RESUMEN
Extracellular diphosphate and triphosphate nucleotides are released from activated or injured cells to trigger vascular and immune P2 purinergic receptors, provoking inflammation and vascular thrombosis. These metabokines are scavenged by ectonucleoside triphosphate diphosphohydrolase-1 (E-NTPDase1 or CD39). Further degradation of the monophosphate nucleoside end products occurs by surface ecto-5'-nucleotidase (NMPase) or CD73. These ectoenzymatic processes work in tandem to promote adenosinergic responses, which are immunosuppressive and antithrombotic. These homeostatic ectoenzymatic mechanisms are lost in the setting of oxidative stress, which exacerbates inflammatory processes. We have engineered bifunctional enzymes made up from ectodomains (ECDs) of CD39 and CD73 within a single polypeptide. Human alkaline phosphatase-ectodomain (ALP-ECD) and human acid phosphatase-ectodomain (HAP-ECD) fusion proteins were also generated, characterized, and compared with these CD39-ECD, CD73-ECD, and bifunctional fusion proteins. Through the application of colorimetrical functional assays and high-performance liquid chromatography kinetic assays, we demonstrate that the bifunctional ectoenzymes express high levels of CD39-like NTPDase activity and CD73-like NMPase activity. Chimeric CD39-CD73-ECD proteins were superior in converting triphosphate and diphosphate nucleotides into nucleosides when compared with ALP-ECD and HAP-ECD. We also note a pH sensitivity difference between the bifunctional fusion proteins and parental fusions, as well as ectoenzymatic property distinctions. Intriguingly, these innovative reagents decreased platelet activation to exogenous agonists in vitro. We propose that these chimeric fusion proteins could serve as therapeutic agents in inflammatory diseases, acting to scavenge proinflammatory ATP and also generate anti-inflammatory adenosine.
Asunto(s)
5'-Nucleotidasa/farmacología , Antiinflamatorios/farmacología , Apirasa/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/efectos de los fármacos , Ingeniería de Proteínas , 5'-Nucleotidasa/química , 5'-Nucleotidasa/genética , 5'-Nucleotidasa/metabolismo , Nucleótidos de Adenina/metabolismo , Antiinflamatorios/química , Antiinflamatorios/metabolismo , Apirasa/química , Apirasa/genética , Apirasa/metabolismo , Proteínas Ligadas a GPI/química , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Proteínas Ligadas a GPI/farmacología , Células HEK293 , Humanos , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Inhibidores de Agregación Plaquetaria/química , Inhibidores de Agregación Plaquetaria/metabolismo , Conformación Proteica , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
OBJECTIVE: The researchers sought to determine what factors might affect the outcomes of remediation, including the likelihood of recidivism, among nurses who had been the subject of disciplinary action and had been put on probation by a state board of nursing. METHODS: Boards of nursing in six states, Arizona, Maryland, Massachusetts, Minnesota, Nebraska, and North Carolina, chose to participate in this exploratory study. A 29-item questionnaire was used to investigate the records of 207 RNs, LPNs, and advanced practice RNs (APRNs) who were disciplined and put on probation by a state nursing board in 2001, as well as to collect data on their employment settings, the boards' actions, and remediation outcomes (the presence or absence of recidivism); 491 nurses who had not been disciplined served as controls. RESULTS: Among the disciplined nurses studied, 57% were RNs, 36% were LPNs, 3% held both RN and LPN licenses, and 3% were APRNs. Of the disciplined group, 39% recidivated between 2001 and 2005. Three factors were shown to influence the recidivism rate: having a history of criminal conviction, having committed more than one violation before the 2001 probation, and changing employers during the probationary period.Data on history of criminal conviction prior to state board disciplinary action were available for 112 (54%) of the 207 nurses. Among those 112, 35% (n = 39) had a history of criminal conviction, whereas only 3% of the control group reported one. The recidivism rate among those with a history of criminal conviction (56%; 22 of 39 nurses) was nearly twice as high as the rate among those without such a history (33%; 24 of 73). Also, 33% of the disciplined nurses changed employers during their probation; the recidivism rate among them was more than twice the rate among the disciplined nurses who stayed with the same employer. The recidivism rate of the 45 disciplined nurses who committed more than one practice-related violation from 1996 through 2001 was twice as high as the rate of those who committed only a single violation.The proportion of men who had been disciplined was more than twice the proportion of men in the national nursing workforce. Younger nurses (both men and women) were more likely to recidivate. CONCLUSIONS: All health care regulators and nursing employers should be aware of the association between a history of criminal conviction and the likelihood of committing a violation that requires state nursing board disciplinary action.