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. 2020 Feb 18;9:e51546. doi: 10.7554/eLife.51546

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© 2020, Lee et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Figure 7. — (A) Rosetting rates in filtered human serum (HS) were lower than those of complete HS (one-way ANOVA with Tukey’s test: p=0.0368; n = 5). IGFBP7 stimulated rosetting in HS-supplied environment (p=0.0276). IGFBP7 did not alter rosetting in filtered HS (p=0.0664). (B) IGFBP7 stimulated rosetting (p=0.0002; n = 8), whereas anti-VWF IgG did not significantly alter rosetting (p=0.3039). No significant changes were found in ‘anti-VWF IgG+IGFBP7’ (p=0.9096). (C) In 20% HS-enriched medium (20% HSM), ‘IGFBP7 and IGFBP7+VWF’ showed higher rosetting than the control (one-way ANOVA with Tukey’s test: p=0.003 for both comparisons; n = 8). VWF did not alter rosetting (p=0.8652). No significant difference was found between ‘IGFBP7’ and ‘IGFBP7+VWF’ (p=0.7853). Rosetting in ‘IGFBP7’ was higher than in ‘VWF’ (p<0.0001). In 2% HSM, ‘IGFBP7’ (p=0.1832) and ‘VWF’ (p=0.9876) did not significantly alter rosetting. Rosetting in ‘IGFBP7+VWF’ was increased (p=0.0014), and was higher than ‘IGFBP7’ and ‘VWF’ (p=0.0030 for both comparisons). No significant difference was found between ‘VWF+IGFBP7’ from both serum settings (p=0.2861). (D) Rosetting (n = 7) in 0.25% Albumax-enriched medium (Alb) supplied with 100 ng/ml IGFBP7 and different concentrations of VWF. No significant difference was found across the VWF concentrations tested (one-way ANOVA with Dunnett’s test: p>0.3 for all comparisons with ‘VWF-free’). (E) In 20% HSM, there was no significant difference between control and ‘anti-TSP-1’ (one-way ANOVA with Tukey’s test: p=0.9961, n = 8). A significant difference was recorded between control and ‘IGFBP7’ (p<0.0001), but not between control and ‘anti-TSP-1 + IGFBP7’ (p=0.9125). A significant difference was found in ‘anti-TSP-1’ vs. ‘IGFBP7’ (p<0.0001), and ‘IGFBP7’ vs. ‘IGFBP7 + anti-TSP-1’ (p=0.0022). (F) Rosetting (n = 8) in Alb was lower than in 20% HSM (p=0.0047). In HSM, IGFBP7 increased rosetting (p=0.0097). No significant changes were seen in comparisons of Alb-control with: IGFBP7 (p=0.7499), VWF (p>0.9999), TSP-1 10 ng/ml (p>0.9999), TSP-1 500 ng/ml (p=0.9491), IGFBP7 + TSP-1 10 ng/ml (p>0.9999), and IGFBP7 + TSP-1 500 ng/ml (p=0.9341). Rosette-stimulation was noted in ‘IGFBP7 + VWF + TSP-1 10 ng/ml’ (p=0.0002) and ‘IGFBP7 + VWF + TSP-1 500 ng/ml’ (p<0.0001). No significant difference was noted between ‘IGFBP7 + VWF + TSP-1 10 ng/ml’ and ‘IGFBP7 + VWF + TSP-1 500 ng/ml’ (p=0.9998), and ‘IGFBP7 + VWF + TSP-1 10 ng/ml’ vs. ‘20%HSM + IGFBP7’ (p>0.9999). ‘IGFBP7 + VWF + TSP-1 500 ng/ml’ was not significantly different from ‘HSM + IGFBP7’ (p=0.9997). (G) In 0.25% Alb supplied with 100 ng/ml IGFBP7, 10 ng/ml TSP-1 and 0.125 IU/ml VWF, rosette-stimulation was noted, as compared to a VWF-free group (one-way ANOVA with Tukey’s test: p=0.0421, n = 8). Rosetting increased with VWF concentrations (p<0.0001 for 0.5 and 2.0 IU/ml compared to VWF-free). No significant difference was seen in rosetting rates between VWF of 0.5 IU/ml and 2.0 IU/ml (p=0.2126).

Figure 7—figure supplement 1. — (A) Rosetting rates in filtered human serum (HS) were lower than those of complete HS (one-way ANOVA with Tukey’s test: p=0.0368; n = 5). IGFBP7 stimulated rosetting in HS-supplied environment (p=0.0276). IGFBP7 did not alter rosetting in filtered HS (p=0.0664). (B) IGFBP7 stimulated rosetting (p=0.0002; n = 8), whereas anti-VWF IgG did not significantly alter rosetting (p=0.3039). No significant changes were found in ‘anti-VWF IgG+IGFBP7’ (p=0.9096). (C) In 20% HS-enriched medium (20% HSM), ‘IGFBP7 and IGFBP7+VWF’ showed higher rosetting than the control (one-way ANOVA with Tukey’s test: p=0.003 for both comparisons; n = 8). VWF did not alter rosetting (p=0.8652). No significant difference was found between ‘IGFBP7’ and ‘IGFBP7+VWF’ (p=0.7853). Rosetting in ‘IGFBP7’ was higher than in ‘VWF’ (p<0.0001). In 2% HSM, ‘IGFBP7’ (p=0.1832) and ‘VWF’ (p=0.9876) did not significantly alter rosetting. Rosetting in ‘IGFBP7+VWF’ was increased (p=0.0014), and was higher than ‘IGFBP7’ and ‘VWF’ (p=0.0030 for both comparisons). No significant difference was found between ‘VWF+IGFBP7’ from both serum settings (p=0.2861). (D) Rosetting (n = 7) in 0.25% Albumax-enriched medium (Alb) supplied with 100 ng/ml IGFBP7 and different concentrations of VWF. No significant difference was found across the VWF concentrations tested (one-way ANOVA with Dunnett’s test: p>0.3 for all comparisons with ‘VWF-free’). (E) In 20% HSM, there was no significant difference between control and ‘anti-TSP-1’ (one-way ANOVA with Tukey’s test: p=0.9961, n = 8). A significant difference was recorded between control and ‘IGFBP7’ (p<0.0001), but not between control and ‘anti-TSP-1 + IGFBP7’ (p=0.9125). A significant difference was found in ‘anti-TSP-1’ vs. ‘IGFBP7’ (p<0.0001), and ‘IGFBP7’ vs. ‘IGFBP7 + anti-TSP-1’ (p=0.0022). (F) Rosetting (n = 8) in Alb was lower than in 20% HSM (p=0.0047). In HSM, IGFBP7 increased rosetting (p=0.0097). No significant changes were seen in comparisons of Alb-control with: IGFBP7 (p=0.7499), VWF (p>0.9999), TSP-1 10 ng/ml (p>0.9999), TSP-1 500 ng/ml (p=0.9491), IGFBP7 + TSP-1 10 ng/ml (p>0.9999), and IGFBP7 + TSP-1 500 ng/ml (p=0.9341). Rosette-stimulation was noted in ‘IGFBP7 + VWF + TSP-1 10 ng/ml’ (p=0.0002) and ‘IGFBP7 + VWF + TSP-1 500 ng/ml’ (p<0.0001). No significant difference was noted between ‘IGFBP7 + VWF + TSP-1 10 ng/ml’ and ‘IGFBP7 + VWF + TSP-1 500 ng/ml’ (p=0.9998), and ‘IGFBP7 + VWF + TSP-1 10 ng/ml’ vs. ‘20%HSM + IGFBP7’ (p>0.9999). ‘IGFBP7 + VWF + TSP-1 500 ng/ml’ was not significantly different from ‘HSM + IGFBP7’ (p=0.9997). (G) In 0.25% Alb supplied with 100 ng/ml IGFBP7, 10 ng/ml TSP-1 and 0.125 IU/ml VWF, rosette-stimulation was noted, as compared to a VWF-free group (one-way ANOVA with Tukey’s test: p=0.0421, n = 8). Rosetting increased with VWF concentrations (p<0.0001 for 0.5 and 2.0 IU/ml compared to VWF-free). No significant difference was seen in rosetting rates between VWF of 0.5 IU/ml and 2.0 IU/ml (p=0.2126).