Figure 5.
ERp5-deficient platelets have increased constitutive secretion of ER proteins. (A) Representative immunoblots of vWF, TSP1, and PF4 in the platelet releasates from control (ERp5fl/fl) and CKO (Pf4Cre+/ERp5fl/fl) mice after treatment of platelets with DMSO (buffer control) or thapsigargin (TG). Densitometry of VWF, TSP1, and PF4 in the releasates from control (gray) or KO (red) platelets treated with DMSO (light box) or TG (dark box). Ratios are presented as fold change over DMSO-treated controls. (B) Proteomic analysis of resting platelet releasate from ERp5fl/fl and Pf4Cre+/ERp5fl/fl mice. Volcano plot for all proteins detected by MS/MS, with the log2 fold change (lfc) plotted on the x-axis vs the negative log10 (P) of a paired test statistic on the y-axis. Proteins in red are significantly increased (positive lfc value) or decreased (negative lfc value) in CKO compared with control mice (P < .05). Color scale represents negative log10 (P), P = .05 corresponds to a negative log10 (P) of 1.3 (upper limit of grayed-out area). (C) Densitometry of PDI, ERp57, and ERp72 on western blots of platelet releasates from control (gray) and ERp5 CKO (red) mice after platelet treatment with buffer (rest) (light box) or thrombin 0.5 U/mL (IIα) (dark box). (D) CKO mice have increased plasma PDI. Densitometry of PDI on a western blot of plasma samples from control (gray) and CKO (red) mice. (E) Relative disulfide reductase activity of thrombin 0.5 U/mL-treated CKO platelets (red) compared with thrombin-treated control platelets (gray) as measured by the dieosin glutathione disulfide assay. (F) Representative immunoblots of TSP1 and PF4 in the platelet releasate after treatment with buffer (rest) or thrombin (IIα). Densitometry of TSP1 and PF4 in the releasates of control (gray) and ERp5 KO (red) platelets treated with buffer (light box) or thrombin (dark box). Ratios are presented as fold change over buffer-treated controls. (G) VWF and serotonin concentration (ng/mL) measured by enzyme-linked immunosorbent assay in the releasates of control (gray) and ERp5 KO (red) platelets treated with buffer (light box) or thrombin (dark box). n = 3 to 6 per genotype, mean ± SEM, Student t test (D, E), 1-way ANOVA with Dunn post hoc analysis (A, C, F, G). ∗P < .05, ∗∗P < .005.

ERp5-deficient platelets have increased constitutive secretion of ER proteins. (A) Representative immunoblots of vWF, TSP1, and PF4 in the platelet releasates from control (ERp5fl/fl) and CKO (Pf4Cre+/ERp5fl/fl) mice after treatment of platelets with DMSO (buffer control) or thapsigargin (TG). Densitometry of VWF, TSP1, and PF4 in the releasates from control (gray) or KO (red) platelets treated with DMSO (light box) or TG (dark box). Ratios are presented as fold change over DMSO-treated controls. (B) Proteomic analysis of resting platelet releasate from ERp5fl/fl and Pf4Cre+/ERp5fl/fl mice. Volcano plot for all proteins detected by MS/MS, with the log2 fold change (lfc) plotted on the x-axis vs the negative log10 (P) of a paired test statistic on the y-axis. Proteins in red are significantly increased (positive lfc value) or decreased (negative lfc value) in CKO compared with control mice (P < .05). Color scale represents negative log10 (P), P = .05 corresponds to a negative log10 (P) of 1.3 (upper limit of grayed-out area). (C) Densitometry of PDI, ERp57, and ERp72 on western blots of platelet releasates from control (gray) and ERp5 CKO (red) mice after platelet treatment with buffer (rest) (light box) or thrombin 0.5 U/mL (IIα) (dark box). (D) CKO mice have increased plasma PDI. Densitometry of PDI on a western blot of plasma samples from control (gray) and CKO (red) mice. (E) Relative disulfide reductase activity of thrombin 0.5 U/mL-treated CKO platelets (red) compared with thrombin-treated control platelets (gray) as measured by the dieosin glutathione disulfide assay. (F) Representative immunoblots of TSP1 and PF4 in the platelet releasate after treatment with buffer (rest) or thrombin (IIα). Densitometry of TSP1 and PF4 in the releasates of control (gray) and ERp5 KO (red) platelets treated with buffer (light box) or thrombin (dark box). Ratios are presented as fold change over buffer-treated controls. (G) VWF and serotonin concentration (ng/mL) measured by enzyme-linked immunosorbent assay in the releasates of control (gray) and ERp5 KO (red) platelets treated with buffer (light box) or thrombin (dark box). n = 3 to 6 per genotype, mean ± SEM, Student t test (D, E), 1-way ANOVA with Dunn post hoc analysis (A, C, F, G). ∗P < .05, ∗∗P < .005.

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