EXTENDED REPORTS

Toll-like receptor and antiphospholipid mediated thrombosis: in vivo studies

Silvia S Pierangeli¹, Mariano E Vega-Ostertag², Elena Raschi³, Xiaowei Liu², Zurina Romay-Penabad¹, Valeria De Micheli⁴, Monica Galli⁵, Marco Moia⁶, Angela Tincani⁷, Maria Orietta Borghi⁸, Tracy Nguyen-Oghalai⁹, Pier Luigi Meroni⁸

¹ Antiphospholipid Standardization Laboratory, Division of Rheumatology, Department of Internal Medicine, University of Texas Medical Branch, Galveston, Texas, USA
² Department of Microbiology, Biochemistry and Immunology, Morehouse School of Medicine, Atlanta, Georgia, USA
³ Allergy, Clinical Immunology and Rheumatology Unit, IRCCS Istituto Auxologico Italiano, Milan, Italy
⁴ Ambulatorio Patologie Trombotiche e Emorragiche, Ospedale San Leopoldo Mandic, Merate, Italy
⁵ Department of Haematology, Ospedali Riuniti, Bergamo, Italy
⁶ Bianchi Bonomi Hemophilia and Thrombosis Center, IRCCS Maggiore Hospital, Mangiagalli and Regina Elena Foundation, Milan, Italy
⁷ Clinical Immunology and Rheumatology Service, Spedali Civili, Brescia, Italy
⁸ Department of Internal Medicine, University of Milan, IRCCS Istituto Auxologico Italiano, Milan, Italy
⁹ Division of Rheumatology, Department of Internal Medicine, University of Texas Medical Branch, Galveston, Texas, USA

Dr Silvia S Pierangeli, Division of Rheumatology/Department of Internal Medicine, University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555-1165, USA; sspieran{at}utmb.edu

Objective: A study was undertaken to investigate the in vivo pathogenic role of Toll-like receptor 4 (TLR-4) in the antiphospholipid syndrome (APS) by studying the thrombogenic antiphospholipid (aPL) activity in lipopolysaccharide (LPS) non-responsive (LPS–/–) mice and the association between tlr4 gene polymorphisms and APS in patients.

Methods: IgGs from two patients with APS, one with aPL negative systemic lupus erythematosus (SLE) and one with normal human serum (NHS), were evaluated for thrombosis, tissue factor (TF) activity and endothelial cell activation in LPS–/– mice displaying a tlr4 spontaneous mutation vs LPS responsive (LPS+/+) mice. Human tlr4 Asp299Gly and Thr399Ile polymorphisms were evaluated by allele-specific PCR in 110 patients with APS with arterial/venous thrombosis and in 220 controls of the same ethnic origin.

Results: IgG-APS produced significantly larger thrombi and more leucocytes (WBC) adhering to endothelial cells in the cremaster muscle microcirculation of LPS+/+ mice than IgG-NHS or aPL negative SLE-IgG. These effects were abrogated after absorption of the anti-ß₂glycoprotein I activity by an affinity column. The two IgG-APS induced significantly smaller thrombi and fewer WBC adhering to endothelial cells in LPS–/– mice than in LPS+/+ mice. IgG-APS induced higher TF activity in carotid artery homogenates of LPS+/+ mice than in LPS–/– mice. The prevalence of Asp299Gly and Thr399Ile tlr4 polymorphisms was significantly lower than in controls.

Conclusions: These findings in LPS–/– mice and the reduction in the "protective" polymorphism in patients with APS with thrombosis suggest that TLR-4 is involved in the interaction of aPL with endothelial cells in vivo.

Abbreviations: aCL, anti-cardiolipin; aPL, antiphospholipid; APS, antiphospholipid syndrome; ß₂GPI, ß₂glycoprotein I; EC, endothelial cells; LPS, lipopolysaccharide; NF-B, nuclear factor-B; NHS, normal human serum; SLE, systemic lupus erythematosus; SNP, single nucleotide polymorphism; TF, tissue factor; TLR, Toll-like receptor

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