RESUMEN
Activated protein C (APC) is an anticoagulant protease that initiates cell signaling via protease-activated receptor 1 (PAR1) to regulate vascular integrity and inflammatory response. In this study, a recombinant APC variant (APC(N329Q)) mimicking the naturally occurring APC-ß plasma glycoform was found to exhibit superior PAR1 proteolysis at a cleavage site that selectively mediates cytoprotective signaling. APC(N329Q) also enhanced integrin αMß2-dependent PAR1 proteolysis to exert significantly improved antiinflammatory activity on macrophages compared with wild-type APC. Recent therapeutic applications of recombinant APC in ischemic stroke models have used APC variants with limited anticoagulant activity to negate potential bleeding side effects. Using a mouse model of ischemic stroke and late t-PA intervention, the neuroprotective activity of a murine APC variant with limited anticoagulant activity (mAPC(PS)) was compared with an identical APC variant except for the absence of glycosylation at the APC-ß sequon (mAPC(PS/N329Q)). Remarkably, mAPC(PS/N329Q) limited cerebral ischemic injury and reduced brain lesion volume significantly more effectively than mAPC(PS). Collectively, this study reveals the importance of APC glycosylation in controlling the efficacy of PAR1 proteolysis by APC and demonstrates the potential of novel APC variants with superior cytoprotective signaling function as enhanced therapeutic agents for the treatment of ischemic stroke.
Asunto(s)
Isquemia Encefálica/metabolismo , Proteína C/metabolismo , Receptor PAR-1/metabolismo , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Isquemia Encefálica/terapia , Catepsinas/genética , Catepsinas/metabolismo , Modelos Animales de Enfermedad , Receptor de Proteína C Endotelial , Variación Genética , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Oligosacáridos , Proteína C/genética , Proteína C/uso terapéutico , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/uso terapéutico , Proteolisis , Receptor PAR-1/genética , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/uso terapéutico , Transducción de SeñalRESUMEN
The anticoagulant-activated protein C (APC) acts not solely as a crucial regulator of thrombus formation following vascular injury, but also as a potent signalling enzyme with important functions in the control of both acute and chronic inflammatory disease. These properties have been exploited to therapeutic effect in diverse animal models of inflammatory disease, wherein recombinant APC administration has proven to effectively limit disease progression. Subsequent clinical trials led to the use of recombinant APC (Xigris) for the treatment of severe sepsis. Although originally deemed successful, Xigris was ultimately withdrawn due to lack of efficacy and an unacceptable bleeding risk. Despite this apparent failure, the problems that beset Xigris usage may be tractable using protein engineering approaches. In this review, we detail the protein engineering approaches that have been utilized to improve the therapeutic characteristics of recombinant APC, from early studies in which the distinct anti-coagulant and signalling activities of APC were separated to reduce bleeding risk, to current attempts to enhance APC cytoprotective signalling output for increased therapeutic efficacy at lower APC dosage. These novel engineered variants represent the next stage in the development of safer, more efficacious APC therapy in disease settings in which APC plays a protective role.
Asunto(s)
Proteína C/metabolismo , Ingeniería de Proteínas/métodos , Animales , Antiinflamatorios/metabolismo , Antiinflamatorios/uso terapéutico , Humanos , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Proteína C/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
The interferon-lambda (IFNL) cytokines have been shown to be important in HCV infection with SNPs in the IFNL3 gene associated with both natural and treatment induced viral clearance. We have recently shown that rs1299860 (an IFNL3 associated SNP) and an NK cell gene, KIR2DS3, synergised to increase the odds of chronic infection in a homogenous cohort of Irish women infected with HCV. To characterise a biological basis for the genetic synergy, we investigated for any evidence that IFNL cytokines regulate NK cell functions. Using a range of functional responses, we did not find any evidence of NK cell activation by IFNL3, IFNL1 or IFNL2 cytokines. Similar results were found using human and murine NK cells. In addition, and in contrast to our preliminary study, we did not find any evidence that IFNL cytokines inhibited NK cell cytokine production; thus, the biological basis for the genetic synergy remains to be discovered.