Platelet Activation In Sitosterolemia Mice

Sitosterolemia is a rare, autosomal recessive disorder characterized by the accumulation of plant sterols in blood and tissues, and is caused by mutations in one of the adenosine triphosphate-binding cassette (ABC) transporter ABCG5 or ABCG8 genes. Patients with mutations in either of these sterol transport proteins, which normally form a heterodimer sterol egress channel, frequently develop tendon and cutaneous xanthomas and, most importantly, are at risk of developing premature coronary atherosclerosis. Other clinical manifestations include hematological abnormalities such as hemolytic anemia, macrothrombocytopenia, and loss of ristocetin-induced platelet agglutination – a measure of the ability of platelet glycoprotein (GP) Ib to function as a adhesion receptor for von Willebrand factor (VWF). Mice genetically deficient in ABCG5 or ABCG8 fully recapitulate the macrothrombocytopenia and loss of platelet function seen in human sitosterolemia, a condition that can be corrected by treatment with the sterol-absorption inhibitor, ezetimibe. Because the mechanism by which accumulated plant sterols affects platelet size, production, and function is incompletely understood, we further analyzed these traits in Abcg5- and Abcg8-deficient mice in an animal model of sitosterolemia.

Blood was collected every 1–2 weeks and CBC monitored in Abcg5- and Abcg8-deficient mice that had been fed either a high or low plant sterol diet. Expression of platelet receptors was analyzed by both flow cytometry and Western blotting. GPIbα null mice were used as controls since they have similarly enlarged platelets. Platelet microparticles were analyzed by labeling with a mAb specific for GPIIb and Annexin V. Plasma samples were analyzed for von Willebrand factor (VWF) antigen and multimer patterns.

Following onset of a high sterol diet, the platelet count decreased over a two week period to less than 30% of that of normal controls in both Abcg5^-/- and Abcg8^-/- mice. At the same time, the mean platelet volume gradually increased over a 4 week period from a normal value of approximately 7 fl to approximately 10 fl. In addition, an increased number of platelet-derived microparticles were detected in Abcg5^-/- and Abcg8^-/- mice kept on high sterol diet over a 9 week period, suggesting that low-grade platelet activation was occurring in these mice. Unexpectedly, the surface expression of the GPIIb integrin subunit was decreased in both Abcg5^-/- and Abcg8^-/- mouse platelets compared to that of similarly large platelets derived from GPIbα-null (Bernard-Soulier) platelets. Intracellular staining revealed that the GPIIb in Abcg8^-/- platelets was being internalized and retained within the cell. Both surface-bound and total platelet fibrinogen was increased in Abcg5^-/- and Abcg8^-/- mice fed a high sterol diet. Finally, plasma VWF antigen levels decreased by approximately 50% within two weeks following switching Abcg5^-/- mice from a low-fat to a high-fat diet. Multimer analysis showed loss of high molecular weight multimers in some of the plasma samples.

This study demonstrates a heightened state of platelet activation in a murine model of sitosterolemia, leading to VWF and fibrinogen binding, with concomitant internalization of GPIIb. This might explain, at least in part, the cause of macrothorombocytopenia and absence of ristocetin-induced platelet aggregation in sitosterolemia patients. Platelet activation may also lead to calpain activation, resulting filamin A and talin degradation and microparticle production. Future studies aims at identifying the mechanism by which plant sterols or their metabolites elicit these effects on platelets may provide important clues to the ability of dietary lipids to affect platelet activation and the development of atherosclerosis.

Newman:New York Blood Center: Membership on an entity's Board of Directors or advisory committees; Children's Hospital of Boston: Membership on an entity's Board of Directors or advisory committees.