Role of albumin in regulating platelet function

Figures

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  FIGURE 1. Human plasma albumin exhibits inhibitory effects on platelet aggregation, integrin αIIbβ3 activation and P-selectin release. (A) Schematic diagram of the experimental procedure. (B–F) Effect of human plasma albumin (PBS, 2.5, 5.0, 7.5, 10.0, 20.0, 30.0, 40.0 mg/mL) on 0.01 U/mL thrombin (B), 200 nM U46619 (C), 1 μg/mL collagen (D), 200 μM ADP (E), 20 μM Trap-6 (F) induced aggregation of human washed platelets (n = 3 independent experimental subjects). (G–I) Effect of different concentrations of human plasma albumin on thrombin (G), U46619 (H) and Trap-6 (I) induced activation of human washed platelet integrin αIIbβ3 (n = 3 independent experimental subjects). (J–L) Effect of human plasma albumin on thrombin (J), U46619 (K) and Trap-6 (L) induced release of P-selectin from human washed platelets (n = 3 independent experimental subjects). Differences between groups were assessed by one-way ANOVA followed by Dunnett’s post hoc test (compared with PBS). Statistics are presented as mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001.

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  FIGURE 2. Albumin inhibited granule release and spread of platelet. (A–D) Effect of human plasma albumin on 0.02 U/mL thrombin (A), 600 nM U46619 (B), 3 μg/mL collagen (C) and 40 μM Trap-6 (D) induced effects on human washed platelet ATP release (n = 5 independent experimental subjects). (E–K) Transmission electron microscopy field of view of platelets induced by 200 nM U46619 at different concentrations of human plasma albumin (20, 30 mg/mL) (E), platelet cell diameters using ImageJ fitting graphs and axes to find the distance of granules from the centre of the cell (F), absolute counts of α-granules (G), absolute counts of dense granules (H), absolute counts of mitochondria (I), absolute distance of granules from the centre of the cell (J), relative distance of granules from the centre of the cell (K) (E–I, n = 3 independent experimental subjects, counted 3 microscope field of view per individual; J,K, n = 3 independent experimental subjects, counted the distance from all granules to the center of the cell and calculated the average). (L–N) Inhibitory effect of human plasma albumin on U46619 induced platelet spreading (L), mean spreading area of a single platelet (M), spreading platelets…

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  FIGURE 3. Albumin inhibited platelet activation by inhibiting PKC and Akt phosphorylation. (A,B) Effect of human plasma albumin (5.0, 10.0 and 20.0 mg/mL) on pleckstrin phosphorylation (A) and Akt Ser473 phosphorylated (B) expression in human washed platelets induced by U46619. (C,D) Effect of human plasma albumin and 2 nM PMA on the expression of pleckstrin phosphorylation (C) and Akt Ser473 phosphorylation (D) in human washed platelets induced by U46619. (E,F) Effect of human plasma albumin and 20 nM PDBu on pleckstrin phosphorylation (E) and Akt Ser473 phosphorylation (F) expression induced by U46619 in human washed platelets. (G,H) Effect of human plasma albumin and 20 nM 740Y-P on pleckstrin phosphorylation (G) and Akt Ser473 phosphorylation (H) expression in human washed platelets induced by U46619. (I,J) Effect of human plasma albumin and 8 μg/mL SC79 on pleckstrin phosphorylation (I) and Akt Ser473 phosphorylation (J) expression in human washed platelets induced by U46619 (A–J, n = 3 independent experimental subjects). (K–M) Effect of 20.0 mg/mL concentration of human plasma albumin and PMA, PDBu, 740Y-P and SC79 on U46619-induced aggregation (K), integrin αIIbβ3 activation (L) and…

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  FIGURE 4. Albumin influenced platelet aggregation, ATP release, granule release and internal granule arrangement via surface charge. (A) Isoelectric point detection of CS albumin and natural albumin (80 mg/mL) and determination of protein secondary structure by circular dichroism. (B–E) Effect of CS albumin and natural albumin (20.0 mg/mL) on human washed platelet aggregation induced by thrombin (B), U46619 (C), collagen (D) and ADP (E). (F–H) Effect of CS albumin and natural albuminon ATP release from human washed platelets induced by 0.02 U/mL thrombin (F), 500 nM U46619 (G) and 3 μg/mL collagen (H) (B–H, n = 5 independent experimental subjects). (I) Transmission electron microscopy was used to observe the effect of CS albumin and natural albumin on the internal granule arrangement in platelets before and after activation induced by U46619. (J–M) Effects of CS albumin and natural albumin on the absolute number of α-granules (J), dense granules (K), mitochondria (L) and the relative distance of granules from the centre of the cell (M) in platelets induced by U46619 (J–L, n = 3 independent experimental subjects, counted 3 microscope field of view per individual; (M) n = 3 independent…

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  FIGURE 5. Development of a murine model of hypoproteinemia induced by adriamycin-associated acute kidney injury. (A) Flow chart of mouse model construction. (B–E) Platelet count (B), MPV (C), PDW (D) and PCT (E) in peripheral blood of 8-week male WT, ADR and ADR Albumin mice. (F–I) Albumin concentration (F), total protein concentration (G), prealbumin concentration (H), globulin concentration (I) in peripheral blood of mice (B–I, n = 5 independent experimental animals). (J) Histopathological staining of kidneys from ADR model mice (4× and 40× magnification), HE staining, Masson staining and PAS staining. (K) Histopathological staining of kidney from WT mice (4× and 40× magnification), HE staining, Masson staining, PAS staining. Differences between groups were assessed by one-way ANOVA followed by Dunnett’s post hoc test. Statistics are presented as mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001.

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  FIGURE 6. Hypoproteinemia was found to promote platelet activation. (A–E) Wash platelet aggregation in WT, ADR and ADR Albumin mice induced by thrombin (A), U46619 (B), PAR4-AP (C), collagen (D) and ADP (E) (n = 5 independent experimental animals). (F–H) Thrombin (F), U46619 (G) and PAR4-AP (H) induced integrin αIIbβ3 activation in washed platelets from mice (n = 5 independent experimental animals). (I–K) Thrombin (I), U46619 (J) and PAR4-AP (K) induced P-selectin release from washed platelets from mice (n = 5 independent experimental animals). (L–O) ATP release from washed platelets in mice was induced by thrombin (L), U46619 (M), PAR4-AP (N) and collagen (O) (n = 5 independent experimental animals). (P–R) Wash platelet spreading in mice induced by U46619 (P), mean spreading area of individual platelets (Q), and proportion of spreading platelets to total platelets in the field of view (R) (n = 3 independent experimental animals). Differences between groups were assessed by one-way ANOVA followed by Dunnett’s post hoc test. Statistics are presented as mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001.

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  FIGURE 7. Albumin exerts an inhibitory effect on thrombus formation and hemostasis. (A) Flow chart of experiments on haemostatic function and thrombosis in mice. (B) Tail bleeding time in mice (n = 6 independent experimental animals). (C–F) Time of thrombosis induced in mouse mesenteric arteries using FeCl3 (C) (n = 3 independent experimental animals), WT (D), ADR (E), ADR Albumin (F) microscopic thrombosis in mice. Differences between groups were assessed by one-way ANOVA followed by Dunnett’s post hoc test. Statistics are presented as mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001.

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  FIGURE 8. Schematic representation of the mechanism by which albumin inhibits platelet activity.