Recurrent Cerebral Hemorrhaging with Platelet Dysfunction Accompanied by Anti-glycoprotein VI Autoantibodies in a Patient with TAFRO Syndrome.

Thrombocytopenia, anasarca, fever, renal dysfunction, and organomegaly (TAFRO) syndrome is an inflammatory disorder with an unclear pathogenesis. We herein report a case of TAFRO syndrome in remission in a patient who experienced recurrent intracranial bleeding despite a normal platelet count and coagulation system. A further investigation suggested the presence of anti-glycoprotein VI (GPVI) autoantibodies in the plasma, which induced platelet dysfunction and bleeding tendency. No new bleeding or relapse of TAFRO syndrome occurred after immunosuppressive therapy was initiated. These findings may help elucidate the autoimmune pathogenesis of TAFRO syndrome.

Phosphorylation of (Ser 291) in the linker insert of Syk negatively regulates ITAM signaling in platelets.

Receptor-induced tyrosine phosphorylation of spleen tyrosine kinase (Syk) has been studied extensively in hematopoietic cells. Metabolic mapping and high-resolution mass spectrometry, however, indicate that one of the most frequently detected phosphorylation sites encompassed S297 (S291 in mice) located within the linker B region of Syk. It has been reported that Protein kinase C (PKC) phosphorylates Syk S297, thus influencing Syk activity. However, conflicting studies suggest that this phosphorylation enhances as well as reduces Syk activity. To clarify the function of this site, we generated Syk S291A knock-in mice. We used platelets as a model system as they possess Glycoprotein VI (GPVI), a receptor containing an immunoreceptor tyrosine-based activation motif (ITAM) which transduces signals through Syk. Our analysis of the homozygous mice indicated that the knock-in platelets express only one isoform of Syk, while the wild-type expresses two isoforms at 69 and 66 kDa. When the GPVI receptor was activated with collagen-related peptide (CRP), we observed an increase in functional responses and phosphorylations in Syk S291A platelets. This potentiation did not occur with AYPGKF or 2-MeSADP, although they also activate PKC isoforms. Although there was potentiation of platelet functional responses, there was no difference in tail bleeding times. However, the time to occlusion in the FeCl3 injury model was enhanced. These data indicate that the effects of Syk S291 phosphorylation represent a significant outcome on platelet activation and signaling in vitro but also reveals its multifaceted nature demonstrated by the differential effects on physiological responses in vivo.

What is the context Spleen tyrosine kinase (Syk) is present a number of cells and important in controlling the functions of various cells and organs.Syk is known to exist in two isoforms Syk L (long form or Syk A) and Syk S (short form or Syk B).It is known that phosphorylation events regulate Syk activation and activity.In several inflammatory disease conditions, Syk mutants are known to play a role.Phosphorylation of the Syk residue Serine 291 is known to occur, but its function in the regulation of Syk activation or activity is not known.What is new In this study, we generated a mutant mouse Syk S291A, which cannot be phosphorylated on serine residue. We evaluated the function of platelets isolated from these mice and compared them to platelets isolated from wild type littermates.We observed that the mutation in Syk L unexpectedly caused Syk S to disappear from a number of tissues.Platelet functions are enhanced in mutant mouse platelets compared to those from wild-type mice.What is the impact These studies enhance our understanding of the impact of Serine 291 phosphorylation on the function of Syk in platelets.

Targeting GPVI with glenzocimab in COVID-19 patients: Results from a randomized clinical trial.

BACKGROUND: Glenzocimab is a novel antithrombotic agent which targets platelet glycoprotein VI (GPVI) and does not induce haemorrhage. SARS-CoV-2 triggers a prothrombotic state and lung injury whose mechanisms include coagulopathy, endothelial dysfunction, and inflammation with dysregulated platelets. METHODS AND PATIENTS: GARDEN was a randomised double-blind, exploratory phase II study of glenzocimab in SARS-CoV-2 respiratory failure (NCT04659109). PCR+ adults in Brazil and France (7 centres) were randomized to standard-of-care (SOC) plus glenzocimab (1000 mg/dayx3 days) or placebo, followed for 40 days. Primary efficacy endpoint was clinical progression at Day 4. All analyses concerned the intention-to-treat population. RESULTS: Between December 2020 and August 2021, 61 patients received at least one dose (30 glenzocimab vs 32 placebo) and 58 completed the study (29 vs 29). Clinical progression of COVID-19 ARDS was not statistically different between glenzocimab and placebo arms (43.3% and 29.0%, respectively; p = 0.245). Decrease in the NEWS-2 category at D4 was statistically significant (p = 0.0290) in the glenzocimab arm vs placebo. No Serious Adverse Event (SAE) was deemed related to study drug; bleeding related events were reported in 6 patients (7 events) and 4 patients (4 events) in glenzocimab and placebo arms, respectively. CONCLUSIONS: Therapeutic GPVI inhibition assessment during COVID-19 was conducted in response to a Public Health emergency. Glenzocimab in coagulopathic patients under therapeutic heparin was neither associated with increased bleeding, nor SAE. Clinical impact of glenzocimab on COVID-19 ARDS was not demonstrated. A potential role for GPVI inhibition in other types of ARDS deserves further experimentation. Glenzocimab is currently studied in stroke (ACTISAVE: NCT05070260) and cardiovascular indications.

Platelets favor the outgrowth of established metastases.

Despite abundant evidence demonstrating that platelets foster metastasis, anti-platelet agents have low therapeutic potential due to the risk of hemorrhages. In addition, whether platelets can regulate metastasis at the late stages of the disease remains unknown. In this study, we subject syngeneic models of metastasis to various thrombocytopenic regimes to show that platelets provide a biphasic contribution to metastasis. While potent intravascular binding of platelets to tumor cells efficiently promotes metastasis, platelets further support the outgrowth of established metastases via immune suppression. Genetic depletion and pharmacological targeting of the glycoprotein VI (GPVI) platelet-specific receptor in humanized mouse models efficiently reduce the growth of established metastases, independently of active platelet binding to tumor cells in the bloodstream. Our study demonstrates therapeutic efficacy when targeting animals bearing growing metastases. It further identifies GPVI as a molecular target whose inhibition can impair metastasis without inducing collateral hemostatic perturbations.

Does Yogurt Enriched with Platelet-Activating Factor Inhibitors from Olive Oil By-Products Affect Gut Microbiota and Faecal Metabolites in Healthy Overweight Subjects? (A randomized, parallel, three arm trial.).

OBJECTIVE: The effect of the daily consumption of a low-fat yogurt (150 g) enriched with Platelet-Activating Factor receptor (PAF-R) antagonists, or the plain one, on gut microbiota and faecal metabolites was investigated in healthy overweight subjects. METHODS: A randomized, three-arm, double-blind, placebo-controlled, parallel-group study was performed that lasted 8 weeks. Blood and stools were collected and analyzed before and after the intervention. RESULTS: Our findings revealed that the intake of the enriched yogurt resulted in a significant increase in the levels of Bifidobacterium spp., Clostridium perfringens group and Firmicutes-to-Bacteroidetes (F/B) ratio. On the other hand, a significant increase in the levels of Lactobacillus and C. perfringens group was detected after the intake of the plain yogurt. The increase in the levels of C. perfringens group was inversely associated with the plasma catabolic enzyme of PAF, namely LpPLA2 (lipoprotein-associated phospholipase A2), a cardiovascular risk marker that has been linked with inflammation and atherosclerosis. Moreover, in the enriched with PAF-R antagonists yogurt group, the increased levels of C. perfringens group were also associated with lower PAF action assessed as ex vivo human platelet-rich plasma (PRP) aggregation. Additionally, a higher % increase in molar ratio of Branched Short Chain Fatty Acids (BSCFAs) was detected for both yogurt groups after the 8 week-intervention compared to control. The consumption of the enriched yogurt also resulted in a significant drop in faecal caproic levels and a trend for lower ratio of butyrate to total volatile fatty acids (VFAs) compared to baseline levels. CONCLUSION: Yogurt consumption seems to favorably affect gut microbiota while its enrichment with PAF-R antagonists from olive oil by-products, may provide further benefits in healthy overweight subjects. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov (NCT02259205).

Antagonism of the Platelet-Activating Factor Pathway Mitigates Inflammatory Adverse Events Driven by Anti-erythrocyte Antibody Therapy in Mice.

Immune thrombocytopenia (ITP) is an autoimmune disease characterized by low platelet counts primarily due to antiplatelet autoantibodies. Anti-D is a donor-derived polyclonal Ab against the rhesus D Ag on erythrocytes used to treat ITP. Unfortunately, adverse inflammatory/hypersensitivity reactions and a Food and Drug Administration-issued black box warning have limited its clinical use. This underscores the imperative to understand the inflammatory pathway associated with anti-erythrocyte Ab-based therapies. TER119 is an erythrocyte-specific Ab with anti-D-like therapeutic activity in murine ITP, while also exhibiting a distinct inflammatory signature involving production of CCL2, CCL5, and CXCL9 but not IFN-γ. Therefore, TER119 has been used to elucidate the potential mechanism underlying the adverse inflammatory activity associated with anti-erythrocyte Ab therapy in murine ITP. Prior work has demonstrated that TER119 administration is associated with a dramatic decrease in body temperature and inflammatory cytokine/chemokine production. The work presented in the current study demonstrates that inhibiting the highly inflammatory platelet-activating factor (PAF) pathway with PAF receptor antagonists prevents TER119-driven changes in body temperature and inhibits the production of the CCL2, CCL5, and CXCL9 inflammatory cytokines in CD-1 mice. Phagocytic cells and a functional TER119 Fc region were found to be necessary for TER119-induced body temperature changes and increases in CXCL9 and CCL2. Taken together, this work reveals the novel requirement of the PAF pathway in causing adverse inflammatory activity associated with anti-erythrocyte Ab therapy in a murine model and provides a strategy of mitigating these potential reactions without altering therapeutic activity.

Redox regulation of platelet function and thrombosis.

Platelets are well-known players in several cardiovascular diseases such as atherosclerosis and venous thrombosis. There is increasing evidence demonstrating that reactive oxygen species (ROS) are generated within activated platelets. Nicotinamide adenine dinucleotide phosphate oxidase (NOX) is a major source of ROS generation in platelets. Ligand binding to platelet receptor glycoprotein (GP) VI stimulates intracellular ROS generation consisting of a spleen tyrosine kinase-independent production involving NOX activation and a following spleen tyrosine kinase-dependent generation. In addition to GPVI, stimulation of platelet thrombin receptors (protease-activated receptors [PARs]) can also trigger NOX-derived ROS production. Our recent study found that mitochondria-derived ROS production can be induced by engagement of thrombin receptors but not by GPVI, indicating that mitochondria are another source of PAR-dependent ROS generation apart from NOX. However, mitochondria are not involved in GPVI-dependent ROS generation. Once generated, the intracellular ROS are also involved in modulating platelet function and thrombus formation; therefore, the site-specific targeting of ROS production or clearance of excess ROS within platelets is a potential intervention and treatment option for thrombotic events. In this review, we will summarize the signaling pathways involving regulation of platelet ROS production and their role in platelet function and thrombosis, with a focus on GPVI- and PAR-dependent platelet responses.

Platelet-Activating Factor Receptor (PAFR) Regulates Retinal Progenitor/Stem Cells Profile in Ciliary Epithelium Cells.

The retina is a central nervous tissue essential to visual perception and highly susceptible to environmental damage. Lower vertebrate retinas activate intrinsic regeneration mechanisms in response to retinal injury regulated by a specialized population of progenitor cells. The mammalian retina does not have populations of progenitor/stem cells available to activate regeneration, but contains a subpopulation of differentiated cells that can be reprogrammed into retinal stem cells, the ciliary epithelium (CE) cells. Despite the regenerative potential, stem cells derived from CE exhibit limited reprogramming capacity probably associated with the expression of intrinsic regulatory mechanisms. Platelet-activating factor (PAF) is a lipid mediator widely expressed in many cells and plays an important role in stem cell proliferation and differentiation. During mammalian development, PAF receptor signaling showed important effects on retinal progenitors' cell cycle regulation and neuronal differentiation that need to be further investigated. In this study, our findings suggested a dynamic role for PAF receptor signaling in CE cells, impacting stem cell characteristics and neurosphere formation. We showed that PAF receptors and PAF-related enzymes are downregulated in retinal progenitor/stem cells derived from PE cells. Blocking PAFR activity using antagonists increased the expression of specific progenitor markers, revealing potential implications for retinal tissue development and maintenance.

Ultra-high throughput-based screening for the discovery of antiplatelet drugs affecting receptor dependent calcium signaling dynamics.

Distinct platelet activation patterns are elicited by the tyrosine kinase-linked collagen receptor glycoprotein VI (GPVI) and the G-protein coupled protease-activated receptors (PAR1/4) for thrombin. This is reflected in the different platelet Ca2+ responses induced by the GPVI agonist collagen-related peptide (CRP) and the PAR1/4 agonist thrombin. Using a 96 well-plate assay with human Calcium-6-loaded platelets and a panel of 22 pharmacological inhibitors, we assessed the cytosolic Ca2+ signaling domains of these receptors and developed an automated Ca2+ curve algorithm. The algorithm was used to evaluate an ultra-high throughput (UHT) based screening of 16,635 chemically diverse small molecules with orally active physicochemical properties for effects on platelets stimulated with CRP or thrombin. Stringent agonist-specific selection criteria resulted in the identification of 151 drug-like molecules, of which three hit compounds were further characterized. The dibenzyl formamide derivative ANO61 selectively modulated thrombin-induced Ca2+ responses, whereas the aromatic sulfonyl imidazole AF299 and the phenothiazine ethopropazine affected CRP-induced responses. Platelet functional assays confirmed selectivity of these hits. Ethopropazine retained its inhibitory potential in the presence of plasma, and suppressed collagen-dependent thrombus buildup at arterial shear rate. In conclusion, targeting of platelet Ca2+ signaling dynamics in a screening campaign has the potential of identifying novel platelet-inhibiting molecules.

Effect of platelet-activating factor on food intake, cloacal temperature, voluntary activity and crop emptying rate in chicks.

Platelet-activating factor (PAF) plays a significant role in several leucocyte functions, including platelet aggregation and inflammation. Additionally, PAF has a role in the behavioral and physiological changes in mammals. However, the effect of PAF has not been well studied in birds. Therefore, the study aimed to determine if PAF affects feeding behavior, voluntary activity, cloacal temperature, and feed passage through the digestive tract in chicks (Gallus gallus). We also studied the involvement of PAF in the innate immune system induced by lipopolysaccharide (LPS), a cell wall component of gram-negative bacteria. Both intraperitoneal (IP) and intracerebroventricular (ICV) injections of PAF significantly decreased food intake. IP injection of PAF significantly decreased voluntary activity and slowed the feed passage from the crop, whereas ICV injection had no effect. Conversely, ICV injection of PAF significantly increased the cloacal temperature, but IP injection had no effect. The IP injection of LPS significantly reduced the mRNA expression of lysophosphatidylcholine acyltransferase 2, an enzyme responsible for PAF production in the heart and pancreas. On the other hand, LPS significantly increased the mRNA expression of the PAF receptor in the peripheral organs. The present study shows that PAF influences behavioral and physiological responses and is related to the response against bacterial infections in chicks.

High factor VIII concentrations interfere with glycoprotein VI-mediated platelet activation in vitro.

BACKGROUND: The recruitment of activated factor VIII (FVIII) at the surface of activated platelets is a key step toward the burst of thrombin and fibrin generation during thrombus formation at the site of vascular injury. It involves binding to phosphatidylserine and, possibly, to fibrin-bound αIIbß3. Seminal work had shown the binding of FVIII to resting platelets, yet without a clear understanding of a putative physiological relevance. OBJECTIVES: To characterize the effects of FVIII-platelet interaction and its potential modulation of platelet function. METHODS: FVIII was incubated with washed platelets. The effects on platelet activation (spontaneously or triggered by collagen and thrombin) were studied by flow cytometry and light transmission aggregometry. We explored the involvement of downstream pathways by studying phosphorylation profiles (Western blot). The FVIII-glycoprotein (GP) VI interaction was investigated by ELISA, confocal microscopy, and proximity ligation assay. RESULTS: FVIII bound to the surface of resting and activated platelets in a dose-dependent manner. FVIII at supraphysiological concentrations did not induce platelet activation but rather specifically inhibited collagen-induced platelet aggregation and altered glycoprotein VI (GPVI)-dependent phosphorylation. FVIII, freed of its chaperone protein von Willebrand factor (VWF), interacted in close proximity with GPVI at the platelet surface. CONCLUSION: We showed that VWF-free FVIII binding to, or close to, GPVI modulates platelet activation in vitro. This may represent an uncharacterized negative feedback loop to control overt platelet activation. Whether locally activated FVIII concentrations achieved during platelet accumulation and thrombus formation at the site of vascular injury in vivo are compatible with such a function remains to be determined.

Antiplatelet effects of the CEACAM1-derived peptide QDTT.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) restricts platelet activation via platelet collagen receptor GPVI/FcRγ-chain. In this study, screening against collagen-induced platelet aggregation was performed to identify functional CEACAM1 extracellular domain fragments. CEACAM1 fragments, including Ala-substituted peptides, were synthesized. Platelet assays were conducted on healthy donor samples for aggregation, cytotoxicity, adhesion, spreading, and secretion. Mice were used for tail bleeding and FeCl3-induced thrombosis experiments. Clot retraction was assessed using platelet-rich plasma. Extracellular segments of CEACAM1 and A1 domain-derived peptide QDTT were identified, while N, A2, and B domains showed no involvement. QDTT inhibited platelet aggregation. Ala substitution for essential amino acids (Asp139, Thr141, Tyr142, Trp144, and Trp145) in the QDTT sequence abrogated collagen-induced aggregation inhibition. QDTT also suppressed platelet secretion and "inside-out" GP IIb/IIIa activation by convulxin, along with inhibiting PI3K/Akt pathways. QDTT curtailed FeCl3-induced mesenteric thrombosis without significantly prolonging bleeding time, implying the potential of CEACAM1 A1 domain against platelet activation without raising bleeding risk, thus paving the way for novel antiplatelet drugs.

What is the context? The study focuses on Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) and its role in platelet activation, particularly through the GPVI/FcRγ-chain pathway.The research aims to identify specific fragments of CEACAM1's extracellular domain that could restrict platelet activation, without increasing bleeding risk.What is new? The researchers identified a peptide called QDTT derived from the A1 domain of CEACAM1's extracellular segment. This peptide demonstrated the ability to inhibit platelet aggregation, secretion, and GP IIb/IIIa activation.The study also revealed that specific amino acids within the QDTT sequence were essential for its inhibitory effects on collagen-induced aggregation.What is the impact? The findings suggest that the A1 domain-derived peptide QDTT from CEACAM1 could serve as a potential basis for developing novel antiplatelet drugs. This peptide effectively limits platelet activation and aggregation without significantly prolonging bleeding time, indicating a promising approach to managing thrombosis and related disorders while minimizing bleeding risks.

Endothelium-mediated regulation of platelet activation: Involvement of multiple protein kinases.

The endothelial regulation of platelet activity is incompletely understood. Here we describe novel approaches to find molecular pathways implicated on the platelet-endothelium interaction. Using high-shear whole-blood microfluidics, employing coagulant or non-coagulant conditions at physiological temperature, we observed that the presence of human umbilical vein endothelial cells (HUVEC) strongly suppressed platelet adhesion and activation, via the collagen receptor glycoprotein VI (GPVI) and the PAR receptors for thrombin. Real-time monitoring of the cytosolic Ca2+ rises in the platelets indicated no major improvement of inhibition by prostacyclin or nitric oxide. Similarly under stasis, exposure of isolated platelets to HUVEC reduced the Ca2+ responses by collagen-related peptide (CRP-XL, GPVI agonist) and thrombin (PAR agonist). We then analyzed the label-free phosphoproteome of platelets (three donors), exposed to HUVEC, CRP-XL, and/or thrombin. High-resolution mass spectrometry gave 5463 phosphopeptides, corresponding to 1472 proteins, with good correlation between biological and technical replicates (R > .86). Stringent filtering steps revealed 26 regulatory pathways (Reactome) and 143 regulated kinase substrates (PhosphoSitePlus), giving a set of protein phosphorylation sites that was differentially (44) or similarly (110) regulated by HUVEC or agonist exposure. The differential regulation was confirmed by stable-isotope analysis of platelets from two additional donors. Substrate analysis indicated major roles of poorly studied protein kinase classes (MAPK, CDK, DYRK, STK, PKC members). Collectively, these results reveal a resetting of the protein phosphorylation profile in platelets exposed to endothelium or to conventional agonists and to endothelium-promoted activity of a multi-kinase network, beyond classical prostacyclin and nitric oxide actors, that may contribute to platelet inhibition.

The subtilisin-like protease furin regulates hemin-induced CD63 surface expression on platelets.

Accumulation of free heme B in the plasma can be the result of severe hemolytic events, when the scavenger system for free hemoglobin and heme B is overwhelmed. Free heme B can be oxidized into toxic hemin, which has been proven to activate platelet degranulation and aggregation and promote thrombosis. In the present study we analyzed the effect of hemin on the activation-mediated lysosomal degranulation and CD63 surface expression on platelets using classic flow cytometry and fluorescence microscopy techniques. Classical platelet activators were used as control to distinguish the novel effects of hemin from known activation pathways. CD63 is a tetraspanin protein, also known as lysosomal-associated membrane protein 3 or LAMP-3. In resting platelets CD63 is located within the membrane of delta granules and lysosomes of platelet, from where it is integrated into the platelet outer membrane upon stimulation. We were able to show that hemin like the endogenous platelet activators ADP, collagen or thrombin does provoke CD63 re-localization. Interestingly, only hemin-induced CD63 externalization is dependent on the subtilisin-like pro-protein convertase furin as shown by inhibitor experiments. Furthermore, we were able to demonstrate that hemin induces lysosome secretion, a source of the hemin-mediated CD63 presentation. Again, only the hemin-induced lysosome degranulation is furin dependent. In summary we have shown that the pro-protein convertase furin plays an important role in hemin-mediated lysosomal degranulation and CD63 externalization.

Safety and efficacy of platelet glycoprotein VI inhibition in acute ischaemic stroke (ACTIMIS): a randomised, double-blind, placebo-controlled, phase 1b/2a trial.

BACKGROUND: Antagonists of glycoprotein VI-triggered platelet activation used in combination with recanalisation therapies are a promising therapeutic approach in acute ischaemic stroke. Glenzocimab is an antibody fragment that inhibits the action of platelet glycoprotein VI. We aimed to determine and assess the safety and efficacy of the optimal dose of glenzocimab in patients with acute ischaemic stroke eligible to receive alteplase with or without mechanical thrombectomy. METHODS: This randomised, double-blind, placebo-controlled study with dose-escalation (1b) and dose-confirmation (2a) phases (ACTIMIS) was done in 26 stroke centres in six European countries. Participants were adults (≥18 years) with disabling acute ischaemic stroke with a National Institutes of Health Stroke Scale score of 6 or higher before alteplase administration. Patients were randomly assigned treatment using a central electronic procedure. Total administered dose at the end of the intravenous administration was 125 mg, 250 mg, 500 mg, and 1000 mg of glenzocimab or placebo in phase 1b and 1000 mg of glenzocimab or placebo in phase 2a. Treatment was initiated 4·5 h or earlier from stroke symptom onset in patients treated with alteplase with or without mechanical thrombectomy. The sponsor, study investigator and study staff, patients, and central laboratories were all masked to study treatment until database lock. Primary endpoints across both phases were safety, mortality, and intracranial haemorrhage (symptomatic, total, and fatal), assessed in all patients who received at least a partial dose of study medication (safety set). The trial is registered on ClinicalTrials.gov, NCT03803007, and is complete. FINDINGS: Between March 6, 2019, and June 27, 2021, 60 recruited patients were randomly assigned to 125 mg, 250 mg, 500 mg, or 1000 mg glenzocimab, or to placebo in phase 1b (n=12 per group) and were included in the safety analysis. Glenzocimab 1000 mg was well tolerated and selected as the phase 2a recommended dose; from Oct 2, 2020, to June 27, 2021, 106 patients were randomly assigned to glenzocimab 1000 mg (n=53) or placebo (n=53). One patient in the placebo group received glenzocimab in error and therefore 54 and 52, respectively, were included in the safety set. In phase 2a, the most frequent treatment-emergent adverse event was non-symptomatic haemorrhagic transformation, which occurred in 17 (31%) of 54 patients treated with glenzocimab and 26 (50%) of 52 patients treated with placebo. Symptomatic intracranial haemorrhage occurred in no patients treated with glenzocimab compared with five (10%) patients in the placebo group. All-cause deaths were lower with glenzocimab 1000 mg (four [7%] patients) than with placebo (11 [21%] patients). INTERPRETATION: Glenzocimab 1000 mg in addition to alteplase, with or without mechanical thrombectomy, was well tolerated, and might reduce serious adverse events, intracranial haemorrhage, and mortality. These findings support the need for future research into the potential therapeutic inhibition of glycoprotein VI with glenzocimab plus alteplase in patients with acute ischaemic stroke. FUNDING: Acticor Biotech.

Double jeopardy, glomangiopericytoma and Glanzmann thrombasthenia resulting in recurrent epistaxis: a case report.

Glanzmann thrombasthenia is a rare bleeding disorder induced by inherited defects of the platelet membrane αIIbß3 glycoprotein. Glomangiopericytoma, on the other hand, is a very rare sinonasal tumor demonstrating a perivascular myoid phenotype. We herein report the first described case in the literature of Glanzmann thrombasthenia and glomangiopericytoma. The patient is a 40-year-old man diagnosed with type 1 Glanzmann thrombasthenia who presented with repetitive and profuse posterior epistaxis initially managed with platelet transfusions and recombinant activated factor VII (rFVIIa). Due to the unresolved epistaxis, nasal endoscopy was performed revealing a vascularized tumor. Subsequently, a sphenopalatine artery embolization followed by a surgical excision of the tumor was performed. The pathology report diagnosis of the tumor was glomangiopericytoma. This case sheds the lights on a very rare cause of epistaxis in a patient with Glanzmann thrombasthenia, with a challenging multidisciplinary management. A local cause of epistaxis should always be considered even in case of a diagnosed bleeding disorder, especially when the bleeding is recurrent.

Trivalent nanobody-based ligands mediate powerful activation of GPVI, CLEC-2, and PEAR1 in human platelets whereas FcγRIIA requires a tetravalent ligand.

BACKGROUND: Clustering of the receptors glycoprotein receptor VI (GPVI), C-type lectin-like receptor 2 (CLEC-2), low-affinity immunoglobulin γ Fc region receptor II-a (FcγRIIA), and platelet endothelial aggregation receptor 1 (PEAR1) leads to powerful activation of platelets through phosphorylation of tyrosine in their cytosolic tails and initiation of downstream signaling cascades. GPVI, CLEC-2, and FcγRIIA signal through YxxL motifs that activate Syk. PEAR1 signals through a YxxM motif that activates phosphoinositide 3-kinase. Current ligands for these receptors have an undefined valency and show significant batch variation and, for some, uncertain specificity. OBJECTIVES: We have raised nanobodies against each of these receptors and multimerized them to identify the minimum number of epitopes to achieve robust activation of human platelets. METHODS: Divalent and trivalent nanobodies were generated using a flexible glycine-serine linker. Tetravalent nanobodies utilize a mouse Fc domain (IgG2a, which does not bind to FcγRIIA) to dimerize the divalent nanobody. Ligand affinity measurements were determined by surface plasmon resonance. Platelet aggregation, adenosine triphosphate secretion, and protein phosphorylation were analyzed using standardized methods. RESULTS: Multimerization of the nanobodies led to a stepwise increase in affinity with divalent and higher-order nanobody oligomers having sub-nanomolar affinity. The trivalent nanobodies to GPVI, CLEC-2, and PEAR1 stimulated powerful and robust platelet aggregation, secretion, and protein phosphorylation at low nanomolar concentrations. A tetravalent nanobody was required to activate FcγRIIA with the concentration-response relationship showing a greater variability and reduced sensitivity compared with the other nanobody-based ligands, despite a sub-nanomolar binding affinity. CONCLUSION: The multivalent nanobodies represent a series of standardized, potent agonists for platelet glycoprotein receptors. They have applications as research tools and in clinical assays.

Elevated plasma levels of specific antiplatelet glycoprotein autoantibodies in patients with primary Sjögren syndrome with thrombocytopenia.

INTRODUCTION: Thrombocytopenia is one of the primary Sjögren's syndrome (pSS) hematological manifestations. The objective of this study was to evaluate the possible roles of antiplatelet glycoprotein autoantibodies in the pathogenesis of thrombocytopenia in primary Sjögren's syndrome (pSS). METHODS: The level of plasma anti-glycoprotein Ib, IIIa and IIb/IIIa autoantibodies in 36 pSS patients without thrombocytopenia and 35 pSS patients with thrombocytopenia, 36 Idiopathic thrombocytopenic purpura (ITP) patients and 39 normal control were measured with enzyme-linked immunosorbent assay (ELISA). RESULTS: The level of anti-GPIb, GPIIIa, GPIIb/IIIa autoantibodies (A490) in the pSS with thrombocytopenia was significantly higher than that of pSS without thrombocytopenia (0.813 ± 0.161 vs 0.688 ± 0.133; 0.917 ± 0.094 vs 0.802 ± 0.070; 0.911 ± 0.125 vs 0.782 ± 0.109). Incidences of the anti-GPIb, GPIIIa, GPIIb/IIIa autoantibodies in the pSS with thrombocytopenia was significantly higher than that of pSS without thrombocytopenia (25.7% vs 0%; 65.7% vs 11.1%; 31.4% vs 0%). In patients with pSS, there was a lower platelet count in anti-GPIb, GPIIIa, GPIIb/IIIa autoantibodies positive patients ((25.67 ± 5.5) × 10^9/L vs (116.8 ± 84.52) × 10^9/L; 29.04 ± 11.33 × 10^9/L vs (152.0 ± 75.47) × 10^9/L; (31.55 ± 14.0) × 10^9/L vs (118.8 ± 85.24) × 10^9/L). CONCLUSION: Elevated plasma levels of anti-platelet glycoprotein autoantibodies may play a role in the pathogenesis of thrombocytopenia in pSS. Key Points • The level of anti-GPIb, GPIIIa, GPIIb/IIIa autoantibodies (A490) in the pSS with thrombocytopenia was increased. • Incidences of the anti-GPIb, GPIIIa, GPIIb/IIIa autoantibodies in the pSS with thrombocytopenia was increased. • In patients with pSS, there was a lower platelet count in anti-GPIb, GPIIIa, GPIIb/IIIa autoantibodies positive patients.