Figure 2: Antibody-mediated platelet desialylation occurs mainly on the GPIbα subunit.

Galactose exposure was detected with RCA-1 binding and measured by flow cytometry in murine (a–d) and human (e–j) platelets following incubation with mAbs (a,b,d,e,f–h,j,k) or antisera (c) n=10–20. (d,g) Co-incubation with sialidase inhibitor DANA prior to addition of mAbs to murine (d) or human (g) platelets; n=8. (h) FcγRII/III blocker IV.3 was incubated with mAbs (9D2, M1 or HUTA B) and platelets, RCA-1 binding was assessed following. Only the healthy donor platelets that were significantly desialylated in the presence of these mAbs were tested; n=4. (i) RCA-1 binding was measured in healthy human platelets following incubation with anti-GPIbα (ITP-1 to ITP-12) or anti-GPIIbIIIa (ITP-a to ITP-l) antibody-positive ITP patient plasma. (j) anti-GPIbα-mediated RCA-1 binding was assessed following removal of GPIbα with OSGE. (k) Representative western blot of RCA-1 binding (left), and probing with commercial anti-GPIbα antibody (right) to confirm the identity of the RCA-1-positive band following incubation with anti-GPIbα mAb (NIT F). RCA-1 binding on GPIbα was also quantified by protein densitometry in the presence of DANA. All flow cytometry data are expressed as fold change from nonspecific murine IgG (murine)- or IVIG (human)-treated control platelets (CTRL). Anti-GPIbα mAbs shown as mean±s.e.m. of individual mAbs. *P<0.05, **P<0.01, ***P<0.001 versus CTRL as analysed by the Student’s t-test (a–c,e,f) or one-way analysis of variance followed by Bonferroni post hoc (d,g–k). NS, not significant.