The role of adenosine diphosphate mediated platelet responsiveness for the stability of platelet integrity in citrated whole blood under ex vivo conditions

Figures

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  Fig 1. Time-dependent CD62P surface expression and fibrinogen binding of platelets in citrated WB. The histograms show the mean fluorescence intensities (MFI) of basal (white columns) and 10 μM TRAP-6 stimulated (shaded columns) CD62P expression (A) or fibrinogen binding (B) in fresh PB and in stored WB units at different time points as indicated. Results are presented in absolute arbitrary units (AU) as mean fluorescence ± SEM; n = 5; *: p < 0.05 (stimulated values compared to basal values); **: p < 0.05 (compared to PB and to WB at day 0).

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  Fig 2. ADP induced platelet aggregation continually decreases in citrated WB. Light transmission aggregometry induced with 10 μM ADP was measured in PRP from PB samples and from stored WB units at different time points as indicated. Mean aggregation traces are presented; n = 5.

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  Fig 3. Platelet purinergic receptor expression in citrated WB is maintained during storage. The histograms show the mean fluorescence intensities (MFI) of basal (white columns) and 10 μM TRAP-6 stimulated (shaded columns) P2Y1- (A), P2Y12- (B), and P2X1- (C) receptor expression in fresh PB samples and in stored WB units at different time points as indicated. Results are presented in absolute arbitrary units (AU) as mean fluorescence ± SEM; n = 6; *: p < 0.05 (compared to basal values).

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  Fig 4. The activity of platelet P2Y1, but not of P2X1 or P2Y12 receptor, continually decreases in citrated WB. Calcium induced fluorescence curves were generated with Fluo-4AM loaded freshly washed platelets from PB samples (black line) and from WB units on Day 0 (dashed line), on Day 2 (dotted line) and on Day 5 (gray line) after stimulation with the P2Y1 agonist MRS2365 (A) or the P2X1 agonist α,β-MeATP (C). Mean fluorescence curves are presented (expressed in relative fluorescence units, R.F.U.); n = 6. For the P2Y12 receptor activity, mean PRI values (B) were determined in PB samples and in stored WB units at different time points as indicated. Results are presented as mean % (PRI) ± SEM; n = 4.

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  Fig 5. Basal VASP phosphorylation and basal cyclic nucleotide levels in citrated WB. Quiescent washed platelets (3 x 108 per mL) from fresh PB samples and from stored WB units were lysed with Laemmli buffer and analyzed on Western blot for VASP Ser239 (A) and VASP Ser157 (B) phosphorylation. After scanning bands were quantified by the Image J program. Results are presented in arbitrary units (AU) as mean ± SEM; n = 8; *: p < 0.05 (compared to values of PB and of WB units on day 0). Cyclic nucleotide concentrations were measured in lysed washed platelets (3 x 108 per mL) from fresh PB samples and from stored WB units. After extraction with ether the contents of cGMP (C) and cAMP (D) were determined with immunoassays. Results are presented as mean (fold stimulation) ± SEM; n = 8; *: p < 0.05 (compared to basal values of PB and of stored WB units on day 0).

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  Fig 6. The content of platelet PDE5A remains stable in citrated WB over the storage period of five days. Quiescent washed platelets (1 x 108 per mL) from PB samples and from stored WB units were lysed with Laemmli buffer and analyzed by Western blot for total PDE5A content. After scanning bands were quantified by the Image J program. The data are presented in arbitrary units (AU) normalized to actin as mean ± SEM; n = 8.