Monocyte response to aPLAs in the presence of anti-TLR2, anti-TLR4, or anti-CD14 antibodies. (A) Monocytes were incubated for 4 hours with 32 aPLA or 19 control IgG (CTL) preparations at 500 μg/mL. Cell activation was assessed by quantification of TNF in cell supernatants by ELISA. The cutoff value (gray line) for monocyte activation was set to 0.44 ng/mL (mean of control IgG ± 3 SD). (B-C) Blocking antibodies to TLR2, TLR4, or CD14 and the corresponding isotype-matched control antibodies (CTL; 10 μg/mL) were incubated with monocytes for 30 minutes, followed by a 4-hour incubation with the 19 activating aPLA or control IgG (CTL) preparations at 500 μg/mL or 2 aPLA preparations immunopurified on β2GP1 (100 μg/mL). (B) TNF secretion was quantified by ELISA in the cell supernatants. (C) Changes in tissue factor mRNA levels were evaluated by qRT-PCR. (D) Tissue factor activity was quantified as described in “Monocyte activation” and is expressed as picomoles of generated FXa. Statistical analysis indicated a significant difference in monocyte stimulation by aPLAs in the presence of compared with in the absence of anti-TLR2 or anti-CD14 antibodies (P < .001 by 2-way ANOVA and paired t test).