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LEUKOCYTES ARE KEY TO THE PRO-THROMBOTIC EFFECTS OF ABACAVIR
Victor Collado-Diaz1, Ainhoa Sanchez-Lopez1, Samuel Orden2, Maria Amparo Blanch-Ruiz1, Maria Angeles Martinez-Cuesta1, Isabel Andujar2, Angeles Alvarez1, Juan V. Esplugues1
1University of Valencia, Valencia, Spain,2FISABIO, Valencia, Spain
Abacavir (ABC) shares a close structural similarity with endogenous purines like ATP and ADP, which are important signalling molecules in vascular physiopathology. ABC induces platelet-leukocyte-endothelial cell interactions and pro-thrombotic effects through a mechanism involving interference with the purinergic system, specifically ATP-P2X7 receptors. In previous in vitro experiments we have determined that the ATP-P2X7 receptors implicated in the actions of ABC are located primarily in leukocytes. Since the recruitment of leukocytes by platelets is an important phase in the formation of thrombi, the present study was performed to evaluate the role of white cells in the pro-thrombotic effects of ABC in an animal model of thrombosis.
Male wild-type C57BL/6 mice were pre-treated with ABC (5–7.5 µg/mL intrascrotally 4h) or rofecoxib (0.1 mg/kg, i.p. 2h). To generate leukopenia, some mice were treated with cyclophosphamide (150 mg/kg, i.p., 96h), which reduced the number of circulating leukocytes by almost 90%. Arterioles of the cremaster muscle were visualized with an intravascular microscope and blood flow was analyzed with a Doppler velocimeter. The endothelium-damaging agent ferric chloride was superfused at a concentration of 25 mM, a dose that does not modify blood flow but predisposes arterioles to thrombosis in the presence of other deleterious vascular agents. In contrast, higher concentrations of ferric chloride (over 75 mM) induced thrombi by themselves, an effect that was maintained in leukopenic mice. Images were recorded until blood flow ceased or for 8 min if no vessel occlusion occurred.
ABC induced dose-dependent vessel occlusion in non-leukopenic mice following superfusion with 25 mM ferric chloride (Figure 1). Rofecoxib – a well characterized vascular deleterious agent - generated levels of thrombosis similar to those produced by ABC when administered in the same setting. However, while the pro-thrombotic effects of rofecoxib were maintained in leukopenic mice, those of ABC were absent.
The pro-thrombotic effect of ABC in vivo depends on the presence of leukocytes, thus demonstrating a key role of these cells in the deleterious vascular effects of this drug. These results support previous research suggesting that ABC induces thrombi formation through a specific mechanism involving leukocyte purinergic P2X7 signalling. This may explain the cardiovascular toxicity associated with the use of ABC in humans.