Figure 4: Anti-GPIbα platelet activation and desialylation is a positive feedback loop.

(a,d) Human platelets were pre-incubated with inhibitors of platelet activation including intracellular Ca²⁺ flux (BAPTA-AM), phosphorylation of P38MAPK (SB203580) and Src kinase (PP1) prior to addition of anti-GPIbα mAbs. Following which, platelet activation (P-selectin expression) (a) or desialylation (RCA-1 binding) (d) was detected via flow cytometry. # and ## indicate comparison with CTRL for a only; n=8. (b) Representative western blots of whole-platelet lysate following incubations with GPIbα mAbs with or without indicated inhibitors. Membranes were probed for phosphorylated p38MAPK (p-p38MAPK) then stripped and re-probed for total pMAP38K. (c) Densitometry protein quantification of p-P38MAPK detected in b. Data representative of three separate experiments. (e) Sialidase inhibitor DANA was added prior to anti-GPIbα mAb incubation. DANA-mediated inhibition of antibody-induced platelet activation was measured by P-selectin expression and detected via flow cytometry. n=6. (f) Schematic flow chart illustrating platelet activation–desialylation-positive feedback loop. Following initial anti-GPIbα antibody crosslinking of GPIbα subunits, activation signalling occurs leading to surface translocation of NEU1. NEU1 cleavage of terminal sialic residues on a GPIbα subunit facilitates receptor clustering, resulting in amplification of platelet activation. (g,h) NIT A, NIT B and NIT F Fab fragments were generated and their effects on platelet activation and desialylation on murine (g) and human platelets (h) were analysed by flow cytometry. n=5. All flow cytometry data are expressed as fold change from nonspecific murine IgG (murine)- or IVIG (human)-treated control platelets (CTRL), unless otherwise indicated. Anti-GPIbα mAbs shown as mean±s.e.m. of individual mAbs. *^,#P<0.05, **^,##P<0.01, ***P<0.001 as assessed by one-way analysis of variance followed by Bonferroni post hoc analysis. NS, not significant.