Figure 5
Figure 5. Aptamer-induced changes in PF4 lead to expression of PF4/heparin-like epitopes. (A) Shown are structural changes in PF4 induced by the 44mer–DNA protein C aptamer. Changes in the CD pattern of PF4 (20 µg/mL) are shown in the absence (n = 4, black line) or presence of either heparin (n = 3, 6.9 µg/mL, ♦) or 44mer–DNA protein C aptamer at different doses (n = 2 each, 2.5 µg/mL, □; 5 µg/mL, Δ; 20 µg/mL, ○). Note that the changes in PF4 spectra are rather similar, regardless whether heparin or the aptamer was used. All data represent mean ± SD of n independent experiments. Each experiment consists of at least 5 measurements. (B-C) Human anti-PF4/heparin antibodies induce platelet activation in the presence of nucleic acids and aptamers. (B) Mean lag time until aggregation of donor platelets (at least n = 15 for each polyanion) and (C) reactivity of donor platelets expressed as the percentage of all tested donor platelets were analyzed in the presence of different anti-PF4/heparin antibody–containing sera (at least 4 for each polyanion) and either reviparin (0.2 µg/mL, bar 1), cellular RNA (0.5 µg/mL, bar 2), 21mer–double-stranded DNA (0.5 µg/mL, bar 3), 21mer–hairpin DNA (0.5 µg/mL, bar 4), deoxynucleotidetriphosphates (0.5 µg/mL, bar 5), 15mer–DNA thrombin aptamer (20 µg/mL, bar 6), 44mer–DNA protein C aptamer (8 µg/mL, bar 7), 57mer–RNA tetracycline aptamer (5 µg/mL, bar 8), or 77mer–RNA FMN–ribozyme (2.5 µg/mL, bar 9). Except for the short 15mer–DNA thrombin aptamer, which induces platelet aggregation after 19.0 ± 0.67 minutes and only in 66.6% of all donors, lag time and reactivity of platelets show only minor differences between the constructs. (D) The graph shows the binding of anti-PF4/heparin antibodies to PF4/aptamer complexes. Anti-PF4/heparin antibodies bind to complexes generated with 20 µg/mL PF4 and either 44mer–DNA protein C aptamer (20 µg/mL, n = 9), 57mer–RNA tetracycline aptamer (10 µg/mL, n = 4), 77mer–RNA FMN aptamer (10 µg/mL, n = 4), or heparin (3.3 µg/mL, n = 9, black bars), while no binding to complexes of PF4 and the 15mer–DNA thrombin aptamer (40 µg/mL, n = 4) occurred. Gray bars show the inhibition of binding by high heparin (660 µg/mL). All data represent mean ± SD of at least 4 independent experiments. (E) Shown is the immune response to PF4/aptamer complexes in mice. Mice were immunized with either mPF4/44mer–DNA protein C aptamer complexes (n = 5), mPF4 alone (n = 6), or 44mer–DNA protein C aptamer alone (n = 6), and the binding of antibodies from the respective sera to mPF4/44mer–DNA protein C aptamer complexes as well as mPF4/heparin complexes was assessed by EIA in the absence (♦) or presence (◊) of 660 µg/mL heparin. Median values are marked by black lines.

Aptamer-induced changes in PF4 lead to expression of PF4/heparin-like epitopes. (A) Shown are structural changes in PF4 induced by the 44mer–DNA protein C aptamer. Changes in the CD pattern of PF4 (20 µg/mL) are shown in the absence (n = 4, black line) or presence of either heparin (n = 3, 6.9 µg/mL, ♦) or 44mer–DNA protein C aptamer at different doses (n = 2 each, 2.5 µg/mL, □; 5 µg/mL, Δ; 20 µg/mL, ○). Note that the changes in PF4 spectra are rather similar, regardless whether heparin or the aptamer was used. All data represent mean ± SD of n independent experiments. Each experiment consists of at least 5 measurements. (B-C) Human anti-PF4/heparin antibodies induce platelet activation in the presence of nucleic acids and aptamers. (B) Mean lag time until aggregation of donor platelets (at least n = 15 for each polyanion) and (C) reactivity of donor platelets expressed as the percentage of all tested donor platelets were analyzed in the presence of different anti-PF4/heparin antibody–containing sera (at least 4 for each polyanion) and either reviparin (0.2 µg/mL, bar 1), cellular RNA (0.5 µg/mL, bar 2), 21mer–double-stranded DNA (0.5 µg/mL, bar 3), 21mer–hairpin DNA (0.5 µg/mL, bar 4), deoxynucleotidetriphosphates (0.5 µg/mL, bar 5), 15mer–DNA thrombin aptamer (20 µg/mL, bar 6), 44mer–DNA protein C aptamer (8 µg/mL, bar 7), 57mer–RNA tetracycline aptamer (5 µg/mL, bar 8), or 77mer–RNA FMN–ribozyme (2.5 µg/mL, bar 9). Except for the short 15mer–DNA thrombin aptamer, which induces platelet aggregation after 19.0 ± 0.67 minutes and only in 66.6% of all donors, lag time and reactivity of platelets show only minor differences between the constructs. (D) The graph shows the binding of anti-PF4/heparin antibodies to PF4/aptamer complexes. Anti-PF4/heparin antibodies bind to complexes generated with 20 µg/mL PF4 and either 44mer–DNA protein C aptamer (20 µg/mL, n = 9), 57mer–RNA tetracycline aptamer (10 µg/mL, n = 4), 77mer–RNA FMN aptamer (10 µg/mL, n = 4), or heparin (3.3 µg/mL, n = 9, black bars), while no binding to complexes of PF4 and the 15mer–DNA thrombin aptamer (40 µg/mL, n = 4) occurred. Gray bars show the inhibition of binding by high heparin (660 µg/mL). All data represent mean ± SD of at least 4 independent experiments. (E) Shown is the immune response to PF4/aptamer complexes in mice. Mice were immunized with either mPF4/44mer–DNA protein C aptamer complexes (n = 5), mPF4 alone (n = 6), or 44mer–DNA protein C aptamer alone (n = 6), and the binding of antibodies from the respective sera to mPF4/44mer–DNA protein C aptamer complexes as well as mPF4/heparin complexes was assessed by EIA in the absence (♦) or presence (◊) of 660 µg/mL heparin. Median values are marked by black lines.

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