Figure 1.
Functional role of ERp46 in human platelets. Expression of ERp46 on the surface of nonactivated platelets (A) and thrombin (1 U/mL)-activated platelets (B). Mean fluorescent intensity (MFI) ± standard error of the mean (SEM), n = 5, ****P < .0001, Student t test. Normal rabbit IgG (nI IgG, 30 μg/mL, red) and anti-ERp46 antibody (30 μg/mL, blue) were both labeled with Alexa-488. (C) SFLLRN (100 μM)-induced platelet activation/aggregation releases ERp46 into the supernatant. Shown is the fold increase of ERp46 protein in the supernatant with platelet activation. ERp46 was immunoprecipitated from the supernatant and analyzed by immunoblotting. (D-E) Platelet activation increases thiol labeling in surface ERp46. After activation by collagen (10 μg/mL) (D) or thrombin (1 U/mL) (E), platelets were labeled with MPB or SSB as described in “Methods.” After immunoblotting with the anti-ERp46 antibody, the band intensities were used to calculate the ratio of MPB/SSB in ERp46 on the surface of activated platelets relative to ERp46 on the surface of nonactivated platelets. (F) The polyclonal anti-ERp46 antibody inhibits platelet aggregation and ATP release. Representative aggregation and ATP release tracings (left) and combined results (right) showing inhibition by the anti-ERp46 antibody of human platelets activated with thrombin (0.05 U/mL); mean ± SEM, n = 3, *P < .05, ***P < .001, analysis of variance (ANOVA). Washed human platelets were preincubated with normal rabbit IgG and polyclonal anti-ERp46 antibody at the indicated concentration. Aggregation and ATP secretion were monitored in the lumi-aggregometer. (G-I) Anti-ERp46 antibody inhibits αIIbβ3 activation measured by PAC1 binding in platelets when added before (G) but not after (H) thrombin (0.025 U/mL) activation. MFI ± SEM, n = 5, ****P < .0001, ANOVA. In these experiments, the anti-ERp46 antibody (30 μg/mL) was incubated with the human platelets for 10 minutes before activation (G) or after activation (H). The red and blue histograms represent thrombin-activated platelets. (I) Addition of ERp46 (2 μM) (blue) potentiates thrombin (0.25 U/mL) activation of αIIbβ3 measured by PAC1 binding on platelets but does not itself induce αIIbβ3 activation. PBS control (red). Left panels, representative histograms; right panels, bar graph of combined results; mean ± SEM, n = 5, ****P < .0001, ANOVA. Non-act, nonactivated platelets.

Functional role of ERp46 in human platelets. Expression of ERp46 on the surface of nonactivated platelets (A) and thrombin (1 U/mL)-activated platelets (B). Mean fluorescent intensity (MFI) ± standard error of the mean (SEM), n = 5, ****P < .0001, Student t test. Normal rabbit IgG (nI IgG, 30 μg/mL, red) and anti-ERp46 antibody (30 μg/mL, blue) were both labeled with Alexa-488. (C) SFLLRN (100 μM)-induced platelet activation/aggregation releases ERp46 into the supernatant. Shown is the fold increase of ERp46 protein in the supernatant with platelet activation. ERp46 was immunoprecipitated from the supernatant and analyzed by immunoblotting. (D-E) Platelet activation increases thiol labeling in surface ERp46. After activation by collagen (10 μg/mL) (D) or thrombin (1 U/mL) (E), platelets were labeled with MPB or SSB as described in “Methods.” After immunoblotting with the anti-ERp46 antibody, the band intensities were used to calculate the ratio of MPB/SSB in ERp46 on the surface of activated platelets relative to ERp46 on the surface of nonactivated platelets. (F) The polyclonal anti-ERp46 antibody inhibits platelet aggregation and ATP release. Representative aggregation and ATP release tracings (left) and combined results (right) showing inhibition by the anti-ERp46 antibody of human platelets activated with thrombin (0.05 U/mL); mean ± SEM, n = 3, *P < .05, ***P < .001, analysis of variance (ANOVA). Washed human platelets were preincubated with normal rabbit IgG and polyclonal anti-ERp46 antibody at the indicated concentration. Aggregation and ATP secretion were monitored in the lumi-aggregometer. (G-I) Anti-ERp46 antibody inhibits αIIbβ3 activation measured by PAC1 binding in platelets when added before (G) but not after (H) thrombin (0.025 U/mL) activation. MFI ± SEM, n = 5, ****P < .0001, ANOVA. In these experiments, the anti-ERp46 antibody (30 μg/mL) was incubated with the human platelets for 10 minutes before activation (G) or after activation (H). The red and blue histograms represent thrombin-activated platelets. (I) Addition of ERp46 (2 μM) (blue) potentiates thrombin (0.25 U/mL) activation of αIIbβ3 measured by PAC1 binding on platelets but does not itself induce αIIbβ3 activation. PBS control (red). Left panels, representative histograms; right panels, bar graph of combined results; mean ± SEM, n = 5, ****P < .0001, ANOVA. Non-act, nonactivated platelets.

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