The alpha1 helix-beta13 strand spanning Leu214 to Val229 of platelet glycoprotein Ibalpha facilitates the interaction with von Willebrand factor: evidence from characterization of the epitope of monoclonal antibody AP1.
The glycoprotein Ib-IX-V (GP Ib-IX-V) complex mediates platelet binding to von Willebrand factor (VWF) through its largest polypeptide, GP Ibalpha. Of the many GP Ibalpha monoclonal antibodies described, AP1 is of particular interest because it blocks static VWF binding induced by 2 modulators, ristocetin and botrocetin, and platelet adhesion to VWF surfaces under flow. We mapped the AP1 binding site to a region encompassing Arg218 to Tyr228, comprising the alpha1 helix and beta13 strand defined by the GP Ibalpha crystal structure. AP1 binding absolutely required Arg218, Asp222, and Glu225. We evaluated the ability of cells expressing mutants of this region to bind VWF under static conditions in the presence of modulators, and to attach to and roll on a VWF matrix under flow. These data indicate that 2 regions within the sequence Arg218 to Tyr228 have important roles in VWF binding: the alpha1 helix has a regulatory role and the beta turn and beta13 strand bind VWF directly. Despite this, the only effect of a synthetic peptide corresponding to Leu214 to Val229 was to slightly increase the rolling velocity of GP Ibalpha-expressing Chinese hamster ovary (CHO) cells on VWF. This region thus appears to be more important for maintaining the regional conformation of GP Ibalpha, thereby facilitating the interaction with VWF.
Thrombosis Research Section, Baylor College of Medicine, BCM 286, N1319, 1 Baylor Plaza, Houston, TX 77030, USA.
Yuandong
Y
Peng
Michael C
MC
Berndt
Miguel A
MA
Cruz
José A
JA
López
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D003435:Crotalid Venoms / Q000494:pharmacology; D000939:Epitopes / Q000737:chemistry; D006801:Humans; D007930:Leucine; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019038:Platelet Glycoprotein GPIb-IX Complex; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012310:Ristocetin / Q000494:pharmacology; D018808:Transcription Factor AP-1 / Q000276:immunology*; D014633:Valine; D014841:von Willebrand Factor / Q000737:chemistry / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D003435:Crotalid Venoms; D000939:Epitopes; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D019038:Platelet Glycoprotein GPIb-IX Complex; D011994:Recombinant Proteins; D018808:Transcription Factor AP-1; C094990:adhesion receptor; D014841:von Willebrand Factor; D012310:Ristocetin; C030961:botrocetin; D007930:Leucine; D014633:Valine