Demonstration of antiphospholipid antibody heterogeneity by phospholipid column chromatography and salt gradient elution techniques

Abstract

Antiphospholipid (aPL) antibodies are associated with thrombosis, recurrent abortions and thrombocytopenia. Studies to determine the mechanisms of action of these antibodies have been hindered by their heterogeneity and limited availability of techniques to isolate and characterize subgroups of the antibodies. We report a new phospholipid affinity chromatography method which enables separation of antiphospholipid positive sera into more than one antibody subpopulation. Sera from five patients with complications of the antiphospholipid syndrome (APS) were studied. Each serum was applied to chromatography columns prepared by coating polystyrene beads (diameter 100 Å) with phosphatidylserine (PS) or cardiolipin (CL). A linear salt gradient (0.03-1.0 M NaCl) was used for elution. Eluates were analyzed for phospholipid binding and for inhibition of the prothrombin-thrombin conversion reaction. Each sample yielded two to three peaks for CL and PS affinity columns. Molarities at which peaks were eluted differed between samples. For individual samples, molarities at which peaks were eluted differed between CL and PS columns. These data suggest that aPL antibodies are heterogenous, with differences existing between patients and even within single serum samples. Subpopulations differed in their avidities for CL and PS but generally all had prothrombinase inhibitory activity.