Abstract 13245: The Pro-Atherogenic DNA-Binding Cytokine HMGB1 Binds to Activated Platelets via the Receptor for Advanced Glycation End Products (RAGE) and Toll-Like Receptor 2 (TLR 2)
Background and Rationale: High-mobility group box1 (HMGB1) facilitates gene transcription as an architectural nuclear protein but may also be secreted, thereby mediating inflammatory and immune responses. HMGB1 has recently been identified in human atherosclerotic lesions and inhibition of HMGB1 reduced atherosclerosis in ApoE-/- mice. Activated platelets are involved in early stages of atherogenesis, therefore we hypothesized an interaction between HMGB1 and platelets.
Methods: Binding of recombinant HMGB1 to platelets was examined in diluted human and mouse whole blood by flow cytometry. Expression of the HMGB1 receptor RAGE was discovered by RT-PCR with mRNA extracted from highly purified platelets and confirmed by Western blot, immunofluorescence microscopy, and flow cytometry. HMGB1 expression in human coronary artery thrombi was studied by immunohistochemistry.
Results: Recombinant HMGB1 [20µg/ml] bound to thrombin-activated human platelets (mean fluorescence intensity MFI 2.49 vs 25.01, p=0.0079). RAGE is one of the known receptors for HMGB1. We identified RAGE in platelets by RT-PCR from highly purified platelets and confirmed expression of RAGE-protein in platelets by Western blot. RAGE expression on platelets increased in response to thrombin (MFI 4.85 vs 6.74, p=0.026). HMGB1 binding to platelets from RAGE -/-, TLR-2 -/-, and TLR-4 -/- mice was analyzed to specify the HMGB1 receptor on platelets. HMGB1 bound to platelets from wild type C57Bl6 (MFI 2.64 vs 20.3, p=0.0079), and TLR-4 -/- mice (MFI 2.11 vs 25.7, p=0.0294) but failed to show binding to platelets from RAGE -/- and TLR-2 -/- mice. The potential relevance of the interaction between HMGB1 and activated platelets was demonstrated by high expression-levels of HMGB1 in platelet rich coronary artery thrombi from patients with acute myocardial infarction.
Conclusions: Platelets express and up-regulate upon activation mRNA and protein of RAGE. Activated platelets bind HMGB1 via RAGE and TLR-2. The high-level expression of HMGB1 in platelet rich coronary artery thrombi points towards a central role of a HMGB1 in atherothrombosis and warrants the further exploration of the potentially HMGB1 mediated monocyte recruitment during atherosclerotic plaque development.
- © 2011 by American Heart Association, Inc.