RESUMEN
Platelet dysregulation is drastically increased with advanced age and contributes to making cardiovascular disorders the leading cause of death of elderly humans. Here, we reveal a direct differentiation pathway from hematopoietic stem cells into platelets that is progressively propagated upon aging. Remarkably, the aging-enriched platelet path is decoupled from all other hematopoietic lineages, including erythropoiesis, and operates as an additional layer in parallel with canonical platelet production. This results in two molecularly and functionally distinct populations of megakaryocyte progenitors. The age-induced megakaryocyte progenitors have a profoundly enhanced capacity to engraft, expand, restore, and reconstitute platelets in situ and upon transplantation and produce an additional platelet population in old mice. The two pools of co-existing platelets cause age-related thrombocytosis and dramatically increased thrombosis in vivo. Strikingly, aging-enriched platelets are functionally hyper-reactive compared with the canonical platelet populations. These findings reveal stem cell-based aging as a mechanism for platelet dysregulation and age-induced thrombosis.
Asunto(s)
Envejecimiento , Plaquetas , Diferenciación Celular , Células Madre Hematopoyéticas , Trombosis , Animales , Células Madre Hematopoyéticas/metabolismo , Plaquetas/metabolismo , Trombosis/patología , Trombosis/metabolismo , Ratones , Humanos , Megacariocitos/metabolismo , Ratones Endogámicos C57BL , Células Progenitoras de Megacariocitos/metabolismo , MasculinoRESUMEN
Red blood cells (RBCs) have been hypothesized to support hemostasis by facilitating platelet margination and releasing platelet-activating factors such as adenosine diphosphate (ADP). Significant knowledge gaps remain regarding how RBCs influence platelet function, especially in (patho)physiologically relevant hemodynamic conditions. Here we present results showing how RBCs affect platelet function and hemostasis in conditions of anemia, thrombocytopenia, and pancytopenia, and how the biochemical and biophysical properties of RBCs regulate platelet function at the blood-vessel wall interface and in the fluid phase under flow conditions. We found that RBCs promoted platelet deposition to collagen under flow conditions in moderate (50 ï´ 103/ïL) but not severe (10 ï´ 103/ïL) thrombocytopenia in vitro. Reduction in hematocrit by 45% led to increased bleeding in mice with hemolytic anemia. In contrast, bleeding diathesis was observed in mice with a 90% but not with a 60% reduction in platelet counts. RBC transfusion improved hemostasis by enhancing fibrin clot formation at the site of vascular injury in mice with severe pancytopenia induced by total body irradiation. Altering membrane deformability changed the ability of RBCs to promote platelet aggregation. RBC-derived ADP contributed to platelet activation and aggregation in vitro under pathologically high shear stresses, as observed in patients supported by left ventricular assist devices. These findings demonstrate that RBCs support platelet function and hemostasis through multiple mechanisms, both at the blood-vessel wall interface and in the fluidic phase of circulation.
RESUMEN
Coronavirus-associated coagulopathy (CAC) is a morbid and lethal sequela of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. CAC results from a perturbed balance between coagulation and fibrinolysis and occurs in conjunction with exaggerated activation of monocytes/macrophages (MO/Mφs), and the mechanisms that collectively govern this phenotype seen in CAC remain unclear. Here, using experimental models that use the murine betacoronavirus MHVA59, a well-established model of SARS-CoV-2 infection, we identify that the histone methyltransferase mixed lineage leukemia 1 (MLL1/KMT2A) is an important regulator of MO/Mφ expression of procoagulant and profibrinolytic factors such as tissue factor (F3; TF), urokinase (PLAU), and urokinase receptor (PLAUR) (herein, "coagulopathy-related factors") in noninfected and infected cells. We show that MLL1 concurrently promotes the expression of the proinflammatory cytokines while suppressing the expression of interferon alfa (IFN-α), a well-known inducer of TF and PLAUR. Using in vitro models, we identify MLL1-dependent NF-κB/RelA-mediated transcription of these coagulation-related factors and identify a context-dependent, MLL1-independent role for RelA in the expression of these factors in vivo. As functional correlates for these findings, we demonstrate that the inflammatory, procoagulant, and profibrinolytic phenotypes seen in vivo after coronavirus infection were MLL1-dependent despite blunted Ifna induction in MO/Mφs. Finally, in an analysis of SARS-CoV-2 positive human samples, we identify differential upregulation of MLL1 and coagulopathy-related factor expression and activity in CD14+ MO/Mφs relative to noninfected and healthy controls. We also observed elevated plasma PLAU and TF activity in COVID-positive samples. Collectively, these findings highlight an important role for MO/Mφ MLL1 in promoting CAC and inflammation.
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COVID-19 , N-Metiltransferasa de Histona-Lisina , Animales , Humanos , Ratones , COVID-19/complicaciones , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Inflamación/metabolismo , Monocitos/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , SARS-CoV-2/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
von Willebrand factor (VWF) mediates primary hemostasis and thrombosis in response to hydrodynamic forces. We previously showed that high shear promoted self-association of VWF into hyperadhesive strands, which can be attenuated by high-density lipoprotein (HDL) and apolipoprotein A-I. In this study, we show that low-density lipoprotein (LDL) binds VWF under shear and enhances self-association. Vortexing VWF in tubes resulted in its loss from the solution and deposition onto tube surfaces, which was prevented by HDL. At a stabilizing HDL concentration of 1.2 mg/mL, increasing concentrations of LDL progressively increased VWF loss, the effect correlating with the LDL-to-HDL ratio and not the absolute concentration of the lipoproteins. Similarly, HDL diminished deposition of VWF in a post-in-channel microfluidic device, whereas LDL increased both the rate and extent of strand deposition, with both purified VWF and plasma. Hypercholesterolemic human plasma also displayed accelerated VWF accumulation in the microfluidic device. The initial rate of accumulation correlated linearly with the LDL-to-HDL ratio. In Adamts13-/- and Adamts13-/-LDLR-/- mice, high LDL levels enhanced VWF and platelet adhesion to the myocardial microvasculature, reducing cardiac perfusion, impairing systolic function, and producing early signs of cardiomyopathy. In wild-type mice, high plasma LDL concentrations also increased the size and persistence of VWF-platelet thrombi in ionophore-treated mesenteric microvessels, exceeding the accumulation seen in similarly treated ADAMTS13-deficient mice that did not receive LDL infusion. We propose that targeting the interaction of VWF with itself and with LDL may improve the course of thrombotic microangiopathies, atherosclerosis, and other disorders with defective microvascular circulation.
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Trombosis , Factor de von Willebrand , Ratones , Humanos , Animales , Factor de von Willebrand/metabolismo , Lipoproteínas LDL , Trombosis/metabolismo , Hemostasis , Adhesividad Plaquetaria , Proteína ADAMTS13RESUMEN
BACKGROUND: Platelets for transfusion are stored for 5 to 7 days. Previous studies have shown that HETE levels in the storage bag negatively correlate with platelet performance in vivo, suggesting that the dysregulation of bioactive lipid mediators may contribute to the storage lesion. In the current study, we sought to understand how genetic deletion and pharmacological inhibition of 12-LOX (12-lipoxygenase) affects platelets during storage and after transfusion. METHODS: Platelets from 12-LOX+/+ (wild-type [WT]) and 12-LOX-/- mice were stored for 24 and 48 hours and profiled using liquid chromatography-tandem mass spectrometry-multiple reaction monitoring or transfused into thrombocytopenic hIL4R (human interleukin 4 receptor)-transgenic mice. Platelet function was assessed by flow cytometry and in vivo thrombosis and hemostasis models. To test the role of the COX-1 (cyclooxygenase-1) pathway, donor mice were treated with acetylsalicylic acid. Human platelets were treated with the 12-LOX inhibitor, VLX-1005, or vehicle, stored, and transfused to NOD/SCID (nonobese diabetic/severe combined immunodeficiency) mice. RESULTS: Polyunsaturated fatty acids increased significantly in stored platelets from 12-LOX-/- mice, whereas oxylipin concentrations were significantly higher in WT platelets. After transfusion to thrombocytopenic mice, we observed significantly more baseline αIIbß3 integrin activation in 12-LOX-/- platelets than in WT platelets. Stored platelets from 12-LOX-/- mice occluded vessels significantly faster than stored WT platelets. In hemostasis models, significantly more stored 12-LOX-/- than WT platelets accumulated at the site of venous injury leading to reduced blood loss. Inhibition of COX-1 abrogated both increased integrin activation and thromboxane generation in stored 12-LOX-/- platelets, highlighting the critical role of this pathway for improved post-transfusion function. Consistent with our mouse studies, human platelets stored with VLX-1005, showed increased integrin activation compared with vehicle-treated platelets after transfusion. CONCLUSIONS: Deleting 12-LOX improves the post-transfusion function of stored murine platelets by increasing thromboxane generation through COX-1-dependent arachidonic acid metabolism. Future studies should determine the feasibility and safety of 12-LOX-inhibited platelets transfused to humans.
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Araquidonato 12-Lipooxigenasa , Plaquetas , Humanos , Ratones , Animales , Araquidonato 12-Lipooxigenasa/genética , Araquidonato 12-Lipooxigenasa/metabolismo , Ratones Endogámicos NOD , Ratones SCID , Plaquetas/metabolismo , Ratones Transgénicos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Tromboxanos/metabolismoRESUMEN
Circulating platelets roll along exposed collagen at vessel injury sites and respond with filipodia protrusion, shape change, and surface area expansion to facilitate platelet adhesion and plug formation. Various glycoproteins were considered to be both collagen responders and mediators of platelet adhesion, yet the signaling kinetics emanating from these receptors do not fully account for the rapid platelet cytoskeletal changes that occur in blood flow. We found the free N-terminal fragment of the adhesion G protein-coupled receptor (GPCR) GPR56 in human plasma and report that GPR56 is the platelet receptor that transduces signals from collagen and blood flow-induced shear force to activate G protein 13 signaling for platelet shape change. Gpr56-/- mice have prolonged bleeding, defective platelet plug formation, and delayed thrombotic occlusion. Human and mouse blood perfusion studies demonstrated GPR56 and shear-force dependence of platelet adhesion to immobilized collagen. Our work places GPR56 as an initial collagen responder and shear-force transducer that is essential for platelet shape change during hemostasis.
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Plaquetas/metabolismo , Colágeno/metabolismo , Hemostasis , Receptores Acoplados a Proteínas G/metabolismo , Animales , Humanos , Integrinas/metabolismo , Ratones , Ratones Noqueados , Adhesividad Plaquetaria , Agregación Plaquetaria , Seudópodos/metabolismo , Receptores Acoplados a Proteínas G/genética , Transducción de Señal , Trombosis/metabolismo , TranscriptomaRESUMEN
OBJECTIVE: Microvascular thrombosis is the hallmark pathology of thrombotic thrombocytopenic purpura (TTP), a rare life-threatening disease. Neurological dysfunction is present in over 90% of patients with TTP, and TTP can cause long-lasting neurological damage or death. However, the pathophysiology of microvascular thrombosis in the brain is not well studied to date. Here, we investigate the formation and resolution of thrombosis in pial microvessels. Approach and Results: Using a cranial intravital microscopy in well-established mouse models of congenital TTP induced by infusion of recombinant VWF (von Willebrand factor), we found that soluble VWF, at high concentration, adheres to the endothelium of the vessel wall, self-associates, and initiates platelet adhesion resulting in the formation of pial microvascular thrombosis in ADAMTS13-/- (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) mice. Importantly, VWF-mediated pial microvascular thrombosis occurred without vascular injury to the brain, and thrombi consisted of resting platelets adhered onto ultra-large VWF without fibrin in the brain in rVWF (recombinant VWF) challenged ADAMTS13-/- mice. Prophylactic treatment with recombinant ADAMTS13 (BAX930) effectively prevented the onset of the VWF-mediated microvascular thrombosis and therapeutic treatment with BAX930 acutely resolved the preexisting or growing thrombi in the brain of ADAMTS13-/- mice after rVWF challenge. The absence of platelet activation and fibrin formation within VWF-mediated thrombi and efficacy of BAX930 was confirmed with an endothelial-driven VWF-mediated microvascular thrombosis model in mice. CONCLUSIONS: Our results provide important insight into the initiation and development of microvascular thrombi in mouse models that mimics TTP and indicate that rADAMTS13 could be an effective interventional therapy for microvascular thrombosis, the hallmark pathology in TTP.
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Piamadre/irrigación sanguínea , Púrpura Trombocitopénica Trombótica/complicaciones , Trombosis/etiología , Proteína ADAMTS13/farmacología , Proteína ADAMTS13/fisiología , Animales , Modelos Animales de Enfermedad , Células Endoteliales/fisiología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Activación Plaquetaria , Adhesividad Plaquetaria , Trombosis/terapia , Factor de von Willebrand/fisiologíaRESUMEN
Objective- Mutations in Krüppel like factor-11 ( KLF11), a gene also known as maturity-onset diabetes mellitus of the young type 7, contribute to the development of diabetes mellitus. KLF11 has anti-inflammatory effects in endothelial cells and beneficial effects on stroke. However, the function of KLF11 in the cardiovascular system is not fully unraveled. In this study, we investigated the role of KLF11 in vascular smooth muscle cell biology and arterial thrombosis. Approach and Results- Using a ferric chloride-induced thrombosis model, we found that the occlusion time was significantly reduced in conventional Klf11 knockout mice, whereas bone marrow transplantation could not rescue this phenotype, suggesting that vascular KLF11 is critical for inhibition of arterial thrombosis. We further demonstrated that vascular smooth muscle cell-specific Klf11 knockout mice also exhibited significantly reduced occlusion time. The expression of tissue factor (encoded by the F3 gene), a main initiator of the coagulation cascade, was increased in the artery of Klf11 knockout mice, as determined by real-time quantitative polymerase chain reaction and immunofluorescence. Furthermore, vascular smooth muscle cells isolated from Klf11 knockout mouse aortas showed increased tissue factor expression, which was rescued by KLF11 overexpression. In human aortic smooth muscle cells, small interfering RNA-mediated knockdown of KLF11 increased tissue factor expression. Consistent results were observed on adenovirus-mediated overexpression of KLF11. Mechanistically, KLF11 downregulates F3 at the transcriptional level as determined by reporter and chromatin immunoprecipitation assays. Conclusions- Our data demonstrate that KLF11 is a novel transcriptional suppressor of F3 in vascular smooth muscle cells, constituting a potential molecular target for inhibition of arterial thrombosis.
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Proteínas Reguladoras de la Apoptosis/fisiología , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Proteínas Represoras/fisiología , Tromboplastina/biosíntesis , Trombosis/prevención & control , Animales , Antitrombina III/análisis , Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Proteínas Reguladoras de la Apoptosis/deficiencia , Proteínas Reguladoras de la Apoptosis/genética , Trasplante de Médula Ósea , Células Cultivadas , Cloruros/toxicidad , Inmunoprecipitación de Cromatina , Regulación hacia Abajo , Femenino , Compuestos Férricos/toxicidad , Regulación de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Péptido Hidrolasas/análisis , Agregación Plaquetaria , Interferencia de ARN , Proteínas Recombinantes/metabolismo , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/deficiencia , Proteínas Represoras/genética , Tromboplastina/genética , Trombosis/inducido químicamente , Transcripción GenéticaRESUMEN
Clinical studies suggest that diets rich in ω-3 polyunsaturated fatty acids (PUFAs) provide beneficial anti-inflammatory effects, in part through their conversion to bioactive metabolites. Here we report on the endogenous production of a previously unknown class of ω-3 PUFA-derived lipid metabolites that originate from the crosstalk between endocannabinoid and cytochrome P450 (CYP) epoxygenase metabolic pathways. The ω-3 endocannabinoid epoxides are derived from docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) to form epoxyeicosatetraenoic acid-ethanolamide (EEQ-EA) and epoxydocosapentaenoic acid-ethanolamide (EDP-EA), respectively. Both EEQ-EAs and EDP-EAs are endogenously present in rat brain and peripheral organs as determined via targeted lipidomics methods. These metabolites were directly produced by direct epoxygenation of the ω-3 endocannabinoids, docosahexanoyl ethanolamide (DHEA) and eicosapentaenoyl ethanolamide (EPEA) by activated BV-2 microglial cells, and by human CYP2J2. Neuroinflammation studies revealed that the terminal epoxides 17,18-EEQ-EA and 19,20-EDP-EA dose-dependently abated proinflammatory IL-6 cytokines while increasing anti-inflammatory IL-10 cytokines, in part through cannabinoid receptor-2 activation. Furthermore the ω-3 endocannabinoid epoxides 17,18-EEQ-EA and 19,20-EDP-EA exerted antiangiogenic effects in human microvascular endothelial cells (HMVEC) and vasodilatory actions on bovine coronary arteries and reciprocally regulated platelet aggregation in washed human platelets. Taken together, the ω-3 endocannabinoid epoxides' physiological effects are mediated through both endocannabinoid and epoxyeicosanoid signaling pathways. In summary, the ω-3 endocannabinoid epoxides are found at concentrations comparable to those of other endocannabinoids and are expected to play critical roles during inflammation in vivo; thus their identification may aid in the development of therapeutics for neuroinflammatory and cerebrovascular diseases.
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Antiinflamatorios/sangre , Endocannabinoides/metabolismo , Compuestos Epoxi/sangre , Etanolaminas/sangre , Ácidos Grasos Omega-3/metabolismo , Amidohidrolasas/metabolismo , Animales , Encéfalo/metabolismo , Bovinos , Citocromo P-450 CYP2J2 , Sistema Enzimático del Citocromo P-450/metabolismo , Evaluación Preclínica de Medicamentos , Epóxido Hidrolasas/metabolismo , Compuestos Epoxi/farmacología , Compuestos Epoxi/uso terapéutico , Etanolaminas/farmacología , Etanolaminas/uso terapéutico , Humanos , Metabolismo de los Lípidos , Ratones , Microglía/metabolismo , Neovascularización Patológica/prevención & control , Agregación Plaquetaria/efectos de los fármacos , Ratas , Vasodilatación/efectos de los fármacosRESUMEN
Integrins are a large family of heterodimeric transmembrane receptors differentially expressed on almost all metazoan cells. Integrin ß subunits contain a highly conserved plexin-semaphorin-integrin (PSI) domain. The CXXC motif, the active site of the protein-disulfide-isomerase (PDI) family, is expressed twice in this domain of all integrins across species. However, the role of the PSI domain in integrins and whether it contains thiol-isomerase activity have not been explored. Here, recombinant PSI domains of murine ß3, and human ß1 and ß2 integrins were generated and their PDI-like activity was demonstrated by refolding of reduced/denatured RNase. We identified that both CXXC motifs of ß3 integrin PSI domain are required to maintain its optimal PDI-like activity. Cysteine substitutions (C13A and C26A) of the CXXC motifs also significantly decreased the PDI-like activity of full-length human recombinant ß3 subunit. We further developed mouse anti-mouse ß3 PSI domain monoclonal antibodies (mAbs) that cross-react with human and other species. These mAbs inhibited αIIbß3 PDI-like activity and its fibrinogen binding. Using single-molecular Biomembrane-Force-Probe assays, we demonstrated that inhibition of αIIbß3 endogenous PDI-like activity reduced αIIbß3-fibrinogen interaction, and these anti-PSI mAbs inhibited fibrinogen binding via different levels of both PDI-like activity-dependent and -independent mechanisms. Importantly, these mAbs inhibited murine/human platelet aggregation in vitro and ex vivo, and murine thrombus formation in vivo, without significantly affecting bleeding time or platelet count. Thus, the PSI domain is a potential regulator of integrin activation and a novel target for antithrombotic therapies. These findings may have broad implications for all integrin functions, and cell-cell and cell-matrix interactions.
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Cadenas beta de Integrinas/inmunología , Proteína Disulfuro Isomerasas/inmunología , Secuencias de Aminoácidos , Animales , Anticuerpos Monoclonales/farmacología , Dominio Catalítico , Moléculas de Adhesión Celular , Humanos , Ratones , Proteínas del Tejido Nervioso , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria , Proteínas Recombinantes , Semaforinas , Trombosis/prevención & controlRESUMEN
Omega-3 polyunsaturated fatty acids (n-3 PUFAs) are associated with a variety of cellular alterations that mitigate cardiovascular disease. However, pinpointing the positive therapeutic effects is challenging due to inconsistent clinical trial results and overly simplistic in vitro studies. Here we aimed to develop realistic models of n-3 PUFA effects on platelet function so that preclinical results can better align with and predict clinical outcomes. Human platelets incubated with the n-3 PUFAs docosahexaenoic acid and eicosapentaenoic acid were stimulated with agonist combinations mirroring distinct regions of a growing thrombus. Platelet responses were then monitored in a number of ex-vivo functional assays. Furthermore, intravital microscopy was used to monitor arterial thrombosis and fibrin deposition in mice fed an n-3 PUFA-enriched diet. We found that n-3 PUFA treatment had minimal effects on many basic ex-vivo measures of platelet function using agonist combinations. However, n-3 PUFA treatment delayed platelet-derived thrombin generation in both humans and mice. This impaired thrombin production paralleled a reduced platelet accumulation within thrombi formed in either small arterioles or larger arteries of mice fed an n-3 PUFA-enriched diet, without impacting P-selectin exposure. Despite an apparent lack of robust effects in many ex-vivo assays of platelet function, increased exposure to n-3 PUFAs reduces platelet-mediated thrombin generation and attenuates elements of thrombus formation. These data support the cardioprotective value of-3 PUFAs and strongly suggest that they modify elements of platelet function in vivo.
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Plaquetas/efectos de los fármacos , Ácidos Docosahexaenoicos/antagonistas & inhibidores , Ácidos Docosahexaenoicos/uso terapéutico , Ácido Eicosapentaenoico/antagonistas & inhibidores , Ácido Eicosapentaenoico/uso terapéutico , Ácidos Grasos Omega-3/uso terapéutico , Animales , Ácidos Docosahexaenoicos/farmacología , Ácido Eicosapentaenoico/farmacología , Ácidos Grasos Omega-3/farmacología , Humanos , Masculino , Ratones , TrombosisRESUMEN
OBJECTIVE: Adequate platelet reactivity is required for maintaining hemostasis. However, excessive platelet reactivity can also lead to the formation of occlusive thrombi. Platelet 12(S)-lipoxygenase (12-LOX), an oxygenase highly expressed in the platelet, has been demonstrated to regulate platelet function and thrombosis ex vivo, supporting a key role for 12-LOX in the regulation of in vivo thrombosis. However, the ability to pharmacologically target 12-LOX in vivo has not been established to date. Here, we studied the effect of the first highly selective 12-LOX inhibitor, ML355, on in vivo thrombosis and hemostasis. APPROACH AND RESULTS: ML355 dose-dependently inhibited human platelet aggregation and 12-LOX oxylipin production, as confirmed by mass spectrometry. Interestingly, the antiplatelet effects of ML355 were reversed after exposure to high concentrations of thrombin in vitro. Ex vivo flow chamber assays confirmed that human platelet adhesion and thrombus formation at arterial shear over collagen were attenuated in whole blood treated with ML355 comparable to aspirin. Oral administration of ML355 in mice showed reasonable plasma drug levels by pharmacokinetic assessment. ML355 treatment impaired thrombus growth and vessel occlusion in FeCl3-induced mesenteric and laser-induced cremaster arteriole thrombosis models in mice. Importantly, hemostatic plug formation and bleeding after treatment with ML355 was minimal in mice in response to laser ablation on the saphenous vein or in a cremaster microvasculature laser-induced rupture model. CONCLUSIONS: Our data strongly support 12-LOX as a key determinant of platelet reactivity in vivo, and inhibition of platelet 12-LOX with ML355 may represent a new class of antiplatelet therapy.
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Hemostasis/efectos de los fármacos , Inhibidores de la Lipooxigenasa/farmacología , Agregación Plaquetaria/efectos de los fármacos , Sulfonamidas/farmacología , Trombosis/prevención & control , Animales , Relación Dosis-Respuesta a Droga , Humanos , Inhibidores de la Lipooxigenasa/administración & dosificación , Inhibidores de la Lipooxigenasa/sangre , Ratones , Adhesividad Plaquetaria/efectos de los fármacos , Sulfonamidas/administración & dosificación , Sulfonamidas/sangre , Trombina/fisiologíaRESUMEN
Thrombosis is the most common underlying pathology responsible for morbidity and mortality in cardiovascular disease (CVD). Platelet adhesion, activation, and aggregation play central roles in hemostasis; however, the same process may also cause thrombosis and vessel occlusion at the site of ruptured atherosclerotic lesions leading to heart attack and stroke. ω-3 and ω-6 polyunsaturated fatty acids (PUFAs) are an essential component of the platelet phospholipid membrane and play a major role in many aspects of platelet function. Dietary supplementation of ω-3 and ω-6 PUFAs has long been used to slow the progression of CVD and to prevent acute cardiovascular events. Despite this, the role of ω-3 and ω-6 PUFAs and their oxylipin metabolites in platelet function remains controversial due to the lack in our understanding of the mechanistic regulation controlling platelet reactivity in vitro and substantial evidence for PUFA regulation of thrombotic events in vivo. In this review, we will outline the role of platelet physiology in hemostasis and the effect of ω-3 and ω-6 PUFAs on platelet function, with special emphasis on in vivo effects on hemostasis and thrombosis due to the role of PUFAs and their bioactive lipids in circulation. Further, recent mechanistic insights and evidence for cardio-protective effects of PUFAs and their bioactive lipids will be discussed.
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Plaquetas/metabolismo , Ácidos Grasos Omega-3/metabolismo , Ácidos Grasos Omega-6/metabolismo , Trombosis/dietoterapia , Plaquetas/efectos de los fármacos , Plaquetas/patología , Cardiotónicos/metabolismo , Cardiotónicos/uso terapéutico , Ácidos Grasos Omega-3/uso terapéutico , Ácidos Grasos Omega-6/uso terapéutico , Humanos , Oxilipinas/metabolismo , Activación Plaquetaria/efectos de los fármacos , Adhesividad Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Trombosis/metabolismo , Trombosis/patologíaRESUMEN
Background: Platelets play an important role in hemostasis, thrombosis, and atherosclerosis. Glycoprotein VI (GPVI) is a major platelet receptor that interacts with exposed collagen on injured vessel walls. Our previous studies have shown that anthocyanins (a type of natural plant pigment) attenuate platelet function; however, whether anthocyanins affect collagen-induced GPVI signaling remains unknown.Objective: The objective of this study was to explore the effects of cyanidin-3-glucoside (Cy-3-g, one of the major bioactive compounds in anthocyanins) on platelet activation and thrombosis and the GPVI signaling pathway.Methods: Platelets from healthy men and women were isolated and incubated with different concentrations (0, 0.5, 5, and 50 µM) of Cy-3-g. The expression of activated integrin αIIbß3, P-selectin, CD63, and CD40L, fibrinogen binding to platelets, and platelet aggregation were evaluated in vitro. Platelet adhesion and aggregation in whole blood under flow conditions were assessed in collagen-coated perfusion chambers. Thrombosis and hemostasis were assessed in 3-4-wk-old male C57BL/6J mice through FeCl3-induced intravital microscopy and tail bleeding time. The effect of Cy-3-g on collagen-induced human platelet GPVI signaling was explored with Western blot.Results: Cy-3-g attenuated platelet function in a dose-dependent manner. The 0.5-µM dose of Cy-3-g inhibited (P < 0.05) human platelet adhesion and aggregation to collagen at both venous (-54.02%) and arterial (-22.90%) shear stresses. The 5-µM dose inhibited (P < 0.05) collagen-induced human platelet activation (PAC-1: -48.21%, P-selectin: -50.63%), secretion (CD63: -73.89%, CD40L: -43.70%), fibrinogen binding (-56.79%), and aggregation (-17.81%). The 5-µM dose attenuated (P < 0.01) thrombus growth (-66.67%) without prolonging bleeding time in mice. The 50-µM dose downregulated (P < 0.05) collagen-induced GPVI signaling in human platelets and significantly decreased phosphorylation of Syk-linker for activation of T cells (LAT)-SLP76 (Syk: -39.08%, LAT: -32.25%, SLP76: -40.00%) and the expression of Lyn (-31.89%), Fyn (-36.27%), and phospholipase C-γ2 (-39.08%).Conclusions: Cy-3-g inhibits human platelet activation, aggregation, secretion, and thrombus formation, and downregulates the collagen-GPVI signaling pathway. Supplementation of Cy-3-g may have protective effects against atherothrombosis.
Asunto(s)
Plaquetas/metabolismo , Fitoterapia , Extractos Vegetales/farmacología , Plantas Comestibles/química , Agregación Plaquetaria/efectos de los fármacos , Glicoproteínas de Membrana Plaquetaria/metabolismo , Trombosis/prevención & control , Proteínas Adaptadoras Transductoras de Señales/sangre , Adulto , Anciano , Animales , Antocianinas/farmacología , Antocianinas/uso terapéutico , Antígenos CD/sangre , Aterosclerosis/sangre , Aterosclerosis/dietoterapia , Aterosclerosis/etiología , Colágeno/sangre , Femenino , Glucósidos/farmacología , Glucósidos/uso terapéutico , Hemostasis/efectos de los fármacos , Humanos , Masculino , Ratones Endogámicos C57BL , Persona de Mediana Edad , Selectina-P/sangre , Fosfoproteínas/sangre , Extractos Vegetales/uso terapéutico , Activación Plaquetaria/efectos de los fármacos , Transducción de Señal , Trombosis/sangre , Trombosis/etiologíaRESUMEN
OBJECTIVE: Dietary supplementation with polyunsaturated fatty acids has been widely used for primary and secondary prevention of cardiovascular disease in individuals at risk; however, the cardioprotective benefits of polyunsaturated fatty acids remain controversial because of lack of mechanistic and in vivo evidence. We present direct evidence that an omega-6 polyunsaturated fatty acid, dihomo-γ-linolenic acid (DGLA), exhibits in vivo cardioprotection through 12-lipoxygenase (12-LOX) oxidation of DGLA to its reduced oxidized lipid form, 12(S)-hydroxy-8Z,10E,14Z-eicosatrienoic acid (12(S)-HETrE), inhibiting platelet activation and thrombosis. APPROACH AND RESULTS: DGLA inhibited ex vivo platelet aggregation and Rap1 activation in wild-type mice, but not in mice lacking 12-LOX expression (12-LOX(-/-)). Similarly, wild-type mice treated with DGLA were able to reduce thrombus growth (platelet and fibrin accumulation) after laser-induced injury of the arteriole of the cremaster muscle, but not 12-LOX(-/-) mice, supporting a 12-LOX requirement for mediating the inhibitory effects of DGLA on platelet-mediated thrombus formation. Platelet activation and thrombus formation were also suppressed when directly treated with 12(S)-HETrE. Importantly, 2 hemostatic models, tail bleeding and arteriole rupture of the cremaster muscle, showed no alteration in hemostasis after 12(S)-HETrE treatment. Finally, the mechanism for 12(S)-HETrE protection was shown to be mediated via a Gαs-linked G-protein-coupled receptor pathway in human platelets. CONCLUSIONS: This study provides the direct evidence that an omega-6 polyunsaturated fatty acid, DGLA, inhibits injury-induced thrombosis through its 12-LOX oxylipin, 12(S)-HETrE, which strongly supports the potential cardioprotective benefits of DGLA supplementation through its regulation of platelet function. Furthermore, this is the first evidence of a 12-LOX oxylipin regulating platelet function in a Gs α subunit-linked G-protein-coupled receptor-dependent manner.
Asunto(s)
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/farmacología , Araquidonato 12-Lipooxigenasa/sangre , Plaquetas/efectos de los fármacos , Cromograninas/sangre , Fibrinolíticos/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gs/sangre , Activación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Trombosis/prevención & control , Ácido 8,11,14-Eicosatrienoico/metabolismo , Animales , Araquidonato 12-Lipooxigenasa/deficiencia , Araquidonato 12-Lipooxigenasa/genética , Plaquetas/metabolismo , Moléculas de Adhesión Celular/sangre , AMP Cíclico/sangre , Proteínas Quinasas Dependientes de AMP Cíclico/sangre , Modelos Animales de Enfermedad , Fibrinolíticos/metabolismo , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos/sangre , Oxidación-Reducción , Fosfoproteínas/sangre , Fosforilación , Agregación Plaquetaria/efectos de los fármacos , Complejo Shelterina , Transducción de Señal/efectos de los fármacos , Proteínas de Unión a Telómeros/sangre , Trombosis/sangre , Trombosis/enzimología , Trombosis/genética , Factores de TiempoRESUMEN
The inhibition of platelet aggregation is key to preventing conditions such as myocardial infarction and ischemic stroke. Aspirin is the most widely used drug to inhibit platelet aggregation. Aspirin absorption can be improved further to increase its permeability across biologic membranes via esterification or converting the carboxylic acid to an anhydride. There are several reports indicating that ω-3 and ω-6 fatty acids such as linoleic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) separately inhibit platelet aggregation. Herein, we synthesize anhydride conjugates of aspirin with linoleic acid, EPA, and DHA to form aspirin anhydrides that are expected to have higher permeability across cellular membranes. These aspirin-fatty acid anhydrides inhibited platelet aggregation in washed human platelets and platelet-rich plasma in a dose-dependent manner. In particular, the aspirin-DHA anhydride displayed similar effectiveness to aspirin. Platelet aggregation studies conducted in the presence of various platelet agonists indicated that the aspirin-lipid conjugates act through inhibition of the cyclooxygenase (COX)-thromboxane synthase (TXAS) pathway. Hence, we performed detailed biochemical studies using purified COX-1 as well as TXAS stabilized in nanoscale lipid bilayers of nanodiscs to confirm results from the platelet aggregation studies. We show that although all of the aspirin conjugates act through the COX-TXAS pathway by inhibiting COX-1, the parent fatty acids do not act via this pathway. Finally, we studied the hydrolysis of these compounds in buffer and human plasma, and we demonstrate that all of the aspirin-fatty acid conjugates hydrolyze to the parent molecules aspirin and fatty acid in a controlled manner.
Asunto(s)
Aspirina/química , Aspirina/farmacología , Plaquetas/efectos de los fármacos , Plaquetas/fisiología , Inhibidores de la Ciclooxigenasa/química , Inhibidores de la Ciclooxigenasa/farmacología , Ácidos Grasos Omega-3/química , Animales , Ciclooxigenasa 1/metabolismo , Descubrimiento de Drogas , Humanos , Agregación Plaquetaria/efectos de los fármacos , Células Sf9 , Spodoptera , Tromboxano-A Sintasa/antagonistas & inhibidoresRESUMEN
BACKGROUND: Blood clots are primarily composed of red blood cells (RBCs), platelets/thrombocytes, and fibrin. Despite the similarities observed between mammals and zebrafish, the composition of fish thrombi is not as well known. OBJECTIVES: To analyze the formation of zebrafish blood clots ex vivo and arterial and venous thrombi in vivo. METHODS: Transgenic zebrafish lines and laser-mediated endothelial injury were used to determine the relative ratio of RBCs and thrombocytes in clots. Scanning electron and confocal microscopy provided high-resolution images of the structure of adult and larval clots. Adult and larval thrombocyte spreading on fibrinogen was evaluated ex vivo. RESULTS: RBCs were present in arterial and venous thrombi, making up the majority of cells in both circulations. However, bloodless mutant fish demonstrated that fibrin clots can form in vivo in the absence of blood cells. Scanning electron and confocal microscopy showed that larval and adult zebrafish thrombi and mammalian thrombi look surprisingly similar externally and internally, even though the former have nucleated RBCs and thrombocytes. Although adult thrombocytes spread on fibrinogen, we found that larval cells do not fully activate without the addition of plasma from adult fish, suggesting a developmental deficiency of a plasma activating factor. Finally, mutants lacking αIIbß3 demonstrated that this integrin mediates thrombocyte spreading on fibrinogen. CONCLUSION: Our data showed strong conservation of arterial and venous and clot/thrombus formation across species, including developmental regulation of thrombocyte function. This correlation supports the possibility that mammals also do not absolutely require circulating cells to form fibrin clots in vivo.
Asunto(s)
Hemostáticos , Tromboembolia , Trombosis , Animales , Pez Cebra , Trombosis/genética , Plaquetas , Fibrina/química , Fibrinógeno/genética , MamíferosRESUMEN
The combination of inflammation and thrombosis is a hallmark of many cardiovascular diseases. Under such conditions, platelets are recruited to an area of inflammation by forming platelet-leukocyte aggregates via interaction of PSGL-1 on leukocytes and P-selectin on activated platelets, which can bind to the endothelium. While particulate drug carriers have been utilized to passively redirect leukocytes from areas of inflammation, the downstream impact of these carriers on platelet accumulation in thromboinflammatory conditions has yet to be studied. Here, we explore the ability of polymeric particles to divert platelets away from inflamed blood vessels both in vitro and in vivo. We find that untargeted and targeted micron-sized polymeric particles can successfully reduce platelet adhesion to an inflamed endothelial monolayer in vitro in blood flow systems and in vivo in a lipopolysaccharide-induced, systemic inflammation murine model. Our data represent initial work in developing cargo-free, anti-platelet therapeutics specifically for conditions of thromboinflammation.
Asunto(s)
Neutrófilos , Trombosis , Humanos , Animales , Ratones , Neutrófilos/metabolismo , Inflamación/metabolismo , Tromboinflamación , Trombosis/metabolismo , Plaquetas/metabolismo , Leucocitos/metabolismo , Selectina-P/metabolismoRESUMEN
Current treatments to prevent thrombosis, namely anticoagulants and platelets antagonists, remain complicated by the persistent risk of bleeding. Improved therapeutic strategies that diminish this risk would have a huge clinical impact. Antithrombotic agents that neutralize and inhibit polyphosphate (polyP) can be a powerful approach towards such a goal. Here, we report a design concept towards polyP inhibition, termed macromolecular polyanion inhibitors (MPI), with high binding affinity and specificity. Lead antithrombotic candidates are identified through a library screening of molecules which possess low charge density at physiological pH but which increase their charge upon binding to polyP, providing a smart way to enhance their activity and selectivity. The lead MPI candidates demonstrates antithrombotic activity in mouse models of thrombosis, does not give rise to bleeding, and is well tolerated in mice even at very high doses. The developed inhibitor is anticipated to open avenues in thrombosis prevention without bleeding risk, a challenge not addressed by current therapies.
Asunto(s)
Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Trombosis , Ratones , Animales , Fibrinolíticos/farmacología , Fibrinolíticos/uso terapéutico , Ligandos , Trombosis/tratamiento farmacológico , Trombosis/prevención & control , Anticoagulantes/efectos adversos , Hemorragia/inducido químicamente , Hemorragia/prevención & control , Hemorragia/tratamiento farmacológico , Inhibidores de Agregación Plaquetaria/farmacología , Inhibidores de Agregación Plaquetaria/uso terapéuticoRESUMEN
Endothelial inflammation is an important pathophysiological driving force in various acute and chronic inflammatory diseases. High-density lipoproteins (HDLs) play critical roles in regulating endothelial functions and resolving endothelial inflammation. In the present study, we developed synthetic HDLs (sHDLs) which actively target inflamed endothelium through conjugating vascular cell adhesion protein 1 (VCAM-1) specific VHPK peptide. The active targeting of VHPK-sHDLs was confirmed in vitro on TNF-α activated endothelial cells. VHPK-sHDLs presented potent anti-inflammatory efficacies in vitro through the reduction of proinflammatory cytokine production and inhibition of leukocyte adhesion to activated endothelium. VHPK-sHDLs showed increased binding on inflamed vessels and alleviated LPS-induced lung inflammation in vivo. The activated endothelium-targeted sHDLs may be further optimized to resolve endothelial inflammation in various inflammatory diseases.