RESUMO
ABSTRACT: Platelet factor 4 (PF4) is an abundant chemokine that is released from platelet α-granules on activation. PF4 is central to the pathophysiology of vaccine-induced immune thrombocytopenia and thrombosis (VITT) in which antibodies to PF4 form immune complexes with PF4, which activate platelets and neutrophils through Fc receptors. In this study, we show that PF4 binds and activates the thrombopoietin receptor, cellular myeloproliferative leukemia protein (c-Mpl), on platelets. This leads to the activation of Janus kinase 2 (JAK2) and phosphorylation of signal transducer and activator of transcription (STAT) 3 and STAT5, leading to platelet aggregation. Inhibition of the c-Mpl-JAK2 pathway inhibits platelet aggregation to PF4, VITT sera, and the combination of PF4 and IgG isolated from VITT patient plasma. The results support a model in which PF4-based immune complexes activate platelets through binding of the Fc domain to FcγRIIA and PF4 to c-Mpl.
Assuntos
Janus Quinase 2 , Trombocitopenia , Humanos , Complexo Antígeno-Anticorpo/metabolismo , Plaquetas/metabolismo , Heparina/efeitos adversos , Fatores Imunológicos/efeitos adversos , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Fator Plaquetário 4 , Receptores de Trombopoetina/metabolismo , Trombocitopenia/induzido quimicamenteRESUMO
S100A8/A9, also known as "calprotectin" or "MRP8/14," is an alarmin primarily secreted by activated myeloid cells with antimicrobial, proinflammatory, and prothrombotic properties. Increased plasma levels of S100A8/A9 in thrombo-inflammatory diseases are associated with thrombotic complications. We assessed the presence of S100A8/A9 in the plasma and lung autopsies from patients with COVID-19 and investigated the molecular mechanism by which S100A8/A9 affects platelet function and thrombosis. S100A8/A9 plasma levels were increased in patients with COVID-19 and sustained high levels during hospitalization correlated with poor outcomes. Heterodimeric S100A8/A9 was mainly detected in neutrophils and deposited on the vessel wall in COVID-19 lung autopsies. Immobilization of S100A8/A9 with collagen accelerated the formation of a fibrin-rich network after perfusion of recalcified blood at venous shear. In vitro, platelets adhered and partially spread on S100A8/A9, leading to the formation of distinct populations of either P-selectin or phosphatidylserine (PS)-positive platelets. By using washed platelets, soluble S100A8/A9 induced PS exposure but failed to induce platelet aggregation, despite GPIIb/IIIa activation and alpha-granule secretion. We identified GPIbα as the receptor for S100A8/A9 on platelets inducing the formation of procoagulant platelets with a supporting role for CD36. The effect of S100A8/A9 on platelets was abolished by recombinant GPIbα ectodomain, platelets from a patient with Bernard-Soulier syndrome with GPIb-IX-V deficiency, and platelets from mice deficient in the extracellular domain of GPIbα. We identified the S100A8/A9-GPIbα axis as a novel targetable prothrombotic pathway inducing procoagulant platelets and fibrin formation, in particular in diseases associated with high levels of S100A8/A9, such as COVID-19.
Assuntos
Plaquetas , COVID-19 , Calgranulina A , Calgranulina B , Complexo Glicoproteico GPIb-IX de Plaquetas , Animais , Camundongos , Plaquetas/metabolismo , Calgranulina A/metabolismo , COVID-19/metabolismo , Fibrina/metabolismo , Fosfatidilserinas/metabolismo , Agregação Plaquetária , Humanos , Calgranulina B/metabolismo , Autopsia , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismoRESUMO
The collagen/fibrin(ogen) receptor, glycoprotein VI (GPVI), is a platelet activating receptor and a promising anti-thrombotic drug target. However, while agonist-induced GPVI clustering on platelet membranes has been shown to be essential for its activation, it is unknown if GPVI dimerisation represents a unique conformation for ligand binding. Current GPVI structures all contain only the two immunoglobulin superfamily (IgSF) domains in the GPVI extracellular region, so lacking the mucin-like stalk, transmembrane, cytoplasmic tail of GPVI and its associated Fc receptor γ (FcRγ) homodimer signalling chain, and provide contradictory insights into the mechanisms of GPVI dimerisation. Here, we utilised styrene maleic-acid lipid particles (SMALPs) to extract GPVI in complex with its two associated FcRγ chains from transfected HEK-293T cells, together with the adjacent lipid bilayer, then purified and characterised the GPVI/FcRγ-containing SMALPs, to enable structural insights into the full-length GPVI/FcRγ complex. Using size exclusion chromatography followed by a native polyacrylamide gel electrophoresis (PAGE) method, SMA-PAGE, we revealed multiple sizes of the purified GPVI/FcRγ SMALPs, suggesting the potential existence of GPVI oligomers. Importantly, GPVI/FcRγ SMALPs were functional as they could bind collagen. Mono-dispersed GPVI/FcRγ SMALPs could be observed under negative stain electron microscopy. These results pave the way for the future investigation of GPVI stoichiometry and structure, while also validating SMALPs as a promising tool for the investigation of human membrane protein interactions, stoichiometry and structure.
Assuntos
Plaquetas , Receptores de IgG , Humanos , Receptores de IgG/metabolismo , Plaquetas/química , Plaquetas/metabolismo , Membrana Celular/metabolismo , Transdução de Sinais , Colágeno/metabolismoRESUMO
Glycoprotein VI (GPVI) is the major signaling receptor for collagen on platelets. We have raised 54 nanobodies (Nb), grouped into 33 structural classes based on their complementary determining region 3 loops, against recombinant GPVI-Fc (dimeric GPVI) and have characterized their ability to bind recombinant GPVI, resting and activated platelets, and to inhibit platelet activation by collagen. Nbs from 6 different binding classes showed the strongest binding to recombinant GPVI-Fc, suggesting that there was not a single dominant class. The most potent 3, Nb2, 21, and 35, inhibited collagen-induced platelet aggregation with nanomolar half maximal inhibitory concentration (IC50) values and inhibited platelet aggregation under flow. The binding KD of the most potent Nb, Nb2, against recombinant monomeric and dimeric GPVI was 0.6 and 0.7 nM, respectively. The crystal structure of monomeric GPVI in complex with Nb2 revealed a binding epitope adjacent to the collagen-related peptide (CRP) binding groove within the D1 domain. In addition, a novel conformation of GPVI involving a domain swap between the D2 domains was observed. The domain swap is facilitated by the outward extension of the C-C' loop, which forms the domain swap hinge. The functional significance of this conformation was tested by truncating the hinge region so that the domain swap cannot occur. Nb2 was still able to displace collagen and CRP binding to the mutant, but signaling was abolished in a cell-based NFAT reporter assay. This demonstrates that the C-C' loop region is important for GPVI signaling but not ligand binding and suggests the domain-swapped structure may represent an active GPVI conformation.
Assuntos
Complexo Antígeno-Anticorpo , Plaquetas , Glicoproteínas da Membrana de Plaquetas , Multimerização Proteica , Anticorpos de Domínio Único , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/metabolismo , Plaquetas/química , Plaquetas/metabolismo , Humanos , Ativação Plaquetária/efeitos dos fármacos , Ativação Plaquetária/genética , Glicoproteínas da Membrana de Plaquetas/química , Glicoproteínas da Membrana de Plaquetas/genética , Glicoproteínas da Membrana de Plaquetas/metabolismo , Domínios Proteicos , Multimerização Proteica/efeitos dos fármacos , Multimerização Proteica/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/farmacologiaRESUMO
The clustering of platelet glycoprotein receptors with cytosolic YxxL and YxxM motifs, including GPVI, CLEC-2 and PEAR1, triggers activation via phosphorylation of the conserved tyrosine residues and recruitment of the tandem SH2 (Src homology 2) domain effector proteins, Syk and PI 3-kinase. We have modelled the clustering of these receptors with monovalent, divalent and tetravalent soluble ligands and with transmembrane ligands based on the law of mass action using ordinary differential equations and agent-based modelling. The models were experimentally evaluated in platelets and transfected cell lines using monovalent and multivalent ligands, including novel nanobody-based divalent and tetravalent ligands, by fluorescence correlation spectroscopy. Ligand valency, receptor number, receptor dimerisation, receptor phosphorylation and a cytosolic tandem SH2 domain protein act in synergy to drive receptor clustering. Threshold concentrations of a CLEC-2-blocking antibody and Syk inhibitor act in synergy to block platelet aggregation. This offers a strategy for countering the effect of avidity of multivalent ligands and in limiting off-target effects.
Assuntos
Glicoproteínas da Membrana de Plaquetas , Domínios de Homologia de src , Simulação por ComputadorRESUMO
The contact system is composed of factor XII (FXII), prekallikrein (PK), and cofactor high-molecular-weight kininogen (HK). The globular C1q receptor (gC1qR) has been shown to interact with FXII and HK. We reveal the FXII fibronectin type II domain (FnII) binds gC1qR in a Zn2+-dependent fashion and determined the complex crystal structure. FXIIFnII binds the gC1qR trimer in an asymmetric fashion, with residues Arg36 and Arg65 forming contacts with 2 distinct negatively charged pockets. gC1qR residues Asp185 and His187 coordinate a Zn2+ adjacent to the FXII-binding site, and a comparison with the ligand-free gC1qR crystal structure reveals the anionic G1-loop becomes ordered upon FXIIFnII binding. Additional conformational changes in the region of the Zn2+-binding site reveal an allosteric basis for Zn2+ modulation of FXII binding. Mutagenesis coupled with surface plasmon resonance demonstrate the gC1qR Zn2+ site contributes to FXII binding, and plasma-based assays reveal gC1qR stimulates coagulation in a FXII-dependent manner. Analysis of the binding of HK domain 5 (HKD5) to gC1qR shows only 1 high-affinity binding site per trimer. Mutagenesis studies identify a critical G3-loop located at the center of the gC1qR trimer, suggesting steric occlusion as the mechanism for HKD5 asymmetric binding. Gel filtration experiments reveal that gC1qR clusters FXII and HK into a higher-order 500-kDa ternary complex. These results support the conclusion that extracellular gC1qR can act as a chaperone to cluster contact factors, which may be a prelude for initiating the cascades that drive bradykinin generation and the intrinsic pathway of coagulation.
Assuntos
Sítio Alostérico , Sítios de Ligação , Proteínas de Transporte/química , Fator XII/química , Cininogênios/química , Glicoproteínas de Membrana/química , Proteínas Mitocondriais/química , Modelos Moleculares , Receptores de Complemento/química , Idoso , Proteínas de Transporte/metabolismo , Fator XII/metabolismo , Feminino , Humanos , Cinética , Cininogênios/metabolismo , Ligantes , Glicoproteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Modelos Biológicos , Simulação de Dinâmica Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Ligação Proteica , Conformação Proteica , Receptores de Complemento/metabolismo , Proteínas Recombinantes , Relação Estrutura-Atividade , Zinco/química , Zinco/metabolismoRESUMO
In specialised cells, the expression of specific tubulin isoforms and their subsequent post-translational modifications drive and coordinate unique morphologies and behaviours. The mechanisms by which ß1-tubulin, the platelet and megakaryocyte (MK) lineage restricted tubulin isoform, drives platelet production and function remains poorly understood. We investigated the roles of two key post-translational tubulin polymodifications (polyglutamylation and polyglycylation) on these processes using a cohort of thrombocytopenic patients, human induced pluripotent stem cell (iPSC) derived MKs, and healthy human donor platelets. We find distinct patterns of polymodification in MKs and platelets, mediated by the antagonistic activities of the cell specific expression of Tubulin Tyrosine Ligase Like (TTLLs) and Cytosolic Carboxypeptidase (CCP) enzymes. The resulting microtubule patterning spatially regulates motor proteins to drive proplatelet formation in megakaryocytes, and the cytoskeletal reorganisation required for thrombus formation. This work is the first to show a reversible system of polymodification by which different cell specific functions are achieved.
Assuntos
Células-Tronco Pluripotentes Induzidas , Tubulina (Proteína) , Plaquetas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Megacariócitos/metabolismo , Processamento de Proteína Pós-Traducional , Trombopoese , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMO
OBJECTIVE: GPVI (glycoprotein VI) is a key molecular player in collagen-induced platelet signaling and aggregation. Recent evidence indicates that it also plays important role in platelet aggregation and thrombus growth through interaction with fibrin(ogen). However, there are discrepancies in the literature regarding whether the monomeric or dimeric form of GPVI binds to fibrinogen at high affinity. The mechanisms of interaction are also not clear, including which region of fibrinogen is responsible for GPVI binding. We aimed to gain further understanding of the mechanisms of interaction at molecular level and to identify the regions on fibrinogen important for GPVI binding. Approach and Results: Using multiple surface- and solution-based protein-protein interaction methods, we observe that dimeric GPVI binds to fibrinogen with much higher affinity and has a slower dissociation rate constant than the monomer due to avidity effects. Moreover, our data show that the highest affinity interaction of GPVI is with the αC-region of fibrinogen. We further show that GPVI interacts with immobilized fibrinogen and fibrin variants at a similar level, including a nonpolymerizing fibrin variant, suggesting that GPVI binding is independent of fibrin polymerization. CONCLUSIONS: Based on the above findings, we conclude that the higher affinity of dimeric GPVI over the monomer for fibrinogen interaction is achieved by avidity. The αC-region of fibrinogen appears essential for GPVI binding. We propose that fibrin polymerization into fibers during coagulation will cluster GPVI through its αC-region, leading to downstream signaling, further activation of platelets, and potentially stimulating clot growth. Graphic Abstract: A graphic abstract is available for this article.
Assuntos
Fibrinogênio/metabolismo , Fragmentos de Peptídeos/sangue , Glicoproteínas da Membrana de Plaquetas/metabolismo , Animais , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Produtos de Degradação da Fibrina e do Fibrinogênio/química , Produtos de Degradação da Fibrina e do Fibrinogênio/metabolismo , Fibrinogênio/química , Humanos , Técnicas In Vitro , Camundongos , Microscopia de Força Atômica , Fragmentos de Peptídeos/química , Peptídeos/química , Peptídeos/metabolismo , Agregação Plaquetária/fisiologia , Glicoproteínas da Membrana de Plaquetas/química , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Transdução de Sinais , Ressonância de Plasmônio de SuperfícieRESUMO
Understanding the pathways involved in the formation and stability of the core and shell regions of a platelet-rich arterial thrombus may result in new ways to treat arterial thrombosis. The distinguishing feature between these two regions is the absence of fibrin in the shell which indicates that in vitro flow-based assays over thrombogenic surfaces, in the absence of coagulation, can be used to resemble this region. In this study, we have investigated the contribution of Syk tyrosine kinase in the stability of platelet aggregates (or thrombi) formed on collagen or atherosclerotic plaque homogenate at arterial shear (1000 s-1). We show that post-perfusion of the Syk inhibitor PRT-060318 over preformed thrombi on both surfaces enhances thrombus breakdown and platelet detachment. The resulting loss of thrombus stability led to a reduction in thrombus contractile score which could be detected as early as 3 min after perfusion of the Syk inhibitor. A similar loss of thrombus stability was observed with ticagrelor and indomethacin, inhibitors of platelet adenosine diphosphate (ADP) receptor and thromboxane A2 (TxA2), respectively, and in the presence of the Src inhibitor, dasatinib. In contrast, the Btk inhibitor, ibrutinib, causes only a minor decrease in thrombus contractile score. Weak thrombus breakdown is also seen with the blocking GPVI nanobody, Nb21, which indicates, at best, a minor contribution of collagen to the stability of the platelet aggregate. These results show that Syk regulates thrombus stability in the absence of fibrin in human platelets under flow and provide evidence that this involves pathways additional to activation of GPVI by collagen.
Assuntos
Resistência ao Cisalhamento , Quinase Syk/metabolismo , Trombose/enzimologia , Colágeno/metabolismo , Humanos , Fosforilação , Adesividade Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas , Glicoproteínas da Membrana de Plaquetas/metabolismo , Anticorpos de Domínio Único/metabolismo , Quinase Syk/antagonistas & inibidores , Temperatura , Trombina/farmacologiaRESUMO
GPVI is a critical signaling receptor responsible for collagen-induced platelet activation and a promising anti-thrombotic target in conditions such as coronary artery thrombosis, ischemic stroke, and atherothrombosis. This is due to the ability to block GPVI while having minimal effects on hemostasis, making it a more attractive target over current dual-antiplatelet therapy (DAPT) with acetyl salicylic acid and P2Y12 inhibitors where bleeding can be a problem. Our current understanding of how the structure of GPVI relates to function is inadequate and recent studies contradict each other. In this article, we summarize the structure-function relationships underlying the activation of GPVI by its major ligands, including collagen, fibrin(ogen), snake venom toxins and charged exogenous ligands such as diesel exhaust particles. We argue that contrary to popular belief dimerization of GPVI is not required for binding to collagen but serves to facilitate binding through increased avidity, and that GPVI is expressed as a mixture of monomers and dimers on resting platelets, with binding of multivalent ligands inducing higher order clustering.
Assuntos
Glicoproteínas da Membrana de Plaquetas/farmacologia , Humanos , Relação Estrutura-AtividadeRESUMO
Charge interactions play a critical role in the activation of the innate immune system by damage- and pathogen-associated molecular pattern receptors. The ability of these receptors to recognize a wide spectrum of ligands through a common mechanism is critical in host defense. In this article, we argue that platelet glycoprotein receptors that signal through conserved tyrosine-based motifs function as pattern recognition receptors (PRRs) for charged endogenous and exogenous ligands, including sulfated polysaccharides, charged proteins and nanoparticles. This is exemplified by GPVI, CLEC-2 and PEAR1 which are activated by a wide spectrum of endogenous and exogenous ligands, including diesel exhaust particles, sulfated polysaccharides and charged surfaces. We propose that this mechanism has evolved to drive rapid activation of platelets at sites of injury, but that under some conditions it can drive occlusive thrombosis, for example, when blood comes into contact with infectious agents or toxins. In this Opinion Article, we discuss mechanisms behind charge-mediated platelet activation and opportunities for designing nanoparticles and related agents such as dendrimers as novel antithrombotics.
Assuntos
Plaquetas/metabolismo , Nanopartículas/metabolismo , Ativação Plaquetária/imunologia , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Humanos , Ligantes , Transdução de SinaisRESUMO
GPVI is the major signalling receptor for collagen on platelets. Dimerization of GPVI is required for collagen binding and initiation of signalling through the associated FcR-γ chain. Recently, fibrin and fibrinogen have been identified as ligands for GPVI and shown to induce signalling in support of thrombus formation and stabilization. Contrasting observations have been reported on whether fibrin binds to monomeric or dimeric GPVI, or to neither form. In this article, we discuss reasons for the contradictory results and how to reconcile these. We conclude that a lack of structural knowledge regarding the GPVI constructs that are being used, along with the use of non-standardized reagents, might be the main cause of the discrepant results. This article aims to highlight some of the key areas that need to be addressed.
Assuntos
Plaquetas/metabolismo , Fibrina/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Humanos , Ligação ProteicaRESUMO
Glycoprotein (GP) VI (GPVI) 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 characterized, and two of these inhibitors, glenzocimab and revacept, have completed Phase II clinical trials in ischaemic stroke. In this review, we summarize mechanisms of GPVI activation and the latest progress of clinically tested GPVI inhibitors, including their mechanisms of action. By focusing on what is known about GPVI activation, we also discuss whether alternate strategies could be used to target GPVI.
Assuntos
Fibrinolíticos , Glicoproteínas da Membrana de Plaquetas , Humanos , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Glicoproteínas da Membrana de Plaquetas/metabolismo , Animais , Fibrinolíticos/uso terapêutico , Fibrinolíticos/efeitos adversos , Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/prevenção & controle , Transdução de Sinais/efeitos dos fármacos , Resultado do Tratamento , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Trombose/prevenção & controle , Trombose/tratamento farmacológico , Trombose/sangue , Inibidores da Agregação Plaquetária/uso terapêutico , Inibidores da Agregação Plaquetária/efeitos adversosRESUMO
BACKGROUND: Clustering of the receptors glycoprotein receptor VI (GPVI), C-type lectin-like receptor 2 (CLEC-2), low-affinity immunoglobulin γ Fc region receptor II-a (FcγRIIA), and platelet endothelial aggregation receptor 1 (PEAR1) leads to powerful activation of platelets through phosphorylation of tyrosine in their cytosolic tails and initiation of downstream signaling cascades. GPVI, CLEC-2, and FcγRIIA signal through YxxL motifs that activate Syk. PEAR1 signals through a YxxM motif that activates phosphoinositide 3-kinase. Current ligands for these receptors have an undefined valency and show significant batch variation and, for some, uncertain specificity. OBJECTIVES: We have raised nanobodies against each of these receptors and multimerized them to identify the minimum number of epitopes to achieve robust activation of human platelets. METHODS: Divalent and trivalent nanobodies were generated using a flexible glycine-serine linker. Tetravalent nanobodies utilize a mouse Fc domain (IgG2a, which does not bind to FcγRIIA) to dimerize the divalent nanobody. Ligand affinity measurements were determined by surface plasmon resonance. Platelet aggregation, adenosine triphosphate secretion, and protein phosphorylation were analyzed using standardized methods. RESULTS: Multimerization of the nanobodies led to a stepwise increase in affinity with divalent and higher-order nanobody oligomers having sub-nanomolar affinity. The trivalent nanobodies to GPVI, CLEC-2, and PEAR1 stimulated powerful and robust platelet aggregation, secretion, and protein phosphorylation at low nanomolar concentrations. A tetravalent nanobody was required to activate FcγRIIA with the concentration-response relationship showing a greater variability and reduced sensitivity compared with the other nanobody-based ligands, despite a sub-nanomolar binding affinity. CONCLUSION: The multivalent nanobodies represent a series of standardized, potent agonists for platelet glycoprotein receptors. They have applications as research tools and in clinical assays.
Assuntos
Glicoproteínas de Membrana , Anticorpos de Domínio Único , Humanos , Camundongos , Animais , Glicoproteínas de Membrana/metabolismo , Ligantes , Fosfatidilinositol 3-Quinases/metabolismo , Anticorpos de Domínio Único/metabolismo , Quinase Syk , Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Agregação Plaquetária , Lectinas Tipo C/metabolismo , Ativação Plaquetária , Receptores de Superfície Celular/metabolismoRESUMO
ABSTRACT: Glycoprotein VI (GPVI) plays a key role in collagen-induced platelet aggregation. Affimers are engineered binding protein alternatives to antibodies. We screened and characterized GPVI-binding Affimers as novel tools to probe GPVI function. Among the positive clones, M17, D22, and D18 bound GPVI with the highest affinities (dissociation constant (KD) in the nanomolar range). These Affimers inhibited GPVI-collagen-related peptide (CRP)-XL/collagen interactions, CRP-XL/collagen-induced platelet aggregation, and D22 also inhibited in vitro thrombus formation on a collagen surface under flow. D18 bound GPVI dimer but not monomer. GPVI binding was increased for D18 but not M17/D22 upon platelet activation by CRP-XL and adenosine 5'-diphosphate. D22 but not M17/D18 displaced nanobody 2 (Nb2) binding to GPVI, indicating similar epitopes for D22 with Nb2 but not for M17/D18. Mapping of binding sites revealed that D22 binds a site that overlaps with Nb2 on the D1 domain, whereas M17 targets a site on the D2 domain, overlapping in part with the glenzocimab binding site, a humanized GPVI antibody fragment antigen-binding fragment. D18 targets a new region on the D2 domain. We found that D18 is a stable noncovalent dimer and forms a stable complex with dimeric GPVI with 1:1 stoichiometry. Taken together, our data demonstrate that Affimers modulate GPVI-ligand interactions and bind different sites on GPVI D1/D2 domains. D18 is dimer-specific and could be used as a tool to detect GPVI dimerization or clustering in platelets. A dimeric epitope regulating ligand binding was identified on the GPVI D2 domain, which could be used for the development of novel bivalent antithrombotic agents selectively targeting GPVI dimer on platelets.
Assuntos
Plaquetas , Agregação Plaquetária , Glicoproteínas da Membrana de Plaquetas , Ligação Proteica , Multimerização Proteica , Glicoproteínas da Membrana de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/química , Humanos , Plaquetas/metabolismo , Ligantes , Agregação Plaquetária/efeitos dos fármacos , Sítios de Ligação , Colágeno/metabolismo , Colágeno/química , Proteínas de Transporte , PeptídeosRESUMO
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.
Assuntos
Colágeno , Glicoproteínas da Membrana de Plaquetas , Humanos , Dimerização , Ligação Proteica , Ligantes , Glicoproteínas da Membrana de Plaquetas/metabolismo , Sítios de Ligação , Colágeno/metabolismo , Plaquetas/metabolismoRESUMO
BACKGROUND: Platelet endothelial aggregation receptor 1 (PEAR1) is a single-transmembrane orphan receptor primarily expressed on platelets and endothelial cells. Genetic variants of PEAR1 have repeatedly and independently been identified to be associated with cardiovascular diseases, including coronary artery disease. OBJECTIVES: We have identified sulfated fucoidans and their mimetics as ligands for PEAR1 and proposed that its endogenous ligand is a sulfated proteoglycan. The aim of this study was to test this hypothesis. METHODS: A heparin proteoglycan-mimetic (HPGM) was created by linking unfractionated heparin (UFH) to albumin. The ability of the HPGM, UFH and selectively desulfated heparins to stimulate platelet aggregation and protein phosphorylation was investigated. Nanobodies against the 12th to 13th epidermal growth factor-like repeat of PEAR1 and phosphoinositide 3-kinase (PI3K) isoform-selective inhibitors were tested for the inhibition of platelet activation. RESULTS: We show that HPGM, heparin conjugated to an albumin protein core, stimulates aggregation and phosphorylation of PEAR1 in washed platelets. Platelet aggregation was abolished by an anti-PEAR1 nanobody, Nb138. UFH stimulated platelet aggregation in washed platelets, but desulfated UFH did not. Furthermore, HPGM, but not UFH, stimulated maximal aggregation in platelet-rich plasma. However, both HPGM and UFH increased integrin αIIbß3 activation in whole blood. By using PI3K isoform-selective inhibitors, we show that PEAR1 activates PI3Kß, leading to Akt phosphorylation. CONCLUSION: Our findings reveal that PEAR1 is a receptor for heparin and HPGM and that PI3Kß is a key signaling molecule downstream of PEAR1 in platelets. These findings may have important implications for our understanding of the role of PEAR1 in cardiovascular disease.
Assuntos
Heparina , Fosfatidilinositol 3-Quinases , Humanos , Heparina/farmacologia , Heparina/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Células Endoteliais/metabolismo , Receptores de Superfície Celular/metabolismo , Plaquetas/metabolismo , Agregação Plaquetária , Proteoglicanas/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Ligantes , AlbuminasRESUMO
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.
Assuntos
Glicoproteínas da Membrana de Plaquetas , Trombose , Humanos , Glicoproteínas da Membrana de Plaquetas/metabolismo , Colágeno/metabolismo , Trombose/tratamento farmacológico , Trombose/etiologia , Trombose/prevenção & controle , Fibrina/metabolismoRESUMO
These illustrated capsules have been prepared by some speakers of State-of-the-Art talks and of original investigations, presented at the 5th European Platelet Network (EUPLAN) International Conference, which was held at the Università degli Studi di Milano (Italy) on September 28-30, 2022. The programme featured various state-of-the-art lectures and a selection of oral presentations covering a broad range of topics in platelet and megakaryocyte biology, from basic science to recent advances in clinical studies. As usual, the meeting brought together senior scientists and trainees in an informal atmosphere to discuss platelet science in person.