The inhibition of protein C anticoagulant activity by anti-β2-glycoprotein I (β2GPI) antibodies isolated from patients with antiphospholipid syndrome by chromatography methods

Abstract

Antiphospholipid antibodies (aPL) are associated with an increased risk of thrombosis; however, the mechanism remains unknown. Recent studies have focused on the impediment of protein C anticoagulant activity by anti-β₂-glycoprotein I (β₂GPI) antibodies (aβ₂GPI Ab). We purified IgG fractions containing a high concentration of aβ₂GPI Ab from patients with antiphospholipid syndrome (APS) and then investigated the effect of purified aβ₂GPI Ab on the activity of activated protein C (APC). Using a three-step chromatography method (DEAE-sepharose column, phosphatidylserine polyacrylamide gel column dependent on the presence of β₂GPI, and protein G column chromatography), we successfully isolated anti-β₂GPI IgG from nine patients with APS. Seven of nine samples inhibited APC activity in a concentration-dependent manner only in the presence of β₂GPI, as observed by a chromogenic assay that was able to determine thrombin activity even in the presence of APC. The extent of APC inhibition by these fractions appeared to be related to aβ₂GPI Ab titers of the purified IgG. However, the inhibitory effect of IgG from patients was not detected in the absence of β₂GPI. IgG purified from three normal subjects did not affect APC activity. Herein, we show a useful method for the isolation of IgG containing a high concentration of aβ₂GPI Ab. Moreover, the present findings indicate that inhibition by aβ₂GPI Ab on APC anticoagulant activity could explain one of the mechanisms for the thrombotic state in APS.

Key words::

• Activated protein C (APC)
• Anticardiolipin antibody (aCL)
• Phosphatidylserine polyacrylamide gel column
• Thrombosis