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

Soluble von Willebrand factor (VWF) has a low affinity for platelet glycoprotein (GP) Ibα and needs immobilization and/or high shear stress to enable binding of its A1 domain to the receptor. The previously described anti-VWF monoclonal antibody 1C1E7 enhances VWF/GPIbα binding and recognizes an epitope in the amino acids 764–1035 region in the N-terminal D′D3 domains. In this study we demonstrated that the D′D3 region negatively modulates the VWF/GPIb-IX-V interaction; (i) deletion of the D′D3 region in VWF augmented binding to GPIbα, suggesting an inhibitory role for this region, (ii) the isolated D′D3 region inhibited the GPIbα interaction of a VWF deletion mutant lacking this region, indicating that intramolecular interactions limit the accessibility of the A1 domain, (iii) using a panel of anti-VWF monoclonal antibodies, we next showed that the D′D3 region is in close proximity with the A1 domain in soluble VWF but not when VWF was immobilized; (iv) destroying the epitope of 1C1E7 resulted in a mutant VWF with an increased affinity for GPIbα. Our results support a model of domain translocation in VWF that allows interaction with GPIbα. The suggested shielding interaction of the A1 domain by the D′D3 region then becomes disrupted by VWF immobilization. Soluble von Willebrand factor (VWF) has a low affinity for platelet glycoprotein (GP) Ibα and needs immobilization and/or high shear stress to enable binding of its A1 domain to the receptor. The previously described anti-VWF monoclonal antibody 1C1E7 enhances VWF/GPIbα binding and recognizes an epitope in the amino acids 764–1035 region in the N-terminal D′D3 domains. In this study we demonstrated that the D′D3 region negatively modulates the VWF/GPIb-IX-V interaction; (i) deletion of the D′D3 region in VWF augmented binding to GPIbα, suggesting an inhibitory role for this region, (ii) the isolated D′D3 region inhibited the GPIbα interaction of a VWF deletion mutant lacking this region, indicating that intramolecular interactions limit the accessibility of the A1 domain, (iii) using a panel of anti-VWF monoclonal antibodies, we next showed that the D′D3 region is in close proximity with the A1 domain in soluble VWF but not when VWF was immobilized; (iv) destroying the epitope of 1C1E7 resulted in a mutant VWF with an increased affinity for GPIbα. Our results support a model of domain translocation in VWF that allows interaction with GPIbα. The suggested shielding interaction of the A1 domain by the D′D3 region then becomes disrupted by VWF immobilization. The plasma protein von Willebrand factor (VWF) 4The abbreviations used are: VWF, von Willebrand factor; GP, glycoprotein; aa, amino acids; moAb, monoclonal antibody; RT, room temperature; scFv, single-chain variable fragment; PBS, phosphate-buffered saline; ON, overnight; TBS, Tris-buffered saline; rrecombinant; b-418, biotinylated moAb 418; WT, wild type. has a central role in normal primary hemostasis (1Ruggeri Z.M. Nat. Med. 2002; 8: 1227-1234Crossref PubMed Scopus (1366) Google Scholar). The interaction of VWF with its platelet receptor glycoprotein (GP) Ibα in the GPIb-IX-V complex mediates platelet adhesion to extracellular matrices exposed at sites of vascular injury. This interaction is essential for thrombus formation at sites of high shear stress, as in microarterioles or in stenosed arteries. Mature VWF comprises a series of multimers that are composed of homodimers interlinked through disulfide bridges. The mature VWF subunit consists of four distinct types of internal homology present in two to three copies in the following order from the N terminus: D′-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK. Native VWF in solution has an extremely low affinity for GPIbα, suggesting the existence of specific regulatory mechanisms that maintain ligand and receptor in the same environment without adverse consequences. The affinity of VWF toward its primary receptor can be increased by artificial means, such as removal of sialic acid side chains (2Vermylen J. Donati M.B. De Gaetano G. Verstraete M. Nature. 1973; 244: 167-168Crossref PubMed Scopus (36) Google Scholar) or the presence of modulators like the bacterial glycopeptide ristocetin (3Vicente V. Houghten R.A. Ruggeri Z.M. J. Biol. Chem. 1990; 265: 274-280Abstract Full Text PDF PubMed Google Scholar) or the snake venom protein botrocetin (4Read M.S. Smith S.V. Lamb M.A. Brinkhous K.M. Blood. 1989; 74: 1031-1035Crossref PubMed Google Scholar). In pathophysiological conditions, however, binding is induced by immobilization of VWF or by elevated fluid shear stress, which is likely due to changes in interdomain interactions (5Siedlecki C.A. Lestini B.J. Kottke-Marchant K.K. Eppell S.J. Wilson D.L. Marchant R.E. Blood. 1996; 88: 2939-2950Crossref PubMed Google Scholar). The VWF A1 domain (amino acids (aa) 1260–1479) contains the only known GPIbα binding site (6Lankhof H. Wu Y.P. Vink T. Schiphorst M.E. Zerwes H.G. de Groot P.G. Sixma J.J. Blood. 1995; 86: 1035-1042Crossref PubMed Google Scholar). A 39/34-kDa dispase fragment of human VWF (aa 1243/1244–1481) (7Andrews R.K. Gorman J.J. Booth W.J. Corino G.L. Castaldi P.A. Berndt M.C. Biochemistry. 1989; 28: 8326-8336Crossref PubMed Scopus (103) Google Scholar) and the recombinant VWF fragment VCL (aa 1267–1491) (8Gralnick H.R. Williams S. McKeown L. Kramer W. Krutzsch H. Gorecki M. Pinet A. Garfinkel L.I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7880-7884Crossref PubMed Scopus (59) Google Scholar), both spanning the A1 domain, bind to platelet GPIb-IX-V in the absence of modulators, in contrast to full-length VWF. Therefore, it is not unlikely to suppose that the remainder of VWF, outside the A1 domain, serves as a masking environment, preventing binding to GPIb-IX-V. Conformational changes in VWF upon immobilization or upon exposure to shear might relieve this possible shielding effect of neighboring domains in VWF and might thereby reveal the functional binding site in the A1 domain. This mode of action is supported by kinetic and crystal studies, which demonstrated a higher binding affinity of the isolated A1 domain for GPIb-IX-V compared with full-length VWF (9Miura S. Li C.Q. Cao Z. Wang H. Wardell M.R. Sadler J.E. J. Biol. Chem. 2000; 275: 7539-7546Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 10Huizinga E.G. Tsuji S. Romijn R.A. Schiphorst M.E. de Groot P.G. Sixma J.J. Gros P. Science. 2002; 297: 1176-1179Crossref PubMed Scopus (501) Google Scholar, 11Dumas J.J. Kumar R. McDonagh T. Sullivan F. Stahl M.L. Somers W.S. Mosyak L. J. Biol. Chem. 2004; 279: 23327-23334Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). Moreover, these crystal studies confirm that conformational changes in both GPIbα and in the A1 domain are required for their mutual interaction. The A1 domain contains a typical α/β-fold that is delimited by a single disulfide bridge (aa 1272–1456) (12Emsley J. Cruz M. Handin R. Liddington R. J. Biol. Chem. 1998; 273: 10396-10401Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). The N-terminal region in the A1 domain (aa 1260–1271), flanking the disulfide bridge, modulates the A1/GPIbα interaction; (i) crystal studies reveal a displacement of this flanking region upon binding to GPIbα (10Huizinga E.G. Tsuji S. Romijn R.A. Schiphorst M.E. de Groot P.G. Sixma J.J. Gros P. Science. 2002; 297: 1176-1179Crossref PubMed Scopus (501) Google Scholar, 11Dumas J.J. Kumar R. McDonagh T. Sullivan F. Stahl M.L. Somers W.S. Mosyak L. J. Biol. Chem. 2004; 279: 23327-23334Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar); (ii) naturally occurring mutations in this region have been reported to induce von Willebrand disease (VWD) type 2B phenotype, characterized by an increased affinity of VWF for GPIbα (13Meyer D. Fressinaud E. Hilbert L. Ribba A.S. Lavergne J.M. Mazurier C. Best. Pract. Res. Clin. Haematol. 2001; 14: 349-364Crossref PubMed Scopus (43) Google Scholar); (iii) Ala substitution mutants in this region show an increased affinity for GPIbα (14Matsushita T. Sadler J.E. J. Biol. Chem. 1995; 270: 13406-13414Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). As mentioned before, it is suggested that neighboring domains in VWF might impede the accessibility of the GPIbα binding region. We have previously described a monoclonal antibody (moAb), 1C1E7, directed against the aa 764–1035 region in the N-terminal D′D3 domains of VWF (15Tornai I. Arnout J. Deckmyn H. Peerlinck K. Vermylen J. J. Clin. Investig. 1993; 91: 273-282Crossref PubMed Scopus (19) Google Scholar). Although this moAb interacts with a region distinct from the VWF A1 domain, it induces von Willebrand disease-type 2B-like alterations (16Ulrichts H. J. L. J. Vermylen J. D. Deckmyn H. I. J. 2004; PubMed Scopus Google Scholar). This that the binding region of 1C1E7 in VWF might have a role for the VWF/GPIbα interaction and be a shielding region for the GPIbα binding domain. The of this study to for the of the D′D3 region in VWF for GPIbα binding using deletion VWF and recombinant 1C1E7 as against VWF described moAb to the VWF domain and VWF binding to types and K. H. Romijn E.G. De M. Deckmyn H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), and moAb 1C1E7 recognizes the N-terminal of VWF (aa (15Tornai I. Arnout J. Deckmyn H. Peerlinck K. Vermylen J. J. Clin. Investig. 1993; 91: 273-282Crossref PubMed Scopus (19) Google Scholar). and of J. P. and the VWF A1 domain H. C. D. Deckmyn H. D. Blood. 1998; 91: PubMed Google Scholar, A. D. Blood. PubMed Google Scholar), moAb interacts with the aa in mature VWF G. Ribba A.S. P. D. Res. 1989; PubMed Scopus Google Scholar). VWF was from the or VWF biotinylated using plasma was from the plasma of K. S. A. Mazurier C. Deckmyn H. 2000; PubMed Scopus Google Scholar). The S. R. P. Biol. PubMed Scopus Google Scholar) was from from of the aa of VWF or the of the VWF the or the as the (aa (aa and and as described E. V. Ribba A.S. E.G. D. de Groot P.G. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). we and as and A of VWF, its and the binding sites of the is in and of 1C1E7 was from using the The and was in the using The N-terminal of the and of 1C1E7 was by these for the and the variable and and used in the S. J. S. I. P. J.M. Deckmyn H. K. Google Scholar, D. A. 1998; PubMed Scopus Google Scholar). of was using and the was in the was with and the was in the the of the 1C1E7 single-chain variable fragment both and such that the following resulted in a This was in the The 1C1E7 was in as a and sites to the and by using The 1C1E7 was in the using the and E. with the and induced for with The for and from the with the protein 1C1E7 was as the scFv, in in phosphate-buffered H. E. R. 1998; PubMed Scopus Google Scholar). The against a series of and at against and of VWF, or VWF to a GPIbα (aa and binding of VWF, or recombinant VWF to a GPIbα fragment was as previously described K. S. A. Mazurier C. Deckmyn H. 2000; PubMed Scopus Google Scholar). at with moAb in and with Tris-buffered A recombinant VWF binding GPIbα fragment (aa was in and as described H. K. S. S. H. S. Deckmyn H. Biol. PubMed Scopus Google Scholar). for at with in TBS, and for at with a series of ristocetin or botrocetin from venom K. M. R. T. H. M. Ruggeri Z.M. Biochemistry. PubMed Scopus Google Scholar) in the presence of a of VWF, or recombinant VWF and in the absence or presence of 1C1E7 VWF, or recombinant VWF was for at room with a of anti-VWF with was with and and the was with which the was at studies in an with at at the using 1C1E7 scFv, was from and at for to plasma was for with and with 1C1E7 or which was induced by the of the using the VWF was acid and in and A platelet of was or to a of and the was for which was induced by the of of for for a recombinant VWF in which the aa region of the human was by the was This recombinant VWF is to as the L. Vink T. de Groot P.G. Sixma J.J. E.G. 2000; PubMed Scopus (36) Google Scholar) was with the and the fragment was in the site of the to the was isolated from a using the and was with the using the specific was by using the and the The was in the A site at was by using the and the The was in the to the the fragment of the was in to the The was with and The fragment was by the of to the of for VWF or for human VWF or for or mutations from the or the and specific the mutations The with and and the fragment was by the of or as described to for full-length VWF. of was in J.J. Schiphorst M.E. H. J. PubMed Scopus Google Scholar) or in as described (6Lankhof H. Wu Y.P. Vink T. Schiphorst M.E. Zerwes H.G. de Groot P.G. Sixma J.J. Blood. 1995; 86: 1035-1042Crossref PubMed Google Scholar). The was in a as previously described K. S. A. Mazurier C. Deckmyn H. 2000; PubMed Scopus Google Scholar). of VWF to moAb at with moAb 1C1E7 in for at with and for at with a series of VWF in VWF was as described of VWF to at with human type in with and with a series of recombinant VWF in VWF was as described S. J. S. I. P. J.M. Deckmyn H. K. Google Scholar). of VWF was as described Z.M. Blood. PubMed Google Scholar). of VWF was and VWF was using with J. Mazurier C. A. M. Biol. Google Scholar) and with the for to at with VWF in and for at with for at with biotinylated moAb at its binding in the presence of the or using as was for at with in was as described for to with moAb in and for at with VWF was with of the anti-VWF for at which this solution was to the a of at was with as described this study and are of was using of VWF the D′D3 has been suggested that the remainder of VWF outside the A1 domain might the GPIbα binding region. the of this shielding has not been The anti-VWF moAb 1C1E7 interacts with the aa 764–1035 region in the N-terminal D′D3 domains and modulates the binding of VWF to GPIbα. This a functional role of the D′D3 region in the binding of the A1 domain to its receptor. Therefore, we to study this role using VWF lacking the D′D3 region. (aa is a composed of two VWF lacking the D′D3 region but contains the N-terminal flanking region of the VWF A1 domain. We the and binding of this to a recombinant GPIbα fragment and compared its affinity to that of recombinant (aa This is a as but the In the presence of ristocetin as a with compared with results with botrocetin results that the D′D3 region has an inhibitory effect the interaction of the A1 domain with GPIbα. The of 1C1E7 to increased binding to to the of not the GPIbα interaction of This that the are likely not due to a affinity of the deletion mutants for the modulators used in the We the inhibitory role by the D′D3 region in VWF/GPIbα interaction in platelet used studies demonstrated the of the of at for platelet M. G. M.E. E. Ruggeri Z.M. Biochemistry. PubMed Scopus Google Scholar). The for of was for for the inhibitory effect of the D′D3 region the interaction of VWF with GPIbα. In with inhibited the supported by effect the supported by results show that the of by deletion of the D′D3 region in be by of these isolated D′D3 domains. the of the D′D3 region in the interaction of VWF with GPIbα, suggesting that this region the GPIbα binding site in VWF, its accessibility and preventing VWF binding to of moAb in the D′D3 region that be for its inhibitory we the binding region of 1C1E7 in VWF. studies demonstrated that 1C1E7 with a fragment the aa 764–1035 in VWF, which is in the D′D3 region (15Tornai I. Arnout J. Deckmyn H. Peerlinck K. Vermylen J. J. Clin. Investig. 1993; 91: 273-282Crossref PubMed Scopus (19) Google Scholar). The of the epitope was by the that 1C1E7 to with but the isolated D′D3 region not 1C1E7 with VWF in this moAb recognizes a Therefore, the of 1C1E7 with VWF of was to in the primary of VWF have an effect the binding of 1C1E7 to plasma VWF but was to with VWF not This that of the in the aa 764–1035 region of human VWF that are not with the VWF might maintain the epitope of these a recombinant VWF in which the aa region of the human was by the was only this of the N-terminal aa 764–1035 region in human VWF was This from the human was in and the interaction with 1C1E7 was 1C1E7 with suggesting that the aa region in VWF the epitope of this of the primary of human VWF in this region with of and VWF and as possible for the epitope of 1C1E7 are human and both and VWF but are the human and VWF mutants in which these for the and in 1C1E7 as with the and mutant as to VWF, suggesting that these are not for the binding was for the this as for the interaction of 1C1E7 of a next the binding of the recombinant full-length VWF and to GPIbα was to the in the D′D3 region might this interaction. VWF, and in which a was in The of both mutants was to VWF with at of the mutants to anti-VWF and was to VWF not suggesting a the functional of the of in the D′D3 region was The interaction with was not for mutant VWF as compared with the WT, in a binding of and for and using VWF as a and to in the presence of ristocetin at of showed a but in binding compared with and results that in the D′D3 region the binding region of 1C1E7, the binding affinity of VWF for GPIbα. the proximity of the D′D3 region with the A1 domain in VWF and in soluble VWF was by binding of moAb with the D′D3 be by and with the A1 This the to the changes in VWF upon immobilization. As a moAb with the was (i) of binding of to VWF by and (ii) the of the of to moAb by the demonstrated that of VWF effect the interaction with or GPIbα, which that the of VWF is not upon H. K. S. Deckmyn H. J. PubMed Scopus Google Scholar). VWF was of the binding of was by of the with as the to with VWF not In and with moAb for the binding to soluble moAb effect This that in solution the A1 domain and the D′D3 region are in close proximity with these when VWF was with the A1 domain to binding of moAb with the D′D3 region, suggesting that these are from and of 1C1E7 the of the moAb 1C1E7, the was as a protein in E. 1C1E7 increased platelet to 1C1E7 that the was Moreover, 1C1E7 inhibited binding of the to VWF, suggesting a same interaction site in VWF not of the primary of the region of the 1C1E7 a with the N-terminal flanking region of the VWF A1 domain As mentioned before, is that this N-terminal flanking region modulates the binding of VWF to GPIbα. we that 1C1E7 might be to this region of the A1 domain. 1C1E7 bind to the D′D3 region in VWF, the N-terminal flanking region of the A1 domain might have binding This interaction of the A1 domain with the D′D3 region the VWF binding to GPIbα. VWF in solution not with its platelet receptor GPIb-IX-V normal however, is induced by immobilization of VWF or by exposure to that the affinity toward GPIb-IX-V is by conformational changes in VWF that are induced by shear and/or immobilization and to exposure of functional In binding of VWF to GPIb-IX-V can be by modulators such as ristocetin or Although in these binding is induced it has been demonstrated that interactions with the Berndt M.C. A. R.K. Blood. 2001; PubMed Scopus Google Scholar). We have previously the moAb 1C1E7 (15Tornai I. Arnout J. Deckmyn H. Peerlinck K. Vermylen J. J. Clin. Investig. 1993; 91: 273-282Crossref PubMed Scopus (19) Google Scholar) which interacts with the aa 764–1035 region in the N-terminal D′D3 domains in VWF, the affinity of VWF for GPIbα, and induces von Willebrand disease type 2B it not bind the GPIb-IX-V binding site (16Ulrichts H. J. L. J. Vermylen J. D. Deckmyn H. I. J. 2004; PubMed Scopus Google Scholar). This a effect for the binding region of 1C1E7 in VWF the GPIbα interaction. Therefore, the of this study was to the functional role of the D′D3 region in binding of VWF to GPIb-IX-V. of the D′D3 region in VWF resulted in a higher affinity for GPIbα as demonstrated in an the deletion mutant lacking the D′D3 region, showed an increased and interaction with a recombinant GPIbα fragment compared with the deletion mutant which the region in VWF. The of 1C1E7 to increased the binding to the binding of This the effect of 1C1E7 the VWF/GPIbα interaction but in that the in affinity of both deletion mutants for GPIbα is not due to a affinity for results in a platelet the of ristocetin to induce of was in the presence of as compared with Moreover, at platelet was to platelet in contrast to not these that the D′D3 region as an inhibitory region, shielding the A1 domain in VWF. Our confirm and studies that demonstrated the shielding of the binding site in the A1 domain by the N-terminal flanking of the A1 domain (aa and the region of the region (aa (i) from these inhibited binding of VWF to human H. M. A. Houghten R.A. Ruggeri Z.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar), (ii) N-terminal in the aa region of a recombinant VWF fragment the A1 domain (aa resulted in increased binding to human M. J. R. J. Ruggeri Z.M. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar); (iii) Ala mutations in the N-terminal flanking region of the A1 domain (aa in full-length VWF resulted in an increased binding to human (14Matsushita T. Sadler J.E. J. Biol. Chem. 1995; 270: 13406-13414Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar); (iv) in the aa region in full-length VWF in which an was resulted in an increased or binding to human when compared with the VWF mutant T. T. Z. Sadler J.E. S. Mazurier C. D. T. H. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar); specific in the aa region negatively the binding to human as demonstrated using VWF A1 domains J. M. J. Haematol. PubMed Scopus Google Scholar). the VWF for the binding region of 1C1E7, we of the that 1C1E7 not with VWF. confirm the role of the in human VWF in the epitope of 1C1E7, a VWF was in which the aa region of human VWF was by the In the recombinant of the with the an was however, in with studies J. Cruz M.A. J. Blood. PubMed Google Scholar), this is a of of resulted in the of the however, as to 1C1E7 as VWF, suggesting that the aa region in VWF the epitope of this of the of the in the VWF domain, as essential for the binding of moAb As this has not been with a functional of VWF. The interaction with of an was but increased at of this compared with VWF. results in platelet studies not in the presence of and of ristocetin to induce compared with VWF. results that this and/or the region might be for the interaction of VWF with GPIbα. studies are to confirm both and platelet in the presence of inhibited the supported by not This is in with that in solution the D′D3 region interacts with in and that these intramolecular interactions in VWF the A1 domain from with GPIbα. is known that immobilization of VWF is a for platelet adhesion at high shear stress, due to conformational changes F. Ruggeri Z.M. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). the of these conformational changes has not been L. Deckmyn H. S. J. Blood. 2002; PubMed Scopus Google Scholar). the that D′D3 with A1 in VWF in solution and when we to against the domains binding or not when VWF was a as it is known that this allows platelet H. K. S. Deckmyn H. J. PubMed Scopus Google Scholar). The moAb with the domain and when VWF was in but not VWF, for a the domains upon immobilization. studies suggested a in the D′D3 region upon immobilization of VWF a affinity for factor J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). be that conformational changes in the D′D3 region are induced by immobilization of VWF a to of the proximity of this region with the A1 domain, thereby the GPIbα binding we a the primary of the region of the 1C1E7 and the N-terminal flanking region of the VWF A1 domain (aa This N-terminal flanking region is in the binding of the A1 domain with GPIbα as or in this region the affinity of VWF for GPIbα (14Matsushita T. Sadler J.E. J. Biol. Chem. 1995; 270: 13406-13414Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, T. T. Z. Sadler J.E. S. Mazurier C. D. T. H. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), and in this might be the region the A1 domain that interacts with the D′D3 region. 1C1E7 then with this N-terminal flanking region this interaction. 1C1E7 interacts with in the domain, it is possible that the N-terminal flanking region interacts with this as In results an inhibitory role for the aa 764–1035 region in VWF for the GPIbα interaction. these the following might be In conditions, the A1 domain and the D′D3 region are in close through an interaction of the N-terminal flanking region of the A1 domain with the with the region of This interaction limit the accessibility of the GPIbα binding VWF is this interaction is disrupted through conformational changes in VWF, in the D′D3 region which allows of through their GPIb-IX-V studies are required to confirm this We and J. P. for the and with the anti-VWF