Background/Purpose:  Recently exclusive IgA anti-b₂Glycoprotein I (ab₂GPI) seropositivity  – in the absence of any other antiphospholipid (aPL) antibodies- has been reported particularly in SLE patients.  A significant proportion of those (70-80%) were found to have Antiphospholipid Syndrome (APS)-clinical manifestations (e.g. thrombosis and/or pregnancy losses).  APL antibodies of the IgG and IgM isotypes have been shown to be pathogenic in vivo, but whether IgA ab₂GPI antibodies are thrombogenic in mice is unknown.   Here we examined the effects of affinity purified IgA ab₂GPI antibodies isolated from patients with exclusive IgA ab₂GPI positivity on thrombus formation and tissue factor (TF) upregulation in mice.  

Methods:

IgA was isolated from pooled sera of four patients (IgA-APS) with isolated IgA ab₂GPI titers (³80 SAU) – two had strokes, one had a confirmed deep vein thrombosis and one had two pregnancy losses – and from normal human serum (IgA-NHS) using a Immobilized Jacalin column (Pierce Biotechnology).  IgA ab₂GPI in the IgA-APS and in the IgA-NHS preparations was determined by ELISA (INOVA Diagnostics), the protein concentration by the Bradford method and the lupus anticoagulant by using a modified silica clotting time (SCT).  CD1 mice weighing between 26-30 g were injected twice with 100 µg/ml of IgA-APS or IgA-NHS. Seventy two hours after the first injection, the size of induced thrombi in the femoral vein was determined as described (Circulation 1996; 94: 1746-1751).  Tissue factor  activity was determined in homogenates of pooled carotid arteries and in peritoneal macrophages using a chromogenic assay.  Student’s t test was used to determine differences in mean thrombus sizes between IgA-APS and IgA-NHS treated mice.  

Results: IgA-APS and IgA-NHS were rendered endotoxin free by the Limulus amebocyte lysate assay, and did not have detectable levels of IgG or IgM.   The IgA-APS preparation but not the IgA-NHS was positive for IgA ab₂GPI (103.7 SAU) and LA  (SCT ratio IgA-APS/IgA-NHS) =2; normal <1.2.  Results of thrombus sizes and TF activities in mice treated with IgA-APS and IgA-NHS are shown in the table.

*statistically significant, p=0.02. 

Conclusion:  This data show for the first time that IgA ab₂GPI antibodies are thrombogenic and upregulate TF in mice.   Detection of IgA ab₂GPI antibodies – currently not included in the classification criteria for APS – may further identify a group of patients with APS-associated clinical manifestations that otherwise would have been missed with tests used routinely in the clinical laboratory to confirm APS.