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1.
Biophys J ; 122(4): 697-712, 2023 02 21.
Artículo en Inglés | MEDLINE | ID: mdl-36635963

RESUMEN

During clotting under flow, thrombin rapidly generates fibrin, whereas fibrin potently sequesters thrombin. This co-regulation was studied using microfluidic whole blood clotting on collagen/tissue factor, followed by buffer wash, and a start/stop cycling flow assay using the thrombin fluorogenic substrate, Boc-Val-Pro-Arg-AMC. After 3 min of clotting (100 s-1) and 5 min of buffer wash, non-elutable thrombin activity was easily detected during cycles of flow cessation. Non-elutable thrombin was similarly detected in plasma clots or arterial whole blood clots (1000 s-1). This thrombin activity was ablated by Phe-Pro-Arg-chloromethylketone (PPACK), apixaban, or Gly-Pro-Arg-Pro to inhibit fibrin. Reaction-diffusion simulations predicted 108 nM thrombin within the clot. Heparin addition to the start/stop assay had little effect on fibrin-bound thrombin, whereas addition of heparin-antithrombin (AT) required over 6 min to inhibit the thrombin, indicating a substantial diffusion limitation. In contrast, heparin-AT rapidly inhibited thrombin within microfluidic plasma clots, indicating marked differences in fibrin structure and functionality between plasma clots and whole blood clots. Addition of GPVI-Fab to blood before venous or arterial clotting (200 or 1000 s-1) markedly reduced fibrin-bound thrombin, whereas GPVI-Fab addition after 90 s of clotting had no effect. Perfusion of AF647-fibrinogen over washed fluorescein isothiocyanate (FITC)-fibrin clots resulted in an intense red layer around, but not within, the original FITC-fibrin. Similarly, introduction of plasma/AF647-fibrinogen generated substantial red fibrin masses that did not penetrate the original green clots, demonstrating that fibrin cannot be re-clotted with fibrinogen. Overall, thrombin within fibrin is non-elutable, easily accessed by peptides, slowly accessed by average-sized proteins (heparin/AT), and not accessible to fresh fibrinogen.


Asunto(s)
Fibrina , Trombina , Trombosis , Humanos , Fibrina/química , Fibrinógeno/metabolismo , Fluoresceína-5-Isotiocianato , Heparina , Microfluídica/métodos , Trombina/química , Trombosis/metabolismo , Glicoproteínas de Membrana Plaquetaria/química
2.
PLoS One ; 17(1): e0262695, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35041713

RESUMEN

OBJECTIVES: Platelet activation underpins thrombus formation in ischemic stroke. The active, dimeric form of platelet receptor glycoprotein (GP) VI plays key roles by binding platelet ligands collagen and fibrin, leading to platelet activation. We investigated whether patients presenting with stroke expressed more GPVI on their platelet surface and had more active circulating platelets as measured by platelet P-selectin exposure. METHODS: 129 ischemic or hemorrhagic stroke patients were recruited within 8h of symptom onset. Whole blood was analyzed for platelet-surface expression of total GPVI, GPVI-dimer, and P-selectin by flow cytometry at admission and day-90 post-stroke. Results were compared against a healthy control population (n = 301). RESULTS: The platelets of stroke patients expressed significantly higher total GPVI and GPVI-dimer (P<0.0001) as well as demonstrating higher resting P-selectin exposure (P<0.0001), a measure of platelet activity, compared to the control group, suggesting increased circulating platelet activation. GPVI-dimer expression was strongly correlated circulating platelet activation [r2 = 0.88, P<0.0001] in stroke patients. Furthermore, higher platelet surface GPVI expression was associated with increased stroke severity at admission. At day-90 post-stroke, GPVI-dimer expression and was further raised compared to the level at admission (P<0.0001) despite anti-thrombotic therapy. All ischemic stroke subtypes and hemorrhagic strokes expressed significantly higher GPVI-dimer compared to controls (P<0.0001). CONCLUSIONS: Stroke patients express more GPVI-dimer on their platelet surface at presentation, lasting at least until day-90 post-stroke. Small molecule GPVI-dimer inhibitors are currently in development and the results of this study validate that GPVI-dimer as an anti-thrombotic target in ischemic stroke.


Asunto(s)
Biomarcadores/sangre , Activación Plaquetaria , Adhesividad Plaquetaria , Glicoproteínas de Membrana Plaquetaria/análisis , Accidente Cerebrovascular/diagnóstico , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Glicoproteínas de Membrana Plaquetaria/química , Glicoproteínas de Membrana Plaquetaria/metabolismo , Pronóstico , Multimerización de Proteína , Accidente Cerebrovascular/metabolismo
3.
Thromb Haemost ; 121(11): 1435-1447, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-33638140

RESUMEN

Collagen has been proposed to bind to a unique epitope in dimeric glycoprotein VI (GPVI) and the number of GPVI dimers has been reported to increase upon platelet activation. However, in contrast, the crystal structure of GPVI in complex with collagen-related peptide (CRP) showed binding distinct from the site of dimerization. Further fibrinogen has been reported to bind to monomeric but not dimeric GPVI. In the present study, we have used the advanced fluorescence microscopy techniques of single-molecule microscopy, fluorescence correlation spectroscopy (FCS) and bioluminescence resonance energy transfer (BRET), and mutagenesis studies in a transfected cell line model to show that GPVI is expressed as a mixture of monomers and dimers and that dimerization through the D2 domain is not critical for activation. As many of these techniques cannot be applied to platelets to resolve this issue, due to the high density of GPVI and its anucleate nature, we used Förster resonance energy transfer (FRET) to show that endogenous GPVI is at least partially expressed as a dimer on resting and activated platelet membranes. We propose that GPVI may be expressed as a monomer on the cell surface and it forms dimers in the membrane through diffusion, giving rise to a mixture of monomers and dimers. We speculate that the formation of dimers facilitates ligand binding through avidity.


Asunto(s)
Plaquetas/metabolismo , Membrana Celular/metabolismo , Colágeno/metabolismo , Glicoproteínas de Membrana Plaquetaria/metabolismo , Sitios de Unión , Transferencia de Energía por Resonancia de Bioluminiscencia , Células HEK293 , Humanos , Ligandos , Microscopía Fluorescente , Mutación , Glicoproteínas de Membrana Plaquetaria/química , Glicoproteínas de Membrana Plaquetaria/genética , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Imagen Individual de Molécula , Espectrometría de Fluorescencia , Relación Estructura-Actividad
4.
Biochimie ; 184: 1-7, 2021 May.
Artículo en Inglés | MEDLINE | ID: mdl-33548391

RESUMEN

Glycoprotein (GP)Ib that binds von Willebrand factor (vWF) and glycoprotein (GP)VI, that binds collagen play a significant role in platelet activation and aggregation, and are potential targets for antithrombotic treatment. They are targeted by snake venom proteinases. The effect of a such proteinase, mutalysin-II, on platelet aggregation was examined using washed human platelets and platelet-rich plasma. Its proteolytic activity on vWF, on its binding partner GPIbα, and on GPVI was analyzed by SDS-PAGE, and immunodetection with the corresponding antibodies after blotting. Dose- and time-dependently, mutalysin-II inhibits aggregation of washed platelets induced by vWF plus ristocetin and by convulxin, but with no significant effect on platelet-rich-plasma. Furthermore, mutalysin-II cleaves vWF into low molecular mass multimers of vWF and a rvWF-A1 domain to realease a ∼27-kDa fragment detectable by SDS-PAGE and blotting with mouse anti-rvWF-A1-domain IgG. Moreover, GPVI was cut by mutalysin-II into a soluble ∼55-kDa ectodomain and a fragment of ∼35-kDa. Thus, mutalysin-II inhibits vWF-induced platelet aggregation via cleavage of bound vWF-A1, and its receptor GPIbα. The additional cleavage of, GPVI, blocks collagen-induced platelets. Our data highlight mutalysin-II as an interesting platelet-directed tool targeting vWF-GPIbα binding and particularly GPVI. Thus, it might be suited for antithrombotic therapy as its combined inactivation of two receptors does not significantly compromise hemostasis, but shows high efficacy and safety. Studies are needed to further develop and demonstrate its potential benefits.


Asunto(s)
Plaquetas/química , Metaloendopeptidasas/química , Inhibidores de Agregación Plaquetaria/química , Complejo GPIb-IX de Glicoproteína Plaquetaria/química , Glicoproteínas de Membrana Plaquetaria/química , Venenos de Serpiente/química , Animales , Plaquetas/metabolismo , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Glicoproteínas de Membrana Plaquetaria/metabolismo
5.
Blood ; 137(24): 3443-3453, 2021 06 17.
Artículo en Inglés | MEDLINE | ID: mdl-33512486

RESUMEN

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.


Asunto(s)
Complejo Antígeno-Anticuerpo , Plaquetas , Glicoproteínas de Membrana Plaquetaria , Multimerización de Proteína , Anticuerpos de Dominio Único , Complejo Antígeno-Anticuerpo/química , Complejo Antígeno-Anticuerpo/metabolismo , Plaquetas/química , Plaquetas/metabolismo , Humanos , Activación Plaquetaria/efectos de los fármacos , Activación Plaquetaria/genética , Glicoproteínas de Membrana Plaquetaria/química , Glicoproteínas de Membrana Plaquetaria/genética , Glicoproteínas de Membrana Plaquetaria/metabolismo , Dominios Proteicos , Multimerización de Proteína/efectos de los fármacos , Multimerización de Proteína/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/farmacología
6.
Arterioscler Thromb Vasc Biol ; 41(3): 1092-1104, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33472402

RESUMEN

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.


Asunto(s)
Fibrinógeno/metabolismo , Fragmentos de Péptidos/sangre , Glicoproteínas de Membrana Plaquetaria/metabolismo , Animales , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Productos de Degradación de Fibrina-Fibrinógeno/química , Productos de Degradación de Fibrina-Fibrinógeno/metabolismo , Fibrinógeno/química , Humanos , Técnicas In Vitro , Ratones , Microscopía de Fuerza Atómica , Fragmentos de Péptidos/química , Péptidos/química , Péptidos/metabolismo , Agregación Plaquetaria/fisiología , Glicoproteínas de Membrana Plaquetaria/química , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Transducción de Señal , Resonancia por Plasmón de Superficie
7.
Nanomedicine ; 29: 102274, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32712174

RESUMEN

Thrombus formation at athero-thrombotic sites is initiated by the exposure of collagen followed by platelet adhesion mediated by the platelet-specific collagen receptor glycoprotein VI (GPVI). Here, dimeric GPVI was used as a targeting motif to functionalize polymeric nanoparticle-based drug carriers and to show that with proper design, such GPVI-coated nanoparticles (GPNs) can efficiently and specifically target arterial injury sites while withstanding physiological flow. In a microfluidic model, under physiological shear levels (1-40 dyne/cm2), 200 nm and 2 µm GPNs exhibited a >60 and >10-fold increase in binding to collagen compared to control particles, respectively. In vitro experiments in an arterial stenosis injury model, subjected to physiological pulsatile flow, showed shear-enhanced adhesion of 200 nm GPNs at the stenosis region which was confirmed in vivo in a mice ligation carotid injury model using intravital microscopy. Altogether, our results illustrate how engineering tools can be harnessed to design nano-carriers that efficiently target cardiovascular disease sites.


Asunto(s)
Aterosclerosis/tratamiento farmacológico , Traumatismos de las Arterias Carótidas/tratamiento farmacológico , Nanopartículas/química , Glicoproteínas de Membrana Plaquetaria/farmacología , Animales , Aterosclerosis/patología , Plaquetas/efectos de los fármacos , Arterias Carótidas/efectos de los fármacos , Arterias Carótidas/patología , Traumatismos de las Arterias Carótidas/patología , Modelos Animales de Enfermedad , Humanos , Ratones , Activación Plaquetaria/efectos de los fármacos , Adhesividad Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Glicoproteínas de Membrana Plaquetaria/química
8.
J Med Chem ; 63(21): 12213-12242, 2020 11 12.
Artículo en Inglés | MEDLINE | ID: mdl-32463237

RESUMEN

Pathogenic thrombus formation accounts for the etiology of many serious conditions including myocardial infarction, stroke, deep vein thrombosis, and pulmonary embolism. Despite the development of numerous anticoagulants and antiplatelet agents, the mortality rate associated with these diseases remains high. In recent years, however, significant epidemiological evidence and clinical models have emerged to suggest that modulation of the glycoprotein VI (GPVI) platelet receptor could be harnessed as a novel antiplatelet strategy. As such, many peptidic agents have been described in the past decade, while more recent efforts have focused on the development of small molecule modulators. Herein the rationale for targeting GPVI is summarized and the published GPVI modulators are reviewed, with particular focus on small molecules. A qualitative pharmacophore hypothesis for small molecule ligands at GPVI is also presented.


Asunto(s)
Inhibidores de Agregación Plaquetaria/uso terapéutico , Glicoproteínas de Membrana Plaquetaria/metabolismo , Trombosis/tratamiento farmacológico , Sitios de Unión , Productos Biológicos/química , Productos Biológicos/metabolismo , Humanos , Ligandos , Losartán/análogos & derivados , Losartán/metabolismo , Losartán/uso terapéutico , Simulación de Dinámica Molecular , Inhibidores de Agregación Plaquetaria/química , Inhibidores de Agregación Plaquetaria/farmacología , Glicoproteínas de Membrana Plaquetaria/química , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Trombosis/patología
9.
ACS Nano ; 14(2): 2024-2035, 2020 02 25.
Artículo en Inglés | MEDLINE | ID: mdl-31927980

RESUMEN

Early diagnosis and treatment of acute ischemic stroke poses a significant challenge due to its suddenness and short therapeutic time window. Human endogenous cells derived biomimetic drug carriers have provided new options for stroke theranostics since these cells have higher biosafety and targeting abilities than artificial carriers. Inspired by natural platelets (PLTs) and their role in targeting adhesion to the damaged blood vessel during thrombus formation, we fabricated a biomimetic nanocarrier comprising a PLT membrane envelope loaded with l-arginine and γ-Fe2O3 magnetic nanoparticles (PAMNs) for thrombus-targeted delivery of l-arginine and in situ generation of nitric oxide (NO). Results demonstrate that the engineered 200 nm PAMNs inherit the natural properties of the PLT membrane and achieve rapid targeting to ischemic stroke lesions under the guidance of an external magnetic field. Subsequent to the release of l-arginine at the thrombus site, endothelial cells produce NO, which promotes vasodilation to disrupt the local PLT aggregation. Rapid targeting of PAMNs to stroke lesions as well as in situ generation of NO prompts vasodilation, recovery of blood flow, and reperfusion of the stroke microvascular. Thus, these PLT membrane derived nanocarriers are diagnostically beneficial for localizing stroke lesions and a promising modality for executing therapies.


Asunto(s)
Materiales Biomiméticos/química , Sistemas de Liberación de Medicamentos , Accidente Cerebrovascular Isquémico/metabolismo , Nanopartículas de Magnetita/química , Óxido Nítrico/biosíntesis , Glicoproteínas de Membrana Plaquetaria/química , Animales , Células Cultivadas , Portadores de Fármacos/química , Células Endoteliales/metabolismo , Humanos , Accidente Cerebrovascular Isquémico/diagnóstico , Campos Magnéticos , Masculino , Ratones , Ratones Endogámicos C57BL , Tamaño de la Partícula , Agregación Plaquetaria , Propiedades de Superficie
10.
Molecules ; 24(19)2019 Sep 26.
Artículo en Inglés | MEDLINE | ID: mdl-31561469

RESUMEN

Atroxlysin-III (Atr-III) was purified from the venom of Bothrops atrox. This 56-kDa protein bears N-linked glycoconjugates and is a P-III hemorrhagic metalloproteinase. Its cDNA-deduced amino acid sequence reveals a multidomain structure including a proprotein, a metalloproteinase, a disintegrin-like and a cysteine-rich domain. Its identity with bothropasin and jararhagin from Bothrops jararaca is 97% and 95%, respectively. Its enzymatic activity is metal ion-dependent. The divalent cations, Mg2+ and Ca2+, enhance its activity, whereas excess Zn2+ inhibits it. Chemical modification of the Zn2+-complexing histidine residues within the active site by using diethylpyrocarbonate (DEPC) inactivates it. Atr-III degrades plasma fibronectin, type I-collagen, and mainly the α-chains of fibrinogen and fibrin. The von Willebrand factor (vWF) A1-domain, which harbors the binding site for GPIb, is not hydrolyzed. Platelets interact with collagen via receptors for collagen, glycoprotein VI (GPVI), and α2ß1 integrin. Neither the α2ß1 integrin nor its collagen-binding A-domain is fragmented by Atr-III. In contrast, Atr-III cleaves glycoprotein VI (GPVI) into a soluble ~55-kDa fragment (sGPVI). Thereby, it inhibits aggregation of platelets which had been stimulated by convulxin, a GPVI agonist. Selectively, Atr-III targets GPVI antagonistically and thus contributes to the antithrombotic effect of envenomation by Bothrops atrox.


Asunto(s)
Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Venenos de Crotálidos/enzimología , Crotalinae , Metaloproteasas/farmacología , Glicoproteínas de Membrana Plaquetaria/biosíntesis , Secuencia de Aminoácidos , Animales , Crotalinae/metabolismo , Matriz Extracelular , Metaloproteasas/química , Metaloproteasas/genética , Metaloproteasas/aislamiento & purificación , Modelos Moleculares , Filogenia , Glicoproteínas de Membrana Plaquetaria/antagonistas & inhibidores , Glicoproteínas de Membrana Plaquetaria/química , Conformación Proteica , Proteolisis , Relación Estructura-Actividad
11.
J Thromb Haemost ; 17(9): 1500-1510, 2019 09.
Artículo en Inglés | MEDLINE | ID: mdl-31145836

RESUMEN

OBJECTIVE: Clinical and laboratory studies have demonstrated that platelets become hyperactive and prothrombotic in conditions of inflammation. We have previously shown that the proinflammatory cytokine interleukin (IL)-6 forms a complex with soluble IL-6 receptor α (sIL-6Rα) to prime platelets for activation by subthreshold concentrations of collagen. Upon being stimulated with collagen, the transcription factor signal transducer and activator of transcription (STAT) 3 in platelets is phosphorylated and dimerized to act as a protein scaffold to facilitate the catalytic action between the kinase Syk and the substrate phospholipase Cγ2 (PLCγ2) in collagen-induced signaling. However, it remains unknown how collagen induces phosphorylation and dimerization of STAT3. METHODS AND RESULTS: We conducted complementary in vitro experiments to show that the IL-6 receptor subunit glycoprotein 130 (GP130) was in physical proximity to the collagen receptor glycoprotein VI (GPVI in membrane lipid rafts of platelets. This proximity allows collagen to induce STAT3 activation and dimerization, and the IL-6-sIL-6Rα complex to activate the kinase Syk and the substrate PLCγ2 in the GPVI signal pathway, resulting in an enhanced platelet response to collagen. Disrupting lipid rafts or blocking GP130-Janus tyrosine kinase (JAK)-STAT3 signaling abolished the cross-activation and reduced platelet reactivity to collagen. CONCLUSION: These results demonstrate cross-talk between collagen and IL-6 signal pathways. This cross-talk could potentially provide a novel mechanism for inflammation-induced platelet hyperactivity, so the IL-6-GP130-JAK-STAT3 pathway has been identified as a potential target to block this hyperactivity.


Asunto(s)
Plaquetas/metabolismo , Receptor gp130 de Citocinas/sangre , Microdominios de Membrana/fisiología , Glicoproteínas de Membrana Plaquetaria/fisiología , Coagulación Sanguínea/efectos de los fármacos , Colágeno/farmacología , Receptor gp130 de Citocinas/química , Hemorreología , Humanos , Inmunoprecipitación , Janus Quinasa 2/antagonistas & inhibidores , Janus Quinasa 2/sangre , Fosfolipasa C gamma/sangre , Fosforilación , Agregación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/fisiología , Glicoproteínas de Membrana Plaquetaria/química , Mapeo de Interacción de Proteínas , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional , Factor de Transcripción STAT3/sangre
12.
Thromb Haemost ; 119(1): 104-116, 2019 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-30597505

RESUMEN

Platelet activators stimulate post-translational modification of signalling proteins to change their activity or their molecular interactions leading to signal propagation. One covalent modification is attachment of the small protein ubiquitin to lysine residues in target proteins. Modification by ubiquitin can either target proteins for degradation by the proteasome or act as a scaffold for other proteins. Pharmacological inhibition of deubiquitylases or the proteasome inhibition of platelet activation by collagen, demonstrating a role for ubiquitylation, but relatively few substrates for ubiquitin have been identified and the molecular basis of inhibition is not established. Here, we report the ubiquitome of human platelets and changes in ubiquitylated proteins following stimulation by collagen-related peptide (CRP-XL). Using platelets from six individuals over three independent experiments, we identified 1,634 ubiquitylated peptides derived from 691 proteins, revealing extensive ubiquitylation in resting platelets. Note that 925 of these peptides show an increase of more than twofold following stimulation with CRP-XL. Multiple sites of ubiquitylation were identified on several proteins including Syk, filamin and integrin heterodimer sub-units. This work reveals extensive protein ubiquitylation during activation of human platelets and opens the possibility of novel therapeutic interventions targeting the ubiquitin machinery.


Asunto(s)
Plaquetas/metabolismo , Glicoproteínas de Membrana Plaquetaria/química , Ubiquitina/química , Ubiquitinación , Plaquetas/efectos de los fármacos , Citometría de Flujo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lisina/química , Espectrometría de Masas , Selectina-P/metabolismo , Activación Plaquetaria , Complejo de la Endopetidasa Proteasomal/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal
13.
J Nat Prod ; 81(9): 1968-1975, 2018 09 28.
Artículo en Inglés | MEDLINE | ID: mdl-30188730

RESUMEN

Three new tetrahydrobenzofuran-6(2 H)-one-type neolignans, heterochromins A-C (1-3), along with a bicyclo[3.2.1]octane neolignan, cinerin C (4), were isolated from an ethanol extract from the leaves of Ocotea heterochroma, a native plant growing in the Colombo-Ecuadorian region of the Andes. The chemical structures of 1-3 were elucidated by spectroscopic methods. The platelet activating factor (PAF) antagonistic activity was tested in vitro for these compounds. Additionally, their binding mode to the PAF receptor was studied by molecular docking and molecular dynamics simulations in order to rationalize such activity. Heterochromin A (1) was found to be a potent PAF antagonist with a favorable molecular profile for interacting with the PAF receptor binding site.


Asunto(s)
Lignanos/aislamiento & purificación , Ocotea/química , Factor de Activación Plaquetaria/antagonistas & inhibidores , Glicoproteínas de Membrana Plaquetaria/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Lignanos/química , Lignanos/metabolismo , Lignanos/farmacología , Espectroscopía de Resonancia Magnética , Simulación del Acoplamiento Molecular , Glicoproteínas de Membrana Plaquetaria/química , Receptores Acoplados a Proteínas G/química , Relación Estructura-Actividad
14.
Nat Struct Mol Biol ; 25(6): 488-495, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-29808000

RESUMEN

Platelet-activating-factor receptor (PAFR) responds to platelet-activating factor (PAF), a phospholipid mediator of cell-to-cell communication that exhibits diverse physiological effects. PAFR is considered an important drug target for treating asthma, inflammation and cardiovascular diseases. Here we report crystal structures of human PAFR in complex with the antagonist SR 27417 and the inverse agonist ABT-491 at 2.8-Å and 2.9-Å resolution, respectively. The structures, supported by molecular docking of PAF, provide insights into the signal-recognition mechanisms of PAFR. The PAFR-SR 27417 structure reveals an unusual conformation showing that the intracellular tips of helices II and IV shift outward by 13 Å and 4 Å, respectively, and helix VIII adopts an inward conformation. The PAFR structures, combined with single-molecule FRET and cell-based functional assays, suggest that the conformational change in the helical bundle is ligand dependent and plays a critical role in PAFR activation, thus greatly extending knowledge about signaling by G-protein-coupled receptors.


Asunto(s)
Glicoproteínas de Membrana Plaquetaria/química , Glicoproteínas de Membrana Plaquetaria/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Sitios de Unión , Cristalografía por Rayos X , Transferencia Resonante de Energía de Fluorescencia , Humanos , Enlace de Hidrógeno , Imidazoles/farmacología , Indoles/farmacología , Ligandos , Simulación del Acoplamiento Molecular , Inhibidores de Agregación Plaquetaria/farmacología , Glicoproteínas de Membrana Plaquetaria/agonistas , Glicoproteínas de Membrana Plaquetaria/antagonistas & inhibidores , Conformación Proteica , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Tiazoles/farmacología
15.
J Thromb Haemost ; 16(2): 389-404, 2018 02.
Artículo en Inglés | MEDLINE | ID: mdl-29210180

RESUMEN

Essentials Glycoprotein VI (GPVI) binds collagen, starting thrombogenesis, and fibrin, stabilizing thrombi. GPVI-dimers, not monomers, recognize immobilized fibrinogen and fibrin through their D-domains. Collagen, D-fragment and D-dimer may share a common or proximate binding site(s) on GPVI-dimer. GPVI-dimer-fibrin interaction supports spreading, activation and adhesion involving αIIbß3. SUMMARY: Background Platelet collagen receptor Glycoprotein VI (GPVI) binds collagen, initiating thrombogenesis, and stabilizes thrombi by binding fibrin. Objectives To determine if GPVI-dimer, GPVI-monomer, or both bind to fibrinogen substrates, and which region common to these substrates contains the interaction site. Methods Recombinant GPVI monomeric extracellular domain (GPVIex ) or dimeric Fc-fusion protein (GPVI-Fc2 ) binding to immobilized fibrinogen derivatives was measured by ELISA, including competition assays involving collagenous substrates and fibrinogen derivatives. Flow adhesion was performed with normal or Glanzmann thrombasthenic (GT) platelets over immobilized fibrinogen, with or without anti-GPVI-dimer or anti-αIIbß3. Results Under static conditions, GPVIex did not bind to any fibrinogen substrate. GPVI-Fc2 exhibited specific, saturable binding to both D-fragment and D-dimer, which was inhibited by mFab-F (anti-GPVI-dimer), but showed low binding to fibrinogen and fibrin under our conditions. GPVI-Fc2 binding to D-fragment or D-dimer was abrogated by collagen type III, Horm collagen or CRP-XL (crosslinked collagen-related peptide), suggesting proximity between the D-domain and collagen binding sites on GPVI-dimer. Under low shear, adhesion of normal platelets to D-fragment, D-dimer, fibrinogen and fibrin was inhibited by mFab-F (inhibitor of GPVI-dimer) and abolished by Eptifibatide (inhibitor of αIIbß3), suggesting that both receptors contribute to thrombus formation on these substrates, but αIIbß3 makes a greater contribution. Notably, thrombasthenic platelets showed limited adhesion to fibrinogen substrates under flow, which was further reduced by mFab-F, supporting some independent GPVI-dimer involvement in this interaction. Conclusion Only dimeric GPVI interacts with fibrinogen D-domain, at a site proximate to its collagen binding site, to support platelet adhesion/activation/aggregate formation on immobilized fibrinogen and polymerized fibrin.


Asunto(s)
Plaquetas/metabolismo , Colágeno/metabolismo , Fibrina/metabolismo , Fibrinógeno/metabolismo , Activación Plaquetaria , Glicoproteínas de Membrana Plaquetaria/metabolismo , Trombastenia/sangre , Trombosis/sangre , Sitios de Unión , Estudios de Casos y Controles , Fibrina/química , Productos de Degradación de Fibrina-Fibrinógeno/metabolismo , Fibrinógeno/química , Humanos , Adhesividad Plaquetaria , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Glicoproteínas de Membrana Plaquetaria/química , Glicoproteínas de Membrana Plaquetaria/genética , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Transducción de Señal , Relación Estructura-Actividad , Trombastenia/genética , Trombosis/genética
16.
SAR QSAR Environ Res ; 28(10): 783-799, 2017 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-29135287

RESUMEN

The GPVI receptor on the platelets plays a major role in inhibiting arterial thrombosis with limited risk of bleeding and is considered a potential anti-thrombotic target for arterial thrombosis. In the reported anti-thrombotics, tetrahydropyridoindoles, the title compound was the best inhibitor of the collagen mediated platelet aggregation by antagonizing the platelet receptor GPVI. Interestingly, the racemic title compound showed better antagonism (IC50 racemate = 6.7 µM) than either of its enantiomers (IC50 S enantiomer = 25.3 µM; IC50 R enantiomer = 126.3 µM). In order to explain this, the molecular modelling approaches viz. site map analysis, protein-protein docking and molecular dynamics simulation were carried out, which led to the identification of a second binding site located near the primary antagonist binding site known to bind losartan. The induced fit docking studies for both the enantiomers at the primary and secondary binding sites showed that the S-enantiomer has better interactions at the primary binding site than the R-enantiomer, while the R-enantiomer has better interactions at the secondary site than the S-enantiomer. Hence, the overall interactions of the racemic compound containing equimolar mixture may be higher than any one of the enantiomers and may explain the higher activity than its enantiomers of the racemic compound.


Asunto(s)
Indoles/química , Losartán/metabolismo , Modelos Moleculares , Glicoproteínas de Membrana Plaquetaria/química , Relación Estructura-Actividad Cuantitativa , Estereoisomerismo
17.
Proc Natl Acad Sci U S A ; 114(43): 11422-11427, 2017 10 24.
Artículo en Inglés | MEDLINE | ID: mdl-29073066

RESUMEN

Tumor necrosis factor (TNF)-receptor associated factor 4 (TRAF4), an adaptor protein with E3-ligase activity, is involved in embryogenesis, cancer initiation and progression, and platelet receptor (GPIb-IX-V complex and GPVI)-mediated signaling for reactive oxygen species (ROS) production that initiates thrombosis at arterial shears. Disruption of platelet receptors and the TRAF4 interaction is a potential target for therapeutic intervention by antithrombotic drugs. Here, we report a crystal structure of TRAF4 (amino acid residues 290∼470) in complex with a peptide from the GPIbß receptor (amino acid residues 177∼181). The GPIbß peptide binds to a unique shallow surface composed of two hydrophobic pockets on TRAF4. Further studies revealed the TRAF4-binding motif Arg-Leu-X-Ala. The TRAF4-binding motif was present not only in platelet receptors but also in the TGF-ß receptor. The current structure will provide a template for furthering our understanding of the receptor-binding specificity of TRAF4, TRAF4-mediated signaling, and related diseases.


Asunto(s)
Complejo GPIb-IX de Glicoproteína Plaquetaria/química , Glicoproteínas de Membrana Plaquetaria/química , Factor 4 Asociado a Receptor de TNF/química , Calorimetría/métodos , Modelos Moleculares , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Glicoproteínas de Membrana Plaquetaria/metabolismo , Conformación Proteica , Transducción de Señal , Factor 4 Asociado a Receptor de TNF/metabolismo
18.
Prep Biochem Biotechnol ; 47(10): 1008-1015, 2017 Nov 26.
Artículo en Inglés | MEDLINE | ID: mdl-28857681

RESUMEN

Glycosylation is one of the most complex post-translational modifications and may have significant influence on the proper function of the corresponding proteins. Bacteria and yeast are, because of easy handling and cost reasons, the most frequently used systems for recombinant protein expression. Bacteria generally do not glycosylate proteins and yeast might tend to hyperglycosylate. Insect cell- and mammalian cell-based expression systems are able to produce complex N-glycosylation structures but are more complex to handle and more expensive. The nonpathogenic protozoa Leishmania tarentolae is an easy-to-handle alternative expression system for production of proteins requiring the eukaryotic protein folding machinery and post-translational modifications. We used and evaluated the system for the secretory expression of extracellular domains from human glycoprotein VI and the receptor for advanced glycation end products from rat. Both proteins were well expressed and homogeneously glycosylated. Analysis of the glycosylation pattern identified the structure as the conserved core pentasaccharide Man3GlcNac2.


Asunto(s)
Leishmania/genética , Glicoproteínas de Membrana Plaquetaria/genética , Receptor para Productos Finales de Glicación Avanzada/genética , Proteínas Recombinantes/genética , Animales , Biotecnología , Clonación Molecular , Expresión Génica , Glicosilación , Humanos , Glicoproteínas de Membrana Plaquetaria/química , Glicoproteínas de Membrana Plaquetaria/aislamiento & purificación , Dominios Proteicos , Ratas , Receptor para Productos Finales de Glicación Avanzada/química , Receptor para Productos Finales de Glicación Avanzada/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación
19.
Mol Med Rep ; 16(5): 7561-7568, 2017 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-28944903

RESUMEN

Cerebral thrombosis is one of the most common causes of cerebral infarction, and anticoagulation therapy is a routine treatment in patients with hemorrhagic cerebral venous thrombosis. The hemostatic function of platelets is important for the anticoagulation therapy of thrombosis. Glycoprotein VI (GPVI) is reported as the major signaling receptor for collagen and is exclusively expressed on platelets and megakaryocytes, initiating platelet recruitment at sites of vascular injury and demonstrating numerous beneficial effects for patients with cerebral thrombosis. In the present study, thrombus formation and platelet adhesion following endothelial injury was monitored in the jugular vein by intra­vital fluorescence microscopy. The morphological and clinical observations of cerebral thrombosis were investigated and analyzed in a mouse model with cerebral thrombosis. In addition, the present study investigated the effect of fusion protein GPVI modified with Fc and PEG, which is specifically linked to the extracellular domain of GPVI (GPVI­Fc­PEG), on thrombus formation following vessel wall injury and on experimental mice with cerebral thrombosis. The maximum tolerated dose (MTD) was identified as 0.18 mg. GPVI­Fc­PEG competitively bound to and prevented von Willebrand Factor­collagen interactions. The results of the present study demonstrated that cerebral thrombosis was greatly relieved and improved functional outcomes treatment with an MTD of GPVI­Fc­PEG following endothelial injury, compared with GPVI­Fc­treated mice. In addition, cerebral edema and infarct size was improved compared with GPVI­Fc­treated mice with ischemic stroke immediately prior to reperfusion. Furthermore, treatment of GPVI­Fc­PEG led to increased reperfusion and improved survival following cerebral thrombosis compared with treatment with either single agent alone. Taken together, GPVI­Fc­PEG relieved cerebral thrombosis following ischemic stroke and improved prognostic preclinical outcomes without intracranial bleeding, which suggested that GPVI­Fc­PEG may be a potential candidate for cerebral thrombosis therapy.


Asunto(s)
Infarto Cerebral/tratamiento farmacológico , Fibrinolíticos/uso terapéutico , Fragmentos Fc de Inmunoglobulinas/química , Trombosis Intracraneal/tratamiento farmacológico , Glicoproteínas de Membrana Plaquetaria/química , Polietilenglicoles/química , Animales , Plaquetas/metabolismo , Bovinos , Adhesión Celular/efectos de los fármacos , Colágeno/metabolismo , Modelos Animales de Enfermedad , Femenino , Fibrinolíticos/química , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/metabolismo , Trombosis Intracraneal/patología , Ratones , Ratones Endogámicos C57BL , Glicoproteínas de Membrana Plaquetaria/genética , Glicoproteínas de Membrana Plaquetaria/metabolismo , Unión Proteica , Accidente Cerebrovascular/tratamiento farmacológico , Accidente Cerebrovascular/patología , Factor de von Willebrand/metabolismo
20.
Proteomics ; 17(6)2017 03.
Artículo en Inglés | MEDLINE | ID: mdl-27452734

RESUMEN

Platelet glycoproteins are known to play central roles in hemostasis and vascular integrity and have pathologic roles in vascular occlusive diseases such as myocardial infarction and stroke. Characterizing glycoproteins within and secreted by platelets can provide insight into the mechanisms that underlie vascular pathologies and the therapeutic benefits or failure of anti-platelet agents. To study the impact of aspirin, which is commonly prescribed for primary and secondary cardiovascular prevention, on the platelet glycoproteome, we evaluated washed platelets from ten donors. The platelet glycoproteome, was studied using an iTRAQ in resting and stimulated states and with and without aspirin treatment. Using solid phase extraction of glycosite-containing peptides (SPEG), we were able to identify 799 unique N-linked glycosylation sites (glycosites) in platelets, representing the largest and the most comprehensive analysis to date. We were able to identity a number of glycoproteins impacted by aspirin treatment, which we validated using global proteomics analysis of platelets and their secreted proteins. In our analyses, metallopeptidase inhibitor 1 (TIMP1) was the single most significantly affected glycoprotein by aspirin treatment. ELISA assays confirmed proteomic results and validated our strategy. Functional analysis demonstrated that TIMP1 levels were highly correlated with platelet reactivity in vitro, with a correlation coefficient of -0.5. The release of TIMP1 from platelets, which was previously unknown to be affected by aspirin treatment, may play important roles in hemostasis and/or vascular integrity. If validated, our findings may be useful for developing assays that assess platelet response to aspirin or other anti-platelet therapies.


Asunto(s)
Aspirina/farmacología , Activación Plaquetaria/efectos de los fármacos , Glicoproteínas de Membrana Plaquetaria/metabolismo , Adulto , Secuencia de Aminoácidos , Cromatografía Liquida , Colágeno/farmacología , Ensayo de Inmunoadsorción Enzimática , Femenino , Glicopéptidos/química , Glicopéptidos/metabolismo , Humanos , Marcaje Isotópico , Masculino , Agregación Plaquetaria/efectos de los fármacos , Glicoproteínas de Membrana Plaquetaria/química , Proteómica , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem , Inhibidor Tisular de Metaloproteinasa-1/metabolismo
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