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Blood, 15 December 2004, Vol. 104, No. 13, pp. 3971-3978.
Prepublished online as a Blood First Edition Paper on August 19, 2004; DOI 10.1182/blood-2004-07-2544.
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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
The  1 helix- 13 strand spanning Leu214 to Val229 of platelet glycoprotein Ib facilitates the interaction with von Willebrand factor: evidence from characterization of the epitope of monoclonal antibody AP1
Yuandong Peng,
Michael C. Berndt,
Miguel A. Cruz, and
José A. López
From the Thrombosis Research Section, Baylor College of Medicine, Houston, TX; and the Vascular Biology Laboratory, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.
The glycoprotein Ib-IX-V (GP Ib-IX-V) complex mediates platelet binding to von Willebrand factor (VWF) through its largest polypeptide, GP Ib . Of the many GP Ib 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 1 helix and  13 strand defined by the GP Ib 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 1 helix has a regulatory role and the turn and 13 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 Ib-expressing Chinese hamster ovary (CHO) cells on VWF. This region thus appears to be more important for maintaining the regional conformation of GP Ib, thereby facilitating the interaction with VWF. (Blood. 2004;104:3971-3978)
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