Fig 1: Platelet depletion reduces DND after SAH.a–c, After SAH, mice depleted of platelets (PF4-DTR + DT), compared with nondepleted (WT + DT) mice, had better neuroscore (a mean with s.e.m.), less DND (b), and fewer brain microthrombi (c) (scale bar = 100 μm, representative images of MSB-stained brain slices with yellow asterisks marking selected microthrombi (best observed zoomed in)). All mice received the entire DT regimen. For neuroscore (a), sham n = 8 per sex, female WT SAH n = 20–30, female (PF4-DTR) SAH n = 27–29, male WT SAH n = 25–30, and male (PF4-DTR) SAH n = 30–31. For DND (b), WT SAH n = 30 per sex, and (PF4-DTR) SAH n = 29 female and n = 31 male. *P < 0.05 WT sham versus WT SAH, #P < 0.05 (PF4-DTR) sham versus (PF4-DTR) SAH, and †P < 0.05 WT SAH versus (PF4-DTR) SAH.
Fig 2: Plasma PAF and TXB2 are elevated on days 1 and 5 post-SAH.a–d Levels of plasma PAF (a), thrombin (b), TXB2 (c), and PF4 (d). Female mice were used (n = 6 per group per time point). *P < 0.05 versus sham, #P < 0.05 versus SAH day 1, †P < 0.05 versus SAH day 3, and ‡P < 0.05 versus SAH day 5.
Fig 3: Platelet activation is increased and Ifnr copy-dependent in Dp16 mice. Platelet activation was measured by flow cytometry. WT NMX, n=10; WT 3D HPX, n=13; Dp16 NMX, n=9; Dp16 3D HPX, n=7; Dp162xIfnrs NMX, n= 13; Dp162xIfnrs 3D HPX, n=13. (A, B) Platelet P-selectin and αIIbβ3 (JONA) activation was increased at baseline and exacerbated by hypoxia in Dp16 mice and normalized in Dp162xIfnrs mice. (C) Activation potential was measured in platelets stimulated with Thrombin. WT NMX and WT + THR, n=9; Dp16 NMX and Dp16 + THR, n=10; NMX, n=13; Dp162xIfnrs NMX + THR, n=13. P-selectin was increased at baseline in Dp16 and Dp162xIfnrs mice. Thrombin-induced P-selectin expression was exacerbated at Dp16 and Dp162xIfnrs mice. (D) αIIbβ3 (JONA) activation was increased at baseline in Dp16 mice. Thrombin-induced αIIbβ3 (JONA) activation is similar between WT, Dp16, and Dp162xIfnrs mice. (E) Circulating PF4 was measured by plasma ELISA. WT NMX, n=7; WT 3D HPX, n=7; Dp16 NMX, n=8; Dp16 3D HPX, n=7; Dp162xIfnrs NMX, n= 5; Dp162xIfnrs 3D HPX, n=7. Plasma PF4 was increased at baseline and exacerbated in hypoxia in Dp16 mice. Baseline and hypoxia-induced plasma PF4 was attenuated in Dp162xIfnrs mice. (F) Soluble GPVI was measured by plasma ELISA. WT NMX, n=7; WT 3D HPX, n=7; Dp16 NMX, n=8; Dp16 3D HPX, n=7; Dp162xIfnrs NMX, n= 5; Dp162xIfnrs 3D HPX, n=7. sGPVI is increased in hypoxic WT mice. Dp16 and Dp162xIfnrs mice have elevated sGPVI at baseline compared to WT mice, which do not increase further in hypoxia. (G) Total platelet count was obtained using a hematologic analyzer. WT NMX, n=10; WT 3D HPX, n=16; Dp16 NMX, n=7; Dp16 3D HPX, n=9; Dp162xIfnrs NMX, n= 4; Dp162xIfnrs 3D HPX, n=10. Total circulating platelet count was similar in WT, Dp16, and Dp162xIfnrs mice under normoxic and hypoxic conditions. Normally distributed data were analyzed using two-way ANOVA with Tukey’s post hoc testing for (A, C, G). Non-parametric data was analyzed using aligned rank transformation (ART) with Holm adjustment (B, D–F). All data are expressed as mean ± SEM. Statistics: *p ≤ 0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Fig 4: Summary model of IFNR-dependent and IFNR-independent mechanisms identified in this study. In the Dp16 mouse model, increased IFNR gene dosage promotes platelet activation and inflammatory signaling, as evidenced by increased platelet P-selectin, activated αIIbβ3, plasma PF4, and lung PF4. These phenotypes, along with baseline pulmonary vascular remodeling and hypoxia-induced RVSP exacerbation are attenuated by normalization of Ifnr copy number in Dp162xIfnrs mice, indicating IFNR dependence. In contrast, elevated baseline RVSPs persists despite Ifnr normalization, and RVH remains elevated at baseline and in hypoxia compared to WT controls, indicating incomplete rescue of cardiopulmonary phenotypes. Together, these findings support a model in which IFNR signaling contributes to platelet activation and structural vascular changes but is not sufficient to determine the full hemodynamic severity of pulmonary hypertension in Trisomy 21. Created in Biorender (https://BioRender.com).
Fig 5: Lung PF4 accumulation is increased in Dp16 mice and attenuated by Ifnr normalization despite similar platelet accumulation. (A) Lung PF4 was measured by ELISA. WT NMX, n=9; WT 3D HPX, n=6; Dp16 NMX, n=10; Dp16 3D HPX, n=7; Dp162xIfnrs NMX, n= 8; Dp162xIfnrs 3D HPX, n=7. Lung PF4 was increased at baseline and exacerbated in hypoxic Dp16 mice. Baseline and hypoxia-induced exacerbation in lung PF4 was normalized in Dp162xIfnrs mice. (B) Lung platelets were assessed using IHC. WT NMX, n=8; WT 3D HPX, n=10; Dp16 NMX, n=6; Dp16 3D HPX, n=6; Dp162xIfnrs NMX, n= 7; Dp162xIfnrs 3D HPX, n=7. Platelets accumulated similarly in the distal lung of hypoxic WT, Dp16 mice, and Dp162xIfnrs mice. (C) Whole slide scans were obtained using the Leica Aperio VERSA brightfield scope (40x objective, 0.5-micron resolution). Representative images of CD41 staining in the distal lung; x20 magnification, scale bars = 100 μm. All images were acquired at identical magnification and processed using consistent scaling parameters; apparent differences reflect underlying changes in lung architecture rather than image scaling. Normally distributed data were analyzed using two-way ANOVA with Tukey’s post hoc testing for (A). Non-parametric data was analyzed using aligned rank transformation (ART) with Holm adjustment (B). All data are expressed as mean ± SEM. Statistics: *p ≤ 0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Supplier Page from Abcam for Mouse PF4 ELISA Kit (CXCL4)