Summary
Anti-platelet autoantibodies may be responsible for hematological complications in
patients with systemic lupus erythematosus (SLE), but the mechanisms by which these
antibodies cause abnormal hemostasis remain unknown. In the present study, using fluorescence
activated cell sorter (FACS) analysis, we demonstrate that an SLE-derived human hybridoma
autoantibody, 9604, recognizes a surface antigen expressed on platelets activated
by ADP, calcium ionophore A23187, or phorbol myristate acetate (PMA), showing saturation
with approximately 2,000 antibody molecules bound per platelet and a Kd of 41 nM. The binding of 9604 to activated platelets was significantly inhibited
by EDTA, indicating partial dependence on divalent cations. It did not appear to be
dependent on platelet secretion, nor did it directly affect α-granule or dense granule
secretion. The protein antigen responsible for the binding of 9604 to activated platelets
was characterized by Western blot and immunoprccipitation and shown to have a native
molecular weight (M. W.) of greater than 400,000, with a 32,000 M. W. subunit (p 32).
Antibody 9604 had little or no effect on the shape change and the initial rate of
primary aggregation of normal platelets. In contrast, 9604 inhibited secondary aggregation
of stirred platelet suspensions (IC50 ≥1 nM) following activation by ADP, thromboxane A2 mimetic U46619, or calcium ionophore
A23187, but not PMA or thrombin. The inhibition of large platelet aggregate formation
(secondary aggregation), with a major shift to smaller microaggregates and singlets,
was confirmed by direct particle count and sizing studies. The functional inhibition
of platelet aggregation by an SLE-derived human hybridoma autoantibody in vitro suggests
one potential mechanism that may play a role in the hemostatic disorders found in
SLE.
