IVIG treatment diminished an antibody response to αIIbβ3. (A) Flow cytometric histograms showed that plasma from mice A and B contained Ig antibodies (shaded peak) that reacted with normal human platelets. Displayed is the relative fluorescence intensity of human platelets incubated with 1:10 diluted murine plasma and an FITC-conjugated F(ab′)2 goat anti–murine IgG Fc secondary antibody. Platelets incubated with secondary antibody and dilution buffer served as a negative control, while a monoclonal antibody to the human αIIbβ3 complex (AP2) was used as a positive control. This result was observed 4 times using platelets from 2 separate human donors analyzed on 2 separate occasions. (B) As in panel A, fluorescence analysis showed that 1:10 diluted plasma from mouse A also reacted with platelets from normal β3+/+, heterozygous β3+/– mice as well as platelets from another human β3-transduced transplant recipient (shaded peak). In striking contrast, plasma did not react with platelets isolated from a β3–/– mouse. Dilution buffer served as the negative control and a PE-conjugated monoclonal antibody to murine GPIbα was used as a positive control. (C) IVIG (0.5 mg/g body weight) was injected each day for 3 days into mouse A. Flow cytometry was then performed with human platelets incubated in plasma from mouse A and secondary antibody as described in panel A. The level of plasma Ig binding to human platelets before and after IVIG treatment of mouse A (black line) was determined by dividing the MFI of platelets incubated with mouse A plasma by the MFI of platelets treated with negative control buffer. The ratio decreased below 2.0 (dotted line), indicating a negligible affinity of plasma Ig for platelet proteins. The overlay graph shows an increase in platelets expressing αIIbβ3 following IVIG treatment of mouse A (orange line). The MFI ratio was calculated from flow-cytometric histograms detecting the binding of an FITC-conjugated antibody against murine αIIb to platelets from mouse A versus antibody binding to β3–/– platelets. These results represent the outcome of IVIG treatment for 3 mice with detectable plasma Ig to human platelets. (D) Following IVIG treatment, mouse A had restored platelet function in an aggregation assay performed at 27 weeks after transplantation. This result was observed using platelets from mouse A and platelets isolated from mice that received a transplant of bone marrow derived from mouse A as second- and third-generation recipients. (E) As in panel C, flow cytometric analysis using the MFI ratio of platelets binding an FITC-conjugated antibody to murine αIIb demonstrated long-term (32 weeks), stable expression of αIIbβ3 on the surface of platelets from mouse A after IVIG and mice B and D. Note: mouse C was killed for the in vivo platelet function assay at week 5.
