Role of glycoprotein Ibα mobility in platelet function

Incubation at 0 degrees C is known to expose beta-N-acetyl-D-glucosamine residues on glycoprotein (GP) Ib alpha inducing receptor clustering and alpha(M)beta(2)-mediated platelet destruction by macrophages. Here we show that incubation at 0/37 degrees C (4 hours at 0 degrees C, followed by 1 hour at 37 degrees C to mimic cold-storage and post-transfusion conditions) triggers a conformational change in the N-terminal flank (NTF, amino acids, aa 1-35) but not in aa 36-282 of GPIb alpha as detected by antibody binding. Addition of the sugar N-acetyl-D-glucosamine (GN) inhibits responses induced by 0/37 degrees C. Incubation at 0 degrees C shifts GPIb alpha from the membrane skeleton to the cytoskeleton. Different GPIb alpha conformations have little effect on VWF/ristocetin-induced aggregation, but arrest of NTF change by GN interferes with agglutination and spreading on a VWF-coated surface under flow. Strikingly, incubation at 0/37 degrees C initiates thromboxane A(2) formation through a von Willebrand factor (VWF)-independent and GPIb alpha-dependent mechanism, as confirmed in VWF- and GPIb alpha-deficient platelets. We conclude that the NTF change induced by 0/37 degrees C incubation reflects clustering of GPIb alpha supports VWF/ristocetin-induced agglutination and spreading and is sufficient to initiate platelet activation in the absence of VWF.