We found that the binding of multimeric vWF to GP Ib under a shear force of 108 dynes/cm2 resulted in the transmembrane flux of Ca2+ ions with a two- to three-fold increase in their intracellular concentration ([Ca2+](i)). The blockage of this event, obtained by inhibiting the vWF-GP Ib interaction, suppressed aggregation. In contrast, the blockage of vWF binding to GP IIb-IIIa, as well as the prevention of activation caused by increased intracellular cAMP levels, inhibited aggregation but had no significant effect on [Ca2+](i) increase. A monomeric recombinant fragment of vWF containing the GP Ib-binding domain of the molecule (residues 445-733) prevented all effects mediated by multimeric vWF but, by itself, failed to support the increase in [Ca2+](i) and aggregation. These results suggest that the binding of multimeric vWF to GP Ib initiates platelets aggregation induced by high shear stress by mediating a transmembrane flux of Ca2+ ions, perhaps through a receptor-dependent calcium channel. The increase in [Ca2+](i) may act as an intracellular message and cause the activation of GP IIb-IIIa; the latter receptor then binds vWF and mediates irreversible aggregation.
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