Title: Identification of a Small Molecule That Modulates Platelet Glycoprotein Ib-von Willebrand Factor Interaction
Abstract: The von Willebrand factor (VWF) A1-glycoprotein (GP) Ibα interaction is of major importance during thrombosis mainly at sites of high shear stress. Inhibitors of this interaction prevent platelet-dependent thrombus formation in vivo, without major bleeding complications. However, the size and/or protein nature of the inhibitors currently in development limit oral bioavailability and clinical development. We therefore aimed to search for a small molecule protein-protein interaction inhibitor interfering with the VWF-GPIbα binding. After determination of putative small molecule binding pockets on the surface of VWF-A1 and GPIbα using site-finding algorithms and molecular dynamics, high throughput molecular docking was performed on both binding partners. A selection of compounds showing good in silico docking scores into the predicted pockets was retained for testing their in vitro effect on VWF-GPIbα complex formation, by which we identified a compound that surprisingly stimulated the VWF-GPIbα binding in a ristocetin cofactor ELISA and increased platelet adhesion in whole blood to collagen under arterial shear rate but in contrast inhibited ristocetin-induced platelet aggregation. The selected compound adhering to the predicted binding partner GPIbα could be confirmed by saturation transfer difference NMR spectroscopy. We thus clearly identified a small molecule that modulates VWF-GPIbα binding and that will now serve as a starting point for further studies and chemical modifications to fully characterize the interaction and to manipulate specific activity of the compound. The von Willebrand factor (VWF) A1-glycoprotein (GP) Ibα interaction is of major importance during thrombosis mainly at sites of high shear stress. Inhibitors of this interaction prevent platelet-dependent thrombus formation in vivo, without major bleeding complications. However, the size and/or protein nature of the inhibitors currently in development limit oral bioavailability and clinical development. We therefore aimed to search for a small molecule protein-protein interaction inhibitor interfering with the VWF-GPIbα binding. After determination of putative small molecule binding pockets on the surface of VWF-A1 and GPIbα using site-finding algorithms and molecular dynamics, high throughput molecular docking was performed on both binding partners. A selection of compounds showing good in silico docking scores into the predicted pockets was retained for testing their in vitro effect on VWF-GPIbα complex formation, by which we identified a compound that surprisingly stimulated the VWF-GPIbα binding in a ristocetin cofactor ELISA and increased platelet adhesion in whole blood to collagen under arterial shear rate but in contrast inhibited ristocetin-induced platelet aggregation. The selected compound adhering to the predicted binding partner GPIbα could be confirmed by saturation transfer difference NMR spectroscopy. We thus clearly identified a small molecule that modulates VWF-GPIbα binding and that will now serve as a starting point for further studies and chemical modifications to fully characterize the interaction and to manipulate specific activity of the compound. IntroductionUpon vascular injury, circulating plasma von Willebrand factor (VWF) 4The abbreviations used are: VWFvon Willebrand factorGPglycoproteinSMPPIIsmall molecule protein-protein interaction inhibitorPPIprotein-protein interactionNHPnormal human plasmaPRPplatelet-rich plasmaSTDsaturation transfer differenceMDmolecular dynamics. binds to the exposed subendothelial collagen. High shear stress conditions induce conformational changes within the VWF that allow binding of the platelet glycoprotein (GP) Ib-V-IX receptor complex to the VWF-A1 domain. The intermolecular contacts are concentrated around two major interaction sites. The largest area is formed by an extended β-sheet, which consists of the β-sheet of the VWF-A1 Rossmann fold on one side and the β-switch of GPIbα on the other side. The C-terminal β-switch, a disordered loop in the unbound conformation, adopts a β-hairpin structure upon binding to VWF-A1. The second and remarkably smaller contact area encompasses interactions between the N-terminal β-finger of GPIbα and the N- and C-terminal bottom of VWF-A1 (1.Dumas J.J. Kumar R. McDonagh T. Sullivan F. Stahl M.L. Somers W.S. Mosyak L. Crystal structure of the wild-type von Willebrand factor A1-glycoprotein Ibα complex reveals conformation differences with a complex bearing von Willebrand disease mutations.J. Biol. Chem. 2004; 279: 23327-23334Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). The formation of this complex gives rise to the initial tethering of platelets over a damaged surface and allows stable platelet binding through additional interactions via the direct platelet collagen receptors. Because of its particular importance in thrombus formation at sites of high shear stress, the VWF-A1-GPIbα interaction is a prime target for the development of new antithrombotics (2.Firbas C. Siller-Matula J.M. Jilma B. Targeting von Willebrand factor and platelet glycoprotein Ib receptor.Expert Rev. Cardiovasc Ther. 2010; 8: 1689-1701Crossref PubMed Scopus (72) Google Scholar, 3.Vanhoorelbeke K. Ulrichts H. Van de Walle G. Fontayne A. Deckmyn H. Inhibition of platelet glycoprotein Ib and its antithrombotic potential.Curr. Pharm. Des. 2007; 13: 2684-2697Crossref PubMed Scopus (43) Google Scholar). Indeed, initial studies showed that Fab fragments of anti-GPIbα monoclonal antibodies (mAbs) that block VWF binding inhibited thrombus formation in vivo without significant prolongation of the bleeding time in non-human primates (4.Cauwenberghs N. Meiring M. Vauterin S. van Wyk V. Lamprecht S. Roodt J.P. Novák L. Harsfalvi J. Deckmyn H. Kotzé H.F. Antithrombotic effect of platelet glycoprotein Ib-blocking monoclonal antibody Fab fragments in nonhuman primates.Arterioscler. Thromb. Vasc. Biol. 2000; 20: 1347-1353Crossref PubMed Scopus (120) Google Scholar, 5.Wu D. Meiring M. Kotze H.F. Deckmyn H. Cauwenberghs N. Inhibition of platelet glycoprotein Ib, glycoprotein IIb/IIIa, or both by monoclonal antibodies prevents arterial thrombosis in baboons.Arterioscler. Thromb. Vasc. Biol. 2002; 22: 323-328Crossref PubMed Scopus (65) Google Scholar, 6.Fontayne A. Meiring M. Lamprecht S. Roodt J. Demarsin E. Barbeaux P. Deckmyn H. The humanized anti-glycoprotein Ib monoclonal antibody h6B4-Fab is a potent and safe antithrombotic in a high shear arterial thrombosis model in baboons.Thromb. Haemost. 2008; 100: 670-677Crossref PubMed Scopus (55) Google Scholar). For other inhibitors of the GPIbα-VWF interaction, such as the VWF-A1 binding aptamer ARC1779 (7.Gilbert J.C. DeFeo-Fraulini T. Hutabarat R.M. Horvath C.J. Merlino P.G. Marsh H.N. Healy J.M. Boufakhreddine S. Holohan T.V. Schaub R.G. First-in-human evaluation of anti-von Willebrand factor therapeutic aptamer ARC1779 in healthy volunteers.Circulation. 2007; 116: 2678-2686Crossref PubMed Scopus (223) Google Scholar, 8.Knöbl P. Jilma B. Gilbert J.C. Hutabarat R.M. Wagner P.G. Jilma-Stohlawetz P. Anti-von Willebrand factor aptamer ARC1779 for refractory thrombotic thrombocytopenic purpura.Transfusion. 2009; 49: 2181-2185Crossref PubMed Scopus (54) Google Scholar, 9.Mayr F.B. Knöbl P. Jilma B. Siller-Matula J.M. Wagner P.G. Schaub R.G. Gilbert J.C. Jilma-Stohlawetz P. The aptamer ARC1779 blocks von Willebrand factor-dependent platelet function in patients with thrombotic thrombocytopenic purpura ex vivo.Transfusion. 2010; 50: 1079-1087Crossref PubMed Scopus (45) Google Scholar) and the humanized bivalent Nanobody® ALX-0081 (10.Bartunek J. Barbato E. Holz J.B. Vercruysse K. Ulrichts H. Heyndrickx G. ALX-0081 a novel anti-thrombotic: results of a single-dose phase 1 study in healthy volunteers and further development in patients with stable angina undergoing PCI.Circulation. 2008; 118: S656Crossref Google Scholar, 11.Ulrichts H. Silence K. Schoolmeester A. de Jaegere P. Rossenu S. Roodt J. Priem S. Lauwereys M. Casteels P. Van Bockstaele F. Verschueren K. Stanssens P. Baumeister J. Holz J.B. Antithrombotic drug candidate ALX-0081 shows superior preclinical efficacy and safety compared with currently marketed antiplatelet drugs.Blood. 2011; 118: 757-765Crossref PubMed Scopus (134) Google Scholar), the antithrombotic potential and safety in initial human clinical trials has been indicated.All these molecules are however rather large, i.e. about 50 kDa for the Fab fragments, 33 kDa for the aptamer, and 28 kDa for the nanobody, and are of a protein or nucleotide nature that limits their oral availability and long term prophylactic use. A small molecule targeting the GPIbα-VWF protein-protein interaction (PPI) would circumvent these problems and might therefore be of particular interest.Targeting a PPI with a small molecule still is challenging and complex. The large interaction surface (1500–3000 Å2) compared with typical protein-small molecule interactions (300–1000 Å2) (12.Jones S. 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A potent and orally bioavailable Bcl-2 family inhibitor.Cancer Res. 2008; 68: 3421-3428Crossref PubMed Scopus (1402) Google Scholar), which has entered phase II clinical trials, and most recently the ledgins that prevent HIV replication by inhibiting the LEDGF-p75-integrase interaction (21.Christ F. Voet A. Marchand A. Nicolet S. Desimmie B.A. Marchand D. Bardiot D. Van der Veken N.J. Van Remoortel B. Strelkov S.V. De Maeyer M. Chaltin P. Debyser Z. Rational design of small molecule inhibitors of the LEDGF/p75-integrase interaction and HIV replication.Nat. Chem. Biol. 2010; 6: 442-448Crossref PubMed Scopus (383) Google Scholar). The emerging numbers of SMPPIIs additionally allowed a more precise description of their physicochemical properties, which differ somewhat from conventional drugs (22.Sperandio O. Reynès C.H. Camproux A.C. Villoutreix B.O. Rationalizing the chemical space of protein-protein interaction inhibitors.Drug Discov. Today. 2010; 15: 220-229Crossref PubMed Scopus (161) Google Scholar).We now aimed to identify such a SMPPII targeting the VWF-GPIbα interaction. Mutagenesis studies and crystal structure analysis of both interaction partners alone or in complex have led to a precise characterization of the VWF-A1-GPIbα interaction surface (1.Dumas J.J. Kumar R. McDonagh T. Sullivan F. Stahl M.L. Somers W.S. Mosyak L. Crystal structure of the wild-type von Willebrand factor A1-glycoprotein Ibα complex reveals conformation differences with a complex bearing von Willebrand disease mutations.J. Biol. Chem. 2004; 279: 23327-23334Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 23.Shimizu A. Matsushita T. Kondo T. Inden Y. Kojima T. Saito H. Hirai M. Identification of the amino acid residues of the platelet glycoprotein Ib (GPIb) essential for the von Willebrand factor binding by clustered charged-to-alanine scanning mutagenesis.J. Biol. Chem. 2004; 279: 16285-16294Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). In this study, we made use of this information to identify a compound capable of influencing binding parameters by strategically targeting potential small molecule binding pockets. Extensive computational docking led to a short list of best scoring compounds, which were tested in vitro for their effect on GPIbα-VWF binding. This resulted in the characterization of a small molecule that increased the ristocetin-induced GPIbα-VWF binding in ELISA and stimulated thrombus formation in flow chambers, but interestingly it inhibited ristocetin-induced platelet aggregation in platelet-rich plasma.DISCUSSIONBecause of its special importance under high shear conditions, the VWF-A1-GPIbα interaction is considered as an interesting target for the development of new antithrombotics for patients with acute coronary syndromes, suffering from thrombotic thrombocytopenic purpura (9.Mayr F.B. Knöbl P. Jilma B. Siller-Matula J.M. Wagner P.G. Schaub R.G. Gilbert J.C. Jilma-Stohlawetz P. The aptamer ARC1779 blocks von Willebrand factor-dependent platelet function in patients with thrombotic thrombocytopenic purpura ex vivo.Transfusion. 2010; 50: 1079-1087Crossref PubMed Scopus (45) Google Scholar), or from ischemic stroke (61.De Meyer S.F. Schwarz T. Deckmyn H. Denis C.V. Nieswandt B. Stoll G. Vanhoorelbeke K. Kleinschnitz C. Binding of von Willebrand factor to collagen and glycoprotein Ibα, but not to glycoprotein IIb/IIIa, contributes to ischemic stroke in mice. Brief report.Arterioscler. Thromb. Vasc. Biol. 2010; 30: 1949-1951Crossref PubMed Scopus (55) Google Scholar). Today clinical trials are ongoing for two compounds targeting the protein partners of this complex (either VWF-A1 or GPIbα) (7.Gilbert J.C. DeFeo-Fraulini T. Hutabarat R.M. Horvath C.J. Merlino P.G. Marsh H.N. Healy J.M. Boufakhreddine S. Holohan T.V. Schaub R.G. First-in-human evaluation of anti-von Willebrand factor therapeutic aptamer ARC1779 in healthy volunteers.Circulation. 2007; 116: 2678-2686Crossref PubMed Scopus (223) Google Scholar, 8.Knöbl P. Jilma B. Gilbert J.C. Hutabarat R.M. Wagner P.G. Jilma-Stohlawetz P. Anti-von Willebrand factor aptamer ARC1779 for refractory thrombotic thrombocytopenic purpura.Transfusion. 2009; 49: 2181-2185Crossref PubMed Scopus (54) Google Scholar, 9.Mayr F.B. Knöbl P. Jilma B. Siller-Matula J.M. Wagner P.G. Schaub R.G. Gilbert J.C. Jilma-Stohlawetz P. The aptamer ARC1779 blocks von Willebrand factor-dependent platelet function in patients with thrombotic thrombocytopenic purpura ex vivo.Transfusion. 2010; 50: 1079-1087Crossref PubMed Scopus (45) Google Scholar, 10.Bartunek J. Barbato E. Holz J.B. Vercruysse K. Ulrichts H. Heyndrickx G. ALX-0081 a novel anti-thrombotic: results of a single-dose phase 1 study in healthy volunteers and further development in patients with stable angina undergoing PCI.Circulation. 2008; 118: S656Crossref Google Scholar, 11.Ulrichts H. Silence K. Schoolmeester A. de Jaegere P. Rossenu S. Roodt J. Priem S. Lauwereys M. Casteels P. Van Bockstaele F. Verschueren K. Stanssens P. Baumeister J. Holz J.B. Antithrombotic drug candidate ALX-0081 shows superior preclinical efficacy and safety compared with currently marketed antiplatelet drugs.Blood. 2011; 118: 757-765Crossref PubMed Scopus (134) Google Scholar), and as anticipated, platelet function is dose-dependently inhibited, importantly without causing bleeding effects or interference with normal hemostasis. Recently, OS1, an 11-mer cysteine-constrained cyclic peptide, has been identified, and by targeting GPIbα it inhibits agonist-induced platelet aggregation by stabilizing the β-switch forming loop region in an alternative α-helical conformation (62.Benard S.A. Smith T.M. Cunningham K. Jacob J. DeSilva T. Lin L. Shaw G.D. Kriz R. Kelleher K.S. Identification of peptide antagonists to glycoprotein Ibα that selectively inhibit von Willebrand factor dependent platelet aggregation.Biochemistry. 2008; 47: 4674-4682Crossref PubMed Scopus (24) Google Scholar, 63.McEwan P.A. Andrews R.K. Emsley J. Glycoprotein Ibα inhibitor complex structure reveals a combined steric and allosteric mechanism of von Willebrand factor antagonism.Blood. 2009; 114: 4883-4885Crossref PubMed Scopus (28) Google Scholar). In addition, four other peptides able to bind to VWF-A1 and to distort its interaction with GPIbα were found by screening a peptide library, whereas subsequent modeling studies investigated the binding of these peptides to VWF-A1 in more detail (64.Munoz Cdel C. Campbell W. Constantinescu I. Gyongyossy-Issa M.I. Rational design of antithrombotic peptides to target the von Willebrand factor (vWf)-GPIb integrin interaction.J. Mol. Model. 2008; 14: 1191-1202Crossref PubMed Scopus (10) Google Scholar). The large molecular weight and/or protein/peptide/nucleotide nature of all these antagonists limits their oral bio-availability. Several groups are therefore attempting to identify smaller molecules interfering with this interaction.By targeting a putative small molecule binding pocket close to the β-switch region, we were able through high throughput molecular docking to select 24 compounds targeted against either VWF-A1 or GPIbα. This computational approach gave us the opportunity to evaluate a diverse and vast small molecule database of about 1.5 106 compounds, without having to deal with the time- and budget-bound hurdles often encountered in classical physical high throughput screening (65.Jorgensen W.L. The many roles of computation in drug discovery.Science. 2004; 303: 1813-1818Crossref PubMed Scopus (1136) Google Scholar, 66.Guido R.V. Oliva G. Andricopulo A.D. Virtual screening and its integration with modern drug design technologies.Curr. Med. Chem. 2008; 15: 37-46Crossref PubMed Scopus (169) Google Scholar). In vitro evaluation of selected compounds resulted in the identification of G6, a small organic molecule (393 Da) that on the one hand stimulated complex formation in a ristocetin-induced GPIbα-VWF binding ELISA but on the other hand inhibited ristocetin-induced platelet aggregation. To circumvent the artifacts that might be encountered by using ristocetin or a recombinant extracellular fragment of GPIbα, the effect of G6 was finally tested in whole blood in a flow chamber, where the VWF-GPIbα interaction is induced more physiologically by unfolding of VWF by binding to collagen and enhanced shear; G6 also stimulated the VWF-GPIbα-dependent platelet deposition.Several docking simulations repetitively generated a consensus pose for this compound in the free and the bound conformation of GPIbα. These two poses correspond well to the calculated electrostatic feature maps for both receptor proteins (Fig. 2, D and E) and are quite similar, suggesting that GPIbα may be initially recognized by G6, followed by a stabilization of G6 bound to GPIbα upon complex formation with VWF-A1. This might result in an overall stabilization of the VWF-A1-GPIbα complex. This idea is strengthened by the fact that the conformations of the docking poses of G6 to free and VWF-A1-bound GPIbα do not differ much (Fig. 2C). To further support this hypothesis, MM/GBSA simulations were performed on the VWF-A1-GPIbα complex with and without G6 docked to GPIbα. We could observe an improvement in the total binding free energy of the complex in the presence of G6. This is mainly caused by the contribution of the van der Waals term. We suspect that the binding of G6 stabilizes the β-switch to a higher degree, because the molecule is predicted to bind in a pocket right below this region, causing VWF-A1 and GPIbα to bind more tightly. A similar mechanism is also observed in the structural binding mechanism of a few plant phytohormones such as auxin, brassinolide, and jasmonate (67.Tan X. Calderon-Villalobos L.I. Sharon M. Zheng C. Robinson C.V. Estelle M. Zheng N. 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It is interesting to note that all these hormone reception systems, being leucine-rich repeats, are small molecule regulated receptors. GPIbα itself has eight leucine-rich repeats and is a member of this same family of proteins.Based on structural analogues of G6 that also activate complex formation between VWF-A1 and GPIbα (Fig. 6, GA13, GA15, and FQC), a pharmacophore could be generated. As expected, compounds not influencing the VWF-GPIbα interaction (Fig. 6, GA6, GA10, GA22, and GA23) did not fit the pharmacophore.Based on all these observations, we assume that the activator G6 presumably binds to its receptor GPIbα, mainly through hydrophobic interactions of its quinoline and methoxybenzyl moieties and through hydrogen bonds with the quinoline's nitrogen and the ester carbonyl in the linker. The presence of a hydrophobic group at the position of the morpholine moiety might also be important but is probably less determining for binding. STD NMR results were also consistent with the above findings. Group epitope mapping by STD NMR further confirmed that the quinoline and methoxybenzene of G6 salt are in close proximity to the rGPIbα compared with the morpholine group and the linker.CONCLUSIONBy using a virtual screening approach, we were able to select a small molecule that increased the GPIbα-VWF binding in ELISA and stimulated thrombus formation under flow but showed unexpectedly strongly reduced ristocetin-induced platelet aggregation. Docking results, inspection of the electrostatic feature maps of GPIbα, and a flexible alignment with other structurally related activators led to the identification of the most important functionalities of the molecule, whereas MM/GBSA studies suggested that binding of the molecule to GPIbα improves the total binding free energy of the VWF-A1-GPIbα complex. Further studies will be required to determine the exact binding position and to provide an explanation for the observed effect. Detailed structural information (e.g. a cocrystal structure) on the binding mode of G6 to GPIbα could offer possibilities for modifying the chemical structure of G6 in an attempt to develop SMPPIIs able to disrupt the VWF-A1-GPIbα interaction. IntroductionUpon vascular injury, circulating plasma von Willebrand factor (VWF) 4The abbreviations used are: VWFvon Willebrand factorGPglycoproteinSMPPIIsmall molecule protein-protein interaction inhibitorPPIprotein-protein interactionNHPnormal human plasmaPRPplatelet-rich plasmaSTDsaturation transfer differenceMDmolecular dynamics. binds to the exposed subendothelial collagen. High shear stress conditions induce conformational changes within the VWF that allow binding of the platelet glycoprotein (GP) Ib-V-IX receptor complex to the VWF-A1 domain. The intermolecular contacts are concentrated around two major interaction sites. The largest area is formed by an extended β-sheet, which consists of the β-sheet of the VWF-A1 Rossmann fold on one side and the β-switch of GPIbα on the other side. The C-terminal β-switch, a disordered loop in the unbound conformation, adopts a β-hairpin structure upon binding to VWF-A1. The second and remarkably smaller contact area encompasses interactions between the N-terminal β-finger of GPIbα and the N- and C-terminal bottom of VWF-A1 (1.Dumas J.J. Kumar R. McDonagh T. Sullivan F. Stahl M.L. Somers W.S. Mosyak L. Crystal structure of the wild-type von Willebrand factor A1-glycoprotein Ibα complex reveals conformation differences with a complex bearing von Willebrand disease mutations.J. Biol. Chem. 2004; 279: 23327-23334Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). The formation of this complex gives rise to the initial tethering of platelets over a damaged surface and allows stable platelet binding through additional interactions via the direct platelet collagen receptors. Because of its particular importance in thrombus formation at sites of high shear stress, the VWF-A1-GPIbα interaction is a prime target for the development of new antithrombotics (2.Firbas C. Siller-Matula J.M. Jilma B. Targeting von Willebrand factor and platelet glycoprotein Ib receptor.Expert Rev. Cardiovasc Ther. 2010; 8: 1689-1701Crossref PubMed Scopus (72) Google Scholar, 3.Vanhoorelbeke K. Ulrichts H. Van de Walle G. Fontayne A. Deckmyn H. Inhibition of platelet glycoprotein Ib and its antithrombotic potential.Curr. Pharm. Des. 2007; 13: 2684-2697Crossref PubMed Scopus (43) Google Scholar). Indeed, initial studies showed that Fab fragments of anti-GPIbα monoclonal antibodies (mAbs) that block VWF binding inhibited thrombus formation in vivo without significant prolongation of the bleeding time in non-human primates (4.Cauwenberghs N. Meiring M. Vauterin S. van Wyk V. Lamprecht S. Roodt J.P. Novák L. Harsfalvi J. Deckmyn H. Kotzé H.F. Antithrombotic effect of platelet glycoprotein Ib-blocking monoclonal antibody Fab fragments in nonhuman primates.Arterioscler. Thromb. Vasc. Biol. 2000; 20: 1347-1353Crossref PubMed Scopus (120) Google Scholar, 5.Wu D. Meiring M. Kotze H.F. Deckmyn H. Cauwenberghs N. Inhibition of platelet glycoprotein Ib, glycoprotein IIb/IIIa, or both by monoclonal antibodies prevents arterial thrombosis in baboons.Arterioscler. Thromb. Vasc. Biol. 2002; 22: 323-328Crossref PubMed Scopus (65) Google Scholar, 6.Fontayne A. Meiring M. Lamprecht S. Roodt J. Demarsin E. Barbeaux P. Deckmyn H. The humanized anti-glycoprotein Ib monoclonal antibody h6B4-Fab is a potent and safe antithrombotic in a high shear arterial thrombosis model in baboons.Thromb. Haemost. 2008; 100: 670-677Crossref PubMed Scopus (55) Google Scholar). For other inhibitors of the GPIbα-VWF interaction, such as the VWF-A1 binding aptamer ARC1779 (7.Gilbert J.C. DeFeo-Fraulini T. Hutabarat R.M. Horvath C.J. Merlino P.G. Marsh H.N. Healy J.M. Boufakhreddine S. Holohan T.V. Schaub R.G. First-in-human evaluation of anti-von Willebrand factor therapeutic aptamer ARC1779 in healthy volunteers.Circulation. 2007; 116: 2678-2686Crossref PubMed Scopus (223) Google Scholar, 8.Knöbl P. Jilma B. Gilbert J.C. Hutabarat R.M. Wagner P.G. Jilma-Stohlawetz P. Anti-von Willebrand factor aptamer ARC1779 for refractory thrombotic thrombocytopenic purpura.Transfusion. 2009; 49: 2181-2185Crossref PubMed Scopus (54) Google Scholar, 9.Mayr F.B. Knöbl P. Jilma B. Siller-Matula J.M. Wagner P.G. Schaub R.G. Gilbert J.C. Jilma-Stohlawetz P. The aptamer ARC1779 blocks von Willebrand factor-dependent platelet function in patients with thrombotic thrombocytopenic purpura ex vivo.Transfusion. 2010; 50: 1079-1087Crossref PubMed Scopus (45) Google Scholar) and the humanized bivalent Nanobody® ALX-0081 (10.Bartunek J. Barbato E. Holz J.B. Vercruysse K. Ulrichts H. Heyndrickx G. ALX-0081 a novel anti-thrombotic: results of a single-dose phase 1 study in healthy volunteers and further development in patients with stable angina undergoing PCI.Circulation. 2008; 118: S656Crossref Google Scholar, 11.Ulrichts H. Silence K. Schoolmeester A. de Jaegere P. Rossenu S. Roodt J. Priem S. Lauwereys M. Casteels P. Van Bockstaele F. Verschueren K. Stanssens P. Baumeister J. Holz J.B. Antithrombotic drug candidate ALX-0081 shows superior preclinical efficacy and safety compared with currently marketed antiplatelet drugs.Blood. 2011; 118: 757-765Crossref PubMed Scopus (134) Google Scholar), the antithrombotic potential and safety in initial human clinical trials has been indicated.All these molecules are however rather large, i.e. about 50 kDa for the Fab fragments, 33 kDa for the aptamer, and 28 kDa for the nanobody, and are of a protein or nucleotide nature that limits their oral availability and long term prophylactic use. A small molecule targeting the GPIbα-VWF protein-protein interaction (PPI) would circumvent these problems and might therefore be of particular interest.Targeting a PPI with a small molecule still is challenging and complex. The large interaction surface (1500–3000 Å2) compared with typical protein-small molecule interactions (300–1000 Å2) (12.Jones S. Thornton J.M. Principles of protein-protein interactions.Proc. Natl. Acad. Sci. 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