Filamin A binding to the cytoplasmic tail of glycoprotein Ibα regulates von Willebrand factor-induced platelet activation

We examined the hypothesis that filamin A binding to the cytoplasmic tail of platelet glycoprotein lbalpha (Gplbalpha) is regulated by pathologic shear stress and modulates von Willebrand factor (VWF)-induced platelet activation. To begin, we examined filamin binding to Gplbalpha in Chinese hamster ovary cells coexpressing mutant human Gplb-IX and wild-type human filamin A. We observed that many different deletions and truncations N-terminal to Gplbalpha's cytoplasmic domain residue 594 disrupted filamin A binding, but that binding was unaffected by 14 different point mutations in hydrophilic residues between amino acids 557 and 593. To try to narrow Gplbalpha's filamin A-binding domain, we next measured the effect of several cytoplasmic domain peptides on human filamin A binding to a GST-Gplbalpha cytoplasmic domain fusion protein. One peptide (residues 557-575; designated "A4 peptide") inhibited filamin A binding to the GST-Gplbalpha cytoplasmic domain fusion protein and competed with Gplbalpha for binding to filamin A. When the A4 peptide was delivered to intact human platelets using a carrier peptide, we observed the dose-dependent inhibition of VWF-induced platelet aggregation in response to both ristocetin and shear stress. The effect of the A4 peptide on shear-induced platelet aggregation was accompanied by the attenuation of shear-induced filamin A binding to Gplbalpha and diminished shear-dependent protein tyrosine phosphorylation. These results suggest that shear-dependent VWF-induced platelet activation affects filamin A binding to Gplb-IX-V, and that filamin A binding to the cytoplasmic tail of Gplbalpha regulates proaggregatory tyrosine kinase signaling. (C) 2003 by The American Society of Hematology.