Platelet Activation Pathways Controlling Reversible Integrin αIIbß3 Activation.

Background Agonist-induced platelet activation, with the integrin αIIbß3 conformational change, is required for fibrinogen binding. This is considered reversible under specific conditions, allowing a second phase of platelet aggregation. The signaling pathways that differentiate between a permanent or transient activation state of platelets are poorly elucidated. Objective To explore platelet signaling mechanisms induced by the collagen receptor glycoprotein VI (GPVI) or by protease-activated receptors (PAR) for thrombin that regulate time-dependent αIIbß3 activation. Methods Platelets were activated with collagen-related peptide (CRP, stimulating GPVI), thrombin receptor-activating peptides, or thrombin (stimulating PAR1 and/or 4). Integrin αIIbß3 activation and P-selectin expression was assessed by two-color flow cytometry. Signaling pathway inhibitors were applied before or after agonist addition. Reversibility of platelet spreading was studied by microscopy. Results Platelet pretreatment with pharmacological inhibitors decreased GPVI- and PAR-induced integrin αIIbß3 activation and P-selectin expression in the target order of protein kinase C (PKC) > glycogen synthase kinase 3 > ß-arrestin > phosphatidylinositol-3-kinase. Posttreatment revealed secondary αIIbß3 inactivation (not P-selectin expression), in the same order, but this reversibility was confined to CRP and PAR1 agonist. Combined inhibition of conventional and novel PKC isoforms was most effective for integrin closure. Pre- and posttreatment with ticagrelor, blocking the P2Y 12 adenosine diphosphate (ADP) receptor, enhanced αIIbß3 inactivation. Spreading assays showed that PKC or P2Y 12 inhibition provoked a partial conversion from filopodia to a more discoid platelet shape. Conclusion PKC and autocrine ADP signaling contribute to persistent integrin αIIbß3 activation in the order of PAR1/GPVI > PAR4 stimulation and hence to stabilized platelet aggregation. These findings are relevant for optimization of effective antiplatelet treatment.

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.

GPVI inhibition: Advancing antithrombotic therapy in cardiovascular disease.

Glycoprotein (GP) VI plays a major role in thrombosis but not haemostasis, making it a promising antithrombotic target. The primary role of GPVI on the surface of platelets is a signalling receptor for collagen, which is one of the most potent thrombotic sub-endothelial components that is exposed by atherosclerotic plaque rupture. Inhibition of GPVI has therefore been investigated as a strategy for treatment and prevention of atherothrombosis, such as during stroke and acute coronary syndromes. A range of specific GPVI inhibitors have been characterised and 2 of these inhibitors, glenzocimab and revacept, have completed phase II clinical trials in ischemic stroke. In this review, we summarise mechanisms of GPVI activation and the latest progress of clinically tested GPVI inhibitors, including their mechanisms of action. By focussing on what is known about GPVI activation, we also discuss whether alternate strategies could also be used to target GPVI.

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.

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.

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.

Platelet Metabolic Profiling Reveals Glycolytic and 1-Carbon Metabolites Are Essential for GP VI-Stimulated Human Platelets-Brief Report.

BACKGROUND: Evolving evidence suggests that besides signaling pathways, platelet activation involves a complex interplay between metabolic pathways to support thrombus growth. Selective targeting of metabolic checkpoints may inhibit platelet activation and provide a novel antiplatelet strategy. We, therefore, examined global metabolic changes that occur during the transition of human platelets from resting to an activated state to identify metabolites and associated pathways that contribute to platelet activation. METHODS: We performed metabolic profiling of resting and convulxin-stimulated human platelet samples. The differential levels, pathway analysis, and PCA (principal component analysis) were performed using Metaboanalyst. Metascape was used for metabolite network construction. RESULTS: Of the 401 metabolites identified, 202 metabolites were significantly upregulated, and 2 metabolites were downregulated in activated platelets. Of all the metabolites, lipids scored highly and constituted ≈50% of the identification. During activation, aerobic glycolysis supports energy demand and provides glycolytic intermediates required by metabolic pathways. Consistent with this, an important category of metabolites was carbohydrates, particularly the glycolysis intermediates that were significantly upregulated compared with resting platelets. We found that lysophospholipids such as 1-palmitoyl-GPA (glycero-3-phosphatidic acid), 1-stearoyl-GPS (glycero-3-phosphoserine), 1-palmitoyl-GPI (glycerophosphoinositol), 1-stearoyl-GPI, and 1-oleoyl-GPI were upregulated in activated platelets. We speculated that platelet activation could be linked to 1-carbon metabolism, a set of biochemical pathways that involve the transfer and use of 1-carbon units from amino acids, for cellular processes, including nucleotide and lysophospholipid synthesis. In alignment, based on pathway enrichment and network-based prioritization, the metabolites from amino acid metabolism, including serine, glutamate, and branched-chain amino acid pathway were upregulated in activated platelets, which might be supplemented by the high levels of glycolytic intermediates. CONCLUSIONS: Metabolic analysis of resting and activated platelets revealed that glycolysis and 1-carbon metabolism are necessary to support platelet activation.

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 α2ß1. 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 αIIbß3 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.

Role of SHP2 (PTPN11) in glycoprotein VI-dependent thrombus formation: Improved platelet responsiveness by the allosteric drug SHP099 in Noonan syndrome patients.

INTRODUCTION: The protein tyrosine phosphatase SHP2 (PTPN11) is a negative regulator of glycoprotein VI (GPVI)-induced platelet signal under certain conditions. Clinical trials with derivatives of the allosteric drug SHP099, inhibiting SHP2, are ongoing as potential therapy for solid cancers. Gain-of-function mutations of the PTPN11 gene are observed in part of the patients with the Noonan syndrome, associated with a mild bleeding disorder. Assessment of the effects of SHP2 inhibition in platelets from controls and Noonan syndrome patients. MATERIALS AND METHODS: Washed human platelets were incubated with SHP099 and stimulated with collagen-related peptide (CRP) for stirred aggregation and flow cytometric measurements. Whole-blood microfluidics assays using a dosed collagen and tissue factor coating were performed to assess shear-dependent thrombus and fibrin formation. Effects on clot formation were evaluated by thromboelastometry. RESULTS: Pharmacological inhibition of SHP2 did not alter GPVI-dependent platelet aggregation under stirring, but it enhanced integrin αIIbß3 activation in response to CRP. Using whole-blood microfluidics, SHP099 increased the thrombus buildup on collagen surfaces. In the presence of tissue factor and coagulation, SHP099 increased thrombus size and reduced time to fibrin formation. Blood from PTPN11-mutated Noonan syndrome patients, with low platelet responsiveness, after ex vivo treatment with SHP099 showed a normalized platelet function. In thromboelastometry, SHP2 inhibition tended to increase tissue factor-induced blood clotting profiles with tranexamic acid, preventing fibrinolysis. CONCLUSION: Pharmacological inhibition of SHP2 by the allosteric drug SHP099 enhances GPVI-induced platelet activation under shear conditions with a potential to improve platelet functions of Noonan syndrome patients.

Association between endometriosis, infertility and autoimmune antiplatelet glycoprotein VI antibodies in two patients.

The association between endometriosis and autoimmune diseases is well known, however no acquired platelet function defect has been described so far. We describe the case of two patients with endometriosis associated with an antiplatelet glycoprotein VI (anti-GPVI) antibody. The two women with deep pelvic endometriosis associated with secondary infertility presented a mild bleeding tendency, a deficient platelet aggregation response to collagen, convulxin or CRP and a severe GPVI deficiency. Immunoblot revealed a combined FcRγ deficiency but no indication of GPVI cleavage. In the first case, platelet count was normal and an anti-GPVI IgG was detected in plasma. A first corticosteroids administration normalized in vitro platelet functions but further administrations were unsuccessful. Three IVF attempts failed. Conservative laparoscopic surgery was carried out after antifibrinolytic treatment without bleeding. The second case presented with a history of moderate thrombocytopenia and a weak anti-GPVI in the context of infertility and autoimmune disease, the Sjögren syndrome resolved after corticosteroids and hydroxychloroquine treatment. Acquired GPVI deficiencies are rare. It would be useful to determine whether the association with endometriosis is coincidental or not by more systematic investigations. It does not seem that in these patients, GPVI deficiency is associated with an increased risk of bleeding.

What is the context? • Evidence for an immune system dysfunction is reported in endometriosis and the association between endometriosis and autoimmune diseases is well known.• No autoimmune platelet function defect has been described so far.What is new?• We report two unrelated patients with endometriosis-associated infertility presenting a platelet glycoprotein VI deficiency due to an autoantibody.• In both cases, a deficient platelet aggregation response to collagen, convulxin or CRP and a severe GPVI deficiency were observed.• Immunoblot revealed no indication of GPVI cleavage.What is the impact? • Our observation raises the question whether GPVI could be a preferential target for the development of anti-GPVI autoantibodies associated with endometriosis.• It does not seem that in these patients, GPVI deficiency is associated with an increased risk of severe bleeding disorder.

An optimized herbal medicine containing Scutellaria baicalensis Georgi, Alisma orientale Juzepzuk, and Atractylodes japonica Koidzumi has potent antiplatelet and antithrombotic activities.

Background and aim: Platelet-derived thrombosis is important in the pathogenesis of cardiovascular diseases. HTB is an optimized herbal medicine including Scutellaria baicalensis Georgi, Alisma orientale Juzepzuk, and Atractylodes japonica Koidzumi. It is widely used in traditional medicine due to its anti-inflammatory and antioxidant effects. However, its antiplatelet and antithrombotic activities have not been completely validated. The current study aimed to examine the inhibitory effect of the novel herb formula HTB against platelet activation and thrombus formation. Experimental procedure: The antiplatelet activities of HTB via platelet aggregation, granule secretion, reactive oxygen species generation, and intracellular calcium mobilization were evaluated. Moreover, the antithrombotic effect of HTB via FeCl3-induced arterial thrombus formation in vivo in mice was assessed. The inhibitory effect of HTB against primary hemostasis was investigated based on transection tail bleeding time. Results and conclusion: HTB treatment significantly inhibited glycoprotein VI-mediated platelet aggregation, granule secretion, reactive oxygen species generation, and intracellular calcium mobilization. Biochemical studies revealed that HTB inhibited glycoprotein VI-mediated platelet signal transduction during cell activation. Further, its antioxidant effect might be derived by reducing the phosphorylation of the p47phox/Hic5 axis signalosome. Oral HTB treatment was effective in decreasing FeCl3-induced arterial thrombus formation without prolonging the tail bleeding time. HTB can be an effective therapeutic agent against thrombotic diseases.

Comprehensive analysis of glycoprotein VI-mediated platelet activation signaling pathway for predicting pan-cancer survival and response to anti-PD-1 immunotherapy.

Platelets play a vital role in cancer and immunity. However, few comprehensive studies have been conducted on the role of platelet-related signaling pathways in various cancers and their responses to immune checkpoint blockade (ICB) therapy. In the present study, we focused on the glycoprotein VI-mediated platelet activation (GMPA) signaling pathway and comprehensively evaluated its roles in 19 types of cancers listed in The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO). Cox regression and meta-analyses showed that for all 19 types of cancers, patients with high GMPA scores tended to have a good prognosis. Furthermore, the GMPA signature score could serve as an independent prognostic factor for patients with skin cutaneous melanoma (SKCM). The GMPA signature was linked to tumor immunity in all 19 types of cancers, and was correlated with SKCM tumor histology. Compared to other signature scores, the GMPA signature scores for on-treatment samples were more robust predictors of the response to anti-PD-1 blockade in metastatic melanoma. Moreover, the GMPA signature scores were significantly negatively correlated with EMMPRIN (CD147) and positively correlated with CD40LG expression at the transcriptomic level in most cancer patient samples from the TCGA cohort and on-treatment samples from anti-PD1 therapy cohorts. The results of this study provide an important theoretical basis for the use of GMPA signatures, as well as GPVI-EMMPRIN and GPVI-CD40LG pathways, to predict the responses of cancer patients to various types of ICB therapy.

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

BACKGROUND: Collagen-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. OBJECTIVES: To determine the importance of GPVI clusters for thrombus formation in whole blood under shear. METHODS: We 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. RESULTS: Automated 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. CONCLUSION: Labeled 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.

Platelet Activation by Antisense Oligonucleotides (ASOs) in the Göttingen Minipig, including an Evaluation of Glycoprotein VI (GPVI) and Platelet Factor 4 (PF4) Ontogeny.

Antisense oligonucleotide (ASO) is a therapeutic modality that enables selective modulation of undruggable protein targets. However, dose- and sequence-dependent platelet count reductions have been reported in nonclinical studies and clinical trials. The adult Göttingen minipig is an acknowledged nonclinical model for ASO safety testing, and the juvenile Göttingen minipig has been recently proposed for the safety testing of pediatric medicines. This study assessed the effects of various ASO sequences and modifications on Göttingen minipig platelets using in vitro platelet activation and aggregometry assays. The underlying mechanism was investigated further to characterize this animal model for ASO safety testing. In addition, the protein abundance of glycoprotein VI (GPVI) and platelet factor 4 (PF4) was investigated in the adult and juvenile minipigs. Our data on direct platelet activation and aggregation by ASOs in adult minipigs are remarkably comparable to human data. Additionally, PS ASOs bind to platelet collagen receptor GPVI and directly activate minipig platelets in vitro, mirroring the findings in human blood samples. This further corroborates the use of the Göttingen minipig for ASO safety testing. Moreover, the differential abundance of GPVI and PF4 in minipigs provides insight into the influence of ontogeny in potential ASO-induced thrombocytopenia in pediatric patients.

Repeated platelet activation and the potential of previously activated platelets to contribute to thrombus formation.

BACKGROUND: Especially in disease conditions, platelets can encounter activating agents in circulation. OBJECTIVES: To investigate the extent to which previously activated platelets can be reactivated and whether in-and reactivation applies to different aspects of platelet activation and thrombus formation. METHODS: Short-and long-term effects of glycoprotein VI (GPVI) and G protein-coupled receptor (GPCR) stimulation on platelet activation and aggregation potential were compared via flow cytometry and plate-based aggregation. Using fluorescence and electron microscopy, we assessed platelet morphology and content, as well as thrombus formation. RESULTS: After 30 minutes of stimulation with thrombin receptor activator peptide 6 (TRAP6) or adenosine diphosphate (ADP), platelets secondarily decreased in PAC-1 binding and were less able to aggregate. The reversibility of platelets after thrombin stimulation was concentration dependent. Reactivation was possible via another receptor. In contrast, cross-linked collagen-related peptide (CRP-XL) or high thrombin stimulation evoked persistent effects in αIIbß3 activation and platelet aggregation. However, after 60 minutes of CRP-XL or high thrombin stimulation, when αIIbß3 activation slightly decreased, restimulation with ADP or CRP-XL, respectively, increased integrin activation again. Compatible with decreased integrin activation, platelet morphology was reversed. Interestingly, reactivation of reversed platelets again resulted in shape change and if not fully degranulated, additional secretion. Moreover, platelets that were previously activated with TRAP6 or ADP regained their potential to contribute to thrombus formation under flow. On the contrary, prior platelet triggering with CRP-XL was accompanied by prolonged platelet activity, leading to a decreased secondary platelet adhesion under flow. CONCLUSION: This work emphasizes that prior platelet activation can be reversed, whereafter platelets can be reactivated through a different receptor. Reversed, previously activated platelets can contribute to thrombus formation.

Characterizing the binding of glycoprotein VI with nanobody 35 reveals a novel monomeric structure of glycoprotein VI where the conformation of D1+D2 is independent of dimerization.

BACKGROUND: The platelet-signaling receptor glycoprotein VI (GPVI) is a promising antithrombotic target. We have previously raised a series of high-affinity nanobodies (Nbs) against GPVI and identified Nb2, Nb21, and Nb35 as potent GPVI inhibitors. The Nb2 binding site has been mapped to the D1 domain, which is directly adjacent to the CRP binding site. Ligand-binding complementary determining region 3 has only 15% conservation between all 3 Nbs. OBJECTIVES: To map the binding sites of Nb21 and Nb35 on GPVI. METHODS: We determined the X-ray crystal structure of the D1 and D2 extracellular domains of the GPVI-Nb35 complex. We then looked at the effects of various GPVI mutations on the ability of Nbs to inhibit collagen binding and GPVI signaling using surface binding assays and transfected cell lines. RESULTS: The crystal structure of GPVI bound to Nb35 was solved. GPVI was present as a monomer, and the D1+D2 conformation was comparable to that in the dimeric structure. Arg46, Tyr47, and Ala57 are common residues on GPVI targeted by both Nb2 and Nb35. Mutating Arg46 to an Ala abrogated the ability of Nb2, Nb21, and Nb35 to inhibit collagen-induced GPVI signaling and blocked the binding of all 3 Nbs. In addition, Arg60 was found to reduce Nb21 inhibition but not the inhibition Nb2 or Nb35. CONCLUSIONS: These findings reveal key residues involved in the high-affinity binding of GPVI inhibitors and negate the idea that GPVI dimerization induces a conformational change required for ligand binding.

Platelets modulate cardiac remodeling via the collagen receptor GPVI after acute myocardial infarction.

Introduction: Platelets play an important role in cardiovascular diseases. After acute myocardial infarction, platelets display enhanced activation and migrate into the infarct zone. Furthermore, platelets trigger acute inflammation and cardiac remodeling leading to alterations in scar formation and cardiac function as observed in thrombocytopenic mice. GPVI is the major collagen receptor in platelets and important for platelet activation and thrombus formation and stability. Antibody induced deletion of GPVI at the platelet surface or treatment of mice with recombinant GPVI-Fc results in reduced inflammation and decreased infarct size in a mouse model of AMI. However, the role of GPVI has not been fully clarified to date. Methods/Results: In this study, we found that GPVI is not involved in the inflammatory response in experimental AMI using GPVI deficient mice that were analyzed in a closed-chest model. However, reduced platelet activation in response to GPVI and PAR4 receptor stimulation resulted in reduced pro-coagulant activity leading to improved cardiac remodeling. In detail, GPVI deficiency in mice led to reduced TGF-ß plasma levels and decreased expression of genes involved in cardiac remodeling such as Col1a1, Col3a1, periostin and Cthrc1 7 days post AMI. Consequently, collagen quality of the scar shifted to more tight and less fine collagen leading to improved scar formation and cardiac function in GPVI deficient mice at 21d post AMI. Conclusion: Taken together, this study identifies GPVI as a major regulator of platelet-induced cardiac remodeling and supports the potential relevance of GPVI as therapeutic target to reduce ischemia reperfusion injury and to improve cardiac healing.

Sinomenium acutum Modulates Platelet Aggregation and Thrombus Formation by Regulating the Glycoprotein VI-Mediated Signalosome in Mice.

Sinomenium acutum (SA) has long been used as a traditional medicine in China, Japan, and Korea to treat a wide range of diseases. It has been traditionally used to ameliorate inflammation and improve blood circulation. However, its role in platelet activation has not been thoroughly investigated. Hence, we conducted this study to assess the potential inhibitory effect of SA on platelet aggregation and thrombus formation. The antiplatelet activities of SA were evaluated by assessing platelet aggregation, granular secretion, intracellular Ca2+ mobilization, and the Glycoprotein (GP) VI-mediated signalosome. The thrombosis and bleeding time assays were used to investigate the effect of SA (orally administered at 50 and 100 mg/kg for seven days) in mice. SA treatment at concentrations of 50, 100, and 200 µg/mL significantly reduced GPVI-mediated platelet aggregation, granular secretion, and intracellular Ca2+ mobilization. Further biochemical studies revealed that SA inhibited spleen tyrosine kinase, phospholipase Cγ2, phosphatidylinositol 3-kinase, and AKT phosphorylation. Interestingly, oral administration of SA efficiently ameliorated FeCl3-induced arterial thrombus formation without prolonging the tail bleeding time. These findings suggest that SA has beneficial effects in thrombosis and hemostasis. Therefore, SA holds promise as an effective therapeutic agent for the treatment of thrombotic diseases.

The Btk inhibitor AB-95-LH34 potently inhibits atherosclerotic plaque-induced thrombus formation and platelet procoagulant activity.

BACKGROUND: New antithrombotic therapies with less effect on bleeding are needed for coronary artery disease. The Btk inhibitor ibrutinib blocks atherosclerotic plaque-mediated thrombus formation. However, it is associated with increased bleeding, possibly due to non-Btk-mediated effects. Btk-deficient patients do not have bleeding issues, suggesting selective Btk inhibition as a promising antithrombotic strategy. OBJECTIVES: To compare the antithrombotic effects of the highly selective Btk inhibitor AB-95-LH34 (LH34) with ibrutinib. METHODS: Glycoprotein VI and G-protein coupled receptor-mediated platelet function and signaling were analyzed in healthy human donor platelets by lumi-aggregometry, flow adhesion, and western blot following 1 h treatment with inhibitors in vitro. RESULTS: LH34 showed similar inhibition of Btk-Y223 phosphorylation as ibrutinib, but had no off-target inhibition of Src-Y418 phosphorylation. Similar dose-dependent inhibition of aggregation to atherosclerotic plaque material was observed for both. However, in response to Horm collagen, which also binds integrin α2ß1, LH34 exhibited less marked inhibition than ibrutinib. Both LH34 and ibrutinib inhibited platelet adhesion and aggregation to plaque material at arterial shear. Ibrutinib demonstrated the most potent effect, with complete blockade at high concentrations. Platelet activation (P-selectin) and procoagulant activity (phosphatidylserine exposure) in thrombi were inhibited by LH34 and completely blocked by ibrutinib at high concentrations. Furthermore, plaque-induced thrombin generation was reduced by higher concentrations of LH34 and ibrutinib. CONCLUSIONS: LH34 potently inhibits atherosclerotic plaque-induced thrombus formation and procoagulant platelet activity in vitro, with less off-target inhibition of Src than ibrutinib, suggesting it is a promising antiplatelet therapy with the potential for reduced bleeding side effects.