Paratope Determination of the Antithrombotic Antibody 82D6A3 Based on the Crystal Structure of Its Complex with the von Willebrand Factor A3-Domain

Abstract

Platelet adhesion, activation, and aggregation are the three major steps in primary hemostasis, a process that is necessary for the cessation of bleeding at sites of vascular injury. The same process is also responsible for pathologic thrombus formation, particularly in arteries at sites of high shear stress. At high shear stress, binding of von Willebrand factor (VWF) 3 to collagen that is exposed in the subendothelial matrix after injury, and the subsequent interaction of the immobilized VWF with the platelet glycoprotein (GP) Ib/IX/V-receptor complex, is a prerequisite for proper adhesion (1-3). As a result of the reversible VWF-GP Ib/IX/V interaction, platelets roll over the injured vessel wall and slow down (3). Subsequent firm adhesion and activation of platelets are mediated by their collagen receptors, integrins ␣2␤1 and GPVI, respectively (4 -8). Activated platelets expose high affinity GPIIb/IIIa receptors that mediate aggregation by binding to fibrinogen and, to a lesser extent, to VWF (9 -11).Collagen type VI and fibrillar collagens types I and III are abundant in the subendothelium (12). VWF interacts with the fibrillar collagens mainly via its A3-domain (13, 14), whereas its A1-domain has been implicated in binding to collagen type VI (15, 16). The latter interaction only mediates platelet adhesion under low shear conditions (17, 18). The A3-domain is homologous in sequence and structure with the I-domains of collagen binding integrin ␣-chains, such as the I-domain of ␣2␤1 (19 -21). These domains adopt a dinucleotide binding fold, or Rossman fold, that consists of a central hydrophobic parallel ␤-sheet flanked on both sites with amphipathic ␣-helices (22-24). Despite their structural similarity, they bind collagen in distinctly different ways. The I-domain of ␣2␤1 binds collagen in a hydrophilic groove at its top face that contains a metal ion-dependent adhesion site (25,26). In contrast, the collagen-binding site of the A3-domain is located at its front face and is rather flat and hydrophobic (23,(27)(28)(29).Our group characterized a monoclonal antibody against human VWF, 82D6A3, that binds with high affinity to the VWF A3-domain and inhibits VWF binding to collagen types I and III (15). Its inhibiting effect on VWF-mediated platelet adhesion becomes more pronounced with increasing shear stress, as is expected because the collagen-VWF-GPIb axis is essential for platelet adhesion under high shear only (30). The discontinuous epitope of 82D6A3, which has recently been unraveled via phage display and binding studies with alanine mutants of the VWF A3-domain, includes several residues of the A3-domain that are important for collagen binding (30). We further demonstrated that 82D6A3 has antithrombotic effects in a baboon arterial thrombosis model (31), proving for the first time that the VWF-collagen interaction is indeed involved in thrombus formation in vivo (32). Interestingly, the antithrombotic effect was not associated with significantly increased bleeding times, even at high doses, demonstrat...

Discovery of novel membrane binding structures and functions

“…The bilayer interaction site of the VWF A3 domain was tested by mapping NMR signal perturbations obtained upon titration with DHPC micelles (Supplementary Figure 1) onto the crystal structure (Staelens et al 2006). This demonstrates a complicating role of conformational flexibility, as it suggests that the C-terminal α -helix of this domain is displaced upon detergent interaction.…”

Implications for collagen I chain registry from the structure of the collagen von Willebrand factor A3 domain complex

“…near the collagen-binding site (2,3,6,12). The β3-α2 loop includes residue W1745 that has been suggested to play a direct role in collagen binding (13,14).…”

Collagen Receptors as Potential Targets for Novel Anti-Platelet Agents

“…Thus, it is generally accepted that VWF binds to collagen via its A3 domain. Detailed studies using recombinant A3 domain [104] or monoclonal antibodies against the A3 domain [105] have shown that both these strategies are capable of inhibiting platelet adhesion to collagen under high shear conditions and, under some circumstances platelet aggregation to collagen. Additional data from a variant von Willebrand's disease Type IIb patient point to a possible (indirect?)…”