Endothelial cell 1-acyl-platelet-activating factor synthesis in response to anticardiolipin antibody-positive serum...

Enhanced endothelial cell 1-acyl platelet-activating factor (1-acylPAF) synthesis has been identified as a consequence of anticardiolipin-antibody-positive serum exposure. While this observation suggests that PAF analogues may play a role in antibody-mediated vasculopathy, it has not been determined whether the antibodies themselves are directly responsible for stimulating 1-acylPAF production. The objective of the current study was to determine whether extracted immunoglobulins or non-immunoglobulin serum constituents are responsible for the increased endothelial cell 1-acylPAF production previously observed. Sera from six ACA-positive, non-pregnant women were precipitated with ammonium sulfate and dialyzed to extract their respective immunoglobulin fractions. IgG, A and M concentrations were measured by nephelometry before and after precipitation and enzyme immunoassay was used to quantify anticardiolipin antibody activity in both whole serum and immunoglobulin suspensions. Paired endothelial cell monolayers were incubated separately with 20% whole serum and the corresponding immunoglobulin fraction from each patient source. After ionophore A23187 stimulation, intracellular PAF production was measured by lipid extraction and radiolabeling. RadylPAF species were distinguished by zonal thin-layer chromatography. Mean radylPAF synthesis was compared between experimental conditions by paired T-test. Significantly more endothelial cell radylPAF synthesis was observed subsequent to whole serum exposure as compared to incubation with the corresponding immunoglobulin fractions (19154 +/- 1916 vs 11785 +/- 1778; dpm, mean +/- SE; p = 0.007); the majority of recovered radylPAF was the sn-1-acyl derivative. Serum samples recovered after immunoglobulin extraction, retained similar stimulatory activity for radylPAF (23045 +/- 4175 dpm) in comparison to intact whole serum (19635 +/- 1266 dpm; p = 0.25). These findings suggest that in ACA-positive serum, non-immunoglobulin constituents provide greater stimulation of 1-acylPAF synthesis than the immunoglobulins themselves.