RESUMEN
Coagulation cofactors profoundly regulate hemostasis and are appealing targets for anticoagulants. However, targeting such proteins has been challenging because they lack an active site. To address this, we isolate an RNA aptamer termed T18.3 that binds to both factor V (FV) and FVa with nanomolar affinity and demonstrates clinically relevant anticoagulant activity in both plasma and whole blood. The aptamer also shows synergy with low molecular weight heparin and delivers potent anticoagulation in plasma collected from patients with coronavirus disease 2019 (COVID-19). Moreover, the aptamer's anticoagulant activity can be rapidly and efficiently reversed using protamine sulfate, which potentially allows fine-tuning of aptamer's activity post-administration. We further show that the aptamer achieves its anticoagulant activity by abrogating FV/FVa interactions with phospholipid membranes. Our success in generating an anticoagulant aptamer targeting FV/Va demonstrates the feasibility of using cofactor-binding aptamers as therapeutic protein inhibitors and reveals an unconventional working mechanism of an aptamer by interrupting protein-membrane interactions.
Asunto(s)
Anticoagulantes/farmacología , Aptámeros de Nucleótidos/farmacología , Coagulación Sanguínea/efectos de los fármacos , Factor V/antagonistas & inhibidores , Factor Va/antagonistas & inhibidores , Secuencia de Aminoácidos , Anticoagulantes/química , Anticoagulantes/metabolismo , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/metabolismo , Emparejamiento Base , Sitios de Unión , COVID-19/sangre , Membrana Celular/química , Membrana Celular/metabolismo , Factor V/química , Factor V/genética , Factor V/metabolismo , Factor Va/química , Factor Va/genética , Factor Va/metabolismo , Heparina de Bajo-Peso-Molecular/química , Heparina de Bajo-Peso-Molecular/metabolismo , Humanos , Sueros Inmunes/química , Sueros Inmunes/metabolismo , Modelos Moleculares , Conformación de Ácido Nucleico , Protaminas , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , SARS-CoV-2/crecimiento & desarrollo , SARS-CoV-2/patogenicidad , Técnica SELEX de Producción de Aptámeros , Especificidad por Sustrato , Tratamiento Farmacológico de COVID-19RESUMEN
Extensive fibrin deposition in the lungs and altered levels of circulating blood coagulation proteins in COVID-19 patients imply local derangement of pathways that limit fibrin formation and/or promote its clearance. We examined transcriptional profiles of bronchoalveolar lavage fluid (BALF) samples to identify molecular mechanisms underlying these coagulopathies. mRNA levels for regulators of the kallikrein-kinin (C1-inhibitor), coagulation (thrombomodulin, endothelial protein C receptor), and fibrinolytic (urokinase and urokinase receptor) pathways were significantly reduced in COVID-19 patients. While transcripts for several coagulation proteins were increased, those encoding tissue factor, the protein that initiates coagulation and whose expression is frequently increased in inflammatory disorders, were not increased in BALF from COVID-19 patients. Our analysis implicates enhanced propagation of coagulation and decreased fibrinolysis as drivers of the coagulopathy in the lungs of COVID-19 patients.
Asunto(s)
Coagulación Sanguínea/genética , COVID-19/patología , Fibrina/genética , Pulmón/patología , SARS-CoV-2 , Anticoagulantes/metabolismo , Líquido del Lavado Bronquioalveolar , COVID-19/genética , COVID-19/metabolismo , Receptor de Proteína C Endotelial/genética , Receptor de Proteína C Endotelial/metabolismo , Fibrina/metabolismo , Expresión Génica , Humanos , Sistema Calicreína-Quinina/genética , Calicreínas/genética , Calicreínas/metabolismo , Cininas/genética , Cininas/metabolismo , Pulmón/metabolismo , ARN Mensajero/metabolismo , Análisis de Secuencia de ARN , Trombomodulina/genética , Trombomodulina/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/genética , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
The COVID-19 pandemic has caused monumental mortality, and there are still no adequate therapies. Most severely ill COVID-19 patients manifest a hyperactivated immune response, instigated by interleukin 6 (IL6) that triggers a so called "cytokine storm" and coagulopathy. Hypoxia is also associated with COVID-19. So far overlooked is the fact that both IL6 and hypoxia depress the abundance of a key anticoagulant, Protein S. We speculate that the IL6-driven cytokine explosion plus hypoxemia causes a severe drop in Protein S level that exacerbates the thrombotic risk in COVID-19 patients. Here we highlight a mechanism by which the IL6-hypoxia curse causes a deadly hypercoagulable state in COVID-19 patients, and we suggest a path to therapy.