Differential expression of protease-activated receptors in monocytes from patients with primary antiphospholipid syndrome.
OBJECTIVE
To investigate the expression of protease-activated receptors (PARs), their potential regulation by anticardiolipin antibodies (aCL), and their association with the expression of other molecules relevant to thrombosis in monocytes obtained from 62 patients with primary antiphospholipid syndrome (APS).


METHODS
Monocytes were isolated from peripheral blood mononuclear cells by magnetic depletion of nonmonocytes. Expression of tissue factor (TF) and PARs 1-4 genes was measured by quantitative real-time reverse transcription-polymerase chain reaction. Cell surface TF and PARs 1-4 expression was analyzed by flow cytometry. For in vitro studies, purified normal monocytes were incubated with purified APS patient IgG, normal human serum IgG, or lipopolysaccharide, in the presence or absence of specific monoclonal antibodies anti-PAR-1 (ATAP2) or anti-PAR-2 (SAM11) to test the effect of blocking the active site of PAR-1 or PAR-2.


RESULTS
Analysis of both mRNA and protein for the 4 PARs revealed significantly increased expression of PAR-2 as compared with the control groups. PAR-1 was significantly overexpressed in APS patients with thrombosis and in the control patients with thrombosis but without APS. PAR-3 expression was not significantly altered. PAR-4 expression was absent in all groups analyzed. In addition, we demonstrated a correlation between the levels of PAR-2 and the titers of IgG aCL, as well as parallel behavior of TF and PAR-2 expression. In vitro, IgG from APS patients significantly increased monocyte expression of PAR-1 and PAR-2. Inhibition studies suggested that there was direct cross-talk between TF and PAR-2, such that inhibition of PAR-2 prevented the aCL-induced expression of TF.


CONCLUSIONS
These results provide the first demonstration of increased expression of PARs in monocytes from patients with APS. Thus, PAR antagonists might have therapeutic potential as antithrombotic agents in APS.
López-Pedrera
Chary
C
Research Unit and Rheumatology Department, University Hospital Reina Sofía, Avenida Menéndez, Pidal s/n, E-14004 Córdoba, Spain. rosario.lopez.exts@juntadeandalucia.es
Aguirre
Maria Angeles
MA
Buendía
Paula
P
Barbarroja
Nuria
N
Ruiz-Limón
Patricia
P
Collantes-Estevez
Eduardo
E
Velasco
Francisco
F
Khamashta
Munther
M
Cuadrado
Maria José
MJ
D000328:Adult; D017153:Antibodies, Anticardiolipin / Q000502:physiology; D000911:Antibodies, Monoclonal / Q000276:immunology; D016736:Antiphospholipid Syndrome / Q000097:blood* / Q000150:complications; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007074:Immunoglobulin G / Q000302:isolation & purification; D008297:Male; D008875:Middle Aged; D009000:Monocytes / Q000737:chemistry*; D012313:RNA / Q000032:analysis; D020239:Receptor Cross-Talk / Q000502:physiology; D044462:Receptors, Proteinase-Activated / Q000097:blood* / Q000235:genetics / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013925:Thromboplastin / Q000032:analysis; D013927:Thrombosis / Q000209:etiology
D017153:Antibodies, Anticardiolipin; D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D044462:Receptors, Proteinase-Activated; D012313:RNA; D013925:Thromboplastin