Amyloid Peptide β 1-42 Induces Integrin α IIb β 3 Activation, Platelet Adhesion, and Thrombus Formation in a NADPH Oxidase-Dependent Manner

Abstract

The progression of Alzheimer's dementia is associated with neurovasculature impairment, which includes inflammation, microthromboses, and reduced cerebral blood flow. Here, we investigate the effects of β amyloid peptides on the function of platelets, the cells driving haemostasis. Amyloid peptide β1-42 (Aβ1-42), Aβ1-40, and Aβ25-35 were tested in static adhesion experiments, and it was found that platelets preferentially adhere to Aβ1-42 compared to other Aβ peptides. In addition, significant platelet spreading was observed over Aβ1-42, while Aβ1-40, Aβ25-35, and the scAβ1-42 control did not seem to induce any platelet spreading, which suggested that only Aβ1-42 activates platelet signalling in our experimental conditions. Aβ1-42 also induced significant platelet adhesion and thrombus formation in whole blood under venous flow condition, while other Aβ peptides did not. The molecular mechanism of Aβ1-42 was investigated by flow cytometry, which revealed that this peptide induces a significant activation of integrin αIIbβ3, but does not induce platelet degranulation (as measured by P-selectin membrane translocation). Finally, Aβ1-42 treatment of human platelets led to detectable levels of protein kinase C (PKC) activation and tyrosine phosphorylation, which are hallmarks of platelet signalling. Interestingly, the NADPH oxidase (NOX) inhibitor VAS2870 completely abolished Aβ1-42-dependent platelet adhesion in static conditions, thrombus formation in physiological flow conditions, integrin αIIbβ3 activation, and tyrosine- and PKC-dependent platelet signalling. In summary, this study highlights the importance of NOXs in the activation of platelets in response to amyloid peptide β1-42. The molecular mechanisms described in this manuscript may play an important role in the neurovascular impairment observed in Alzheimer's patients.

Figure 1

β amyloid peptides in static conditions support platelet adhesion. Human platelet suspensions were plated on glass coverslips coated with 10 μM scrambled Aβ1-42 (scAβ42), Aβ1-42, Aβ1-40, and Aβ25-35. The adhered platelets after 30 minutes shown in (a) were fixed, permeabilized, and stained with TRITC-conjugated phalloidin and are representative images at 10x magnification. The quantification of the adhered platelets evaluated as the mean number of platelets per optical field is shown in (b). Statistical significance for 4 independent experiments was analysed by one-way ANOVA with Bonferroni posttest (^∗P < 0.05), with bars representing standard error of the mean (SEM).

Figure 2

β amyloid peptides in static conditions support platelet spreading. Human platelet suspensions were plated on glass coverslips coated with 10 μM scrambled Aβ1-42 (scAβ42), Aβ1-42, Aβ1-40, and Aβ25-35. The adhered platelets shown in (a) were fixed, permeabilized, and stained with TRITC-conjugated phalloidin and are representative images at 100x magnification. The quantification of the mean surface area of the adhered platelets (μm²/plt) is shown in (b). Statistical significance for 4 independent experiments was analysed by one-way ANOVA with Bonferroni posttest (^∗P < 0.05), with bars representing standard error of the mean (SEM).

Figure 3

Effect of NOX inhibitor VAS2870 on platelet adhesion to Aβ1-42. Human platelet suspension was preincubated with NOX inhibitor VAS2870 (10 μM) for 30 mins then plated on glass coverslips coated with 10 μM Aβ1-42 and 5 mg/ml BSA in PBS. The numbers of the adhered platelets fixed, permeabilized, and stained with TRITC-conjugated phalloidin are shown in (a), and representative images at 10x magnification are displayed. The quantification of the adhered platelets evaluated as the mean number of the adhered platelets per optical field is shown (b). Statistical significance for 4 independent experiments was analysed by one-way ANOVA with Bonferroni posttest (^∗P < 0.05), with bars representing standard error of the mean (SEM).

Figure 4

Effect of NOX inhibitor VAS2870 on platelet spreading on Aβ1-42. Human platelet suspension was preincubated with NOX inhibitor VAS2870 (10 μM) for 30 mins then plated on glass coverslips coated with 10 μM Aβ1-42 and 5 mg/ml BSA in PBS. The surface area of the adhered platelets fixed, permeabilized, and stained with TRITC-conjugated phalloidin is shown in (a), and representative images at 100x magnification are displayed. The quantification of the surface area of the adhered platelets (μm²/plt) is shown in (b). Statistical significance for 4 independent experiments was analysed by one-way ANOVA with Bonferroni posttest (^∗P < 0.05), with bars representing standard error of the mean (SEM).

Figure 5

Adhesion of platelets to amyloid peptides under physiological shear stress. Flow biochips (ibidi Vena8 Fluoro+) were coated with 0.1 mg/ml fibrillary collagen or 10 μM scrambled Aβ1-42 (scAβ42), Aβ1-42, Aβ1-40, and Aβ25-35. Platelet adhesion was tested in human whole blood at shear rates of 1,000 sec⁻¹ and 200 sec⁻¹. Where indicated, 10 μM VAS2870 was added to the blood to inhibit NOXs. Pictures shown here are representative of 3 independent experiments. Surface coverage analysis was performed using ImageJ and statistically analysed by one-way ANOVA with Bonferroni posttest (^∗P < 0.05).

Figure 6

Activation of integrin αIIbβ3 by Aβ1-42. Washed platelets were stimulated as indicated with Aβ1-42, Aβ1-40, Aβ25-35, or scrambled Aβ1-42 or 0.5 units/ml thrombin for 10 minutes and then labelled with FITC-PAC1 (b-g) and PE-Cy5-P-selectin (h) for a further 10 minutes. A side-scattering (SSC)/forward-scattering (FSC) dot plot is shown in (a) and suggests the high purity of the platelet preparation. The histograms for the intensity of PAC1 staining in the different conditions are shown in (b-f) (representative of 3 independent experiments). Data analyses are shown in (g) and (h). Statistical analysis by one-way ANOVA with Bonferroni posttest is shown in (g) and (h) (n = 3, ^∗P < 0.05).

Figure 7

Aβ42-induced signalling in platelets. (a, b) Unstimulated and Aβ42-stimulated (20 μM) human platelets were treated with 10 μM DMSO or VAS2870. Total proteins were separated by SDS-PAGE as described in the Materials and Methods, and protein phosphorylation was analysed by immunoblotting with the indicated antibodies: (a) anti-p-Tyr and anti-pleckstrin antibody and (b) anti-PKC phosphosubstrate and anti-pleckstrin antibody. The figure represents blots from three independent experiments.