PMC

Protein localization of Rap1, talin, and GPIIIa. PLT-rich plasma from freshly drawn PLTs was either left untreated (resting PLTs) or activated with 10 μmol/L ADP for 30 minutes. Samples from stored PLT concentrates were taken on Days 1 and 7 of storage. The localization of the respective proteins is visualized by type-specific secondary antibodies labeled with Alexa Fluor 488 in a white-field epifluorescence microscope. A representative image (marked by an arrow) with a threefold magnification is displayed on top of each sample.

Influence of the PI3-kinase inhibitor LY294002 on the expression of selected surface proteins. Determinations of (A) CD62P, (B) active state of αIIbβ3 monitored by Pac-1 binding, and (C) CD61 on different days of PLT storage were made in the absence ( ) or presence ( ) of the PI3-kinase inhibitor LY294002. The right bar (7*) indicates addition of 10 μmol/L LY294002 to the previously untreated control sample on Day 7 (light gray). The results represent the mean ± 1 SD of four independent samples (n = 4). *p < 0.05 and **p < 0.01 comparing results with versus without the inhibitor LY294002.

Influence of LY294002 on the amount of activated and total Rap1 during storage of PLTs. Pairs of untreated and treated supernatants after pull-down (top panel) or an aliquot of the total lysate (bottom panel), respectively, were loaded from Storage Days 1, 4, and 7 without and with the inhibitor. The last lane (+*) represents data from control PLTs stored for 7 days and then treated with fresh 10 μmol/L LY294002 for 45 minutes. Representative Western blots from experiments performed five times with similar results are shown. Band intensities were determined by densitometry and values normalized to the Day 1 sample for active and total Rap1, respectively.

Influence of the PI3-kinase inhibitor LY294002 on PLT quality over time of storage. In vitro measures were determined on Day 1 (control) and on Days 2, 4, and 7 during storage (n = 5). Metabolic activity was monitored by assessing glucose and lactate levels. PLT integrity was assessed using the ESC and HSR as well as morphology using a modification of the Kunicki morphology score. On Day 7, 10 μmol/L fresh inhibitor was added 45 minutes before the morphologic analysis (day 7*). n.d. = not determined. The results represent the mean ± 1 SD of four independent samples (n = 4). *p < 0.05 comparing results with versus without the inhibitor LY294002.

PLT and Rap1 activation during storage. (A) Fresh PRP ( ) was left untreated (resting PLTs) or activated with 10 μmol/L ADP and labeled with CD62P-PE, as were PLT concentrates (□) sampled on Days 1 and 7 of storage. The graph shows the mean of at least four separate samples ± 1 SD. (B) The activated form of Rap1 was purified using glutathione-Sepharose beads coated with GST-Rap1 binding domain of RalGDS. Bar graph (top) represents the amount of activated Rap1 in the pull-down assay determined by quantification of the intensity of the bands on Western blots against Rap1. The data represent the mean ± 1 SD of four individual experiments and values are relative to the resting sample, which was set to 1. A representative blot is shown (below). An aliquot of the lysates taken prior to the pull-down assay was analyzed for the total Rap1 amount, with actin as a loading control. This analysis was repeated on at least four different donors of fresh PLTs or at least four different PLT units.