Fig 1.
Experimental set-up to compare the effect of different anticoagulants on plasma levels of platelet-stored factors.
(A) Plasma preparation. Anticoagulated blood of 8 healthy volunteers was centrifuged at 1,000 x g and 4°C for 10 minutes. Supernatants were transferred to new tubes and centrifuged at 10,000 x g and 4°C for 10 minutes to generate platelet-free plasma. Supernatants of the second centrifugation step were aliquoted and stored at –80°C until further use. (B) Sampling. Blood of each donor was collected using CTAD (blue), ACD (pink), citrate (green), EDTA (red) and heparin (yellow) as anticoagulant. Half of the samples were kept either at 4°C or at room temperature. Plasma was generated at 0.5 h, 2 h, 6 h, and 24 h. To analyze the effect of in vitro platelet activation a subgroup of samples was stimulated with thrombin at a submaximal concentration and plasma was generated after 0.5 h and 6 h.
Fig 2.
Plasma levels of platelet-stored factors vary among different anticoagulants.
Plasma levels of PF4 (A, C) and TSP-1 (B, D) were determined in 8 healthy individuals. Plasma was prepared from CTAD (black bar), ACD (dark grey bar), citrate (grey bar), heparin (white bar) or EDTA (patterned bar) blood at 4°C (A-B) or at room temperature (C-D) within 30 min after blood draw. Plasma concentration was measured using ELISA. Significant differences were analyzed using one-way ANOVA with Dunnett correction and were depicted as *p<0.05, **p<0.01, ***p<0.001 (in comparison to CTAD).
Table 1.
Fig 3.
Time-dependent differences in plasma levels of platelet-stored factors.
Blood from 8 healthy donors was anticoagulated with CTAD (black bar), ACD (dark grey bar), citrate (grey bar), heparin (white bar) or EDTA (patterned bar) and stored at 4°C (A-B) or at room temperature (C-D) for 0.5 h, 2 h, 6 h or 24 h until plasma preparation. Concentrations of PF4 (A, C) and TSP-1 (B, D) were determined for each time point. Significant differences were analyzed using two-way ANOVA with Dunnett correction (with CTAD as reference) and were depicted as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.
Table 2.
Fig 4.
Fold-increase of platelet-stored factors due to suboptimal plasma generation.
Plasma concentrations of PF4 (A, C) and TSP-1 (B, D) were calculated as fold of plasma levels relative to CTAD plasma 30 min 4°C (8 healthy donors). Fold-increase is depicted for ACD (dark grey bar), citrate (grey bar), heparin (white bar) or EDTA (patterned bar) plasma at 0.5 h, 2 h, 6 h, and 24 h. Samples were either processed at 4°C (A-B) or at ambient temperature (C-D). Significant differences were analyzed using one-way ANOVA with Dunnett correction and were depicted as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 (in comparison to CTAD 30 min 4°C).
Fig 5.
Effect of in vitro platelet activation on concentration of plasma factors.
Blood of 8 healthy donors was anticoagulated with CTAD, ACD, citrate, heparin or EDTA (grey bar) and stimulated with the platelet activator thrombin (dark grey bar) at a submaximal dose for 0.5 h and 6 h at room temperature. Subsequently, plasma was prepared and analyzed for PF4 (A, C) and TSP-1 (B, D) levels. Significant differences were analyzed using two-way ANOVA with Bonferroni correction and were depicted as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 (in comparison to untreated).