Platelet hyperreactivity explains the bleeding abnormality and macrothrombocytopenia in a murine model of sitosterolemia

Abstract

Sitosterolemia is a rare, autosomal recessive disease caused by mutations in the adenosine triphosphate-binding cassette transporter genes ABCG5 or ABCG8 that result in accumulation of xenosterols in the body. Clinical manifestations include tendon xanthomas, premature coronary artery disease, hemolytic anemia, macrothrombocytopenia, and bleeding. Although the effect of sterol accumulation on the predisposition for atherosclerosis is evident, how xenosterol accumulation leads to defects in platelet physiology is unknown. Sitosterolemia induced in Abcg5- and Abcg8-deficient mice fed a high plant sterol diet resulted in accumulation of free sterols in platelet plasma membranes, leading to hyperactivatable platelets characterized by constitutive binding of fibrinogen to its αIIbβ3 integrin receptor, internalization of the αIIbβ3 complex, generation of platelet-derived microparticles, and changes in the quantity and subcellular localization of filamin. The latter was associated with macrothrombocytopenia, shedding of GPIbα, impaired platelet adhesion to von Willebrand factor, and inability to form stable thrombi. Plasma levels of soluble GPIbα were strongly correlated with plasma sitosterol levels in samples from human sitosterolemic patients, implicating a similar mechanism of sterol-induced platelet passivation in the human disease. Intercalation of plant sterols into the plasma membrane therefore results in dysregulation of multiple platelet activation pathways, leading to macrothrombocytopenia and bleeding.

Figure 1

Hematologic parameters of Abcg8^−/− and Abcg5^−/− mice fed an Abcg8^−/− HS or Abcg8^−/− LS diet. (A) Platelet counts, (B) mean platelet volumes, and (C) hemoglobin values. Samples were analyzed using a Vet ABC Counter (n = 6 in each group); **P < .001. (D) Tail bleeding time assays of Abcg8^−/− mice fed an HS or LS diet. When bleeding did not cease within 5 minutes, the tail was cauterized and bleeding time was recorded as 300 seconds.

Figure 2

Sterol accumulation and increased platelet-derived microparticle (PMP) generation in sitosterolemic mouse platelets. (A) Whole blood samples were stained with phycoerythrin (PE)-conjugated anti-mouse αIIb mAb and platelet fraction was identified by αIIb positivity and forward-side scatter. Platelets from Abcg5^−/− HS become large and are compared with GPIb-deficient (Bernard Soulier) platelets, which exhibit αIIb positivity in a larger forward-side scatter gate. Cells were incubated with filipin to detect sterol-laden membranes. The degree of filipin staining in those specific gates is shown (n = 6 in each group). (B) Comparison of cholesterol and plant sterol levels in platelets of Abcg5^−/− HS and Abcg5^−/− LS were determined as described in “Methods.” Whole blood samples were fixed with 2% paraformaldehyde and αIIb-positive platelets were isolated using anti-PE antibody-conjugated magnetic immunobeads. Sterol levels were analyzed by GC-MS. (C) Representative dot plot analyses of Annexin V–positive platelets and PMPs. Whole blood samples were double-stained with PE-anti-mouse αIIb mAb and fluorescein isothiocyanate-Annexin V. The αIIb-positive platelets were gated as in (A) and analyzed for Annexin V positivity. Note the increased number of Annexin V–positive PMPs in the blood of Abcg8^−/− mice fed a high, but not in low plant sterol diet. (D-E) Statistical analyses of αIIb-positive, Annexin V–positive PMPs (D) and platelets (E) found in Abcg8^−/− HS and Abcg8^−/− LS blood samples (n = 8 for Abcg8^−/− LS mice and n = 10 for Abcg8^−/− HS mice). Though the increase in PMPs in the circulation of Abcg8^−/− HS mice was significantly higher than in Abcg8^−/− LS mice, the difference in Annexin V–positive platelets did not reach statistical significance.

Figure 3

Constitutive activation and internalization of the integrin αIIbβ3 complex in Abcg8^−/− HS platelets. (A) Elevated constitutively bound fibrinogen on the surface of Abcg8^−/− HS platelets. Platelets in whole blood were double-stained with a PE-anti-mouse αIIb mAb and fluorescein isothiocyanate–anti-fibrinogen antibody. (B) Summary of fluorescence-activated cell sorter analysis of integrin αIIb expression on the surface (left: nonpermeabilized) or inside (right: permeabilized) of platelets from Abcg8^−/− mice fed a high plant sterol diet. Mean fluorescence intensity/forward scatter were compared with those obtained from similarly sized GPIbα-null platelets, the latter of which was normalized to 100% over 5 different experiments. Surface expression of integrin αIIb and integrin β3 was decreased in Abcg8^−/− HS platelets (left), whereas total cellular integrin αIIb and integrin β3 were similar to that of GPIbα-null platelets (right). FSC, forward scatter.

Figure 4

Decreased fibrinogen binding and increased proteolysis of GPIb and filamin in sitosterolemic platelets. Blood from Abcg8^−/− LS and Abcg8^−/− HS mice were incubated with 10 μg/mL of collagen-related peptide in the presence of antigen-presenting cell (APC)-labeled fibrinogen for 10 minutes at room temperature. Blood samples were fixed, permeabilized, and stained with anti-FlnA antibody followed by AlexaFluor405-labeled goat anti-rabbit immunoglobulin G. Note that ∼40% of Abcg8^−/− HS platelets failed to bind fibrinogen (A-B). (C) Western blot analysis of platelet lysate confirmed decrease of cellular filamin and other known µ-calpain substrates β actin and septin 5. (D) Analysis of intracellular filamin content by flow cytometry shows selective cleavage and degradation of filamins in the Abcg8^−/− HS platelet population that become refractory to agonist stimulation. (E) Surface expression of GPIbα in platelets double stained with PE-labeled anti-mouse αIIb mAb and a mAb specific for the N-terminal 45 kDa domain of GPIbα and analyzed by flow cytometry. GPIbα shedding was expressed as the percentage of GPIbα-negative platelets in total platelets. Inset: western blot analysis of platelets from the same preparation, demonstrating loss of GPIbα from Abcg8^−/− HS platelets. (F) Subcellular localization of FlnA in Abcg8^−/− HS and Abcg8^−/− LS platelets analyzed by confocal microscopy reveals marked reduction in the GPIb/filamin complex from the cell periphery.

Figure 5

Impaired adhesion and thrombus formation of Abcg8^−/− HS platelets. (A-B) PPACK-anticoagulated whole blood from Abcg8^−/− fed a low or high sterol diet was labeled and perfused on mouse VWF immobilized surface at a shear rate of 2000 s⁻¹. Representative images of adherent platelets (A); percent surface coverage for 11 independent experiments (n = 6 for Abcg8^−/− LS and n = 5 for Abcg8^−/− HS) (B). (C-D) Pooled blood samples of Abcg8^−/− LS (n = 4), Abcg8^−/− HS (n = 4), and mouse GPIbα-null platelets were perfused over type I collagen for 120 seconds at 2000 s⁻¹. (C) Representative image of adhesion and thrombus formation at 120 seconds (D) quantitates the time-course of surface coverage (%) as they accumulate in the field of view. Platelet counts and hematocrit of these pooled Abcg8^−/− LS, Abcg8^−/− HS, and control mGPIbα-null samples were 498 × 10⁹/L, 38.2%; 76 × 10⁹/L, 31.3%; and 194 × 10⁹/L, 31.3%, respectively.

Figure 6

Correlation between circulating plasma glycocalicin and sitosterol levels. Plasma glycocalicin in sitosterolemia patients was analyzed using a sandwich enzyme-linked immunosorbent assay that employs 2 different mAbs against human GPIbα. The Scientific and Standardization Committee/International Society on Thrombosis and Haemostasis Secondary Coagulation Standard was assigned as 1 U/mL and used to construct a standard curve.

Figure 7

Analyses of Abcg8^−/− HS and Abcg8^−/− LS megakaryocytes. (A-C) BM from Abcg8^−/− LS (A) and Abcg8^−/− HS mice (B-C) was harvested for histologic analysis. The number of multinucleated mature megakaryocytes (black arrowheads) was visually identified and counted in hematoxylin and eosin–stained sections. (C, black arrows) Emperipolesis of neutrophils into megakaryocytes. (D-E) BM megakaryocytes isolated by discontinuous gradient centrifugation over bovine serum albumin were stained with anti-αIIb mAb (red) and 4,6 diamidino-2-phenylindole (blue). Large αIIb-positive cytoplasmic extrusions (arrowhead) and “bare” megakaryocyte nuclei (asterisks) were noted in the BM samples from Abcg8^−/− HS (E) but not in Abcg8^−/− LS mice. (F-H) DNA ploidy profiles and integrin αIIb expression in BM megakaryocytes isolated using anti-CD41 magnetic beads and cultured in the presence of thrombopoietin for 60 hours. An HS diet resulted in a greater total number of CD41+ cells (7.6% vs 3.9%), with a corresponding proportional increase in both the high (>128N) and low (2N-4N) ploidy cells (F-G). Expression of integrin αIIb was similar in Abcg8^−/− HS and Abcg8^−/− LS cells regardless of ploidy (H). (I) Western blot analysis of purified, CD41-positive megakaryocytes. Expression of filamin A (FlnA), GPIbα, and µ-calpain in megakaryocytes derived from Abcg8^−/− HS mice was slightly decreased compared with that of Abcg8^−/− LS mice. No significant differences were observed in the expression of HSC70 and β actin.