Abstract
Binding of the von Willebrand factor (vWF) A1 domain to the glycoprotein (GP) Ib-IX-V complex mediates platelet adhesion to reactive substrates under high shear stress conditions, a key event in hemostasis and thrombosis. We have now used the known three-dimensional structure of the A1 domain to model the interaction with the GP Ibalpha sequence 271-279, which has previously been implicated in ligand binding. Docking procedures suggested that A1 domain residues in strand beta3 and preceding loop (residues 559-566) as well as in helix alpha3 (residues 594-603) interact with Asp residues 272, 274, 277 and sulfated Tyr residues 278 and 279 in GP Ibalpha. To verify this model, 14 mutant A1 domain fragments containing single or multiple side chain substitutions were tested for their ability to mediate platelet adhesion under flow. Each of the vWF residues Tyr(565), Glu(596), and Lys(599) proved to be strictly required for A1 domain function, which, in agreement with previous findings, was also dependent on Gly(561). Moreover, an accessory functional role was apparent for a group of positively charged residues, including Arg at positions 629, 632, 636 and Lys at positions 643 and 645, possibly acting in concert. There was, however, no evidence from the model that these residues directly participate in forming the complex with GP Ibalpha. These results provide a partial model of the vWF-GP Ibalpha interaction linked to the manifestation of functional activity in platelet adhesion.
Highlights
Binding of von Willebrand factor1 to the platelet membrane glycoprotein (GP) Ib␣, a component of the GP Ib-IX-V complex [1], supports the arrest of bleeding after tissue trauma [2, 3] and may contribute to the acute thrombotic occlusion of diseased arteries [4, 5]
This method may be used to evaluate the consequences of specific von Willebrand factor (vWF) mutations without the confounding presence of exogenous modulators and, complemented by modeling procedures based on the available A1 domain crystal structure [24], may provide initial information on individual residues involved in receptor binding
Residues Asp560, His563, Tyr565, and Glu596 and Lys599 in the A1 domain appeared to play a prominent role in contacting the receptor (Fig. 2)
Summary
Modeling of the vWF-GP Ib␣ Interaction—The coordinates of the complex between vWF A1 domain and the monoclonal antibody NMC-4 are deposited in the Protein Data Bank with accession number 1OAK. The remaining portion of the GP Ib␣ peptide was built one residue at a time, and the conformation was optimized by energy minimization using X-PLOR [28] In these calculations, each new peptide unit was treated as completely flexible, whereas only 2– 4 torsion angles at a time were allowed to vary in the YDYY sequence. The vWF A1 domain molecule was kept fixed throughout the simulation while the GP Ib␣ peptide was subjected to random movement in the space around the protein accompanied by changes in its conformation. The platelet count in the cell suspension was lowered to a value between 9,000 and 12,000/l to decrease interacting platelets on the surface and facilitate quantitative image analysis This was achieved by centrifuging the cell suspension at 150 ϫ g for 15 min, removing the platelet-rich supernatant fluid, and replacing it with an equivalent volume of HEPES-Tyrode buffer. Regardless of the duration of their transient interaction with immobilized vWF A1 domain, were counted on an area of 25,600 m2
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