Filamin A binding to the cytoplasmic tail of glycoprotein Ibalpha regulates von Willebrand factor-induced platelet activation.
We examined the hypothesis that filamin A binding to the cytoplasmic tail of platelet glycoprotein Ibalpha (GpIbalpha) is regulated by pathologic shear stress and modulates von Willebrand factor (VWF)-induced platelet activation. To begin, we examined filamin binding to GpIbalpha in Chinese hamster ovary cells coexpressing mutant human GpIb-IX and wild-type human filamin A. We observed that many different deletions and truncations N-terminal to GpIbalpha'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 GpIbalpha's filamin A-binding domain, we next measured the effect of several cytoplasmic domain peptides on human filamin A binding to a GST-GpIbalpha cytoplasmic domain fusion protein. One peptide (residues 557-575; designated "A4 peptide") inhibited filamin A binding to the GST-GpIbalpha cytoplasmic domain fusion protein and competed with GpIbalpha 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 GpIbalpha and diminished shear-dependent protein tyrosine phosphorylation. These results suggest that shear-dependent VWF-induced platelet activation affects filamin A binding to GpIb-IX-V, and that filamin A binding to the cytoplasmic tail of GpIbalpha regulates proaggregatory tyrosine kinase signaling.
Feng
Shuju
S
Thrombosis Research (151), VA Medical Center, 2002 Holcombe Blvd, Houston, TX 77030, USA.
Reséndiz
Julio C
JC
Lu
Xin
X
Kroll
Michael H
MH
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D003285:Contractile Proteins / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D003599:Cytoskeleton / Q000502:physiology; D064448:Filamins; D006801:Humans; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000502:physiology; D015539:Platelet Activation / Q000502:physiology*; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics; D015398:Signal Transduction / Q000502:physiology; D014841:von Willebrand Factor / Q000378:metabolism / Q000494:pharmacology*
D003285:Contractile Proteins; D064448:Filamins; D008840:Microfilament Proteins; D019038:Platelet Glycoprotein GPIb-IX Complex; D011994:Recombinant Proteins; D014841:von Willebrand Factor