Glycoprotein VI Research Articles

Cathelicidin-HG Alleviates Psoriasis by Targeting Glycoprotein VI to Inhibit Platelet-Neutrophil Complexes Formation.

Psoriasis, a chronic inflammatory skin disorder, is characterized by a complex interplay among immune cells. Although the specific mechanisms of platelet involvement in psoriasis are not fully elucidated, platelet activation is considered a major pathogenic factor. In this study, we investigated the role of Cathelicidin-HG (Cath-HG), a peptide derived from Hylarana guentheri skin that exhibits potent inhibitory activity against platelet glycoprotein VI (GPVI), in an imiquimod-induced psoriasis mouse model. Our experimental findings demonstrated that Cath-HG significantly alleviates psoriasis symptoms, reduces platelet-neutrophil complexes (PNCs) formation, and attenuates neutrophil activation at lesion sites. In vitro studies confirmed that Cath-HG inhibits collagen-induced platelet activation via GPVI, thereby disrupting PNCs formation and suppressing subsequent inflammatory responses. These findings not only establish GPVI as a key regulator of PNC-mediated inflammation in psoriasis but also identify Cath-HG as a promising therapeutic candidate. This study provides a novel mechanistic insight into platelet-neutrophil interactions in psoriasis and highlights the potential of GPVI as a therapeutic target for innovative psoriasis treatments.

Investigation of Genomic and Transcriptomic Risk Factors of Clopidogrel Response in African Americans.

Clopidogrel, an anti-platelet drug, is used to prevent thrombosis after percutaneous coronary intervention. Clopidogrel resistance results in recurring ischemic events, with African Americans (AA) suffering disproportionately. The aim of this study was to discover novel biomarkers of clopidogrel resistance in African Americans using genome and transcriptome data. We conducted a genome-wide association study (GWAS), including local ancestry adjustment, in 141 AA on clopidogrel to identify genetic associations with high on-treatment platelet reactivity (HTPR), with validation of genome-wide significant and suggestive loci in an independent cohort of AA clopidogrel patients (N = 823) from the Million Veteran's Program (MVP) along with in vitro functional analysis. We performed differential gene expression (DGE) analysis in whole blood to identify transcriptomic predictors of response, followed by functional validation in MEG-01 cells. GWAS identified one signal on Chromosome 7 as significantly associated with increasing risk of HTPR. The lead single-nucleotide polymorphism (SNP), rs7807369, within thrombospondin 7A (THSD7A) was associated with an increased risk of HTPR (odds ratio (OR) = 4.02, P = 4.56 × 10-9). Higher THSD7A gene expression was associated with HTPR in an independent cohort of clopidogrel-treated patients (P = 0.004) and carrying a risk allele showed increased gene expression in primary human endothelial cells. Notably, the CYP2C19*2 variants showed no association with clopidogrel response in the discovery or MVP cohorts. DGE analysis identified an association with decreased LAIR1 and AP3B2 expression to HTPR. LAIR1 knockdown in MEG-01 cells resulted in increased expression of SYK and AKT1, suggesting an inhibitory role of LAIR1 in the Glycoprotein VI pathway. In summary, these findings suggest that other variants and genes outside of CYP2C19 star alleles play an important role in clopidogrel response in AA.

Comparative Effects of Glenzocimab and Eptifibatide on Bleeding Severity in 2 Mouse Models of Intracranial Hemorrhage.

Antiplatelet drugs represent potential candidates for protecting the penumbral microcirculation during cerebral ischemia and improving the benefits of arterial recanalization in ischemic stroke. Yet while the efficacy of such adjuvant strategies has been shown to be highly time dependent, antiplatelet therapy at the acute phase of ischemic stroke cannot be envisioned until the diagnosis of stroke and its ischemic nature have been confirmed because of the presumed risk of worsening bleeding in case of intracranial hemorrhage (ICH). Here, we investigated this risk for 2 antiplatelet drugs currently being tested in clinical trials for ischemic stroke, glenzocimab and eptifibatide, in 2 mouse models of ICH. The severity of ICH was assessed in mice humanized for glycoprotein VI treated or not with glenzocimab or eptifibatide at effective dose, in a model of primary ICH caused by unilateral striatal injection of collagenase type VII, and in a model of hyperglycemia-induced hemorrhagic transformation of cerebral ischemia-reperfusion injury. Glenzocimab had no impact on bleeding severity in either model of ICH. Conversely, eptifibatide caused a significant increase in intracranial bleeding in both models, and a drastic increase in death after hyperglycemia-induced hemorrhagic transformation of cerebral ischemia-reperfusion injury. Unlike eptifibatide, glenzocimab is safe in the setting of ICH. These results suggest that glenzocimab could be administered upon suspicion of ischemic stroke, before assessment of its ischemic nature, thus opening the way to hastening of treatment initiation.

Sibanin, a novel black fly-derived Kunitz protease inhibitor, prevents thrombus formation in mice by anticoagulation-antiplatelet duality.

Most Kunitz inhibitors exhibit serine protease inhibitory activity, but limited information is available on the regulation of platelet function. Herein, we report the purification and characterization of a novel single Kunitz domain inhibitor (Sibanin) from the salivary glands of the black fly Simulium bannaense. Recombinant Sibanin prolonged activated partial thromboplastin time and prothrombin time, and exhibited high-affinity binding to FXa and elastase with a KD of 5.0 nM and 1.67 nM, respectively. Moreover, Sibanin also shows strong anti-inflammatory and analgesic functions, which would facilitate blood-feeding. Of note, Sibanin markedly suppressed platelet spreading and aggregation, as well as clot retraction. Further studies showed that Sibanin dose-dependently inhibited ADP-induced platelet aggregation by acting on the P2Y12 receptor and blocking its downstream PI3K/AKT/ERK signal pathway. Furthermore, Sibanin also suppressed collagen-induced platelet aggregation by blocking the glycoprotein VI (GPVI) receptor and attenuating the activation of RAP1 signaling pathways. In addition, Sibanin prevented FeCl3-induced arterial thrombosis and carrageenan-induced tail vein thrombosis in mice without inducing a bleeding tendency. Our findings provide new insights into the molecular and functional of Kunitz inhibitors, and will contribute to the understanding of the molecular mechanisms that mediate hematophagous lifestyle of Simulium bannaense.

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.

Platelet-derived extracellular vesicles induced through different activation pathways drive melanoma progression by functional and transcriptional changes

BackgroundBeyond their conventional roles in hemostasis and wound healing, platelets have been shown to facilitate hematogenous metastasis by interacting with cancer cells. Depending on the activation route, platelets also generate different platelet-derived extracellular vesicles (PEVs) that may educate cancer cells in the circulation or within the tumor microenvironment. We engaged different platelet-activating receptors, including glycoprotein VI and C-type lectin-like receptor 2, to generate a spectrum of PEV types. This allowed us to investigate the differential capacity of PEVs to alter cancer hallmark functions such as proliferation, invasion, and pro-angiogenic potential using melanoma as a model. Additionally, we analyzed changes in the cell transcriptomes and cancer EV profiles.MethodsTwo human melanoma cell lines (MV3 and A2058) with differential metastatic potential were studied in the 3D spheroid cultures. Human platelets were activated with collagen related peptide (CRP), fucoidan from Fucus vesiculosus (FFV), thrombin & collagen co-stimulus and Ca2+ ionophore, and PEVs were isolated by size-exclusion chromatography followed by ultrafiltration. Spheroids or cells were treated with PEVs and used in functional assays of proliferation, invasion, and endothelial tube formation as well as for the analysis of cancer EV production and their tetraspanin profiles. Differentially expressed genes and enriched signaling pathways in the PEV-treated spheroids were analyzed at 6 h and 24 h by RNA sequencing.ResultsAmong the studied PEVs, those generated by CRP and FFV exhibited the most pronounced effects on altering cancer hallmark functions. Specifically, CRP and FFV PEVs increased proliferation in both MV3 and A2058 spheroids. Distinct tetraspanin signatures of melanoma EVs were induced by all PEV types. While the PI3K-Akt and MAPK signaling pathways were activated by both CRP and FFV PEVs, they differently upregulated the immunomodulatory TGF-β and type-I interferon signaling pathways, respectively.ConclusionsOur study revealed both shared and distinct, cancer-promoting functions of PEVs, which contributed to the transcriptome and metastatic capabilities of the melanoma spheroids. Inhibiting the platelet receptors that modulate the PEVs’ cancer-promoting properties may open up new strategies for identifying promising treatment targets for cancer therapy.

Isorhynchophylline Inhibits Platelet Activation and Thrombus Formation.

Isorhynchophylline is a Chinese herbal medicine and has multiple effects such as anti-inflammatory and neuroprotective effects. Whether isorhynchophylline has antithrombotic property is unknown. This study aims to evaluate its role in platelet function. Human platelets were incubated with isorhynchophylline (0, 10, 20, and 40 μM) at 37°C for 1 hour to detect platelet aggregation and activation, receptors level, spreading, and calcium mobilization. In addition, isorhynchophylline (5 mg/kg) was injected into mice to measure in vivo hemostasis and thrombosis. Isorhynchophylline dose-dependently reduced platelet aggregation, adenosine triphosphate secretion, P-selectin expression, and α IIb β 3 activation induced by collagen-related peptide or thrombin without affecting surface level of receptors α IIb β 3 , GPIbα, and glycoprotein VI. Meanwhile, isorhynchophylline-treated platelets showed reduced spreading. Moreover, isorhynchophylline reduced platelet calcium mobilization, phosphatidylserine exposure, and the phosphorylation of PLCγ2 and PKCα. Furthermore, administration of isorhynchophylline into mice impaired platelet hemostatic function and arterial/venous thrombosis without affecting coagulation. In conclusion, isorhynchophylline impairs platelet function and arterial/venous thrombosis, implying its potential to be a novel agent for treating thrombotic or cardiovascular diseases.

Protein disulfide isomerase 1 (PDIA1) regulates platelet-derived extracellular vesicle release

BackgroundProtein disulfide isomerase 1 (PDIA1) and 3 (PDIA3) regulate platelet activation and thrombus formation. However, their role in the formation of platelet-derived extracellular vesicles (pEVs) remains unknown. AimTo characterise the effects of PDIA1 and PDIA3 inhibition on pEV formation in washed murine platelets in response to platelet glycoprotein VI (GPVI) receptor or intracellular calcium signal activation. MethodsWashed platelets were isolated from C57BL/6 mice and activated using convulxin or the calcium ionophore A23187. Then, the resulting pEVs were analysed using nano flow cytometry (FC), platelet aggregation was measured by FC and a 96-well plate-based assay, and intracellular free calcium concentration ([Ca2+]i) was measured by indicator fluorescence. Platelet PDIs were blocked by a classic selective PDIA1 inhibitor (bepristat 2a) and sulphonamides of aziridine-2-carboxylic acid derivatives, novel PDI inhibitors relatively selective for PDIA1 or PDIA3 (C-3389 and C-3399, respectively). Clinically relevant antiplatelet drugs were used for comparison. ResultsConvulxin and A23187 concentration-dependently induced pEV formation. However, unlike convulxin, platelet activation by A23187 did not stimulate their aggregation. Bepristat 2a, C-3389 and C-3399 inhibited convulxin-induced pEV release accompanied by the reduction of [Ca2+]i. In contrast, only bepristat 2a inhibited A23187-induced pEV release, but without effect on [Ca2+]i. Cangrelor and tirofiban, but not acetylsalicylic acid (ASA), inhibited convulxin-induced pEV release, but neither of them inhibited A23187-induced pEV release. ConclusionThe inhibition of PDIA1 represents a novel approach to inhibit pEV formation by a mechanism independent of platelet aggregation and calcium signaling.

GP VI-Mediated Platelet Activation and Procoagulant Activity Aggravate Inflammation and Aortic Wall Remodeling in Abdominal Aortic Aneurysm.

Platelets play an important role in cardiovascular and cerebrovascular diseases. Abdominal aortic aneurysm (AAA) is a highly lethal, atherosclerosis-related disease with characteristic features of progressive dilatation of the abdominal aorta and degradation of the vessel wall, accompanied by chronic inflammation. Platelet activation and procoagulant activity play a decisive role in the AAA pathology as they might trigger AAA development in both mice and humans. The present study investigated the impact of the major platelet collagen receptor GP (platelet glycoprotein) VI in pathophysiological processes underlying AAA initiation and progression. For experimental AAA induction in mice, PPE (porcine pancreatic elastase) and the external PPE model were used. Genetic deletion of GP VI offered protection of mice against aortic diameter expansion in experimental AAA. Mechanistically, GP VI deficiency resulted in decreased inflammation with reduced infiltration of neutrophils and platelets into the aortic wall. Furthermore, remodeling of the aortic wall was improved in the absence of GP VI, as indicated by reduced MMP (matrix metalloproteinase)-2/9 and OPN (osteopontin) plasma levels and an enhanced α-SMA (α-smooth muscle actin) content within the aortic wall, accompanied by reduced cell apoptosis. Consequently, an elevation in intima/media thickness and elastin content was observed in GP VI-deficient PPE mice, resulting in a significantly reduced aortic diameter expansion and reduced aneurysm incidence. In patients with AAA, enhanced plasma levels of soluble GP VI and fibrin, as well as fibrin accumulation within the intraluminal thrombus might serve as new biomarkers to detect AAA early. Moreover, we hypothesize that GP VI might play a role in procoagulant activity and thrombus stabilization via binding to fibrin. In conclusion, our results emphasize the potential need for a GP VI-targeted antiplatelet therapy to reduce AAA initiation and progression, as well as to protect patients with AAA from aortic rupture.

Suppressed ORAI1-STIM1-dependent Ca2+ entry by protein kinase C isoforms regulating platelet procoagulant activity

Agonist-induced rises in cytosolic Ca2+ control most platelet responses in thrombosis and hemostasis. In human platelets we earlier demonstrated that the ORAI1-STIM1 pathway is a major component of extracellular Ca2+ entry, in particular when induced via the ITAM-linked collagen receptor, glycoprotein VI (GPVI). In the present paper, using functionally defective platelets from patients with a loss-of-function mutation in ORAI1 or STIM1, we show that Ca2+ entry induced by the endoplasmic reticulum ATPase inhibitor, thapsigargin, fully relies on this pathway. We demonstrate that both the GPVI-induced and thapsigargin-induced Ca2+ entry is strongly suppressed by protein kinase C (PKC) activation, while leaving intracellular Ca2+ mobilization unchanged. Comparing effects of a PKC inhibitory panel pointed to redundant roles of beta and theta PKC isoforms in Ca2+-entry suppression. In contrast, tyrosine kinases positively regulated GPVI-induced Ca2+ entry and mobilization. Label-free and stable isotope phosphoproteome analysis of GPVI-stimulated platelets suggested a regulatory role of bridging integrator-2 (BIN2), known as important mediator of the ORAI1-STIM1 pathway in mouse platelets. Identified were 25-45 regulated phospho- sites in BIN2 and 16-18 in STIM1. Five of these were characterized as direct substrates of the expressed PKC isoforms alpha, beta delta and theta. Functional platelet testing indicated that the downregulation of Ca2+ entry by PKC resulted in suppressed phosphatidylserine exposure and plasmatic thrombin generation. Conclusively, our results indicate that in platelets multiple PKC isoforms constrain the store-regulated Ca2+ entry via ORAI1-BIN2-STIM1, and hence downregulate platelet-dependent coagulation.

Association of primary and secondary hemostasis biomarkers with acute ischemic stroke outcome in patients undergoing thrombectomy, with or without thrombolytics: post hoc analysis of the Multicenter Randomized Clinical Trial of Endovascular Treatment for Acute Ischemic Stroke in the Netherlands-NO IV

BackgroundIntravenous thrombolysis (IVT) using recombinant tissue plasminogen activator prior to endovascular thrombectomy treatment (EVT) failed to improve treatment effect in acute ischemic stroke (AIS) patients compared with EVT alone. ObjectivesWe investigated whether primary and secondary hemostasis biomarkers are associated with the effect of intravenous thrombolytics on clinical and radiological outcomes after EVT. MethodsIn the Multicenter Randomized Clinical Trial of Endovascular Treatment for Acute Ischemic Stroke in the Netherlands (MR CLEAN)-NO IV, AIS patients were randomized to receive IVT plus EVT or EVT alone. We measured hemostatic biomarkers before and 24 hours postreperfusion to determine changes in biomarkers and the association of the biomarkers with short term stroke severity on National Institutes of Health Stroke Scale score, long-term functional outcome (modified Rankin scale [mRS] score), post-EVT extended Thrombolysis in Cerebral Infarction score, and final infarct size. ResultsThis substudy included 214 of the 539 AIS patients who underwent IVT + EVT (n = 108/266) or EVT alone (n = 106/273). In the EVT group, low soluble glycoprotein VI (sGPVI) and high factor (F)VIII levels before treatment were associated with severe National Institutes of Health Stroke Scale score at 24 hours and poor mRS score at 90 days posttreatment, respectively. Also, in this group, sGPVI levels 24 hours after treatment were negatively associated with final infarct size. In the IVT + EVT group, high fibrinogen before treatment was associated with good extended Thrombolysis in Cerebral Infarction score, and low a disintegrin and metalloprotease with thrombospondin motif repeats 13 activity 24 hours posttreatment was associated with an unfavorable mRS score at 90 days. ConclusionOur findings suggest that patients with high FVIII and fibrinogen and low sGPVI levels might be the most suitable candidates for IVT + EVT and that patients with low a disintegrin and metalloprotease with thrombospondin motif repeats 13 activity might be suitable for EVT alone.

Soluble Glycoprotein VI Levels Assessed Locally within the Extra- and Intracerebral Circulation in Hyper-Acute Thromboembolic Stroke: A Pilot Study.

Background: Severe acute ischemic stroke (AIS) is mainly caused by thromboembolism originating from symptomatic carotid artery (ICA) stenosis or in the heart due to atrial fibrillation. Glycoprotein VI (GPVI), a principal platelet receptor, facilitates platelet adherence and thrombus formation at sites of vascular injury such as symptomatic ICA stenosis. The shedding of GPVI from the platelet surface releases soluble GPVI (sGPVI) into the circulation. Here, we aimed to determine whether sGPVI can serve as a local biomarker to differentiate between local atherosclerotic and systemic cardiac thromboembolism in AIS. Methods: We conducted a cohort study involving 105 patients undergoing emergency endovascular thrombectomy (EVT) for anterior circulation stroke. First, sGPVI concentrations were measured in systemic arterial plasma samples collected at the ipsilateral ICA level, including groups with significantly (≥50%) stenotic and non-stenotic arteries. A second sample, taken from the intracerebral pial circulation, was used to assess GPVI shedding locally within the ischemic brain. Results: Our analysis revealed no significant increase in systemic sGPVI levels in patients with symptomatic ≥ 50% ICA stenosis (3.2 [95% CI 1.5-5.0] ng/mL; n = 33) compared with stroke patients without significant ICA stenosis (3.2 [95% CI 2.3-4.2] ng/mL; n = 72). Additionally, pial blood samples, reflecting intravascular molecular conditions during collateral flow, showed similar sGPVI levels when compared to the systemic ICA samples in both groups. Conclusions: Our findings indicate that GPVI is not locally cleaved and shed into the bloodstream in significant amounts during hyper-acute ischemic stroke, neither at the level of symptomatic ICA nor intracranially during collateral blood supply. Therefore, sGPVI does not appear to be suitable as a local stroke biomarker despite strong evidence of a major role for GPVI-signaling in stroke pathophysiology.

Patients with Waldenström macroglobulinemia have impaired platelet and coagulation function

AbstractClinical features in patients with the B-cell lymphoma, Waldenström macroglobulinemia (WM), include cytopenias, immunoglobulin M (IgM)–mediated hyperviscosity, fatigue, bleeding, and bruising. Therapeutics such as Bruton's tyrosine kinase inhibitors (BTKis) exacerbate bleeding risk. Abnormal hemostasis arising from platelet dysfunction, altered coagulation or vascular impairment have not yet been investigated in patients with WM. Therefore, this study aimed to evaluate hemostatic dysfunction in samples from these patients. Whole blood (WB) samples were collected from 14 patients with WM not receiving therapy, 5 patients receiving BTKis and 15 healthy donors (HDs). Platelet receptor levels and reticulation were measured by flow cytometry, plasma thrombin generation with or without platelets by fluorescence resonance energy transfer assay, WB clotting potential by rotational thromboelastometry, and plasma soluble glycoprotein VI (sGPVI) and serum thrombopoietin (TPO) by enzyme-linked immunosorbent assay. Donor platelet spreading, aggregation, and ability to accelerate thrombin generation in the presence of WM-derived IgM were assessed. WM platelet receptor levels, responses to physiological agonists, and plasma sGPVI were within normal ranges. WM platelets had reduced reticulation (P = .0012) whereas serum TPO levels were increased (P = .0040). WM plasma displayed slower thrombin generation (P = .0080) and WM platelets contributed less to endogenous thrombin potential (ETP; P = .0312). HD plasma or platelets incubated with IgM (50-60 mg/mL) displayed reduced spreading (P = .0002), aggregation (P < .0001), and ETP (P = .0081). Thus, alterations to thrombin potential and WB coagulation were detected in WM samples. WM IgM significantly impaired hemostasis in vitro. Platelet and coagulation properties are disturbed in patients with well-managed WM.

Mendelian Randomization and Bayesian Colocalization Analysis Implicate Glycoprotein VI as a Potential Drug Target for Cardioembolic Stroke in South Asian Populations.

Circulating plasma proteins are clinically useful biomarkers for stroke risk. We examined the causal links between plasma proteins and stroke risk in individuals of South Asian ancestry. We applied proteome-wide Mendelian randomization and colocalization approaches to understand causality of 2922 plasma proteins on stroke risk in individuals of South Asian ancestry. We obtained genetic instruments (proxies) for plasma proteins from the UK Biobank (N=920). Genome-wide association studies summary data for strokes (N≤11 312) were sourced from GIGASTROKE consortium. Our primary approach involved the Wald ratio or inverse-variance-weighted methods, with statistical significance set at false discovery rate <0.1. Additionally, a Bayesian colocalization approach assessed shared causal variants among proteome, transcriptome, and stroke phenotypes to minimize bias from linkage disequilibrium. We found evidence of a potential causal effect of plasma GP6 (glycoprotein VI) levels on cardioembolic stroke (odds ratio [OR]Wald ratio=2.53 [95% CI, 1.59-4.03]; P=9.2×10-5, false discovery rate=0.059). Generalized Mendelian randomization accounting for correlated single nucleotide polymorphisms (SNPs), with the P value threshold at P<5×10-8 and clumped at r2=0.3, showed consistent direction of effect of GP6 on cardioembolic stroke (ORgeneralized inverse-variance-weighted=2.21 [95% CI, 1.46-3.33]; P=1.6×10-4). Colocalization analysis indicated that plasma GP6 levels colocalize with cardioembolic stroke (posterior probability=91.4%). Multitrait colocalization combining transcriptome, proteome, and cardioembolic stroke showed moderate to strong evidence that these 2 traits colocalize with GP6 expression in the coronary artery and brain tissues (multitrait posterior probability>50%). The potential causal effect of GP6 on cardioembolic stroke was not significant in European populations (ORinverse-variance-weighted=1.08 [95% CI, 0.93-1.26]; P=0.29). Our joint Mendelian randomization and colocalization analyses suggest that genetically predicted GP6 is potentially causally associated with cardioembolic stroke risk in individuals of South Asian ancestry. As genetic data on individuals of South Asian ancestry increase, future Mendelian randomization studies with larger sample size for plasma GP6 levels should be implemented to further validate our findings. Additionally, clinical studies will be necessary to verify GP6 as a therapeutic target for cardioembolic stroke in South Asians.

The humanized platelet glycoprotein VI Fab inhibitor EMA601 protects from arterial thrombosis and ischaemic stroke in mice.

Glycoprotein VI (GPVI) is a platelet collagen/fibrin(ogen) receptor and an emerging pharmacological target for the treatment of thrombotic and thrombo-inflammatory diseases, notably ischaemic stroke. A first anti-human GPVI (hGPVI) antibody Fab-fragment (ACT017/glenzocimab, KD: 4.1 nM) recently passed a clinical phase 1b/2a study in patients with acute ischaemic stroke and was found to be well tolerated, safe, and potentially beneficial. In this study, a novel humanized anti-GPVI antibody Fab-fragment (EMA601; KD: 0.195 nM) was developed that inhibits hGPVI function with very high potency in vitro and in vivo. Fab-fragments of the mouse anti-hGPVI IgG Emf6.1 were tested for functional GPVI inhibition in human platelets and in hGPVI expressing (hGP6tg/tg) mouse platelets. The in vivo effect of Emf6.1Fab was assessed in a tail bleeding assay, an arterial thrombosis model and the transient middle cerebral artery occlusion (tMCAO) model of ischaemic stroke. Using complementary-determining region grafting, a humanized version of Emf6.1Fab (EMA601) was generated. Emf6.1Fab/EMA601 interaction with hGPVI was mapped in array format and kinetics and quantified by bio-layer interferometry. Emf6.1Fab (KD: 0.427 nM) blocked GPVI function in human and hGP6tg/tg mouse platelets in multiple assays in vitro at concentrations ≥5 µg/mL. Emf6.1Fab (4 mg/kg)-treated hGP6tg/tg mice showed potent hGPVI inhibition ex vivo and were profoundly protected from arterial thrombosis as well as from cerebral infarct growth after tMCAO, whereas tail-bleeding times remained unaffected. Emf6.1Fab binds to a so far undescribed membrane proximal epitope in GPVI. The humanized variant EMA601 displayed further increased affinity for hGPVI (KD: 0.195 nM) and fully inhibited the receptor at 0.5 µg/mL, corresponding to a >50-fold potency compared with ACT017. EMA601 is a conceptually novel and promising anti-platelet agent to efficiently prevent or treat arterial thrombosis and thrombo-inflammatory pathologies in humans at risk.

Soluble glycoprotein VI predicts abdominal aortic aneurysm growth rate and is a novel therapeutic target

AbstractA common feature in patients with abdominal aortic aneurysms (AAAs) is the formation of a nonocclusive intraluminal thrombus (ILT) in regions of aortic dilation. Platelets are known to maintain hemostasis and propagate thrombosis through several redundant activation mechanisms, yet the role of platelet activation in the pathogenesis of AAA-associated ILT is still poorly understood. Thus, we sought to investigate how platelet activation affects the pathogenesis of AAA. Using RNA sequencing, we identified that the platelet-associated transcripts are significantly enriched in the ILT compared with the adjacent aneurysm wall and healthy control aortas. We found that the platelet-specific receptor glycoprotein VI (GPVI) is among the top enriched genes in AAA ILT and is increased on the platelet surface of patients with AAAs. Examination of a specific indicator of platelet activity, soluble GPVI (sGPVI), in 2 independent cohorts of patients with AAAs is highly predictive of an AAA diagnosis and associates more strongly with aneurysm growth rate than D-dimer in humans. Finally, intervention with the anti-GPVI antibody (JAQ1) in mice with established aneurysms blunted the progression of AAA in 2 independent mouse models. In conclusion, we show that the levels of sGPVI in humans can predict a diagnosis of AAA and AAA growth rate, which may be critical in the identification of high-risk patients. We also identify GPVI as a novel platelet-specific AAA therapeutic target, with minimal risk of adverse bleeding complications, for which none currently exists.

Screening for biomarkers of tuberous sclerosis complex-associated epilepsy: a bioinformatics analysis.

The optimal biomarkers for early diagnosis, treatment, and prognosis of tuberous sclerosis complex (TSC)-associated epilepsy are not yet clear. This study identifies the crucial genes involved in the pathophysiology of TSC-associated epilepsy via a bioinformatics analysis. These genes may serve as novel therapeutic targets. Gene chip data sets (GSE62019 and GSE16969) comprising the data of patients with TSC-associated epilepsy and healthy control participants were obtained from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) in the GEO database were identified using the GEO2R gene expression analysis tool. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, Gene Ontology function, and protein-protein interaction (PPI) network analyses were then conducted. The results were analyzed using R language, and are presented in volcano plots, Venn diagrams, heatmaps, and enrichment pathway bubble charts. A gene set enrichment analysis (GSEA), was conducted to examine the KEGG pathways and crucial genes linked to TSC-associated epilepsy. The potential genes were compared with the genes listed in the Online Mendelian Inheritance in Man (OMIM) database and analyzed against the literature to determine their clinical significance. Finally, the expression of the key genes in the TSC-associated epilepsy mice cerebral cortex was examined through immunohistochemical staining. The intersection of the GSE62019 and GSE16969 data sets revealed 151 commonly upregulated DEGs. The KEGG enrichment analysis indicated that these DEGs affected the occurrence and development of TSC-associated epilepsy by modulating complement and coagulation cascades, glycosaminoglycans in cancer, and extracellular matrix-receptor interactions. Four high-scoring clusters emerged, and podoplanin (PDPN) was identified as a key gene through the construction of a PPI network of the common DEGs using the Cytoscape software. A GSEA of the DEGs revealed that the common DEG PDPN was enriched in both data sets in pathways related to platelet activation, aggregation, and the glycoprotein VI (GPVI)-mediated activation cascade. Immunohistochemical staining revealed a significant elevation in PDPN expression in the cerebral cortex of mice with TSC-associated epilepsy. Conversely, the control group mice did not display any significantly positive neurons. The discovery of these crucial genes and signaling pathways extends understanding of the molecular processes underlying the development of TSC-associated epilepsy. Additionally, our findings may provide a theoretical basis for research into targeted clinical treatments.

Physiological platelet aggregation assay to mitigate drug-induced thrombocytopenia using a microphysiological system

Developing a reliable method to predict thrombocytopenia is imperative in drug discovery. Here, we establish an assay using a microphysiological system (MPS) to recapitulate the in-vivo mechanisms of platelet aggregation and adhesion. This assay highlights the role of shear stress on platelet aggregation and their interactions with vascular endothelial cells. Platelet aggregation induced by soluble collagen was detected under agitated, but not static, conditions using a plate shaker and gravity-driven flow using MPS. Notably, aggregates adhered on vascular endothelial cells under gravity-driven flow in the MPS, and this incident increased in a concentration-dependent manner. Upon comparing the soluble collagen-induced aggregation activity in platelet-rich plasma (PRP) and whole blood, remarkable platelet aggregate formation was observed at concentrations of 30 µg/mL and 3 µg/mL in PRP and whole blood, respectively. Moreover, ODN2395, an oligonucleotide, induced platelet aggregation and adhesion to vascular endothelial cells. SYK inhibition, which mediated thrombogenic activity via glycoprotein VI on platelets, ameliorated platelet aggregation in the system, demonstrating that the mechanism of platelet aggregation was induced by soluble collagen and oligonucleotide. Our evaluation system partially recapitulated the aggregation mechanisms in blood vessels and can contribute to the discovery of safe drugs to mitigate the risk of thrombocytopenia.

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

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. 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. 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. 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.

The LAT Rheostat as a Regulator of Megakaryocyte Activation.

Specifically positioned negatively charged residues within the cytoplasmic domain of the adaptor protein, linker for the activation of T cells (LAT), have been shown to be important for efficient phosphorylation of tyrosine residues that function to recruit cytosolic proteins downstream of immunoreceptor tyrosine-based activation motif (ITAM) receptor signaling. LAT tyrosine 132-the binding site for PLC-γ2-is a notable exception, preceded instead by a glycine, making it a relatively poor substrate for phosphorylation. Mutating Gly131 to an acidic residue has been shown in T cells to enhance ITAM-linked receptor-mediated signaling. Whether this is generally true in other cell types is not known. To examine whether LAT Gly131 restricts ITAM signaling in cells of the megakaryocyte lineage, we introduced an aspartic acid at this position in human induced pluripotent stem cells (iPSCs), differentiated them into megakaryocytes, and examined its functional consequences. iPSCs expressing G131D LAT differentiated and matured into megakaryocytes normally, but exhibited markedly enhanced reactivity to glycoprotein VI (GPVI)-agonist stimulation. The rate and extent of LAT Tyr132 and PLC-γ2 phosphorylation, and proplatelet formation on GPVI-reactive substrates, were also enhanced. These data demonstrate that a glycine residue at the -1 position of LAT Tyr132 functions as a kinetic bottleneck to restrain Tyr132 phosphorylation and signaling downstream of ITAM receptor engagement in the megakaryocyte lineage. These findings may have translational applications in the burgeoning field of in vitro platelet bioengineering.