Modeling and Functional Analysis of the Interaction between von Willebrand Factor A1 Domain and Glycoprotein Ibα

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 Ib␣ sequence 271-279, which has previously been implicated in ligand binding. Docking procedures suggested that A1 domain residues in strand ␤3 and preceding loop (residues 559 -566) as well as in helix ␣3 (residues 594 -603) interact with Asp residues 272, 274, 277 and sulfated Tyr residues 278 and 279 in GP Ib␣. 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 Ib␣. These results provide a partial model of the vWF-GP Ib␣ interaction linked to the manifestation of functional activity in platelet adhesion. Binding of von Willebrand factor (vWF)1 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 also contribute to the acute thrombotic occlusion of diseased arteries (4, 5). The interaction between GP Ib␣ and immobilized vWF can tether platelets to surfaces in rapidly flowing blood but is inherently short-lived, mediating transient periods of arrest that alternate with slow translocation (6). During this rolling motion, however, additional synergistic bonds can form through specific integrin receptors and establish firm platelet adhesion to extravascular matrix constituents (6, 7). The rapid association of GP Ib␣ with vWF not only initiates platelet deposition at sites of vessel injury but is also essential for maintaining thrombus growth in blood flowing with high shear rates (8). A detailed explanation of the molecular mechanisms underlying these processes may, therefore, elucidate key regulatory aspects of normal and pathological platelet function as well as indicate new approaches for antithrombotic therapy.The notion that the GP Ib␣ binding site is located between vWF residues 449 and 728 (9, 10), comprising the entire A1 domain and the preceding carboxyl-terminal portion of domain D3 (11, 12), was established over a decade ago. Since then, studies attempting to identify A1 domain residues involved in interacting with the receptor have yielded inconclusive and sometimes conflicting results (13-16). One reason for this outcome is that the assays employed to e...

Shielding the front-strand β3 of the von Willebrand factor A1 domain inhibits its binding to platelet glycoprotein Ibα

“…These mutations include those generating type 2M von Willebrand disease (VWF database) and those shown in different studies to abolish platelet interactions with the VWF A1 domain in flow. 16,19,20 The present localization of these mutations on the VWF A1 domain supports the model proposed by Vasudevan et al 16 Among the 21 mutated residues identified, 14 are located at the front of the A1 domain. Eleven of these frontal mutations were located either in strand ␤3 and flanking loops (His559Phe, Asp560Arg, Gly561Ser/Ala, His563Thr, Tyr565Ala, and Lys572Ala) or in helix ␣3 and flanking loops (Glu596Ala, Lys599Thr, Gln604Arg, Phe606Ile, and Ser607Arg).…”

“…Based on the GPIb␣-peptide docking model, 16 residues Gly561, Tyr565, Glu596, and Lys599 were claimed to play a direct role in GPIb binding. These residues are represented in Figure 8A.…”

“…15 Peptide-docking studies have anticipated a central front groove on the A1 domain, next to strand ␤3, to be part of the binding site for GPIb␣. 16 On the other hand, spontaneously occurring missense mutations in VWF have been found, associated with decreased affinity of VWF for GPIb␣. These so-called type 2M loss-offunction mutations, with preserved degree of VWF-multimerization, have identified mutations located on the front of the VWF A1 domain (International Society on Thrombosis and Haemostasis, Scientific and Standardization Committee (ISTH SSC VWF) database: www.shef.ac.uk/vwf/).…”

“…These so-called type 2M loss-offunction mutations, with preserved degree of VWF-multimerization, have identified mutations located on the front of the VWF A1 domain (International Society on Thrombosis and Haemostasis, Scientific and Standardization Committee (ISTH SSC VWF) database: www.shef.ac.uk/vwf/). Likewise, mutagenesis studies using recombinant full-length VWF 17,18 or recombinant VWF A1 domain mutants 16,19,20 have further identified a number of front residues involved in VWF binding to GPIb␣ (see "Discussion").…”

Platelet adhesion involves a novel interaction between vimentin and von Willebrand factor under high shear stress

“…Our understanding of the molecular basis of platelet adhesion via the interaction of the GPIb/XI/V complex with VWF has advanced primarily through the use of purified fragments of the GPIba and the A1 domain of VWF. 16,17,34 For this reason, the observation that the translocation velocity of platelets over the A1A2A3 domains protein was lower than over the single A1 domain was intriguing and unexpected. .…”