Platelet Glycoprotein VI Research Articles

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.

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.

Multi-phased Kinetics and Interaction of Protein Kinase Signaling in Glycoprotein VI-Induced Platelet αIIbβ3 Integrin Activation and Degranulation.

Platelet glycoprotein VI (GPVI) stimulation activates the tyrosine kinases Syk and Btk, and the effector proteins phospholipase Cγ 2 (PLCγ2) and protein kinase C (PKC). Here, the activation sequence, crosstalk, and downstream effects of this Syk-Btk-PKC signalosome in human platelets were analyzed. Using immunoblotting, we quantified 14 regulated phospho-sites in platelets stimulated by convulxin with and without inhibition of Syk, Btk, or PKC. Convulxin induced fast, reversible tyrosine phosphorylation (pY) of Syk, Btk, LAT, and PLCγ2, followed by reversible serine/threonine phosphorylation (pS/T) of Syk, Btk, and downstream kinases MEK1/2, Erk1/2, p38, and Akt. Syk inhibition by PRT-060318 abolished all phosphorylations, except Syk pY352. Btk inhibition by acalabrutinib strongly decreased Btk pY223/pS180, Syk pS297, PLCγ2 pY759/Y1217, MEK1/2 pS217/221, Erk1/2 pT202/Y204, p38 pT180/Y182, and Akt pT308/S473. PKC inhibition by GF109203X abolished most pS/T phosphorylations except p38 pT180/Y182 and Akt pT308, but enhanced most Y-phosphorylations. Acalabrutinib, but not GF109203X, suppressed convulxin-induced intracellular Ca2+ mobilization, whereas all three protein kinase inhibitors abolished degranulation and αIIbβ3 integrin activation assessed by flow cytometry. Inhibition of autocrine ADP effects by AR-C669931 partly diminished convulxin-triggered degranulation. Kinetic analysis of GPVI-initiated multisite protein phosphorylation in human platelets demonstrates multiple phases and interactions of tyrosine and serine/threonine kinases with activation-altering feedforward and feedback loops partly involving PKC. The protein kinase inhibitor effects on multisite protein phosphorylation and functional readouts reveal that the signaling network of Syk, Btk, and PKC controls platelet granule exocytosis and αIIbβ3 integrin activation.

Abstract 3094: Traditional Chinese Medicine Buyang Huanwu Decoction Regulates The Platelet Gpvi And Its Downstream Protein Signaling Pathways,inhibiting Thrombosis And Preventing Cardiovascular Disease.

Background: Coronary heart disease Qi-Deficiency and Blood Stasis Syndrome will lead to the body of platelets disorders, blood viscosity, conducive to the formation of thrombosis, Buyang Huanwu Decoction (BYHWD) is a classic Chinese medicine treatment of Qi-Deficiency and Blood Stasis Syndrome. Platelet glycoprotein VI (GPVI) plays a crucial role in collagen-induced activation and aggregation of platelets. GPVI has become the focus of new approaches to antithrombotic therapy. Methods: After 6 weeks of irregular sleep deprivation in rats with syndrome of deficiency of qi and blood stasis formed, the coronary heart disease model was established by ligation of the left anterior descending coronary artery in rats. The dosage of 1 mL/100 g was given by intragastric administration for 14 days after operation. Results: The purpose of this study was to investigate the effects of BYHWD on platelet granule secretion and platelet function in vitro. We found that BYHWD can significantly reduce the platelet aggregation induced by ADP and collagen, significantly reduce the median expression of CD62p and the ratio of activated platelets, and reduce the concentration of fluorescent calcium ions in platelets. Under the electron microscope observation found that under the action of BYHWD, the ultrastructure of rat platelets pseudopodia reduced, aggregation reduced, rat coronary inner wall platelets, thrombus mass adhesion reduced, and the inner wall was smoother. In addition, BYHWD can regulate GPVI and its downstream proteins. Compared with the coronary heart disease group with syndrome of blood stasis, BYHWD can significantly reduce the expression of GPVI. Significantly reduced the expression of Syk, CALDAG-GEFI, significantly reduced the expression of IP 3 and significantly reduced the degree of PLCγ 2 of rats. Decreased the degranulation reaction of the rat blood, significantly decreased the level of TXA 2 , 5-HT in the rat blood, and significantly decreased the content of the rat blood COX-1 in the rat serum. Conclusion: Therefore, BYHWD can regulate GPVI and its downstream protein signaling pathways, reduce platelet over-activation, degranulation reaction, thereby inhibiting thrombosis, protecting heart function and preventing cardiovascular disease.

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

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. 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. 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). 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. Acticor Biotech.

Platelet glycoprotein VI inhibition: a promising therapeutic avenue in acute ischaemic stroke

Platelet glycoprotein VI inhibition: a promising therapeutic avenue in acute ischaemic stroke

Diphenyl-tetrazol-propanamide Derivatives Act as Dual-Specific Antagonists of Platelet CLEC-2 and Glycoprotein VI.

Platelet C-type lectin-like receptor 2 (CLEC-2) induces platelet activation and aggregation after clustering by its ligand podoplanin (PDPN). PDPN, which is not normally expressed in cells in contact with blood flow, is induced in inflammatory immune cells and some malignant tumor cells, thereby increasing the risk of venous thromboembolism (VTE) and tumor metastasis. Therefore, small-molecule compounds that can interfere with the PDPN-CLEC-2 axis have the potential to become selective antiplatelet agents. Using molecular docking analysis of CLEC-2 and a PDPN-CLEC-2 binding-inhibition assay, we identified a group of diphenyl-tetrazol-propanamide derivatives as novel CLEC-2 inhibitors. A total of 12 hit compounds also inhibited PDPN-induced platelet aggregation in humans and mice. Unexpectedly, these compounds also fit the collagen-binding pocket of the glycoprotein VI molecule, thereby inhibiting collagen interaction. These compounds also inhibited collagen-induced platelet aggregation, and one compound ameliorated collagen-induced thrombocytopenia in mice. For clinical use, these compounds will require a degree of chemical modification to decrease albumin binding. Nonetheless, as dual activation of platelets by collagen and PDPN-positive cells is expected to occur after the rupture of atherosclerotic plaques, these dual antagonists could represent a promising pharmacophore, particularly for arterial thrombosis, in addition to VTE and metastasis.

PB0751 Platelet Glycoprotein VI Drives Acute Pulmonary Inflammation by Promoting Neutrophil Recruitment, Clustering and NETosis

PB0751 Platelet Glycoprotein VI Drives Acute Pulmonary Inflammation by Promoting Neutrophil Recruitment, Clustering and NETosis

Regulation of Glycoprotein VI-Dependent Platelet Activation and Thrombus Formation by Heparan Sulfate Proteoglycan Perlecan.

Proteoglycans form a heterogeneous family of proteins with covalently bound sulfated glycosaminoglycans. The extracellular matrix proteoglycan perlecan has been proposed to bind to the platelet- and megakaryocyte-specific receptor G6bB, co-regulating platelet glycoprotein VI (GPVI) signaling. The derived non-sulfate proteoglycan endorepellin was previously shown to enhance platelet adhesion via the collagen receptor, integrin α2β1. Here, we compared the roles of perlecan and other matrix proteoglycans in platelet responses and thrombus formation. We used multi-color flow cytometry to measure the degranulation and integrin αIIbβ3 activation of washed platelets in response to various proteoglycans and collagen-related peptide (CRP), the GPVI agonist. Perlecan, but not endorepellin, enhanced the CRP-induced activation of platelets in a time- and concentration-dependent manner. Similar to collagen, immobilized perlecan, but not other proteoglycans, supported static platelet adhesion and spreading. In-flowed whole-blood perlecan diminished shear-dependent platelet adhesion, while it enforced GPVI-dependent thrombus formation-to a larger extent than endorepellin-to induce more contracted aggregates of activated platelets. We concluded that the sulfated proteoglycan perlecan enhances GPVI-dependent platelet responses extending to thrombus formation, but it does so at the expense of reduced adhesion of platelets under flow.

A key role for platelet GPVI in neutrophil recruitment, migration, and NETosis in the early stages of acute lung injury

AbstractAcute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are associated with high morbidity and mortality. Excessive neutrophil infiltration into the pulmonary airspace is the main cause for the acute inflammation and lung injury. Platelets have been implicated in the pathogenesis of ALI/ARDS, but the underlying mechanisms are not fully understood. Here, we show that the immunoreceptor tyrosine-based activation motif–coupled immunoglobulin-like platelet receptor, glycoprotein VI (GPVI), plays a key role in the early phase of pulmonary thrombo-inflammation in a model of lipopolysaccharide (LPS)-induced ALI in mice. In wild-type (WT) control mice, intranasal LPS application triggered severe pulmonary and blood neutrophilia, hypothermia, and increased blood lactate levels. In contrast, GPVI-deficient mice as well as anti-GPVI–treated WT mice were markedly protected from pulmonary and systemic compromises and showed no increased pulmonary bleeding. High-resolution multicolor microscopy of lung sections and intravital confocal microcopy of the ventilated lung revealed that anti-GPVI treatment resulted in less stable platelet interactions with neutrophils and overall reduced platelet–neutrophil complex (PNC) formation. Anti-GPVI treatment also reduced neutrophil crawling and adhesion on endothelial cells, resulting in reduced neutrophil transmigration and alveolar infiltrates. Remarkably, neutrophil activation was also diminished in anti-GPVI–treated animals, associated with strongly reduced formation of PNC clusters and neutrophil extracellular traps (NETs) compared with that in control mice. These results establish GPVI as a key mediator of neutrophil recruitment, PNC formation, and NET formation (ie, NETosis) in experimental ALI. Thus, GPVI inhibition might be a promising strategy to reduce the acute pulmonary inflammation that causes ALI/ARDS.

Platelet glycoprotein VI cluster size is related to thrombus formation and phosphatidylserine exposure in collagen-adherent platelets under arterial shear

BackgroundCollagen-induced platelet activation is predominantly mediated by glycoprotein (GP) VI through formation of receptor clusters that coincide with the accumulation of signaling molecules and are hypothesized to drive strong and sustained platelet activation. ObjectivesTo determine the importance of GPVI clusters for thrombus formation in whole blood under shear. MethodsWe utilized whole blood microfluidics and an anti-GPVI nanobody (Nb), Nb28, labeled with AlexaFluor 488, to assess the distribution of GPVI on the surface of platelets adhering to a range of collagen-like substrates with different platelet activation potentials. ResultsAutomated analysis of GPVI surface distribution on platelets supported the hypothesis that there is a relationship between GPVI cluster formation, thrombus size, and phosphatidylserine (PS) exposure. Substrates that supported the formation of macroclusters also induced significantly bigger aggregates, with increased amounts of PS-exposing platelets in comparison to substrates where no GPVI clusters were detected. Furthermore, we demonstrate that only direct inhibition of GPVI binding, but not of downstream signaling, is able to disrupt cluster formation. ConclusionLabeled anti-GPVI Nb28 permits visualization of GPVI clustering under flow conditions. Furthermore, whilst inhibition of downstream signaling does not affect clustering, it does prevent thrombus formation. Therefore, GPVI macroclustering is a prerequisite for thrombus formation and platelet activation, namely, PS exposure, on highly GPVI-dependent collagen surfaces.

Targeting platelet GPVI with glenzocimab: a novel mechanism for inhibition.

Platelet glycoprotein VI (GPVI) is attracting interest as a potential target for the development of new antiplatelet molecules with a low bleeding risk. GPVI binding to vascular collagen initiates thrombus formation and GPVI interactions with fibrin promote the growth and stability of the thrombus. In this study, we show that glenzocimab, a clinical stage humanized antibody fragment (Fab) with a high affinity for GPVI, blocks the binding of both ligands through a combination of steric hindrance and structural change. A cocrystal of glenzocimab with an extracellular domain of monomeric GPVI was obtained and its structure determined to a resolution of 1.9 Å. The data revealed that (1) glenzocimab binds to the D2 domain of GPVI, GPVI dimerization was not observed in the crystal structure because glenzocimab prevented D2 homotypic interactions and the formation of dimers that have a high affinity for collagen and fibrin; and (2) the light variable domain of the GPVI-bound Fab causes steric hindrance that is predicted to prevent the collagen-related peptide (CRP)/collagen fibers from extending out of their binding site and preclude GPVI clustering and downstream signaling. Glenzocimab did not bind to a truncated GPVI missing loop residues 129 to 136, thus validating the epitope identified in the crystal structure. Overall, these findings demonstrate that the binding of glenzocimab to the D2 domain of GPVI induces steric hindrance and structural modifications that drive the inhibition of GPVI interactions with its major ligands.

Minimal Collagen-Binding Epitope of Glycoprotein VI in Human and Mouse Platelets

Glycoprotein VI (GPVI) is a platelet-specific receptor for collagen and fibrin, regulating important platelet functions such as platelet adhesion and thrombus growth. Although the blockade of GPVI function is widely recognized as a potent anti-thrombotic approach, there are limited studies focused on site-specific targeting of GPVI. Using computational modeling and bioinformatics, we analyzed collagen- and CRP-binding surfaces of GPVI monomers and dimers, and compared the interacting surfaces with other mammalian GPVI isoforms. We could predict a minimal collagen-binding epitope of GPVI dimer and designed an EA-20 antibody that recognizes a linear epitope of this surface. Using platelets and whole blood samples donated from wild-type and humanized GPVI transgenic mice and also humans, our experimental results show that the EA-20 antibody inhibits platelet adhesion and aggregation in response to collagen and CRP, but not to fibrin. The EA-20 antibody also prevents thrombus formation in whole blood, on the collagen-coated surface, in arterial flow conditions. We also show that EA-20 does not influence GPVI clustering or receptor shedding. Therefore, we propose that blockade of this minimal collagen-binding epitope of GPVI with the EA-20 antibody could represent a new anti-thrombotic approach by inhibiting specific interactions between GPVI and the collagen matrix.

Radiolabeled GPVI-Fc for PET Imaging of Multiple Extracellular Matrix Fibers: A New Look into Pulmonary Fibrosis Progression.

Invariably fatal and with a particularly fast progression, pulmonary fibrosis (PF) is currently devoid of curative treatment options. Routine clinical diagnosis relies on breathing tests and visualizing the changes in lung structure by CT, but anatomic information is often not sufficient to identify early signs of progressive PF. For more efficient diagnosis, additional imaging techniques were investigated in combination with CT, such as 18F-FDG PET, although with limited success because of lack of disease specificity. Therefore, novel molecular targets enabling specific diagnosis are investigated, in particular for molecular imaging techniques. Methods: In this study, we used a 64Cu-radiolabeled platelet glycoprotein VI fusion protein (64Cu-GPVI-Fc) targeting extracellular matrix (ECM) fibers as a PET tracer to observe longitudinal ECM remodeling in a bleomycin-induced PF mouse model. Results: 64Cu-GPVI-Fc showed significant uptake in fibrotic lungs, matching histology results. Contrary to 18F-FDG PET measurements, 64Cu-GPVI-Fc uptake was linked entirely to the fibrotic activity of tissue and not was susceptible to inflammation. Conclusion: Our study highlights 64Cu-GPVI-Fc as a specific tracer for ECM remodeling in PF, with clear therapy-monitoring and clinical translation potential.

The severity and duration of Hypoglycemia affect platelet-derived protein responses in Caucasians

ObjectiveSevere hypoglycemia is associated with increased cardiovascular death risk, and platelet responses to hypoglycemia (hypo) have been described. However, the impact of deep transient hypo (deep-hypo) versus prolonged milder hypo (mild-hypo) on platelet response is unclear.Research Design and methodsTwo hypo studies were compared; firstly, mild-hypo in 18-subjects (10 type-2-diabetes (T2D), 8 controls), blood glucose to 2.8mmoL/L (50 mg/dL) for 1-hour; secondly deep-hypo in 46-subjects (23 T2D, 23 controls), blood glucose to < 2.2mmoL/L (< 40 mg/dL) transiently. Platelet-related protein (PRP) responses from baseline to after 1-hour of hypo (mild-hypo) or at deep-hypo were compared, and at 24-hours post-hypo. Slow Off-rate Modified Aptamer (SOMA)-scan plasma protein measurement was used to determine PRP changes for 13 PRPs.ResultsIn controls, from baseline to hypo, differences were seen for four PRPs, three showing increased %change in deep-hypo (Plasminogen activator inhibitor-1(PAI-1), CD40 ligand (CD40LG) and Protein-S), one showing increased %change in mild-hypo (von Willebrand factor (vWF)); at 24-hours in controls, %change for Protein-S remained increased in deep-hypo, whilst % change for vWF and plasminogen were increased in mild-hypo. In T2D, from baseline to hypo, differences were seen for 4 PRPs, three showing increased %change in deep-hypo (PAI-1, platelet glycoprotein VI and Tissue factor), one showing increased %change in mild-hypo (CD40LG); at 24-hours in T2D, %change for CD40LG remained increased, together with vWF, in deep-hypo.ConclusionBoth mild-hypo and deep-hypo showed marked PRP changes that continued up to 24-hours, showing that both the severity and duration of hypoglycemia are likely important and that any degree of hypoglycemia may be detrimental for increased cardiovascular risk events through PRP changes.

Glenzocimab: A GPVI (Glycoprotein VI)-Targeted Potential Antiplatelet Agent for the Treatment of Acute Ischemic Stroke.

It has previously been shown in several animal experiments that platelet GPVI (glycoprotein VI) contributes to thrombosis, particularly in ischemic stroke. Moreover, GPVI levels are upregulated in stroke patients. This review describes the therapeutic roles of anti-GPVI antibody in preclinical models of ischemic stroke and provides the current evidence for potential benefits of glenzocimab, a Fab fragment of humanized anti-GPVI monoclonal antibody, in stroke patients. Anti-GPVI antibody, JAQ1, significantly decreased infarct volume and improved neurological function in mice with transient middle cerebral artery occlusion, a model of ischemic stroke, with no or minor bleeding tendency. Intravenous injection of glenzocimab in nonhuman primates produced rapid inhibition of ex vivo platelet aggregation induced by collagen (a GPVI ligand). Complete platelet inhibition is observed at 30 minutes following administration without increasing the risk of bleeding. In humans, glenzocimab is well tolerated and produces dose-dependent antiplatelet activity. More importantly, glenzocimab (125-1000 mg) was safe when administered as soon as possible (<3 hours) following reperfusion with the r-tPA (recombinant tissue-type plasminogen activator) in patients with acute ischemic stroke. Although glenzocimab 1000 mg (a selected dose) did not demonstrate a significant improvement in overall clinical outcomes, it appeared to provide benefits in severe cases and in patients who required thrombectomy. This promising efficacy together with a good safety profile of glenzocimab warrant further investigation in phase III (ACTISAVE [Adaptive Efficacy and Safety Study of Glenzocimab Used as an Add-On Therapy on Top of Standard of Care in the 4.5 Hours Following an Acute Ischemic Stroke]) clinical study.

Platelet glycoprotein VI genetic polymorphism T13254C in neonatal sepsis

Background Neonatal sepsis is a global burden, being a leading cause of neonatal morbidity and mortality worldwide. Platelet glycoprotein VI (GPVI) affects sepsis at multiple stages of the inflammatory response. The expression of the GPVI receptor is genetically determined, thus influencing the coagulation processes. The authors focused in this study on the role of platelet GPVI genetic polymorphism T13254C (rs1613662) in neonatal sepsis in relation to other risk factors, laboratory tests, sepsis progression, and outcome. Methods The authors studied 50 neonates with early-onset sepsis. The authors detected platelet GPVI T13254C polymorphism using the TaqMan allelic discrimination method by the real-time polymerase chain reaction technique. Results The results showed that GPVI mutant polymorphic group was associated with higher D-dimer levels (P=0.032). Moreover, septic neonates with mutant homozygous type showed poor survival (P=0.047). However, GPVI mutant polymorphic types were not significantly related to other demographic, laboratory data, and different scoring systems, such as sepsis-induced coagulopathy score, International Society on Thrombosis and Hemostasis score, and Score for Neonatal Acute Physiology. Conclusion The authors found a relation between platelet GPVI T13254C polymorphism and D-dimer levels, hence suggesting a relation with neonatal sepsis-associated coagulopathy, which might further affect patients’ outcome.

237-OR: Platelet-Derived Protein Responses Differ According to Severity of Hypoglycemia

Introduction: Severe hypoglycemia is associated with an increased risk of cardiovascular (CV) death, and platelet responses to hypoglycemia (hypo) have been described. The impact of a transient severe hypo versus prolonged less severe hypo is unclear. Methods: 2 hypo studies were compared; firstly, in 18 subjects (type 2 diabetes (T2D) : 8 controls) blood glucose lowered to 3.5mmoL/L (90mg/dL) for 1-hour; secondly, in 46 subjects (23 T2D: 23 controls) blood glucose lowered to &amp;lt;2.2mmoL/L (&amp;lt;40mg/dL) transiently. The platelet-related protein (PRP) responses from baseline to after 1-hour of hypo (study-1) or at severe transient hypo (study-2) were compared, and at 24-hours post-hypo. Slow Off-rate Modified Aptamer (SOMA) -scan plasma protein measurement was used to determine platelet-related protein changes for 13 proteins. Results: In T2D, from baseline to hypo, differences were seen for 4 PRPs, three showing increased percent change in study-2 (plasminogen activator inhibitor-1 (PAI-1) , platelet glycoprotein VI (PGVI) and Tissue factor) , one showing increased percent change in study-1 (CD40 ligand (CD40LG)) ; at 24-hours in T2D, percent change for CD40LG remained increased, together with von Willebrand factor (vWF) , in study-2. In controls, from baseline to hypo, differences were seen for four PRPs, three showing increased percent change in study-2 (PAI-1, CD40LG and Protein S) , one showing increased percent change in study-1 (vWF) ; at 24-hours in controls, percent change for Protein S remained increased in study-2, whilst percent change for vWF and plasminogen were increased in study-1. Conclusion: Thrombogenic potential was enhanced in T2D versus controls, as evidenced by an increased thrombogenic protein expression profile in both mild and severe hypoglycemia, that showed persistent changes at 24-hours in severe hypo. Disclosure A. Moin: None. H. Kahal: None.

Procoagulant platelet sentinels prevent inflammatory bleeding through GPIIBIIIA and GPVI

AbstractImpairment of vascular integrity is a hallmark of inflammatory diseases. We recently reported that single immune-responsive platelets migrate and reposition themselves to sites of vascular injury to prevent bleeding. However, it remains unclear how single platelets preserve vascular integrity once encountering endothelial breaches. Here we demonstrate by intravital microscopy combined with genetic mouse models that procoagulant activation (PA) of single platelets and subsequent recruitment of the coagulation cascade are crucial for the prevention of inflammatory bleeding. Using a novel lactadherin-based compound, we detect phosphatidylserine (PS)-positive procoagulant platelets in the inflamed vasculature. We identify exposed collagen as the central trigger arresting platelets and initiating subsequent PA in a CypD- and TMEM16F-dependent manner both in vivo and in vitro. Platelet PA promotes binding of the prothrombinase complex to the platelet membrane, greatly enhancing thrombin activity and resulting in fibrin formation. PA of migrating platelets is initiated by costimulation via integrin αIIbβ3 (GPIIBIIIA)/Gα13-mediated outside-in signaling and glycoprotein VI signaling, leading to an above-threshold intracellular calcium release. This effectively targets the coagulation cascade to breaches of vascular integrity identified by patrolling platelets. Platelet-specific genetic loss of either CypD or TMEM16F as well as combined blockade of platelet GPIIBIIIA and glycoprotein VI reduce platelet PA in vivo and aggravate pulmonary inflammatory hemorrhage. Our findings illustrate a novel role of procoagulant platelets in the prevention of inflammatory bleeding and provide evidence that PA of patrolling platelet sentinels effectively targets and confines activation of coagulation to breaches of vascular integrity.

Galectin‐9 activates platelet ITAM receptors glycoprotein VI and C‐type lectin‐like receptor‐2

BackgroundPlatelets are multifunctional cellular mediators in many physiological and pathophysiological processes such as thrombosis, angiogenesis, and inflammation. Several members of galectins, a family of carbohydrate‐binding proteins with a broad range of immunomodulatory actions, have been reported to activate platelets. ObjectiveIn this study, we investigated the role of galectin‐9 (Gal‐9) as a novel ligand for platelet glycoprotein VI (GPVI) and C‐type lectin‐like receptor 2 (CLEC‐2). MethodsPlatelet spreading, aggregation, and P‐selectin expression in response to Gal‐9 were measured in washed platelet suspensions via static adhesion assay, light transmission aggregometry, and flow cytometry, respectively. Solid‐phase binding assay and protein phosphorylation studies were utilized to validate the interaction between Gal‐9 and GPVI, and immunoprecipitation for detecting CLEC‐2 phosphorylation. Wild‐type (WT), GPVI‐knockout (Gp6−/−), and GPVI and CLEC‐2‐double knockout (Gp6−/−/Gp1ba‐Cre‐Clec1bfl/fl) mice were used. ResultsWe have shown that recombinant Gal‐9 stimulates aggregation in human and mouse washed platelets dose‐dependently. Platelets from both species adhere and spread on immobilized Gal‐9 and express P‐selectin. Gal‐9 competitively inhibited the binding of human recombinant D1 and D2 domains of GPVI to collagen. Gal‐9 stimulated tyrosine phosphorylation of CLEC‐2 and proteins known to lie downstream of GPVI and CLEC‐2 including spleen tyrosine kinase and linker of activated T cells in human platelets. GPVI‐deficient murine platelets exhibited significantly impaired aggregation in response to Gal‐9, which was further abrogated in GPVI and CLEC‐2‐double‐deficient platelets. ConclusionsWe have identified Gal‐9 as a novel platelet agonist that induces activation through interaction with GPVI and CLEC‐2.