A3 Domain Is Essential for Interaction of von Willebrand Factor with Collagen Type III

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189

To explore the molecular basis of von Willebrand factor (VWF) clearance, an experimental model employing VWF-deficient mice was developed. Biodistribution was examined by the injection of radiolabeled VWF, which was primarily directed to the liver with minor amounts in other organs. Disappearance of VWF from plasma was characterized by a rapid initial phase (t1 ⁄2 ␣ ‫؍‬ 13 min) and a slow secondary phase (t1 ⁄2 ␤ ‫؍‬ 3 h), with a mean residence time (MRT) of 2.8 h. A similar clearance was observed for VWF consisting of only high or low molecular weight multimers, indicating that, in our experimental model, clearance is independent of multimeric distribution. This allowed us to compare the survival of fulllength VWF to truncated variants. Deletion of both the amino-terminal D -D3 and carboxyl-terminal D4-CK domains resulted in a fragment with a similar clearance to wild-type VWF. Deletion of only the D -D3 region was associated with an almost 2-fold lower recovery and increased clearance (MRT ‫؍‬ 1.6 h), whereas deletion of only the D4-CK region resulted in a significantly reduced clearance (MRT ‫؍‬ 4.5 h, p < 0.02). These results point to a role of the D -D3 region in preventing clearance of VWF. Furthermore, replacement of D3 domain residue Arg-1205 by His resulted in a markedly increased clearance (MRT ‫؍‬ 0.3 h; p ‫؍‬ 0.004). Therefore, this mutation seems to abrogate the protective effect of the D -D3 region. In vitro analysis of this mutant also revealed a 2-fold reduced affinity for VWF propeptide at low pH, showing that mutation of Arg-1205 results not only in an increased clearance rate but is also associated with an impaired pH-dependent interaction with VWF propeptide.von Willebrand factor (VWF) 1 is a multimeric plasma protein that participates in the hemostatic process. The absence of functional VWF is associated with an abnormal bleeding tendency known as von Willebrand Disease (VWD) (1, 2). VWF contributes to hemostasis in a dual manner. First, it promotes the adhesion of platelets at sites of vascular injury by acting as a molecular bridge between the sub-endothelial collagen matrix and the platelet-surface receptor complex glycoprotein (Gp) Ib␣/IX/V (3). In addition, VWF serves as a carrier protein for coagulation factor VIII (FVIII) in the circulation. This chaperone function results in stabilization of the FVIII heterodimeric structure (4) and protection of FVIII from premature clearance by the low density lipoprotein receptor-related protein (5, 6).VWF is produced and stored in endothelial cells and megakaryocytes. It is synthesized as pre-pro-VWF, a single chain polypeptide with the domain structure

Section: Methodsmentioning

confidence: 99%

An Experimental Model to Study the in Vivo Survival of von Willebrand Factor

Lenting

Westein

Terraube

et al. 2004

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189

“…Both ␣ 2 ␤ 1 and VWF bind to collagen through their I-domains, in VWF known as A-domains (13)(14)(15)(16)(17)(18)(19). A-domains form independent globular modules of some 200 amino acid residues.…”

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confidence: 99%

“…The A1-domain contains the binding site for glycoprotein Ib (20,21), sulfatides (22), heparin (23), and collagen VI (24,25), which constitutes the main reactive collagen in the extracellular matrix of endothelial cells. The A2-domain has no clear binding function but is sensitive to protease ADAMTS13-mediated enzymatic degradation (26,27), whereas the A3-domain (residues 920 -1111) contains the main binding site for fibrillar collagens such as type I and III (19,28). Recombinant A3-domain also binds to collagen (28), whereas deletion of A3 results in a VWF that binds 40 times less to collagen (19).…”

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confidence: 99%

“…The A2-domain has no clear binding function but is sensitive to protease ADAMTS13-mediated enzymatic degradation (26,27), whereas the A3-domain (residues 920 -1111) contains the main binding site for fibrillar collagens such as type I and III (19,28). Recombinant A3-domain also binds to collagen (28), whereas deletion of A3 results in a VWF that binds 40 times less to collagen (19). By using synthetic triple helical collagen-related peptides, the VWF-binding site has been localized to residues 541-558 of the ␣1CB4(III) fragment of collagen type III (29).…”

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confidence: 99%

See 1 more Smart Citation

A Consensus Tetrapeptide Selected by Phage Display Adopts the Conformation of a Dominant Discontinuous Epitope of a Monoclonal Anti-VWF Antibody That Inhibits the von Willebrand Factor-Collagen Interaction

Vanhoorelbeke

Depraetere

Romijn

et al. 2003

Monoclonal antibody (mAb) 82D6A3 is an anti-vonIn conclusion, to our knowledge, this is the first report where a modeled peptide containing a consensus sequence could be fitted onto the three-dimensional structure of the antigen, indicating that it might adopt the conformation of the discontinuous epitope.Platelet adhesion to subendothelial structures, more specifically to the thrombogenic compound collagen, is one of the first steps in a sequence of reactions that can lead to arterial thrombosis. Platelets interact with collagen both in a direct manner via their collagen receptors (e.g. ␣ 2 ␤ 1 (1, 2) and glycoproteins IV (3) and VI (4, 5)) and indirectly with VWF, 1 forming the bridge between collagen and its platelet receptor glycoprotein Ib/IX/V (6). Binding via both ␣ 2 ␤ 1 and VWF is necessary to sustain platelet adhesion under high shear forces (7-9); VWF-mediated interaction results in rolling of the platelets over the collagen surface (10), upon which the collagen receptors can interact with the damaged vessel wall, leading to firm adhesion. This is the result of platelet activation by the signal-transducing glycoprotein VI (11), leading to a gain-in-affinity of ␣ 2 ␤ 1 (12) and activation of ␣ IIb ␤ 3 with platelet aggregation as a consequence.Both ␣ 2 ␤ 1 and VWF bind to collagen through their I-domains, in VWF known as A-domains (13-19). A-domains form independent globular modules of some 200 amino acid residues. In VWF three such domains have been identified. The A1-domain contains the binding site for glycoprotein Ib (20,21), sulfatides (22), heparin (23), and collagen VI (24, 25), which constitutes the main reactive collagen in the extracellular matrix of endothelial cells. The A2-domain has no clear binding function but is sensitive to protease ADAMTS13-mediated enzymatic degradation (26,27), whereas the A3-domain (residues 920 -1111) contains the main binding site for fibrillar collagens such as type I and III (19,28). Recombinant A3-domain also binds to collagen (28), whereas deletion of A3 results in a VWF that binds 40 times less to collagen (19). By using synthetic triple helical collagen-related peptides, the VWF-binding site has been localized to residues 541-558 of the ␣1CB4(III) fragment of collagen type III (29). Recently, we identified the collagen binding site by cocrystallization of the A3-domain with an inhibitory anti-A3 antibody, RU5, which was confirmed by showing that especially an H1023A mutant abolished binding of VWF to collagen (30). This study was further extended by the analysis of a series of 27 VWF-A3 mutants, which defined the collagen binding site of the VWF-A3-domain to the "front" face of the domain (31), an observation confirmed by Nishida et al. (32).We raised a monoclonal antibody (mAb), 82D6A3, against human VWF that prevents the binding of VWF to collagen (24) and that is antithrombotic in a baboon arterial thrombosis model (33). Since a previous effort to determine the epitope of 82D6A3 using phage display, was not successful (34, 35), we repeated this study us...

“…Resting platelets using several receptors adhering to subendothelium of damaged blood vessels are activated and spread to provide finally a new nonthrombogenic surface until vasculature regeneration occurs. Reversible binding between GPIb-V-IX 1 and von Willebrand factor, associated with collagen, is crucial to slow down the platelet (especially under high shear) so that it can bind more firmly via other receptors (3,4). This mechanism strongly parallels that of the selectins in leukocyte adhesion (5).…”

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confidence: 99%

Aggretin, a Heterodimeric C-type Lectin from Calloselasma rhodostoma (Malayan Pit Viper), Stimulates Platelets by Binding to α2β1 Integrin and Glycoprotein Ib, Activating Syk and Phospholipase Cγ2, but Does Not Involve the Glycoprotein VI/Fc Receptor γ Chain Collagen Receptor

Navdaev

Clemetson

Polgár

et al. 2001

Aggretin, a potent platelet activator, was isolated from Calloselasma rhodostoma venom, and 30-amino acid N-terminal sequences of both subunits were determined. Aggretin belongs to the heterodimeric snake Ctype lectin family and is thought to activate platelets by binding to platelet glycoprotein ␣ 2 ␤ 1 . We now show that binding to glycoprotein (GP) Ib is also required. Aggretin-induced platelet activation was inhibited by a monoclonal antibody to GPIb as well as by antibodies to ␣ 2 ␤ 1 . Binding of both of these platelet receptors to aggretin was confirmed by affinity chromatography. No binding of other major platelet membrane glycoproteins, in particular GPVI, to aggretin was detected. Aggretin also activates platelets from Fc receptor ␥ chain (Fc␥)-deficient mice to a greater extent than those from normal control mice, showing that it does not use the GPVI/Fc␥ pathway. Platelets from Fc␥-deficient mice expressed fibrinogen receptors normally in response to collagen, although they did not aggregate, indicating that these platelets may partly compensate via other receptors including ␣ 2 ␤ 1 or GPIb for the lack of the Fc␥ pathway. Signaling by aggretin involves a dose-dependent lag phase followed by rapid tyrosine phosphorylation of a number of proteins. Among these are p72 SYK , p125 FAK , and PLC␥2, whereas, in comparison with collagen and convulxin, the Fc␥ subunit neither is phosphorylated nor coprecipitates with p72 SYK . This supports an independent, GPIb-and integrin-based pathway for activation of p72 SYK not involving the Fc␥ receptor.Platelet-collagen interactions are integral to primary hemostasis (1, 2). Resting platelets using several receptors adhering to subendothelium of damaged blood vessels are activated and spread to provide finally a new nonthrombogenic surface until vasculature regeneration occurs. Reversible binding between GPIb-V-IX 1 and von Willebrand factor, associated with collagen, is crucial to slow down the platelet (especially under high shear) so that it can bind more firmly via other receptors (3, 4). This mechanism strongly parallels that of the selectins in leukocyte adhesion (5). Another important receptor is the ␣ 2 ␤ 1 integrin, which is essential for anchoring the platelet to collagen in the subendothelium (6) and for linking to the platelet cytoskeleton to prevent the receptor being torn from the membrane by the forces that it has to withstand. Activation induces the release of storage granules and the expression of new receptors on the platelet surface (7) as well as changes in other receptors such as the fibrinogen receptor, ␣ IIb ␤ 3 , which is critical for spreading. Although GPIb-V-IX and ␣ 2 ␤ 1 also participate in signaling to the platelet interior (8, 9), recent studies, particularly in patients with platelet receptor deficiencies, have implicated GPVI/Fc␥ as a major collagen receptor for platelet activation (10 -12). Patients with platelets lacking any one of these receptors (GPIb-V-IX, ␣ 2 ␤ 1 , or GPVI/Fc␥) have increased bleeding times, and platelet adhesio...