CD36 signaling activates endothelial release of FVIII. (A) Left: silencing of CD36 in CD32+ HLEC measured by qRT-PCR. Right: silencing CD36 decreases the basal release of FVIII from CD32+ cells into the media, as measured by ELISA. CD32+ HLEC were transfected with oligonucleotides directed against CD36 or a control sequence, mRNA for CD36 was measured by qRT-PCR (N = 5, mean ± SD, ∗P < .05), and FVIII released into the media was measured by ELISA (N = 4-8, mean ± SD, ∗P < .05). (B) Silencing CD36 increases FVIII levels inside CD32+ cells. CD36 was silenced and FVIII levels in cell lysates were measured by ELISA (N = 7-11, mean ± SD, ∗P < .05). (C) The CD36 agonist oxLDL increases FVIII release from CD32+ HLEC. CD32+ HLEC were treated with increasing concentrations of oxLDL 0.1, 1, and 10 μg/mL for 16 hours and FVIII levels in the media were measured (N = 8-9, mean ± SD, ∗P < .05). (D) The CD36 agonist oxLDL increases FVIII release from CD32+ HLEC over time. CD32+ HLEC were treated with 10 μg/mL oxLDL, and FVIII levels in the media were measured over time (N = 3, mean ± SD, ∗P < .05). (E) Silencing CD36 decreases the ability of oxLDL to increase FVIII release from CD32+ HLEC. CD32+ HLEC were transfected with siCD36 oligonucleotides or siControl oligonucleotides as above for 48 hours and then treated with oxLDL or vehicle at 10 μg/mL for 16 hours, and FVIII release into the media was measured by ELISA (N = 12-20, mean ± SD, ∗P < .05). (F) OxLDL activates p38. CD32+ HLEC were treated with vehicle or oxLDL10 μg/mL, and cell lysates were immunoblotted for phospho-p38 and total p38 (N = 12, mean ± SD, ∗P < .05). (G) OxLDL activates p38 signaling. CD32+ HLEC were treated with vehicle or 10 μg/mL oxLDL and cell lysates were immunoblotted for the p38 target HSP27 (N = 4, mean ± SD, ∗P < .05). (H) Inhibiting p38 decreases the effect of oxLDL on the release of FVIII from CD32+ HLEC. CD32+ HLEC were pretreated with vehicle or the p38 inhibitor SB203580 (10 μM), stimulated with vehicle or oxLDL, and FVIII release into the media was measured by ELISA (N = 6-9, ± SD, ∗P < .05). (I) Scheme for CD36 regulation of FVIII release.

CD36 signaling activates endothelial release of FVIII. (A) Left: silencing of CD36 in CD32+ HLEC measured by qRT-PCR. Right: silencing CD36 decreases the basal release of FVIII from CD32+ cells into the media, as measured by ELISA. CD32+ HLEC were transfected with oligonucleotides directed against CD36 or a control sequence, mRNA for CD36 was measured by qRT-PCR (N = 5, mean ± SD, ∗P < .05), and FVIII released into the media was measured by ELISA (N = 4-8, mean ± SD, ∗P < .05). (B) Silencing CD36 increases FVIII levels inside CD32+ cells. CD36 was silenced and FVIII levels in cell lysates were measured by ELISA (N = 7-11, mean ± SD, ∗P < .05). (C) The CD36 agonist oxLDL increases FVIII release from CD32+ HLEC. CD32+ HLEC were treated with increasing concentrations of oxLDL 0.1, 1, and 10 μg/mL for 16 hours and FVIII levels in the media were measured (N = 8-9, mean ± SD, ∗P < .05). (D) The CD36 agonist oxLDL increases FVIII release from CD32+ HLEC over time. CD32+ HLEC were treated with 10 μg/mL oxLDL, and FVIII levels in the media were measured over time (N = 3, mean ± SD, ∗P < .05). (E) Silencing CD36 decreases the ability of oxLDL to increase FVIII release from CD32+ HLEC. CD32+ HLEC were transfected with siCD36 oligonucleotides or siControl oligonucleotides as above for 48 hours and then treated with oxLDL or vehicle at 10 μg/mL for 16 hours, and FVIII release into the media was measured by ELISA (N = 12-20, mean ± SD, ∗P < .05). (F) OxLDL activates p38. CD32+ HLEC were treated with vehicle or oxLDL10 μg/mL, and cell lysates were immunoblotted for phospho-p38 and total p38 (N = 12, mean ± SD, ∗P < .05). (G) OxLDL activates p38 signaling. CD32+ HLEC were treated with vehicle or 10 μg/mL oxLDL and cell lysates were immunoblotted for the p38 target HSP27 (N = 4, mean ± SD, ∗P < .05). (H) Inhibiting p38 decreases the effect of oxLDL on the release of FVIII from CD32+ HLEC. CD32+ HLEC were pretreated with vehicle or the p38 inhibitor SB203580 (10 μM), stimulated with vehicle or oxLDL, and FVIII release into the media was measured by ELISA (N = 6-9, ± SD, ∗P < .05). (I) Scheme for CD36 regulation of FVIII release.

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