RESUMEN
Platelets play a critical role in hemostasis and thrombosis; therefore, in vitro assays that measure platelet reactivity are fundamental tools to gain insight into these physiologic processes, to diagnose platelet disorders, and to develop antithrombotic therapies. However, conventional platelet assays such as aggregometry, the clinical gold standard for assessing platelet function, are low throughput and require specialized equipment. Since platelets have a finite life span ex vivo, processes to miniaturize and multiplex assays allow a much broader overview of platelet function in significantly less time than conventional assays. Several groups have developed simplified, high-throughput approaches to quantify platelet activation with standard laboratory equipment to lower the barrier of entry to study platelet biology. This article describes a panel of optimized and validated high-throughput microplate assays to comprehensively assess platelet functionality, independently or in combination, to increase throughput and reduce costs. Specifically, following stimulation of platelets, a plate reader can be used to measure light transmission aggregation via absorbance; dense-granule secretion based on ATP-dependent luminescence generation; and cytosolic calcium levels with a cell-permeant, fluorescent Ca2+ -sensitive dye. Additionally, platelets are an easily accessible component of the blood that share signaling pathways with other cells, making them ideal for high-throughput drug screens. The highly adaptable and complementary assays presented in this article can be used to decipher the molecular mechanism underlying platelet activation or to identify novel inhibitors. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Microtiter plate-based light transmission aggregometry Basic Protocol 2: Measuring dense-granule secretion in high-throughput microplate assays Basic Protocol 3: Microtiter plate-based calcium mobilization Support Protocol: Platelet isolation and enumeration.
Asunto(s)
Agregación Plaquetaria , Pruebas de Función Plaquetaria , Pruebas de Función Plaquetaria/métodos , Calcio/metabolismo , Plaquetas/metabolismo , Activación PlaquetariaRESUMEN
The antiplatelet effect of polyunsaturated fatty acids is primarily attributed to its metabolism to bioactive metabolites by oxygenases, such as lipoxygenases (LOX). Platelets have demonstrated the ability to generate 15-LOX-derived metabolites (15-oxylipins); however, whether 15-LOX is in the platelet or is required for the formation of 15-oxylipins remains unclear. This study seeks to elucidate whether 15-LOX is required for the formation of 15-oxylipins in the platelet and determine their mechanistic effects on platelet reactivity. In this study, 15-HETrE, 15-HETE, and 15-HEPE attenuated collagen-induced platelet aggregation, and 15-HETrE inhibited platelet aggregation induced by different agonists. The observed anti-aggregatory effect was due to the inhibition of intracellular signaling including αIIbß3 and protein kinase C activities, calcium mobilization, and granule secretion. While 15-HETrE inhibited platelets partially through activation of peroxisome proliferator-activated receptor ß (PPARß), 15-HETE also inhibited platelets partially through activation of PPARα. 15-HETrE, 15-HETE, or 15-HEPE inhibited 12-LOX in vitro, with arachidonic acid as the substrate. Additionally, a 15-oxylipin-dependent attenuation of 12-HETE level was observed in platelets following ex vivo treatment with 15-HETrE, 15-HETE, or 15-HEPE. Platelets treated with DGLA formed 15-HETrE and collagen-induced platelet aggregation was attenuated only in the presence of ML355 or aspirin, but not in the presence of 15-LOX-1 or 15-LOX-2 inhibitors. Expression of 15-LOX-1, but not 15-LOX-2, was decreased in leukocyte-depleted platelets compared to non-depleted platelets. Taken together, these findings suggest that 15-oxylipins regulate platelet reactivity; however, platelet expression of 15-LOX-1 is low, suggesting that 15-oxylipins may be formed in the platelet through a 15-LOX-independent pathway.
Asunto(s)
Ácidos Grasos , Oxilipinas , Araquidonato 15-Lipooxigenasa , Eicosanoides , Inhibidores de la Lipooxigenasa/farmacología , Receptores Depuradores de Clase ERESUMEN
Immune thrombocytopenia (ITP) is an acquired bleeding disorder characterized by immunoglobulin G (IgG)-mediated platelet destruction. Current therapies primarily focus on reducing antiplatelet antibodies using immunosuppression or increasing platelet production with thrombopoietin mimetics. However, there are no universally safe and effective treatments for patients presenting with severe life-threatening bleeding. The IgG-degrading enzyme of Streptococcus pyogenes (IdeS), a protease with strict specificity for IgG, prevents IgG-driven immune disorders in murine models, including ITP. In clinical trials, IdeS prevented IgG-mediated kidney transplant rejection; however, the concentration of IdeS used to remove pathogenic antibodies causes profound hypogammaglobulinemia, and IdeS is immunogenic, which limits its use. Therefore, this study sought to determine whether targeting IdeS to FcγRIIA, a low-affinity IgG receptor on the surface of platelets, neutrophils, and monocytes, would be a viable strategy to decrease the pathogenesis of antiplatelet IgG and reduce treatment-related complications of nontargeted IdeS. We generated a recombinant protein conjugate by site-specifically linking the C-terminus of a single-chain variable fragment from an FcγRIIA antibody, clone IV.3, to the N-terminus of IdeS (scIV.3-IdeS). Platelets treated with scIV.3-IdeS had reduced binding of antiplatelet IgG from patients with ITP and decreased platelet phagocytosis in vitro, with no decrease in normal IgG. Treatment of mice expressing human FcγRIIA with scIV.3-IdeS reduced thrombocytopenia in a model of ITP and significantly improved the half-life of transfused platelets expressing human FcγRIIA. Together, these data suggest that scIV.3-IdeS can selectively remove pathogenic antiplatelet IgG and may be a potential treatment for patients with ITP and severe bleeding.
Asunto(s)
Púrpura Trombocitopénica Idiopática , Trombocitopenia , Animales , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/uso terapéutico , Plaquetas/metabolismo , Humanos , Inmunoglobulina G , Ratones , Púrpura Trombocitopénica Idiopática/tratamiento farmacológico , Streptococcus pyogenes/metabolismo , Trombocitopenia/tratamiento farmacológicoRESUMEN
The association between inflammation, infection, and venous thrombosis has long been recognized; yet, only in the last decades have we begun to understand the mechanisms through which the immune and coagulation systems interact and reciprocally regulate one another. These interconnected networks mount an effective response to injury and pathogen invasion, but if unregulated can result in pathological thrombosis and organ damage. Neutrophils, monocytes, and platelets interact with each other and the endothelium in host defense and also play critical roles in the formation of venous thromboembolism. This knowledge has advanced our understanding of both human physiology and pathophysiology, as well as identified mechanisms of anticoagulant resistance and novel therapeutic targets for the prevention and treatment of thrombosis. In this review, we discuss the contributions of inflammation and infection to venous thromboembolism.
Asunto(s)
Infecciones/complicaciones , Inflamación/complicaciones , Tromboembolia Venosa/etiología , Inmunidad Adaptativa , Coagulación Sanguínea/fisiología , Plaquetas/fisiología , Endotelio Vascular/fisiología , Trampas Extracelulares , Vesículas Extracelulares/fisiología , Fibrinólisis , Hematopoyesis , Hemostasis/fisiología , Humanos , Sistema Inmunológico/fisiología , Leucocitos/fisiología , Monocitos/fisiología , Neutrófilos/fisiología , Tromboembolia Venosa/prevención & control , Tromboembolia Venosa/terapiaRESUMEN
The oxylipins, 5S,12S-dihydroxy-6E,8Z,10E,14Z-eicosatetraenoic acid (5S,12S-diHETE) and 5S,15S-dihydroxy-6E,8Z,11Z,13E-eicosatetraenoic acid (5S,15S-diHETE), have been identified in cell exudates and have chemotactic activity toward eosinophils and neutrophils. Their biosynthesis has been proposed to occur by sequential oxidations of arachidonic acid (AA) by lipoxygenase enzymes, specifically through oxidation of AA by h5-LOX followed by h12-LOX, h15-LOX-1, or h15-LOX-2. In this work, h15-LOX-1 demonstrates altered positional specificity when reacting with 5S-HETE, producing 90% 5S,12S-diHETE, instead of 5S,15S-diHETE, with kinetics 5-fold greater than that of h12-LOX. This is consistent with previous work in which h15-LOX-1 reacts with 7S-HDHA, producing the noncanonical, DHA-derived, specialized pro-resolving mediator, 7S,14S-diHDHA. It is also determined that oxygenation of 5S-HETE by h15-LOX-2 produces 5S,15S-diHETE and its biosynthetic kcat/KM flux is 2-fold greater than that of h15-LOX-1, suggesting that h15-LOX-2 may have a greater role in lipoxin biosynthesis than previously thought. In addition, it is shown that oxygenation of 12S-HETE and 15S-HETE by h5-LOX is kinetically slow, suggesting that the first step in the in vitro biosynthesis of both 5S,12S-diHETE and 5S,15S-diHETE is the production of 5S-HETE.
Asunto(s)
Araquidonato 15-Lipooxigenasa/metabolismo , Lipoxinas/metabolismo , Ácido Araquidónico/metabolismo , Ácidos Araquidónicos/metabolismo , Humanos , Ácidos Hidroxieicosatetraenoicos/metabolismoRESUMEN
The two oxylipins 7S,14S-dihydroxydocosahexaenoic acid (diHDHA) and 7S,17S-diHDHA [resolvin D5 (RvD5)] have been found in macrophages and infectious inflammatory exudates and are believed to function as specialized pro-resolving mediators (SPMs). Their biosynthesis is thought to proceed through sequential oxidations of DHA by lipoxygenase (LOX) enzymes, specifically, by human 5-LOX (h5-LOX) first to 7(S)-hydroxy-4Z,8E,10Z,13Z,16Z,19Z-DHA (7S-HDHA), followed by human platelet 12-LOX (h12-LOX) to form 7(S),14(S)-dihydroxy-4Z,8E,10Z,12E,16Z,19Z-DHA (7S,14S-diHDHA) or human reticulocyte 15-LOX-1 (h15-LOX-1) to form RvD5. In this work, we determined that oxidation of 7(S)-hydroperoxy-4Z,8E,10Z,13Z,16Z,19Z-DHA to 7S,14S-diHDHA is performed with similar kinetics by either h12-LOX or h15-LOX-1. The oxidation at C14 of DHA by h12-LOX was expected, but the noncanonical reaction of h15-LOX-1 to make over 80% 7S,14S-diHDHA was larger than expected. Results of computer modeling suggested that the alcohol on C7 of 7S-HDHA hydrogen bonds with the backbone carbonyl of Ile399, forcing the hydrogen abstraction from C12 to oxygenate on C14 but not C17. This result raised questions regarding the synthesis of RvD5. Strikingly, we found that h15-LOX-2 oxygenates 7S-HDHA almost exclusively at C17, forming RvD5 with faster kinetics than does h15-LOX-1. The presence of h15-LOX-2 in neutrophils and macrophages suggests that it may have a greater role in biosynthesizing SPMs than previously thought. We also determined that the reactions of h5-LOX with 14(S)-hydroperoxy-4Z,7Z,10Z,12E,16Z,19Z-DHA and 17(S)-hydroperoxy-4Z,7Z,10Z,13Z,15E,19Z-DHA are kinetically slow compared with DHA, suggesting that these reactions may be minor biosynthetic routes in vivo. Additionally, we show that 7S,14S-diHDHA and RvD5 have anti-aggregation properties with platelets at low micromolar potencies, which could directly regulate clot resolution.
Asunto(s)
Araquidonato 15-Lipooxigenasa/metabolismo , Ácidos Docosahexaenoicos/biosíntesis , Plaquetas/metabolismo , Ácidos Docosahexaenoicos/química , HumanosRESUMEN
Human reticulocyte 15-lipoxygenase-1 (h15-LOX-1 or ALOX15) and platelet 12-lipoxygenase (h12-LOX or ALOX12) catalysis of docosahexaenoic acid (DHA) and the maresin precursor, 14S-hydroperoxy-4Z,7Z,10Z,12E,16Z,19Z-docosahexaenoic acid (14S-HpDHA), were investigated to determine their product profiles and relative rates in the biosynthesis of the key maresin intermediate, 13S,14S-epoxy-4Z,7Z,9E,11E,16Z,19Z-docosahexaenoic acid (13S,14S-epoxy-DHA). Both enzymes converted DHA to 14S-HpDHA, with h12-LOX having a 39-fold greater kcat/KM value (14.0 ± 0.8 s-1 µM-1) than that of h15-LOX-1 (0.36 ± 0.08 s-1 µM-1) and a 1.8-fold greater 14S-HpDHA product selectivity, 81 and 46%, respectively. However, h12-LOX was markedly less effective at producing 13S,14S-epoxy-DHA from 14S-HpDHA than h15-LOX-1, with a 4.6-fold smaller kcat/KM value, 0.0024 ± 0.0002 and 0.11 ± 0.006 s-1 µM-1, respectively. This is the first evidence of h15-LOX-1 to catalyze this reaction and reveals a novel in vitro pathway for maresin biosynthesis. In addition, epoxidation of 14S-HpDHA is negatively regulated through allosteric oxylipin binding to h15-LOX-1 and h12-LOX. For h15-LOX-1, 14S-HpDHA (Kd = 6.0 µM), 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12S-HETE) (Kd = 3.5 µM), and 14S-hydroxy-7Z,10Z,12E,16Z,19Z-docosapentaenoic acid (14S-HDPAω-3) (Kd = 4.0 µM) were shown to decrease 13S,14S-epoxy-DHA production. h12-LOX was also shown to be allosterically regulated by 14S-HpDHA (Kd = 3.5 µM) and 14S-HDPAω-3 (Kd = 4.0 µM); however, 12S-HETE showed no effect, indicating for the first time an allosteric response by h12-LOX. Finally, 14S-HpDHA inhibited platelet aggregation at a submicrololar concentration, which may have implications in the benefits of diets rich in DHA. These in vitro biosynthetic pathways may help guide in vivo maresin biosynthetic investigations and possibly direct therapeutic interventions.
Asunto(s)
Araquidonato 12-Lipooxigenasa/metabolismo , Araquidonato 15-Lipooxigenasa/metabolismo , Ácidos Docosahexaenoicos/biosíntesis , Ácidos Docosahexaenoicos/metabolismo , Regulación Alostérica , Araquidonato 12-Lipooxigenasa/aislamiento & purificación , Araquidonato 15-Lipooxigenasa/aislamiento & purificación , Ácidos Docosahexaenoicos/análogos & derivados , Ácidos Docosahexaenoicos/química , Humanos , Estructura Molecular , Agregación Plaquetaria , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Deep vein thrombosis (DVT), caused by alterations in venous homeostasis is the third most common cause of cardiovascular mortality; however, key molecular determinants in venous thrombosis have not been fully elucidated. Several lines of evidence indicate that DVT occurs at the intersection of dysregulated inflammation and coagulation. The enzyme ectonucleoside tri(di)phosphohydrolase (ENTPD1, also known as CD39) is a vascular ecto-apyrase on the surface of leukocytes and the endothelium that inhibits intravascular inflammation and thrombosis by hydrolysis of phosphodiester bonds from nucleotides released by activated cells. Here, we evaluated the contribution of CD39 to venous thrombosis in a restricted-flow model of murine inferior vena cava stenosis. CD39-deficiency conferred a >2-fold increase in venous thrombogenesis, characterized by increased leukocyte engagement, neutrophil extracellular trap formation, fibrin, and local activation of tissue factor in the thrombotic milieu. This was orchestrated by increased phosphorylation of the p65 subunit of NFκB, activation of the NLRP3 inflammasome, and interleukin-1ß (IL-1ß) release in CD39-deficient mice. Substantiating these findings, an IL-1ß-neutralizing antibody attenuated the thrombosis risk in CD39-deficient mice. These data demonstrate that IL-1ß is a key accelerant of venous thrombo-inflammation, which can be suppressed by CD39. CD39 inhibits in vivo crosstalk between inflammation and coagulation pathways, and is a critical vascular checkpoint in venous thrombosis.
Asunto(s)
Antígenos CD/metabolismo , Apirasa/metabolismo , Inflamasomas/metabolismo , Interleucina-1beta/metabolismo , Trombosis de la Vena/metabolismo , Animales , Antígenos CD/genética , Apirasa/genética , Modelos Animales de Enfermedad , Trampas Extracelulares/genética , Trampas Extracelulares/metabolismo , Humanos , Inflamasomas/genética , Proteína Antagonista del Receptor de Interleucina 1/farmacología , Interleucina-1beta/genética , Ratones , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Neutrófilos/metabolismo , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismo , Trombosis de la Vena/genética , Trombosis de la Vena/patologíaRESUMEN
The reaction of 5 S,15 S-dihydroperoxyeicosatetraenoic acid (5,15-diHpETE) with human 5-lipoxygenase (LOX), human platelet 12-LOX, and human reticulocyte 15-LOX-1 was investigated to determine the reactivity and relative rates of producing lipoxins (LXs). 5-LOX does not react with 5,15-diHpETE, although it can produce LXA4 when 15-HpETE is the substrate. In contrast, both 12-LOX and 15-LOX-1 react with 5,15-diHpETE, forming specifically LXB4. For 12-LOX and 5,15-diHpETE, the kinetic parameters are kcat = 0.17 s-1 and kcat/ KM = 0.011 µM-1 s-1 [106- and 1600-fold lower than those for 12-LOX oxygenation of arachidonic acid (AA), respectively]. On the other hand, for 15-LOX-1 the equivalent parameters are kcat = 4.6 s-1 and kcat/ KM = 0.21 µM-1 s-1 (3-fold higher and similar to those for 12-HpETE formation by 15-LOX-1 from AA, respectively). This contrasts with the complete lack of reaction of 15-LOX-2 with 5,15-diHpETE [Green, A. R., et al. (2016) Biochemistry 55, 2832-2840]. Our data indicate that 12-LOX is markedly inferior to 15-LOX-1 in catalyzing the production of LXB4 from 5,15-diHpETE. Platelet aggregation was inhibited by the addition of 5,15-diHpETE, with an IC50 of 1.3 µM; however, LXB4 did not significantly inhibit collagen-mediated platelet activation up to 10 µM. In summary, LXB4 is the primary product of 12-LOX and 15-LOX-1 catalysis, if 5,15-diHpETE is the substrate, with 15-LOX-1 being 20-fold more efficient than 12-LOX. LXA4 is the primary product with 5-LOX but only if 15-HpETE is the substrate. Approximately equal proportions of LXA4 and LXB4 are produced by 12-LOX but only if LTA4 is the substrate, as described previously [Sheppard, K. A., et al. (1992) Biochim. Biophys. Acta 1133, 223-234].
Asunto(s)
Araquidonato 12-Lipooxigenasa/metabolismo , Araquidonato 15-Lipooxigenasa/metabolismo , Araquidonato 5-Lipooxigenasa/metabolismo , Leucotrienos/metabolismo , Peróxidos Lipídicos/metabolismo , Lipoxinas/biosíntesis , Biocatálisis , Vías Biosintéticas , Plaquetas/metabolismo , Humanos , Ácidos Hidroxieicosatetraenoicos/metabolismo , Cinética , Leucocitos/metabolismo , Reticulocitos/metabolismo , Especificidad por SustratoRESUMEN
OBJECTIVE: Platelet activation after stimulation of PAR (protease-activated receptor) 4 is heightened in platelets from blacks compared with those from whites. The difference in PAR4 signaling by race is partially explained by a single-nucleotide variant in PAR4 encoding for either an alanine or threonine at amino acid 120 in the second transmembrane domain. The current study sought to determine whether the difference in PAR4 signaling by this PAR4 variant is because of biased Gq signaling and whether the difference in PAR4 activity results in resistance to traditional antiplatelet intervention. APPROACH AND RESULTS: Membranes expressing human PAR4-120 variants were reconstituted with either Gq or G13 to determine the kinetics of G protein activation. The kinetics of Gq and G13 activation were both increased in membranes expressing PAR4-Thr120 compared with those expressing PAR4-Ala120. Further, inhibiting PAR4-mediated platelet activation by targeting COX (cyclooxygenase) and P2Y12 receptor was less effective in platelets from subjects expressing PAR4-Thr120 compared with PAR4-Ala120. Additionally, ex vivo thrombus formation in whole blood was evaluated at high shear to determine the relationship between PAR4 variant expression and response to antiplatelet drugs. Ex vivo thrombus formation was enhanced in blood from subjects expressing PAR4-Thr120 in the presence or absence of antiplatelet therapy. CONCLUSIONS: Together, these data support that the signaling difference by the PAR4-120 variant results in the enhancement of both Gq and G13 activation and an increase in thrombus formation resulting in a potential resistance to traditional antiplatelet therapies targeting COX-1 and the P2Y12 receptor.
Asunto(s)
Aspirina/uso terapéutico , Coagulación Sanguínea/efectos de los fármacos , Plaquetas/efectos de los fármacos , Clopidogrel/uso terapéutico , Inhibidores de la Ciclooxigenasa/uso terapéutico , Resistencia a Medicamentos , Inhibidores de Agregación Plaquetaria/uso terapéutico , Agregación Plaquetaria/efectos de los fármacos , Antagonistas del Receptor Purinérgico P2Y/uso terapéutico , Receptores de Trombina/sangre , Negro o Afroamericano/genética , Coagulación Sanguínea/genética , Plaquetas/metabolismo , Ciclooxigenasa 1/sangre , Resistencia a Medicamentos/genética , Subunidades alfa de la Proteína de Unión al GTP G12-G13/sangre , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/sangre , Genotipo , Humanos , Cinética , Variantes Farmacogenómicas , Fenotipo , Agregación Plaquetaria/genética , Polimorfismo de Nucleótido Simple , Receptores Purinérgicos P2Y12/sangre , Receptores Purinérgicos P2Y12/efectos de los fármacos , Receptores de Trombina/genética , Transducción de Señal/efectos de los fármacos , Población Blanca/genética , Proteína de Unión al GTP rhoA/sangreRESUMEN
Platelets are key contributors to the formation of occlusive thrombi; the major underlying cause of ischemic heart disease and stroke. Antiplatelet therapy has reduced the morbidity and mortality associated with thrombotic events; however, the utility of current antiplatelet therapies is limited by the concomitant risk of an adverse bleeding event. Novel antiplatelet therapies that are more efficacious at inhibiting thrombosis while minimally affecting hemostasis are required. Platelet-type 12-(S)-lipoxygenase (12-LOX), an oxygenase shown to potentiate platelet activation, represents a novel antiplatelet target. Recently, a selective 12-LOX inhibitor, ML355, was shown to decrease thrombosis without prolonging hemostasis. While published data suggests targeting 12-LOX is a viable approach, further work is required to determine the safety and effectiveness of 12-LOX inhibitors in humans.
Asunto(s)
Araquidonato 12-Lipooxigenasa/sangre , Inhibidores de la Lipooxigenasa/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Animales , Plaquetas/efectos de los fármacos , Plaquetas/enzimología , Humanos , Terapia Molecular DirigidaRESUMEN
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.
Asunto(s)
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
The dihomo-γ-linolenic acid (DGLA)-derived metabolite of 12-lipoxygenase, 12-hydroxy-eicosatrienoic acid (12-HETrE), was recently shown to potently inhibit thrombus formation without prolonging bleeding in murine models. Although 12-HETrE was found to inhibit platelet activation via the Gαs signaling pathway, the Gαs-coupled receptor by which 12-HETrE mediates its antiplatelet effects has yet to be identified. Defining the receptor by which 12-HETrE exerts its effects is key to determining its therapeutic potential as an antiplatelet drug. Therefore, the goal of this study was to determine the Gαs-coupled platelet receptor through which 12-HETrE exerts its antiplatelet effects. In this study, we showed that pharmacological inhibition of the prostacyclin (IP) receptor in human platelets or genetic ablation of IP in murine platelets prevented 12-HETrE from blocking aggregation in vitro. Furthermore, the antithrombotic effects of 12-HETrE were significantly diminished in IP knockout mice in vivo. Together these data demonstrate that the antiplatelet effects of 12-HETrE are at least partially dependent on IP signaling. Importantly, this work identified 12-HETrE as a novel regulator of IP signaling that may aid in the rationale for design of novel therapeutics to inhibit platelet function. Additionally, this study provides further insight into the mechanism by which DGLA supplementation inhibits platelets function.
RESUMEN
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
OBJECTIVE: Black individuals are at an increased risk of myocardial infarction and stroke, 2 vascular diseases with strong thrombotic components. Platelet activation is a key step in platelet clot formation leading to myocardial infarction and stroke, and recent work supports a racial difference in platelet aggregation through the thrombin protease-activated receptors (PARs). The underlying mechanism for this racial difference, however, has not been established. Determining where in the signaling cascade these racial differences emerge will aid in understanding why individuals of differing racial ancestry may possess an inherent difference in their responsiveness to antiplatelet therapies. APPROACH AND RESULTS: Washed human platelets from black volunteers were hyperaggregable in response to PAR4-mediated platelet stimulation compared with whites. Interestingly, the racial difference in PAR4-mediated platelet aggregation persisted in platelets treated ex vivo with aspirin and 2MeSAMP (2-methylthioadenosine 5'-monophosphate triethylammonium salt hydrate), suggesting that the racial difference is independent of secondary feedback. Furthermore, stimulation of platelets from black donors with PAR4-activating peptide showed a potentiated level of activation through the Gq pathway compared with platelets from white donors. Differences in signaling included increased Ca(2+) mobilization, Rap1 (Ras-related protein 1) activation, and integrin αIIbß3 activation with no observed difference in platelet protein expression between the groups tested. CONCLUSIONS: Our study is the first to demonstrate that the Gq pathway is differentially regulated by race after PAR4 stimulation in human platelets. Furthermore, the racial difference in PAR4-mediated platelet aggregation persisted in the presence of cyclooxygenase and P2Y12 receptor dual inhibition, suggesting that current antiplatelet therapy may provide less protection to blacks than whites.
Asunto(s)
Población Negra , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/sangre , Activación Plaquetaria/fisiología , Receptores de Trombina/sangre , Población Blanca , Adulto , Señalización del Calcio , Inhibidores de la Ciclooxigenasa/farmacología , Femenino , Humanos , Masculino , Activación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/fisiología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Prostaglandina-Endoperóxido Sintasas/sangre , Proteína Quinasa C/sangre , Antagonistas del Receptor Purinérgico P2Y/farmacología , Receptores Purinérgicos P2Y12/sangre , Complejo Shelterina , Transducción de Señal , Proteínas de Unión a Telómeros/sangreRESUMEN
Human platelets express 2 thrombin receptors: protease-activated receptor (PAR)-1 and PAR4. Recently, we reported 3.7-fold increased PAR4-mediated aggregation kinetics in platelets from black subjects compared with white subjects. We now show that platelets from blacks (n = 70) express 14% more PAR4 protein than those from whites (n = 84), but this difference is not associated with platelet PAR4 function. Quantitative trait locus analysis identified 3 common single nucleotide polymorphisms in the PAR4 gene (F2RL3) associated with PAR4-induced platelet aggregation. Among these single nucleotide polymorphisms, rs773902 determines whether residue 120 in transmembrane domain 2 is an alanine (Ala) or threonine (Thr). Compared with the Ala120 variant, Thr120 was more common in black subjects than in white subjects (63% vs 19%), was associated with higher PAR4-induced human platelet aggregation and Ca2+ flux, and generated greater inositol 1,4,5-triphosphate in transfected cells. A second, less frequent F2RL3 variant, Phe296Val, was only observed in blacks and abolished the enhanced PAR4-induced platelet aggregation and 1,4,5-triphosphate generation associated with PAR4-Thr120. PAR4 genotype did not affect vorapaxar inhibition of platelet PAR1 function, but a strong pharmacogenetic effect was observed with the PAR4-specific antagonist YD-3 [1-benzyl-3(ethoxycarbonylphenyl)-indazole]. These findings may have an important pharmacogenetic effect on the development of new PAR antagonists.
Asunto(s)
Plaquetas/fisiología , Activación Plaquetaria/genética , Agregación Plaquetaria/genética , Polimorfismo de Nucleótido Simple , Grupos Raciales , Receptores de Trombina/genética , Población Negra/genética , Plaquetas/metabolismo , Femenino , Genotipo , Células HEK293 , Humanos , Masculino , Pruebas de Función Plaquetaria , Grupos Raciales/genética , Receptores de Trombina/metabolismo , Población Blanca/genéticaRESUMEN
Platelets are essential in maintaining hemostasis following inflammation or injury to the vasculature. Dysregulated platelet activity often results in thrombotic complications leading to myocardial infarction and stroke. Activation of the FcγRIIa receptor leads to immune-mediated thrombosis, which is often life threatening in patients undergoing heparin-induced thrombocytopenia or sepsis. Inhibiting FcγRIIa-mediated activation in platelets has been shown to limit thrombosis and is the principal target for prevention of immune-mediated platelet activation. In this study, we show for the first time that platelet 12(S)-lipoxygenase (12-LOX), a highly expressed oxylipin-producing enzyme in the human platelet, is an essential component of FcγRIIa-mediated thrombosis. Pharmacologic inhibition of 12-LOX in human platelets resulted in significant attenuation of FcγRIIa-mediated aggregation. Platelet 12-LOX was shown to be essential for FcγRIIa-induced phospholipase Cγ2 activity leading to activation of calcium mobilization, Rap1 and protein kinase C activation, and subsequent activation of the integrin αIIbß3. Additionally, platelets from transgenic mice expressing human FcγRIIa but deficient in platelet 12-LOX, failed to form normal platelet aggregates and exhibited deficiencies in Rap1 and αIIbß3 activation. These results support an essential role for 12-LOX in regulating FcγRIIa-mediated platelet function and identifies 12-LOX as a potential therapeutic target to limit immune-mediated thrombosis.
Asunto(s)
Araquidonato 12-Lipooxigenasa/metabolismo , Plaquetas/metabolismo , Receptores de IgG/metabolismo , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/química , Animales , Calcio/metabolismo , Activación Enzimática , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosfolipasa C gamma/metabolismo , Fosforilación , Activación Plaquetaria , Agregación Plaquetaria , Proteína Quinasa C/metabolismo , Transducción de Señal , Trombosis/inmunologíaRESUMEN
The prevalence of cardiovascular disease (CVD), the leading cause of death in the US, is predicted to increase due to the shift in age of the general population and increase in CVD risk factors such as obesity and diabetes. New therapies are required to decrease the prevalence of CVD risk factors (obesity and diabetes) as well as reduce atherothrombosis, the major cause of CVD related mortality. Oxylipins, bioactive metabolites derived from the oxygenation of polyunsaturated fatty acids, play a role in the progression of CVD risk factors and thrombosis. Aspirin, a cyclooxygenase-1 inhibitor, decreases atherothrombotic associated mortality by 25%. These potent effects of aspirin have shown the utility of modulating oxylipin signaling pathways to decrease CVD mortality. The role of many oxylipins in the progression of CVD, however, is still uncertain or controversial. An increased understanding of the role oxylipins play in CVD risk factors and thrombosis could lead to new therapies to decrease the prevalence of CVD and its associated mortality.
RESUMEN
Immune complexes consisting of heparin, platelet factor 4 (PF4), and PF4/heparin-reactive antibodies are central to the pathogenesis of heparin-induced thrombocytopenia (HIT). It is as yet unclear what triggers the initial induction of pathogenic antibodies. We identified B cells in peripheral blood of healthy adults that produce PF4/heparin-specific antibodies following in vitro stimulation with proinflammatory molecules containing deoxycytosine-deoxyguanosine (CpG). Similarly, B cells from unmanipulated wild-type mice produced PF4/heparin-specific antibodies following in vitro or in vivo CpG stimulation. Thus, both healthy humans and mice possess preexisting inactive/tolerant PF4/heparin-specific B cells. The findings suggest that breakdown of tolerance leads to PF4/heparin-specific B-cell activation and antibody production in patients developing HIT. Consistent with this concept, mice lacking protein kinase Cδ (PKCδ) that are prone to breakdown of B-cell tolerance produced anti-PF4/heparin antibodies spontaneously. Therefore, breakdown of tolerance can lead to PF4/heparin-specific antibody production, and B-cell tolerance may play an important role in HIT pathogenesis.