPVI receptor, where we know the phosphoinositide pathway is particularly active. To shed light on regulatotory steps that are often opaque to experimentation we use this data within a mathematical and computational framework. We construct and assess eleven mathematical models that represent competing interpretations of the dominant mechanisms that regulate the pathway. We find that while four of the models can generate the available data only one model, that incorporates an additional pool of PtdIns, is consistent with the data and is able to successfully predict the effects of an inhibitor. We publish all models openly in a form that is easily usable and adaptable for other researchers to use alongside our or their own data. We studied how changes in the shape and magnitude of events that stimulate the phosphoinositide pathway affect its dynamics. Despite these perturbations, the abundance of Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) remained stable, consistent with findings reported in the literature.

Background:We have reported that trivalent and tetravalent nanobodies against glycoprotein (GP)VI, C-type lectin-like receptor (CLEC)-2, and platelet endothelial aggregation receptor (PEAR)1 stimulate powerful aggregation and adenosine triphosphate secretion in human platelets. Objectives:This study aimed to evaluate changes in platelet surface GPs elicited by activation of GPVI, CLEC-2, and PEAR1 using trivalent and tetravalent nanobodies. Methods:The effect of the crosslinked nanobodies on P-selectin was measured in whole blood and washed platelets with and without secondary mediator inhibitors using classical flow cytometry and on 16 platelet surface GPs in whole blood using multispectral flow cytometry. Results:Trivalent nanobodies to GPVI and CLEC-2 stimulated modest (<60% of collagen-related peptide) expression of P-selectin in whole blood (10-fold dilution) and washed platelets (2 × 107 mL), whereas tetravalent nanobodies induced a response approaching that of collagen-related peptide. Stimulation of P-selectin expression was partially reduced by inhibitors of adenosine diphosphate (ADP) and thromboxane A2, indicating secondary platelet activation despite the low platelet concentration. By multispectral flow cytometry, tetravalent nanobodies to GPVI and CLEC-2 stimulated similar maximal fibrinogen binding and platelet surface α-granule (TLT-1 and CD154) and δ-granule (CD63) markers, but lower levels of the lysosomal marker CD107a. The tetravalent PEAR1 nanobody showed partial agonist activity in some donors but full activity in others. Conclusion:Tetravalent nanobodies to GPVI and CLEC-2 stimulate powerful activation of platelets at low nanomolar concentrations in flow cytometry. In contrast, trivalent nanobodies are partial agonists. The defined stoichiometry of the nanobodies will aid development of standardized platelet flow cytometry assays.

BACKGROUND:Sickle cell disease (SCD) is a challenging genetic disorder characterized by hemolytic anemia, vaso-occlusive crises (VOC), and progressive organ damage. Despite its severity, effective treatments are limited. The recent withdrawal of promising therapies, such as the anti-P-selectin antibody Crizanlizumab and the hemoglobin polymerization inhibitor Voxelotor, highlights the urgent need for innovative approaches to alleviate vaso-occlusion and thromboinflammation. METHODS:In this study, we used advanced techniques, including intravital microscopy, laser speckle contrast imaging, and histological analysis, to examine the role of syk (spleen tyrosine kinase) in platelet and neutrophil recruitment, and blood perfusion in the lung, kidney, liver, and spleen of SCD mice. RESULTS:In the Berkeley SCD model, hemin-induced vaso-occlusion and impairment in pulmonary blood perfusion were independent of red cell congestion and fibrin deposition. Hypoperfusion was driven by adhesion of neutrophils and platelets in the microcirculation and exacerbated by pulmonary emboli. Hemin-induced cell adhesion and hypoperfusion were also observed in the renal microcirculation, whereas it was limited in the liver and spleen of SCD mice, suggesting that organ-specific mechanisms drive hypoperfusion and vaso-occlusion. To explore therapeutic options, we investigated the potential of Syk inhibition in improving blood perfusion and reducing thrombo-inflammation in different organs. Selective Syk inhibition, using BI-1002494, reduced cellular adhesion in the pulmonary and renal microvasculature, effectively restoring blood perfusion and reducing thrombo-inflammation. Low-dose Syk inhibitor was effective in reducing neutrophil adhesion and improving blood perfusion without inducing bleeding. Increasing the dose exacerbated hemin-induced bleeding in the lungs, likely due to off-target activity againt other kinases, including Src. CONCLUSIONS:These findings underscore the critical role of Syk in platelet and neutrophil mediated-thrombo-inflammation and hypoperfusion in SCD, suggesting that Syk inhibition is a promising strategy to reduce organ-specific vaso-occlusion, improve renal and pulmonary perfusion, and reduce organ damage.

Background Thrombosis with thrombocytopenia syndrome is a rare condition known to occur spontaneously or after heparin use. With the advent of COVID-19 vaccines during the pandemic, thrombosis with thrombocytopenia syndrome cases emerged post administration of adenoviral vaccines, termed vaccine-induced immune thrombosis and thrombocytopenia. In response, the thrombosis with thrombocytopenia syndrome consortium was formed to deepen our understanding of this syndrome post vaccination. Methods The consortium employed a comprehensive approach across five work packages. This included designing cohort studies covering the entire English population and analysing local linked regional data sets to detect thrombosis with thrombocytopenia syndrome occurrences in real time. Various patient and healthy control specimens, including those from vaccinated individuals, underwent testing for antiplatelet factor 4 antibodies using three different assays. Patients who developed vaccine-induced immune thrombosis and thrombocytopenia after the AstraZeneca (AZD1222) COVID-19 vaccine underwent whole-genome and ribonucleic acid sequencing to identify genetic susceptibility factors. Multiple studies were conducted to investigate the mechanism of antiplatelet factor 4 antibody formation, including assessments of adenoviral vector structure and binding to platelet factor 4. Detailed studies were also conducted to understand the immune response to vaccines, the role of immune complexes involving platelet factor 4 and their effects on proinflammatory cytokines, neutrophil extracellular traps and platelets in the pathogenesis of the syndrome. Results Cohort studies revealed a higher risk of arterial and venous thromboses after COVID-19 infection compared to vaccination. Specifically, regarding vaccines, the risk of thrombosis and/or thrombocytopenia was higher after the first dose of the AZD1222 vaccine but not with subsequent doses of. Regional linked data indicated that real-time ascertainment of diseases across multiple acute hospital sites’ secure data environments is not yet feasible at scale. The overall background seroprevalence of antiplatelet factor 4 antibodies was low in healthy individuals, vaccinated individuals and those infected with COVID-19. Whole-genome sequencing did not identify significant variants predisposing to vaccine-induced immune thrombosis and thrombocytopenia, with ongoing work on ribonucleic acid sequencing. An electrostatic interaction between the hexon hypervariable regions of the ChAdOx1 capsid and platelet factor 4 was suggested as a possible mechanism for antiplatelet factor 4 antibody development. Strong immune response drove the formation of neutrophil extracellular traps, significant inflammatory responses and clot formation in distant organs. Platelet activation post immune complex formation against platelet factor 4 was dependent on FcγRIIa but independent of complement, also occurring through binding with c-Mpl. T-cell reactivity against the AZD1222 vaccine indicates potential cross-reactivity with prevalent human adenoviruses. Conclusions The consortium’s comprehensive work has uncovered new potential mechanisms of vaccine-induced immune thrombosis and thrombocytopenia and identified novel biomarkers and therapeutic strategies for further development and validation. This is crucial, as the combination of thrombosis and thrombocytopenia, alongside antiplatelet factor 4 antibodies, can occur without exposure to heparin or adenovirus vaccines. Future considerations Recommendations include the development of a national reference laboratory and registry for diagnosis and further study of thrombosis with thrombocytopenia syndrome; future vaccine development using the adenoviral vector platform to focus on the reduction of the electrostatic interaction between viral hexons and platelet factor 4; international genomics collaboration; and studies focused on understanding the symptoms suffered by patients as well as strategies to ameliorate them. Limitations Direct identification of vaccine-induced immune thrombosis and thrombocytopenia patients was hindered by poor recording. The rarity of vaccine-induced immune thrombosis and thrombocytopenia limited the number of patients recruited for genomic and mechanistic studies. Funding This synopsis presents independent research funded by the National Institute for Health and Care Research (NIHR) Efficacy and Mechanism Evaluation (EME) programme as award number NIHR135073. Plain language summary Thrombosis with thrombocytopenia syndrome is rare: it is characterised by thrombosis and lowered platelet counts together with the development of an antibody against a protein called platelet factor 4. This syndrome has been linked to heparin use or can occur spontaneously. With COVID-19 vaccines, a new form called vaccine-induced immune thrombosis and thrombocytopenia appeared. The thrombosis with thrombocytopenia syndrome consortium formed to better understand this syndrome. The consortium used various methods, like studying the data of the entire English population and analysing local data in real time. They tested patient and healthy control samples for antiplatelet factor 4 antibodies and sequenced genes from patients who got vaccine-induced immune thrombosis and thrombocytopenia after the AZD1222 COVID-19 vaccine. They also studied how these antibodies form and their effects, including changes in cytokines and platelet involvement. Our studies showed a higher thrombosis risk after COVID-19 infection compared to vaccination. The first dose of the AZD1222 vaccine had higher risks of thrombosis and lowered platelets (occurring separately), but subsequent doses or mRNA vaccines were safer. Identifying vaccine-induced immune thrombosis and thrombocytopenia patients directly was difficult due to poor records. Real-time tracking of diseases across hospitals was not yet possible at scale. The prevalence of antiplatelet factor 4 antibodies was low in healthy, vaccinated and COVID-19-infected individuals. Genetic sequencing didn’t find significant variants causing vaccine-induced immune thrombosis and thrombocytopenia, but there are ongoing ribonucleic acid studies. Our studies found a possible mechanism for antiplatelet factor 4 antibody development involving the AZD1222 vaccine. The immune response caused generalised inflammation and clotting in distant organs. Platelet activation was influenced by certain factors. T-cell reactivity against the AZD1222 vaccine hinted at potential cross-reactivity with common human viruses. The consortium’s work has uncovered new insights into vaccine-induced immune thrombosis and thrombocytopenia, suggesting potential new diagnostic and treatment strategies. This is crucial, as thrombosis with thrombocytopenia syndrome can occur without exposure to heparin or adenovirus vaccines.

Sickle cell disease (SCD) leads to hemolytic anemia, vaso-occlusive crisis (VOC), hypoperfusion, and progressive organ damage. Hemin, released during hemolysis in SCD, induces platelet activation through CLEC-2, endothelial activation through TLR4, neutrophil adhesion and NETosis, all of which are regulated by spleen tyrosine kinase (Syk). In this study, we assessed neutrophil and platelet recruitment to the pulmonary, renal, splenic, and hepatic microvasculature in control and SCD mice following hemin injection and the effect of Syk inhibition on cell recruitment and organ perfusion. Compared to controls, SCD mice exhibited higher baseline neutrophil and platelet recruitment to the lungs without alterations in lung perfusion as measured by laser speckle contrast imaging. Injection of hemin increased cell recruitment to the pulmonary and renal vasculature with a concomitant reduction in organ perfusion. However, hemin injection did not change cell recruitment or organ perfusion in the spleen and liver, both of which were altered at baseline in SCD mice. Pretreatment of SCD mice with the Syk inhibitor BI-1002494 mitigated baseline and hemin-induced neutrophil and platelet adhesion in the pulmonary and renal microvasculature, with a corresponding normalization of perfusion. Syk regulates vascular integrity in the lung of SCD mice; whilst high concentrations of BI-1002494 increased bleeding, lowering drug concentrations preserved the inhibitory effect on platelet and neutrophil recruitment and lung perfusion and protected from bleeding complications. These data substantiate Syk as a mediator of vascular thrombo-inflammation and hypoperfusion in the lung and kidney of SCD and provide a rationale for pharmacological inhibition as a therapeutic strategy.### Competing Interest StatementPLRN has received a research grant from Rigel. No other authors have conflicts of interest to declare.

Platelet factor 4 (PF4) is an abundant chemokine that is released from platelet alpha-granules on activation. PF4 is central to the pathophysiology of vaccine-induced immune thrombocytopenia and thrombosis (VITT) in which antibodies to PF4 form immune complexes with PF4, which activate platelets and neutrophils through Fc receptors. In this study, we show that PF4 binds and activates the thrombopoietin receptor, cellular myeloproliferative leukemia protein (c-Mpl), on platelets. This leads to the activation of Janus kinase 2 (JAK2) and phosphorylation of signal transducer and activator of transcription (STAT) 3 and STAT5, leading to platelet aggregation. Inhibition of the c-Mpl-JAK2 pathway inhibits platelet aggregation to PF4, VITT sera, and the combination of PF4 and IgG isolated from VITT patient plasma. The results support a model in which PF4-based immune complexes activate platelets through binding of the Fc domain to Fc gamma RIIA and PF4 to c-Mpl.

The collagen/fibrin(ogen) receptor, glycoprotein VI (GPVI), is a platelet activating receptor and a promising anti-thrombotic drug target. However, while agonist-induced GPVI clustering on platelet membranes has been shown to be essential for its activation, it is unknown if GPVI dimerisation represents a unique conformation for ligand binding. Current GPVI structures all contain only the two immunoglobulin superfamily (IgSF) domains in the GPVI extracellular region, so lacking the mucin-like stalk, transmembrane, cytoplasmic tail of GPVI and its associated Fc receptor γ (FcRγ) homodimer signalling chain, and provide contradictory insights into the mechanisms of GPVI dimerisation. Here, we utilised styrene maleic-acid lipid particles (SMALPs) to extract GPVI in complex with its two associated FcRγ chains from transfected HEK-293T cells, together with the adjacent lipid bilayer, then purified and characterised the GPVI/FcRγ-containing SMALPs, to enable structural insights into the full-length GPVI/FcRγ complex. Using size exclusion chromatography followed by a native polyacrylamide gel electrophoresis (PAGE) method, SMA-PAGE, we revealed multiple sizes of the purified GPVI/FcRγ SMALPs, suggesting the potential existence of GPVI oligomers. Importantly, GPVI/FcRγ SMALPs were functional as they could bind collagen. Mono-dispersed GPVI/FcRγ SMALPs could be observed under negative stain electron microscopy. These results pave the way for the future investigation of GPVI stoichiometry and structure, while also validating SMALPs as a promising tool for the investigation of human membrane protein interactions, stoichiometry and structure.

Current microscopy approaches applied to platelet aggregates in both haemostatic and thrombotic settings indicate their structure has important implications in efficient haemostasis and in clinical treatment of thrombosis. However, current fluorescence microscopy approaches are not amenable to volumetric imaging of platelet aggregate structures. This is largely due to the small size of individual platelets and the tight packing of platelets within aggregates, resulting in optical opacity. Here we demonstrate that expansion microscopy, applied to platelet aggregates, can reveal multi-scale information about the structure of platelet aggregates. We produced volumetric images at nanoscale resolution of >700 platelet aggregates under normal and perturbed conditions, stained for cytoskeletal and membrane components. We demonstrate our custom analysis workflow provides quantitative description of platelet numbers, volumes and morphology within entire platelet aggregates. Additionally, we quantitatively describe subcellular organisation of F-actin. By comparing these measurements following treatment with the actin inhibitors, cytochalasin D and latrunculin A, we can robustly detect structural disruptions in platelet aggregates. Together these data provide a workflow to qualitatively and quantitatively describe the architecture of platelet aggregates at a range of scales (whole aggregates down to sub-cellular features within individual platelets). ### Competing Interest Statement The authors have declared no competing interest.

MOONS is the Multi-Object Optical and Near-IR Spectrograph for ESO's Very Large Telescope. The instrument will use similar to 1000 optical fibres which can be individually aligned to on-sky targets across a field of view of 500 square arcmin. Each fibre is positioned using a dual arm theta-phi fibre positioning unit (FPU). The MOONS metrology system must be able to simultaneously measure the position of each fibre to a high accuracy (similar to 15 micrometres) as well as measuring the orientation of the FPU arms. In this paper, we present a description of photogrammetry-based metrology system design and its implementation in the instrument. We also report on the integration, testing, and performance of the system within the instrument.

Glycoprotein VI (GPVI) plays a key role in collagen-induced platelet aggregation. Affimers are engineered binding protein alternatives to antibodies. We screened and characterized GPVIbinding Affimers as novel tools to probe GPVI function. Among the positive clones, M17, D22, and D18 bound GPVI with the highest affinities (dissociation constant (KD) D ) in the nanomolar range). These Affimers inhibited GPVI-collagen-related peptide (CRP)-XL/collagen interactions, CRP-XL/collagen-induced platelet aggregation, and D22 also inhibited in vitro thrombus formation on a collagen surface under flow. D18 bound GPVI dimer but not monomer. GPVI binding was increased for D18 but not M17/D22 upon platelet activation by CRP-XL and adenosine 5 '-diphosphate. '-diphosphate. D22 but not M17/D18 displaced nanobody 2 (Nb2) binding to GPVI, indicating similar epitopes for D22 with Nb2 but not for M17/D18. Mapping of binding sites revealed that D22 binds a site that overlaps with Nb2 on the D1 domain, whereas M17 targets a site on the D2 domain, overlapping in part with the glenzocimab binding site, a humanized GPVI antibody fragment antigen-binding fragment. D18 targets a new region on the D2 domain. We found that D18 is a stable noncovalent dimer and forms a stable complex with dimeric GPVI with 1:1 stoichiometry. Taken together, our data demonstrate that Affimers modulate GPVI-ligand interactions and bind different sites on GPVI D1/D2 domains. D18 is dimer-specific and could be used as a tool to detect GPVI dimerization or clustering in platelets. A dimeric epitope regulating ligand binding was identified on the GPVI D2 domain, which could be used for the development of novel bivalent antithrombotic agents selectively targeting GPVI dimer on platelets.

Invasive non-typhoidal Salmonella infections are responsible for >75 000 deaths/year and >500 000 cases/year globally. Seventy-five percent of these cases occur in Sub-Saharan Africa, an increasing number of which are from multi-drug resistant strains. Interactions between bacteria and platelets can lead to thrombus formation, which can be beneficial for control of infection (immunothrombosis), or harmful through uncontrolled inflammation and organ damage (thromboinflammation). It is unknown whether Salmonella Typhimurium can activate human platelets. To assess this, light transmission aggregometry was used to measure platelet activation by two different Salmonella Typhimurium strains in 26 healthy donors in platelet-rich plasma and washed platelets. In platelet-rich plasma, but not in washed platelets, Salmonella Typhimurium activated platelets in a donor- and strain-dependent manner mediated through the low affinity immune receptor Fc gamma RIIA and the feedback agonists, ADP and thromboxane A(2). Plasma swap studies between strong and weak responders demonstrated a plasma component was responsible for the variation between donors. Depletion of anti-Salmonella antibodies from plasma abolished Salmonella-induced platelet aggregation responses, and addition of polyclonal anti-Salmonella antibody allowed aggregation in washed platelets. Correlating levels of anti-Salmonella total IgG or the IgG1, IgG2, IgG3 and IgG4 subclasses to platelet responses revealed total IgG levels, rather than levels of individual subclasses, positively correlated with maximum platelet aggregation results, and negatively with lag times. Overall, we show that anti-Salmonella IgG antibodies are responsible for donor variation in platelet aggregation responses to Salmonella and mediate this activity through Fc gamma RIIA.

Introduction Heparin is a negatively charged, heavily sulfated polysaccharide that is used in medicine as a highly effective anticoagulant. In about 1% of patients who receive unfractionated heparin, heparin-induced thrombocytopenia (HIT) can occur. This is a disorder characterized by an intensely prothrombotic phenotype and thrombocytopenia that typically occurs 5-10 days after heparin exposure. In HIT, heparin binds to positively charged platelet factor 4 (PF4), exposing neoepitopes on PF4 to which heparin-dependent anti-PF4 antibodies can bind. This results in immune complexes which activate platelets through the low affinity Fc receptor, FcγRIIA. Recently, heparin was shown to activate platelets by binding to platelet endothelial aggregation receptor 1 (PEAR1). We therefore hypothesized that heparin itself may have a role in platelet activation in HIT in cooperation with activation through FcγRIIA. Methods We assessed activation of healthy donor washed platelets (2 x 108/mL) to the HIT-like monoclonal antibody (mAb) 5B9 and HIT sera in the presence of heparin by light transmission aggregometry. Experiments were performed in the presence or absence of the PEAR1 nanobody, Nb138, which was raised against the heparin-binding domain of PEAR1. Results Heparin (0.5 IU/mL) but not the HIT-like mAb 5B9 (20 µg/mL) stimulated slow and sustained aggregation of washed platelets with a second, more rapid phase at ~20 min. The response to heparin was blocked by the PEAR1 nanobody, Nb138 (100 nM). In contrast, the combination of heparin and mAb 5B9 stimulated robust aggregation of washed platelets within 15 min (n=7). In 3/7 donors, the response was blocked by Nb138 and was delayed by over 30 seconds in two others. We then tested the effect of Nb138 on two HIT sera that also induce strong activation of platelets in the presence of heparin. Nb138 delayed aggregation to the first HIT serum in all 7 donors. Nb138 blocked aggregation to the second HIT serum in 4 donors and significantly delayed aggregation in 2 others. Conclusions The present results demonstrate that the platelet heparin receptor, PEAR1, potentiates the response to HIT-like mAb and HIT sera and that this is an important variable in diagnostic assays using HIT sera. Further studies are required to establish whether activation of PEAR1 contributes to the pathogenesis of HIT and whether this is due solely to binding of heparin to PEAR1 or to binding of heparin to PEAR1 when present in an immune complex. Acknowledgements RJB is supported by a British Heart Foundation Accelerator Award (AA/18/2/34218) and SJM by a British Heart Foundation Project grant (PG/23/11230). SPW holds a BHF Chair (CH03/003).

Platelet CLEC-2 is a hemITAM-containing receptor which has a critical role in venous thrombosis, but minimal involvement in haemostasis. CLEC-2 can be blocked by Btk inhibitors. Treatment with ibrutinib is associated with increased bleeding due to off-target inhibition of Src family kinases (SFKs). Patients with X-linked agammaglobulinemia (XLA) who lack Btk however do not bleed, suggesting selective Btk inhibition is a viable antithrombotic strategy. We assessed the effects of selective Btk inhibitors PRN1008 (rilzabrutinib) and PRN473 on platelet signalling and function mediated by CLEC-2 and GPVI. We used healthy donor and XLA platelets to determine off-target inhibitor effects. Inferior vena cava (IVC) stenosis and Salmonella infection mouse models were used to assess antithrombotic effects of PRN473 in vivo. PRN1008 and PRN473 potently inhibited CLEC-2-mediated platelet activation to rhodocytin. No off-target inhibition of SFKs was seen. PRN1008 treatment of Btk-deficient platelets resulted in minor additional inhibition of aggregation and tyrosine phosphorylation, likely reflecting inhibition of Tec. No effect on GPCR-mediated platelet function was observed. PRN473 significantly reduced the number of thrombi in podoplanin positive vessels following Salmonella infection and the presence of IVC thrombosis following vein stenosis. The potent inhibition of human platelet CLEC-2, and reduced thrombosis in in vivo models, together with the lack of off-target SFK inhibition and absence of bleeding reported in rilzabrutinib treated immune thrombocytopenia patients, suggest Btk inhibition as a promising antithrombotic strategy.

Vaccination has proven to be a valuable tool to combat SARS-CoV-2. However, reports of rare adverse reactions such as thrombosis/thrombocytopenia syndrome after ChAdOx1 nCoV-19 vaccination have caused scientific, public and media concern. ChAdOx1 was vectorised from the Y25 chimpanzee adenovirus, which was selected due to low human seroprevalence to circumvent pre-existing immunity. In this study, we aimed to explore patterns of T-cell activation after SARS-CoV-2 COVID-19 vaccine exposure in vitro using PBMCs collected from pre-pandemic ChAdOx1 nCoV-19 naïve healthy donors (HDs), and ChAdOx1 nCoV-19 and Pfizer vaccinated controls. PBMCs were assessed for T-cell proliferation using the lymphocyte transformation test (LTT) following exposure to SARS-CoV-2 COVID-19 vaccines. Cytokine analysis was performed via intracellular cytokine staining, ELISpot assay and LEGENDplex immunoassays. T-cell assays performed in pre-pandemic vaccine naïve HDs, revealed widespread lymphocyte stimulation after exposure to ChAdOx1 nCoV-19 (95%), ChAdOx-spike (90%) and the Ad26.COV2. S vaccine, but not on exposure to the BNT162b2 vaccine. ICS analysis demonstrated that CD4+ CD45RO+ memory T-cells are activated by ChAdOx1 nCoV-19 in vaccine naïve HDs. Cytometric immunoassays showed ChAdOx1 nCoV-19 exposure was associated with the release of proinflammatory and cytotoxic molecules, such as IFN-γ, IL-6, perforin, granzyme B and FasL. These studies demonstrate a ubiquitous T-cell response to ChAdOx1 nCoV-19 and Ad26.COV2. S in HDs recruited prior to the SARS-CoV-2 pandemic, with T-cell stimulation also identified in vaccinated controls. This may be due to underlying T-cell cross-reactivity with prevalent human adenoviruses and further study will be needed to identify T-cell epitopes involved.