The antithrombotic monoclonal antibody 82D6A3 is directed against amino acids Arg-963, Pro-981, Asp-1009, Arg-1016, Ser-1020, Met-1022, and His-1023 of the von Willebrand factor A3-domain (Vanhoorelbeke, K., Depraetere, H., Romijn, R. A., Huizinga, E., De Maeyer, M., and Deckmyn, H. (2003) J. Biol. Chem. 278, 37815-37821). By this, it potently inhibits the interaction of von Willebrand factor to collagens, which is a prerequisite for blood platelet adhesion to the injured vessel wall at sites of high shear. To fully understand the mode of action of 82D6A3 at the molecular level, we resolved its crystal structure in complex with the A3-domain and fine mapped its paratope by construction and characterization of 13 mutants. The paratope predominantly consists of two short sequences in the heavy chain CDR1 (Asn-31 and Tyr-32) and CDR3 (Asp-99, Pro-101, Tyr-102 and Tyr-103), forming one patch on the surface of the antibody. Trp-50 of the heavy and His-49 of the light chain, both situated adjacent to the patch, play ancillary roles in antigen binding. The crystal structure furthermore confirms the epitope location, which largely overlaps with the collagen binding site deduced from mutagenesis of the A3-domain ( Romijn, R. A., Westein, E., Bouma, B., Schiphorst, M. E., Sixma, J. J., Lenting, P. J., and Huizinga, E. G. (2003) J. Biol. Chem. 278, 15035-15039). We herewith further consolidate the location of the collagen binding site and reveal that the potent action of the antibody is due to direct competition for the same interaction site. This information allows the design of a paratope-mimicking peptide with antithrombotic properties.

Summary.  Background: Bitiscetin, a heterodimeric snake venom protein purified from Bitis arietans, binds to the A1 domain of von Willebrand factor (VWF) and induces binding of this domain to platelet glycoprotein (GP) Ib. We previously purified a distinct form of dimeric bitiscetin (herein called bitiscetin‐2) that also induces the VWF A1 domain‐GPIb interaction, but does not bind to the A1 domain. Instead, it interacts with the collagen‐binding A3 domain of VWF. Methods: In the current study we identify the amino terminal sequence of the bitiscetin‐2 as DEGCLPDDSSRT, showing conclusively that the protein is distinct form the originally described bitiscetin. We further studied the interaction of bitiscetin‐2 and VWF using DeltaA3 VWF and a series of 33 VWF point mutants previously prepared to map the collagen‐binding site. Results: Our results confirm that DeltaA3 VWF, even though containing the A1‐domain, is unable to interact with bitiscetin‐2. Mutation of VWF‐A3 residues Ile975, Asp979, Pro981, Ser1020 and His1023 reduces binding by 80% while mutation of residues Val980, Glu1001 and Arg1021 reduces binding by 30–60%. A 2‐ to 6‐fold increase of binding is caused by mutation of residues Val985, Glu987, and Arg1016. Conclusion: Nearly all of these mutations also affect collagen binding showing that the binding sites for bitiscetin‐2 and collagen type III in the VWF‐A3 domain closely overlap.

The interaction of the plasma protein von Willebrand factor (VWF) with subendothelial collagen initiates adhesion of blood platelets to the damaged vessel wall or ruptured atherosclerotic plaque. A detailed molecular description of the VWF-collagen interaction may facilitate development of a novel class of antithrombotic drugs that inhibits this vital step in platelet thrombus formation. We have previously used site-directed mutagenesis to map the collagen-binding site in the VWF A3 domain. Here, we report the identification of a 9-aminoacid sequence in collagen type III which mediates VWF binding. We have synthesized a set of 57 peptides, each containing 27 amino acids of native collagen sequence flanked at each end by five GPP (standard amino acid nomenclature) triplets which support the triple helical structure that is essential for ligand recognition by collagen. The sequence of each peptide overlaps by 9 amino acids with that of each adjacent peptide. A single peptide from this set (#23) was shown to bind VWF in a solid-phase binding assay. The affinity of peptide #23 for VWF was comparable to that of native collagen type III. The peptide #23-VWF interaction was abolished by a monoclonal antibody directed against the collagen-binding site on the VWF A3 domain. Furthermore, recombinant VWF variants that were previously shown to lack collagen-binding capacity (delta A3, His1023Ala) were not able to bind to the peptide. Using surface plasmon resonance, we showed that the peptide bound strongly to the isolated VWF A3 domain (Kd 300 nM). Immobilized peptide #23 also supported platelet adhesion from whole blood under flow conditions and washed platelet adhesion under static conditions. Platelet adhesion to peptide #23 could be abrogated by a monoclonal antibody directed against the VWF A3 domain, which inhibits the interaction of VWF with full-length collagen. We subsequently synthesized a set of truncated and alanine-modified triple helical peptides based on the sequence of #23, which were all tested for VWF and platelet binding from whole blood under flow conditions. Modified peptides either strongly interacted with both VWF and platelets, or lacked both VWF and platelet binding. Based on these experiments, we identified the sequence RGQOGVMGF (O is hydroxyproline) as the minimal VWF binding sequence in collagen type III. Mutation of either Q or M to alanine (A) did not affect VWF binding, whereas replacement of R, O, V, and F by A completely abolished VWF binding. Glycine residues were not replaced, as they are essential for triple helix formation. A model of the VWF A3 domain with this nonapeptide collagen sequence was constructed to give detailed insight into the VWF-collagen interaction. In conclusion, we have identified a 9 amino acid sequence in collagen type III that is entirely responsible for high affinity binding to VWF. The detailed molecular description of the VWF-collagen interaction described here may facilitate development of agents disrupting this interaction, which may have potential as antithrombotic drugs.

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 ( t(1/2) alpha = 13 min) and a slow secondary phase (t(1/2) alpha = 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 full-length 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.

The interaction of thrombin with platelet glycoprotein (GP)-Ibα has been well demonstrated. However, recent data have provided new insights into the GPIb–thrombin interaction. GPIb-clustering, which seems to be required for signal transduction, might be achieved by removal of GPV from the complex. In addition, the GPIbα subunits might need to be relatively mobile, as would occur in rafts or with GPIbα that has dissociated from the cytoskeleton. Finally, by resolving the crystal structures, two groups have identified different interaction sites in both thrombin and GPIbα that could be involved in cross-linking. Our direct comparison of the two structures reveals that, whereas one thrombin molecule binds to exactly the same site in GPIbα in both crystals, the other does not. Nevertheless, present biochemical and structural data complement each other well and help to clarify how GPIb might facilitate platelet activation by thrombin.

Monoclonal antibody (mAb) 82D6A3 is an anti-von Willebrand factor (VWF) mAb directed against the A3-domain of VWF that inhibits the VWF binding to fibrillar collagens type I and III in vitro and in vivo. To identify the discontinuous epitope of this mAb, we used phage display, mutant analysis, and peptide modeling. All 82D6A3-binding phages displayed peptides containing the consensus sequence SPWR that could be aligned with P981W982 in the VWF A3-domain. Next, the binding of mAb 82D6A3 to 27 Ala mutants with mutations in the A3-domain of VWF revealed that amino acids Arg963, Pro981, Asp1009, Arg1016, Ser1020, Met1022, and His1023 are part of the epitope of mAb 82D6A3. Inspection of residues Ser1020, Arg1016, Pro981, and Trp982 in the three-dimensional structure of the A3-domain demonstrated that these residues are close together in space, pointing out that the structure of the SPWR consensus sequence might mimic this discontinuous epitope. Modeling of a cyclic 6-mer peptide containing the consensus sequence and superposition of its three-dimensional structure onto the VWF A3-domain demonstrated that the Ser and Arg in the peptide matched the Ser1020 and Arg1016 in the A3-domain. The Pro residue of the peptide served as a spacer, and the side chain of the Trp pointed in the direction of Trp982. In 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.

The multimeric glycoprotein von Willebrand factor (VWF) mediates platelet adhesion to collagen at sites of vascular damage. The binding site for collagen types I and III is located in the VWF-A3 domain. Recently, we showed that His(1023), located near the edge between the "front" and "bottom" faces of A3, is critical for collagen binding (Romijn, R. A., Bouma, B., Wuyster, W., Gros, P., Kroon, J., Sixma, J. J., and Huizinga, E. G. (2001) J. Biol. Chem. 276, 9985-9991). To map the binding site in detail, we introduced 22 point mutations in the front and bottom faces of A3. The mutants were expressed as multimeric VWF, and binding to collagen type III was evaluated in a solid-state binding assay and by surface plasmon resonance. Mutation of residues Asp(979), Ser(1020), and His(1023) nearly abolished collagen binding, whereas mutation of residues Ile(975), Thr(977), Val(997), and Glu(1001) reduced binding affinity about 10-fold. Together, these residues define a flat and rather hydrophobic collagen-binding site located at the front face of the A3 domain. The collagen-binding site of VWF-A3 is distinctly different from that of the homologous integrin alpha(2) I domain, which has a hydrophilic binding site located at the top face of the domain. Based on the surface characteristics of the collagen-binding site of A3, we propose that it interacts with collagen sequences containing positively charged and hydrophobic residues. Docking of a collagen triple helix on the binding site suggests a range of possible engagements and predicts that at most eight consecutive residues in a collagen triple helix interact with A3.

Collagen in the subendothelium is suggested as a Major binding partner of von Willebrand factor (vWF), thereby providing its function in primary hemostasis. The lack of the functionally Most active vWF high Molecular weight Multimers in von Willebrand disease (vWd) type 2A correlates with significantly decreased vWF binding to collagen type I and III, respectively, and offers a possible explanation for the bleeding symptoms in such patients. Consequently, one would expect a similar clinical phenotype in persons with an isolated defect of vWF collagen binding (vWF:CB) but with normal vWF Multimers. A Mutation in the vWF A3 domain that is regarded as the Major vWF:CB site [1], was recently identified in a family with Mother and daughter suffering from a significant bleeding tendency [2]. However, vWF:CB seemed normal in the vWF:CB assay. Only after identification of a Mutation in the vWF A3 domain and recombinant expression of the Mutant protein, a reduced VWF:CB was demonstrated by the collagen binding curve [2]. Here we report on three different novel Mutations in the A3 domain which do not correlate with bleeding symptoms, although they significantly decrease vWF collagen binding.

Transient interactions of platelet-receptor glycoprotein Ibα (GpIbα) and the plasma protein von Willebrand factor (VWF) reduce platelet velocity at sites of vascular damage and play a role in haemostasis and thrombosis. Here we present structures of the GpIbα amino-terminal domain and its complex with the VWF domain A1. In the complex, GpIbα wraps around one side of A1, providing two contact areas bridged by an area of solvated charge interaction. The structures explain the effects of gain-of-function mutations related to bleeding disorders and provide a model for shear-induced activation. These detailed insights into the initial interactions in platelet adhesion are relevant to the development of antithrombotic drugs.

Rhamnogalacturonan (RG), known as the hairy portion of the pectin network of the primary plant cell wall, is composed of repeating dimeric units of -(1-2)αa-L-rhamnopyranosyl-(1,4)-αa-D-galactopyranosyluronidewith varying degrees of branching of Rha residues and acetyl or methyl esterification of GalUA residues.Rhamnogalacturonan lyase (RG-lyase) cleaves the α 1-4 glycosidic bond of the RG polysaccharide backbone leaving an α-∆-(4,5)unsaturated D-GalUA at the non-reducing end and L-Rha at the reducing end.A unique feature of this enzyme is its requirement for very large substrates.We have determined the structure of RG-lyase from Aspergillus aculeatus by MIR and refined it to 1.5 Å resolution.This is the first known structure of a CAZy Family 4 polysaccharide lyase.RG-lyase exists as a monomer with 508 residues (M.W. = 54.2 kDa).A three-domain structure made up of mostly βsheet with an overall shape of a flattened rugby ball is observed.No closely homologous structures have been found using the DALI server suggesting that RG-lyase has an overall unique structural fold.The three intimately interacting domains consist of a 251 residue N-terminal domain, an 86 residue mid domain, and the remaining 171 residues make up the C-terminal domain.The proposed active site is situated in the deepest pocket on the surface of the enzyme within a groove made up from all three domains that is large enough to accommodate the polysaccharide substrate.It is proposed that the catalytic machinery of RG-lyase is similar to that of other polysaccharide lyases.

Von Willebrand factor (vWF) is a multimeric glycoprotein that mediates platelet adhesion and thrombus formation at sites of vascular injury. vWF functions as a molecular bridge between collagen and platelet receptor glycoprotein Ib. The major collagen-binding site of vWF is contained within the A3 domain, but its precise location is unknown. To localize the collagen-binding site, we determined the crystal structure of A3 in complex with an Fab fragment of antibody RU5 that inhibits collagen binding. The structure shows that RU5 recognizes a nonlinear epitope consisting of residues 962-966, 981-997, and 1022-1026. Alanine mutants were constructed of residues Arg(963), Glu(987), His(990), Arg(1016), and His(1023), located in or close to the epitope. Mutants were expressed as fully processed multimeric vWF. Mutation of His(1023) abolished collagen binding, whereas mutation of Arg(963) and Arg(1016) reduced collagen binding by 25-35%. These residues are part of loops alpha3beta4 and alpha1beta2 and alpha-helix 3, respectively, and lie near the bottom face of the domain. His(1023) and flanking residues display multiple conformations in available A3-crystal structures, suggesting that binding of A3 to collagen involves an induced-fit mechanism. The collagen-binding site of A3 is located distant from the top face of the domain where collagen-binding sites are found in homologous integrin I domains.