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Platelet amyloid precursor protein is a modulator of venous thromboembolism in mice

Ilaria Canobbio, Caterina Visconte, Stefania Momi, Gianni Francesco Guidetti, Marta Zarà, Jessica Canino, Emanuela Falcinelli, Paolo Gresele and Mauro Torti

Article Figures & Data

Figures

  • Figure 1.

    Characterization of platelets from APP-KO mice. In all the graphs, red bars refer to WT mice, and blue bars refer to APP-KO mice. Data are expressed as mean ± SEM and have been obtained from 3 different experiments, unless otherwise specified. (A) Analysis of APP and tubulin (as control) expression in platelets from WT and APP-KO mice by immunoblotting with specific antibodies, as indicated. (B) Analysis of glycoprotein expression by flow cytometry. (C) Platelets and white blood cells count in whole blood. *P < .05. (D) Blood was withdrawn from WT and APP-KO mice at the indicated points after injection of AlexaFluor488-labeled antibodies to GPIX, and the fraction of labeled to unlabeled platelets was determined (n = 5). (E) Electron microscopy analysis of platelet morphology. (i) Representative images at different magnitude (5600×, upper; and 28 000×, lower). Quantification of the mean platelet diameter and number of internal α and dense (δ) granules is reported in (ii) and (iii), respectively. Data have been obtained from the analysis of 50 different platelets from 5 different slides. *P < .05. (F) Analysis of megakaryocytes and proplatelets formation. (i) Representative images of proplatelets forming megakaryocytes from WT and APP-KO mice on staining with anti-tubulin antibody (green) and with Hoechst (blue). Quantification of total megakaryocytes (MKs) and percentage of cells protruding proplatelets (PP) is reported in (ii) and (iii), respectively. ***P < .005.

  • Figure 2.

    Analysis of platelet function. (A) Aggregation of washed platelets from WT and APP-KO mice induced by the indicated agonists. Aggregation induced by adenosine 5′-diphosphate (ADP) was measured on addition of purified fibrinogen, as indicated. (B-C) Flow cytometry analysis of agonist-induced integrin αIIbβ3 activation, measured as binding of fluorescein isothiocyanate-labeled fibrinogen (B) and granule secretion as P-selectin exposure (C). Red bars, WT platelets; blue bars, APP-deficient platelets. Results are the mean ± SEM of 3 different experiments. (D) Tail bleeding time determined in groups of 10 mice for each genotype and expressed as time required for bleeding cessation (i) and amount of blood hemoglobin lost (ii). Each symbol represents 1 animal.

  • Figure 3.

    Analysis of thrombus formation in vivo. (A) Photochemical-induced arterial thrombosis assessed as time required for occlusion of femoral artery measured by laser Doppler in WT (red bars) and APP-KO (blue bars) mice. Data are the mean ± SEM of measurements performed on 5 animals for both genotypes. (B) Analysis of deep vein thrombosis. Thrombus formation in the IVC was induced by vein ligation. Thrombi were extracted 24 or 48 hours after surgery. (i) Representative image showing the different size of thrombi from WT and APP-KO mice after 24 hours of ligation. Quantifications of thrombus length and weight at 24 and 48 hours are reported in (ii) and (iii), respectively. **P < .01; ***P < .005. (C) Analysis of thrombus embolization. (i) Staining of lung sections from WT or APP-KO with phosphotungstic acid–hematoxylin. Samples were prepared 48 hours on IVC ligation. Fibrin-occluded vessels are indicated by the arrows. (ii) Analysis of the percentage of occluded vessel observed in the 2 genotypes. Data are presented as mean ± SEM. n = 5. **P < .01. (D) Comparison of venous thrombosis in WT mice transplanted with bone marrow cells from WT animals (WT/WT) and in WT mice transplanted with bone marrow cells from APP-KO mice (WT/APPKO). Thrombus length is reported in (i) and thrombus weight in (ii). *P < .05; **P < .001.

  • Figure 4.

    Analysis of pulmonary thromboembolism. (A) Representative lung histology images on staining with hematoxylin/eosin (H&E) and phosphotungstic acid–hematoxylin (PTAH). Arrows indicate occluded vessels. Quantification of the percentage of occluded vessels is reported in (B), and analysis of the mean diameter of occluded vessels is reported in (C). Data have been collected by examining 10 microscopic fields for each lung section, prepared from 5 different animals, and are presented as mean ± SEM. **P < .01.

  • Figure 5.

    Blood clotting assays. In all panels, red bars refer to WT mice, and blue bars to APP-KO mice. Data are expressed as mean ± SEM of 10 different determinations. Quantification of the level of plasma fibrinogen in the 2 genotypes is reported in (A). PT (B,D) and APTT (C,E) was measured in PPP (B-C) and PRP (D-E). *P < .05. (F) Measurement of APTT in reconstituted samples obtained by mixing PPP from 1 genotype with washed platelets from the other genotype, as indicated. *P < .05. (G) Quantification of the activity of FXIa (i) and FXIIa (ii) in PRP from WT and APP-KO mice. *P < .05.

  • Figure 6.

    Analysis of NETs formation. In all the panels, red bars refer to WT mice, and blue bars to APP-KO mice. Data are expressed as mean ± SEM of at least 5 different determinations. (A) NETs formation in purified neutrophils from WT and APP-KO mice was visualized by staining for DNA with Hoechst (blue) and for citrullinate Histone H3 (red). Representative images of resting and PMA-stimulated neutrophils are reported in (i), and quantification of the NETs formation expressed as percentage of neutrophils extruding NETs is reported in (ii). ***P < .005. (B) Analysis of NETs formation induced by incubation of WT and APP-KO neutrophils with WT platelets, APP-deficient platelets, or no cells (none), as indicate in the bottom. (C) Flow cytometry analysis of circulating platelet–leukocyte aggregates in blood from WT and APP-KO mice untreated (none) or on stimulation with 0.5 U/mL thrombin, as indicated. *P < .05; **P < .01. (D) Venous thrombi were isolated from WT and APP-KO mice 24 hours on IVC ligation, and stained for the NETs-related markers CRAMP and citrullinated histone H3 (Cit-H3), as indicated. (i) Some representative images, whereas (ii) reports the quantification of the positive cells counted form 5 positive stained fields. Results are reported as mean ± SEM. *P < .05; **P < .01.