Shedding of either GPVI or FcγRIIa induced by Ig from patients with HIT is blocked by an anti–Fc receptor antibody. Washed platelets were incubated with either VM58 or Ig from control or HIT patient serum. Some samples also contained the blocking anti–FcγRIIa mAb, IV.3 (10 μg/mL, final concentration). Levels of (A) GPVI on platelets (top) or GPVI fragment in supernatant (bottom) or (B) FcγRIIa (top) and β3 integrin (bottom) on platelets were assessed by Western blotting with anti–GPVI mAb (6B-12), anti–FcγRIIa tail antiserum, or the anti-β3 mAb (CRC54) as indicated. Equivalent amounts of VM58 and IV.3 alone or mixed with platelets in the absence of agonist, were run in parallel lanes to distinguish bands corresponding to Ig heavy chains. PL indicates platelet lysate, containing full-length GPVI for reference. Vertical lines have been inserted to indicate a repositioned gel lane. All lanes within each figure came from the same experiment and the same gel/Western blot.
