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

Abstract

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...

Shielding of the A1 Domain by the D′D3 Domains of von Willebrand Factor Modulates Its Interaction with Platelet Glycoprotein Ib-IX-V

“…moAb 82D6A3 binds to the VWF A3 domain and inhibits VWF binding to collagen types I, III, and IV (17), and moAb 1C1E7 recognizes the N-terminal part of VWF (aa 764 -1035) (15). moAbs 724, 701, and 418 were kind gifts of Dr. J. P. Girma (INSERM U143, le Kremlin-Bicê-tre, Paris, France).…”

Cochlin in the eye: Functional implications

“…These deposits were observed around the Schlemm's canal and in pseudoendothelial cells (Bhattacharya et al, 2005c). The two vWFA domains of cochlin have been implicated in adherence and aggregation for platelets, macrophages and leukocytes (Shankaran et al, 2003) through interaction with a number of proteins such as collagen, GpIbα, and integrin αIibβ3 (Eble and Tuckwell, 2003;Vanhoorelbeke et al, 2003).…”

“…The vWFA domains have also been implicated in host defense mechanisms and in hemostasis as complement factor C, factor B, integrins LFA-1, Mac-1, VLA-1 and 2, p150 and p95 are part of the immune system that harbor vWF domains (Colombatti and Bonaldo, 1991;Colombatti et al, 1993). A number of proteins such as collagen, GpIbα, and integrin αIibβ3 are known to interact with vWF domains (Ruggeri et al, 1983;Eble and Tuckwell, 2003;Vanhoorelbeke et al, 2003). Many of these interactions play a role in different regimes of fluid dynamics (Shankaran et al, 2003) and have been implicated in adherence to platelets and possibly also to macrophages.…”

Cochlin and glaucoma: A mini-review

“…vWFA domains have been implicated in defense mechanisms, including hemostasis, and many components of the immune system (complement factor C, factor B, integrins LFA-1, Mac-1, VLA-1 and 2, p150, and p95) harbor vWFA domains (Colombatti et al, 1993). Furthermore, vWFA domains are known to interact with proteins such as collagen, GpIbα, and integrin α Iib β 3 (Ruggeri et al, 1983;Eble & Tuckwell, 2003;Vanhoorelbeke et al, 2003). It is interesting to note that many of these interactions are involved in different regimes of fluid dynamics (Shankaran et al, 2003) and have been implicated in adherence to platelets and possibly also to macrophages.…”