Identification of the Regulatory Elements of the Human von Willebrand Factor for Binding to Platelet GPIb

Nakayama, Takayuki; Matsushita, Tadashi; Dong, Zhengyu; Sadler, J. Evan; Jorieux, Sylvie; Mazurier, Claudine; Meyer, Dominique; Kojima, Tetsuhito; Saito, Hidehiko

doi:10.1074/jbc.m201327200

Cited by 29 publications

(6 citation statements)

References 32 publications

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“…One possible explanation is that the amino acid sequence 1238 -1247 from the C terminal end of the D3 domain, which is included in our A1A2A3 construct, forms part of the molecular mechanism that masks the binding site for GPIb␣. In fact, others have proposed that segment as a regulatory element of VWF-GPIb␣ binding (37,38).…”

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confidence: 99%

Destabilization of the A1 Domain in von Willebrand Factor Dissociates the A1A2A3 Tri-domain and Provokes Spontaneous Binding to Glycoprotein Ibα and Platelet Activation under Shear Stress

Auton

Sowa

Smith

et al. 2010

Journal of Biological Chemistry

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This study used recombinant A1A2A3 tri-domain proteins to demonstrate that A domain association in von Willebrand factor (VWF) regulates the binding to platelet glycoprotein Ib␣ (GPIb␣). We performed comparative studies between wild type (WT) A1 domain and the R1450E variant that dissociates the tri-domain complex by destabilizing the A1 domain. Using urea denaturation and differential scanning calorimetry, we demonstrated the destabilization of the A1 domain structure concomitantly results in a reduced interaction among the three A domains. This dissociation results in spontaneous binding of R1450E to GPIb␣ without ristocetin with an apparent K D of 85 ؎ 34 nM, comparable with that of WT (36 ؎ 12 nM) with ristocetin. The mutant blocked 100% ristocetin-induced platelet agglutination, whereas WT failed to inhibit. The mutant enhanced shear-induced platelet aggregation at 500 and 5000 s ؊1 shear rates, reaching 42 and 66%, respectively. Shear-induced platelet aggregation did not exceed 18% in the presence of WT. A1A2A3 variants were added before perfusion over a fibrin(ogen)-coated surface. At 1500 s ؊1 , platelets from blood containing WT adhered <10% of the surface area, whereas the mutant induced platelets to rapidly bind, covering 100% of the fibrin(ogen) surface area. Comparable results were obtained with multimeric VWF when ristocetin (0.5 mg/ml) was added to blood before perfusion. EDTA or antibodies against GPIb␣ and ␣IIb␤3 blocked the effect of the mutant and ristocetin on platelet activation/adhesion. Therefore, the termination of A domain association within VWF in solution results in binding to GPIba and platelet activation under high shear stress.Platelet adhesion at sites of vascular injury contributes to the arrest of bleeding as well as to the pathologic occlusion of diseased vessels under elevated shear stress. Under this high shear stress, the platelet-von Willebrand factor (VWF) 3 interaction is essential for platelet adhesion. The interaction between VWF and the exposed subendothelium permits the VWF to interact with circulating platelets via the receptor glycoprotein (GP)Ib/ IX/V complex (1). Mature VWF consists of a 2050-residue subunit that contains domains that are arranged in the order DЈ-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK (cystine knot) (2-4). The VWF contains a triplicate repeat sequence or A domains in the central portion of the subunit. Binding sites for platelet GPIb␣, heparin, sulfatides, and collagen (5-9) are within the A1 domain, whereas its homologous A3 domain only binds to collagen, and the A2 domain contains the cleavage site for the metalloprotease ADAMTS-13 (10 -12). The functions of these A domains relevant to the biology of VWF have been characterized by individual recombinant expression of each of the A domains (9, 10, 13).The interaction between VWF and GPIb␣ occurs when the binding site for GPIb␣ in the A1 domain of VWF has been exposed by the influence of high fluid forces or when the multimeric protein has been immobilized (14). This activation can also be induced by nat...

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confidence: 99%

Destabilization of the A1 Domain in von Willebrand Factor Dissociates the A1A2A3 Tri-domain and Provokes Spontaneous Binding to Glycoprotein Ibα and Platelet Activation under Shear Stress

Auton

Sowa

Smith

et al. 2010

Journal of Biological Chemistry

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“…The recent findings of Borthaker et al showed that sulfatide inhibits the binding of beads covalently coupled to the fragment of GPIbα to the VWF-A1 domain [13]. The binding of VWF to GPIb requires conformational change of VWF, induced by shear stress under irregular blood flow [24, 31]. Therefore, it is likely that VWF–sulfatide binding competes with VWF–GPIb binding, and that sulfatides possess antithrombotic properties under some pathological conditions, such as sites of arteriosclerosis or vascular injury where the VWF–GPIb interaction initiates thrombus formation [32].…”

Section: Discussionmentioning

confidence: 99%

“…The preparation of recombinant VWF (rVWF) was described previously [24]. Briefly, human 293T cells, growing in Dulbecco’s modified Eagle’s medium (Gibco-BRL, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco-BRL), were transfected by the lipofection method using TransFast transfection kit (Promega, Madison, MI, USA) according to the manufacture’s instructions.…”

Section: Methodsmentioning

confidence: 99%

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Identification of amino acid residues responsible for von Willebrand factor binding to sulfatide by charged-to-alanine-scanning mutagenesis

et al. 2008

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von Willebrand factor (VWF) performs its hemostatic functions through binding to various proteins. The A1 domain of VWF contains binding sites of not only physiologically important ligands, but also exogenous modulators that induce VWF-platelet aggregation. Sulfatides, 3-sulfated galactosyl ceramides, that are expressed on oligodendrocytes, renal tubular cells, certain tumor cells and platelets, have been suggested to interact with VWF under some pathological conditions. The binding of VWF to sulfatide requires the A1 domain, but its binding sites have not been precisely identified. Here, we report that alanine mutations at Arg1392, Arg1395, Arg1399 and Lys1423 led to decreased VWF–sulfatide binding. These sites have been reported to be the binding sites for platelet membrane glycoprotein (GP) Ib and/or snake venom botrocetin, and, interestingly, are identical to the monoclonal antibody (mAb) NMC4 epitope previously reported to inhibit the VWF-GPIb interaction. We observed that NMC4 also inhibited VWF interaction with sulfatides in a dose-dependent manner. Thus, we conclude that VWF binding sites of sulfatide overlap those of platelet GPIb and botrocetin.

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“…The A1 domain of VWF contains a single intramolecular disulfide bond between C1272 and C1458 that may optimize its structure for platelet binding [8], [9]. The residues N-terminal to C1272 have been proposed to allosterically hinder binding between the A1 domain and GPIb [10], [11], [12]. The contribution of other VWF regions to GPIb binding has been less characterized.…”

Section: Introductionmentioning

confidence: 99%

Functional Display of Platelet-Binding VWF Fragments on Filamentous Bacteriophage

2013

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von Willebrand factor (VWF) tethers platelets to sites of vascular injury via interaction with the platelet surface receptor, GPIb. To further define the VWF sequences required for VWF-platelet interaction, a phage library displaying random VWF protein fragments was screened against formalin-fixed platelets. After 3 rounds of affinity selection, DNA sequencing of platelet-bound clones identified VWF peptides mapping exclusively to the A1 domain. Aligning these sequences defined a minimal, overlapping segment spanning P1254–A1461, which encompasses the C1272–C1458 cystine loop. Analysis of phage carrying a mutated A1 segment (C1272/1458A) confirmed the requirement of the cystine loop for optimal binding. Four rounds of affinity maturation of a randomly mutagenized A1 phage library identified 10 and 14 unique mutants associated with enhanced platelet binding in the presence and absence of botrocetin, respectively, with 2 mutants (S1370G and I1372V) common to both conditions. These results demonstrate the utility of filamentous phage for studying VWF protein structure-function and identify a minimal, contiguous peptide that bind to formalin-fixed platelets, confirming the importance of the VWF A1 domain with no evidence for another independently platelet-binding segment within VWF. These findings also point to key structural elements within the A1 domain that regulate VWF-platelet adhesion.

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Identification of the Regulatory Elements of the Human von Willebrand Factor for Binding to Platelet GPIb

Cited by 29 publications

References 32 publications

Destabilization of the A1 Domain in von Willebrand Factor Dissociates the A1A2A3 Tri-domain and Provokes Spontaneous Binding to Glycoprotein Ibα and Platelet Activation under Shear Stress

Destabilization of the A1 Domain in von Willebrand Factor Dissociates the A1A2A3 Tri-domain and Provokes Spontaneous Binding to Glycoprotein Ibα and Platelet Activation under Shear Stress

Identification of amino acid residues responsible for von Willebrand factor binding to sulfatide by charged-to-alanine-scanning mutagenesis

Functional Display of Platelet-Binding VWF Fragments on Filamentous Bacteriophage

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