Effect of Nitric Oxide on the Cryopreservation of Platelets / 대한진단검사의학회지

BACKGROUND: To determine whether nitric oxide (NO) could inhibit activation of platelets stored in a cold or frozen state, we measured platelet P-selectin expression and platelet-bound fibrinogen in platelet-rich plasma (PRP) with S-nitrosoglutathione (GSNO) (Sigma, USA) by flow cytometry. METHODS: PRP was prepared by centrifuging venous blood collected in a 3.2% sodium citrate tube from 10 healthy donors. It was aliquotted into 4 groups (no cryoprotectant, GSNO, GSNO/dimethyl sulfoxide [DMSO] [Sigma], and DMSO), and stored at room, cold and freezing temperatures for 24 hrs. We performed a flow cytometric analysis of all specimens stained with FITC-fibrinogen and PE-CD62P monoclonal antibodies (Becton Dickinson, USA). The results were compared according to the storage temperature and agonist among 4 groups. RESULTS: GSNO inhibited significantly the activation of frozen platelets, but not in the presence of DMSO. GSNO was also shown to preserve the aggregability of frozen platelets because in the presence of GSNO the delta percent change of P-selectin expression and fibrinogen binding of frozen platelets increased significantly irrelevant to DMSO. CONCLUSIONS: GSNO inhibited the activation of frozen platelets and preserved the platelet aggregability; therefore, it may be used as a protectant for platelet cryopreservation.

Efecto de los leucocitos sobre la lesión por almacenamiento en plaquetas de aferesis / Effect of leukocytes on the storage lesion in apheresis platelet

En el Banco de Sangre , las plaquetas sufren una serie de cambios físicos, metabólicos y fisiológicos que se denominan "lesión por almacenamiento" (LA), que depende de varios factores: métodos de preparación del concentrado, tipo de bolsa utilizada, concentración de plaquetas, número de leucocitos presentes en la unidad y acumulación de citoquinas. Todos ellos podrían producir activación plaquetaria y así afectar la calidad del producto, lo cual se reflejaría en una menor sobrevida de las plaquetas transfundidas. Basándose en lo anterior, se plantea que la remoción precoz de leucocitos aminoraría la LA en los concentrados plaquetarios (CPs) obtenidos por aféresis. Se estudiaron veinte CPs obtenidos mediante dos métodos de aféresis que difieren en el número de leucocitos residuales que permanecen en el producto final; un CP leucoreducido (Cobe Spectra) y otro estándar (Baxter CS 3000 plus). Las determinaciones se realizaron el día cero (prealmacenamiento) y al quinto día de almacenamiento. La evaluación de la LA incluyó marcadores de menbrana plaquetaria: p-selectina (CD62-P), glicoproteína Ib (CD42b), fosfatidilserina (Ax V.), factor tisular (CD142), formación de micropartículas (MPs), los cuales se analizaron por citometría de flujo, y citoquinas liberadas por los leucocitos y/o plaquetas activadas (IL-1. IL-6,FNT y RANTES), las cuales se analizaron por ELISA. El principal marcador de activación de las plaquetas (p-selectina) se encontró significativamente aumentado en los CP leucorreducidos (P: 0.001) y en los obtenidos en forma estándar (p: 0.02). La expresión de GPIb disminuyó significativamente solo en las plaquetas no leucorreducidas (p: 0.01). En relación a la actividad procoagulante de las plaquetas, se observó un aumento significativo en la expresión de fosfatidilserina sobre la cara externa de la membrana (p: 0.019) y de MPs plasmáticas (p: 0.025) solo en las plaquetas leucorreducidas y un muy leve aumento de la expresión de factor tisular...

Under Blood Bank storage conditions, platelet undergo a series of physical, metabolic and physiological changes that are denominated "platelet storage lesion" (PSL). This condition depends on several factors: the platelets number and the methodology used for the preparations of platelet concentrates (PC), type of storage bag, the number of leukocytes present in the cell unit, cytokines release, among others. All these factors may produce platelet activation and thus affect the quality of the product, which would be reflected in a shorter survival of the transfused platelets. Based on the previous knowledge, we hypothesized that early removal of leukocytes from the apheresis concentrate will diminish platelets "activation/lesion" during storage. We studied twenty PC obtained by two methods of apheresis that differed in the number of residual leukoreduced PC (Cobe Spectra) and a standard PC (3000 Baxter CS extra). The determinations were made at day zero (pre-storage) and at the fifth day of storage. The evaluation included markers present in platelets membrane, such as, p-selectin (CD62-P), glycoprotein Ib (CD42b), phosphatydilserine expression (PS). Tissue Factor (CD142) and microparticles (MPs) generation, that were analyzed by flow cytometry. Cytokines released by leucocytes or activated platelet (IL-1). IL-6, TNF and RANTES), were analysed by the ELISA technique. The most important marker of platelets activation, CD62-P, was significantly more increased in leukoreduced CP (P: 0.001) than in the standard method (p: 0.02). The expression of GPIb diminished significantly only in non-leukoreduced platelets (p: 0.01). With regard to the procoagulant activity of platelets, a significant increase in the PS expression was observed on the external face of the platelet membranes (p: 0.019) and on MPs (p: 0.025) only in leukoreduced preparations, changes that were accompanied by a very slight increase of tissue factor expression (p: 0.055). The determinations...

Vibrio vulnificus cytolysin induces hyperadhesiveness of pulmonary endothelial cells for neutrophils through endothelial P-selectin: a mechanism for pulmonary damage by Vibrio vulnificus cytolysin

Vibrio vulnificus cytolysin forms transmembrane pores that are permeable to calcium ions in pulmonary endothelial cells, and has been suggested as an important virulence factor that sequestrate neutrophils primarily in the lung. To elucidate the mechanism we investigated whether the cytolysin affect the expression of endothelial P-selectin and adhesiveness of pulmonary endothelial cells for neutrophils. The cytolysin increased the adhesiveness of CPAE cell, a pulmonary endothelial cell line, for neutrophils in a concentrationand time-dependent manner. The increase of adhesiveness occurred within several minutes after the cytolysin exposure, persisted up to 90 min, and was not affected by cycloheximide. Furthermore, flow cytometric analyses showed that cytolysin enhanced the level of P-selectin on CPAE cell surface. Therefore, these results suggest that the cytolysin-induced hyperadhesiveness of pulmonary endothelial cells for neutrophils is mediated by the mobilization of endothelial P-selectin to the cell surface.

Vibrio vulnificus cytolysin induces hyperadhesiveness of pulmonary endothelial cells for neutrophils through endothelial P-selectin: a mechanism for pulmonary damage by Vibrio vulnificus cytolysin

Vibrio vulnificus cytolysin forms transmembrane pores that are permeable to calcium ions in pulmonary endothelial cells, and has been suggested as an important virulence factor that sequestrate neutrophils primarily in the lung. To elucidate the mechanism we investigated whether the cytolysin affect the expression of endothelial P-selectin and adhesiveness of pulmonary endothelial cells for neutrophils. The cytolysin increased the adhesiveness of CPAE cell, a pulmonary endothelial cell line, for neutrophils in a concentrationand time-dependent manner. The increase of adhesiveness occurred within several minutes after the cytolysin exposure, persisted up to 90 min, and was not affected by cycloheximide. Furthermore, flow cytometric analyses showed that cytolysin enhanced the level of P-selectin on CPAE cell surface. Therefore, these results suggest that the cytolysin-induced hyperadhesiveness of pulmonary endothelial cells for neutrophils is mediated by the mobilization of endothelial P-selectin to the cell surface.