Leucine-rich Repeats 2-4 (Leu60-Glu128) of Platelet Glycoprotein Ibα Regulate Shear-dependent Cell Adhesion to von Willebrand Factor
2006; Elsevier BV; Volume: 281; Issue: 36 Linguagem: Inglês
10.1074/jbc.m604296200
ISSN1083-351X
AutoresYang Shen, Susan L. Cranmer, Andrea Aprico, James C. Whisstock, Shaun P. Jackson, Michael C. Berndt, Robert K. Andrews,
Tópico(s)Blood properties and coagulation
ResumoGlycoprotein (GP) Ib-IX-V binds von Willebrand factor (VWF), initiating thrombosis at high shear stress. The VWF-A1 domain binds the N-terminal domain of GPIbα (His1-Glu282); this region contains seven leucine-rich repeats (LRR) plus N- and C-terminal flanking sequences and an anionic sequence containing three sulfated tyrosines. Our previous analysis of canine/human and human/canine chimeras of GPIbα expressed on Chinese hamster ovary (CHO) cells demonstrated that LRR2-4 (Leu60-Glu128) were crucial for GPIbα-dependent adhesion to VWF. Paradoxically, co-crystal structures of the GPIbα N-terminal domain and GPIbα-binding VWF-A1 under static conditions revealed that the LRR2-4 sequence made minimal contact with VWF-A1. To resolve the specific functional role of LRR2-4, we compared wild-type human GPIbα with human GPIbα containing a homology domain swap of canine for human sequence within Leu60-Glu128 and a reverse swap (canine GPIbα with human Leu60-Glu128) for the ability to support adhesion to VWF under flow. Binding of conformation-specific anti-GPIbα antibodies and VWF binding in the presence of botrocetin (which does not discriminate between species) confirmed equivalent expression of wild-type and mutant receptors in a functional form competent to bind ligand. Compared with CHO cells expressing wild-type GPIbα, cells expressing GPIbα, where human Leu60-Glu128 sequence was replaced by canine sequence, supported adhesion to VWF at low shear rates but became increasingly ineffective as shear increased from 50 to 2000 s-1. Together, these data demonstrate that LRR2-4, encompassing a pronounced negative charge patch on human GPIbα, is essential for GPIbα·VWF-dependent adhesion as hydrodynamic shear increases. Glycoprotein (GP) Ib-IX-V binds von Willebrand factor (VWF), initiating thrombosis at high shear stress. The VWF-A1 domain binds the N-terminal domain of GPIbα (His1-Glu282); this region contains seven leucine-rich repeats (LRR) plus N- and C-terminal flanking sequences and an anionic sequence containing three sulfated tyrosines. Our previous analysis of canine/human and human/canine chimeras of GPIbα expressed on Chinese hamster ovary (CHO) cells demonstrated that LRR2-4 (Leu60-Glu128) were crucial for GPIbα-dependent adhesion to VWF. Paradoxically, co-crystal structures of the GPIbα N-terminal domain and GPIbα-binding VWF-A1 under static conditions revealed that the LRR2-4 sequence made minimal contact with VWF-A1. To resolve the specific functional role of LRR2-4, we compared wild-type human GPIbα with human GPIbα containing a homology domain swap of canine for human sequence within Leu60-Glu128 and a reverse swap (canine GPIbα with human Leu60-Glu128) for the ability to support adhesion to VWF under flow. Binding of conformation-specific anti-GPIbα antibodies and VWF binding in the presence of botrocetin (which does not discriminate between species) confirmed equivalent expression of wild-type and mutant receptors in a functional form competent to bind ligand. Compared with CHO cells expressing wild-type GPIbα, cells expressing GPIbα, where human Leu60-Glu128 sequence was replaced by canine sequence, supported adhesion to VWF at low shear rates but became increasingly ineffective as shear increased from 50 to 2000 s-1. Together, these data demonstrate that LRR2-4, encompassing a pronounced negative charge patch on human GPIbα, is essential for GPIbα·VWF-dependent adhesion as hydrodynamic shear increases. Binding of platelet glycoprotein (GP) 2The abbreviations used are: GP, glycoprotein; VWF, von Willebrand factor; LRR, leucine-rich repeats; CHO, Chinese hamster ovary; WT, wild-type. Ib-IX-V to von Willebrand factor (VWF) in plasma, subendothelial matrix, or on endothelium initiates thrombus formation at high shear stress in normal hemostasis and thrombotic diseases, such as heart attack or stroke (1Andrews R.K. Gardiner E.E. Shen Y. Whisstock J.C. Berndt M.C. Int. J. Biochem. Cell Biol. 2003; 35: 1170-1174Crossref PubMed Scopus (168) Google Scholar, 2Kroll M.H. Hellums J.D. McIntire L.V. Schafer A.I. Moake J.L. Blood. 1996; 88: 1525-1541Crossref PubMed Google Scholar, 3Ozaki Y. Asazuma N. Suziki-Inoue K. Berndt M.C. J. Thromb. Haemost. 2005; 3: 1745-1751Crossref PubMed Scopus (109) Google Scholar, 4Bhatt D.L. Topol E.J. Nat. Rev. Drug Discov. 2003; 2: 15-28Crossref PubMed Scopus (402) Google Scholar, 5Phillips D.R. Conley P.B. Sinha U. Andre P. J. Thromb. Haemost. 2005; 3: 1577-1589Crossref PubMed Scopus (73) Google Scholar). GPIb-IX-V consists of four members of the leucine-rich repeat (LRR) family: GPIbα disulfide-linked to GPIbβ and associated with GPIX and GPV (2:2:2:1) (1Andrews R.K. Gardiner E.E. Shen Y. Whisstock J.C. Berndt M.C. Int. J. Biochem. Cell Biol. 2003; 35: 1170-1174Crossref PubMed Scopus (168) Google Scholar). VWF binds to the N-terminal domain of GPIbα (His1-Glu282), consisting of seven leucine-rich repeats (Leu36-Ala200), N- and C-terminal flanking sequences (His1-Ile35 and Phe201-Gly268), and an anionic sequence (Asp269-Glu282) containing three sulfotyrosines at positions 276, 278, and 279. We previously expressed a series of human/canine and canine/human chimeras of GPIbα and mapped binding sites for VWF and a panel of inhibitory anti-GPIbα antibodies to precise structural domains (6Shen Y. Romo G.M. Dong J.-F. Schade A. McIntire L.V. Kenny D. Whisstock J.C. Berndt M.C. López J.A. Andrews R.K. Blood. 2000; 95: 903-910Crossref PubMed Google Scholar, 7Shen Y. Dong J.-F. Romo G.M. Arceneaux W. Aprico A. Gardiner E.E. López J.A. Berndt M.C. Andrews R.K. Blood. 2002; 99: 145-150Crossref PubMed Scopus (27) Google Scholar). This approach is based on the specificity of human VWF and murine antibodies for human (not canine) GPIbα (6Shen Y. Romo G.M. Dong J.-F. Schade A. McIntire L.V. Kenny D. Whisstock J.C. Berndt M.C. López J.A. Andrews R.K. Blood. 2000; 95: 903-910Crossref PubMed Google Scholar). Chimeras consisted of human sequence incrementally replaced by canine sequence from the N terminus at domain boundaries and canine sequence His1-Glu282 rehumanized from the N terminus (human replacing canine sequence). LRR2-4, spanning residues Leu60-Glu128, was identified as crucial for GPIbα-dependent adhesion to VWF under shear conditions. Paradoxically, co-crystal structures subsequently reported for GPIbα N-terminal domain and VWF-A1 domain fragments (8Sadler J.E. Science. 2002; 297: 1128-1129Crossref PubMed Scopus (40) Google Scholar, 9Huizinga 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 (503) Google Scholar, 10Dumas 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) revealed major contact sites clustered N- and C-terminally to LRR2-4, whereas the Leu60-Glu128 sequence made minimal contact with VWF-A1 (with the exception of a single water-mediated contact between Asp63 of GPIbα and Arg571 of VWF-A1) (10Dumas 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). This discrepancy between structure and function requires resolution to understand the molecular basis for shear-dependent platelet adhesion, especially if the GPIbα-VWF interaction is considered as an anti-thrombotic target (4Bhatt D.L. Topol E.J. Nat. Rev. Drug Discov. 2003; 2: 15-28Crossref PubMed Scopus (402) Google Scholar, 5Phillips D.R. Conley P.B. Sinha U. Andre P. J. Thromb. Haemost. 2005; 3: 1577-1589Crossref PubMed Scopus (73) Google Scholar). Notably, LRR2-4 in human but not canine GPIbα has a pronounced negative charge patch at the concave surface of the repeats, complementary to a positive patch on VWF, implying electrostatic interactions are critical for GPIbα-mediated adhesion to VWF (1Andrews R.K. Gardiner E.E. Shen Y. Whisstock J.C. Berndt M.C. Int. J. Biochem. Cell Biol. 2003; 35: 1170-1174Crossref PubMed Scopus (168) Google Scholar, 11Whisstock J.C. Shen Y. López J.A. Andrews R.K. Berndt M.C. Thromb. Haemostasis. 2002; 87: 329-333Crossref PubMed Scopus (13) Google Scholar), even though this sequence makes minimal contact with VWF-A1 under static conditions used for crystallography (8Sadler J.E. Science. 2002; 297: 1128-1129Crossref PubMed Scopus (40) Google Scholar, 9Huizinga 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 (503) Google Scholar, 10Dumas 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). Here we analyzed GPIbα human/canine homology domain swaps designed to maintain N- and C-terminal contact sites (based on crystal structures) (9Huizinga 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 (503) Google Scholar, 10Dumas 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) but altered the intervening electrostatic region (6Shen Y. Romo G.M. Dong J.-F. Schade A. McIntire L.V. Kenny D. Whisstock J.C. Berndt M.C. López J.A. Andrews R.K. Blood. 2000; 95: 903-910Crossref PubMed Google Scholar), to establish the specific functional role of Leu60-Glu128. Comparing wild-type (WT) human GPIbα and a homology swap with canine instead of human sequence within Leu60-Glu128 (HUMCAN60-128) expressed on CHO cells shows that LRR2-4 of GPIbα is essential for GPIbα·VWF-dependent adhesion as hydrodynamic shear increases. Antibodies—Murine anti-GPIbα monoclonal antibodies have been characterized elsewhere: AN51 maps to His1-Ile35, 6D1 to Leu104-Glu128 (LRR4), VM16d to Val226-Gly268, and WM23 to the macroglycopeptide region downstream of Glu282 (6Shen Y. Romo G.M. Dong J.-F. Schade A. McIntire L.V. Kenny D. Whisstock J.C. Berndt M.C. López J.A. Andrews R.K. Blood. 2000; 95: 903-910Crossref PubMed Google Scholar, 7Shen Y. Dong J.-F. Romo G.M. Arceneaux W. Aprico A. Gardiner E.E. López J.A. Berndt M.C. Andrews R.K. Blood. 2002; 99: 145-150Crossref PubMed Scopus (27) Google Scholar). CR1 against VWF-A1 (12De Luca M. Facey D.A. Favaloro E.J. Hertzberg M.S. Whisstock J.C. McNally T. Andrews R.K. Berndt M.C. Blood. 2000; 95: 164-172Crossref PubMed Google Scholar) was used as a negative control. Molecular Modeling—Models of canine GPIbα N-terminal domain and human/canine homology swaps, based on the GPIbα crystal structure, were built as described previously (6Shen Y. Romo G.M. Dong J.-F. Schade A. McIntire L.V. Kenny D. Whisstock J.C. Berndt M.C. López J.A. Andrews R.K. Blood. 2000; 95: 903-910Crossref PubMed Google Scholar, 11Whisstock J.C. Shen Y. López J.A. Andrews R.K. Berndt M.C. Thromb. Haemostasis. 2002; 87: 329-333Crossref PubMed Scopus (13) Google Scholar). GPIb-IX-transfected CHO Cells—Human/canine GPIbα Leu60-Glu128 homology domain swap constructs were generated by PCR using previously described human/canine chimeras as templates (6Shen Y. Romo G.M. Dong J.-F. Schade A. McIntire L.V. Kenny D. Whisstock J.C. Berndt M.C. López J.A. Andrews R.K. Blood. 2000; 95: 903-910Crossref PubMed Google Scholar) and cloned into F460 vector (13Hobbs S. Jitrapakdee S. Wallace J.C. Biochem. Biophys. Res. Commun. 1998; 252: 368-372Crossref PubMed Scopus (222) Google Scholar) with puromycin selection. WT/mutant GPIbα vectors were transfected into CHO cells stably transfected with GPIbβ and GPIX (CHOβIX cells) (6Shen Y. Romo G.M. Dong J.-F. Schade A. McIntire L.V. Kenny D. Whisstock J.C. Berndt M.C. López J.A. Andrews R.K. Blood. 2000; 95: 903-910Crossref PubMed Google Scholar, 7Shen Y. Dong J.-F. Romo G.M. Arceneaux W. Aprico A. Gardiner E.E. López J.A. Berndt M.C. Andrews R.K. Blood. 2002; 99: 145-150Crossref PubMed Scopus (27) Google Scholar, 14Cranmer S.L. Ulsemer P. Cooke B.M. Salem H.H. de la Salle C. Lanza F. Jackson S.P. J. Biol. Chem. 1999; 274: 6097-6106Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 15Williamson D. Pikovski I. Cranmer S.L. Mangin P. Mistry N. Domagala T. Chehab S. Lanza F. Salem H.H. Jackson S.P. J. Biol. Chem. 2002; 277: 2151-2159Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 16Cranmer S.L. Pikovski I. Mangin P. Thompson P.E. Domagala T. Frazzetto M. Salem H.H. Jackson S.P. Biochem. J. 2005; 387: 849-858Crossref PubMed Scopus (39) Google Scholar) and selected using WM23 (6Shen Y. Romo G.M. Dong J.-F. Schade A. McIntire L.V. Kenny D. Whisstock J.C. Berndt M.C. López J.A. Andrews R.K. Blood. 2000; 95: 903-910Crossref PubMed Google Scholar). GPV is not required for GPIb-IX expression. Flow cytometry of cells (106/ml) in 0.01 m phosphate, 0.15 m NaCl, pH 7.4, probed with anti-GPIbα (WM23, AN51, 6D1, or VM16d) or control (CR1) antibodies (2 μg/ml) and fluorescein isothiocyanate-conjugated secondary antibodies (Silenus, Hawthorn, Australia) was performed as described previously (6Shen Y. Romo G.M. Dong J.-F. Schade A. McIntire L.V. Kenny D. Whisstock J.C. Berndt M.C. López J.A. Andrews R.K. Blood. 2000; 95: 903-910Crossref PubMed Google Scholar, 7Shen Y. Dong J.-F. Romo G.M. Arceneaux W. Aprico A. Gardiner E.E. López J.A. Berndt M.C. Andrews R.K. Blood. 2002; 99: 145-150Crossref PubMed Scopus (27) Google Scholar). Adhesion Assays—Flow-based CHO cell adhesion assays were performed as described elsewhere (14Cranmer S.L. Ulsemer P. Cooke B.M. Salem H.H. de la Salle C. Lanza F. Jackson S.P. J. Biol. Chem. 1999; 274: 6097-6106Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 15Williamson D. Pikovski I. Cranmer S.L. Mangin P. Mistry N. Domagala T. Chehab S. Lanza F. Salem H.H. Jackson S.P. J. Biol. Chem. 2002; 277: 2151-2159Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 16Cranmer S.L. Pikovski I. Mangin P. Thompson P.E. Domagala T. Frazzetto M. Salem H.H. Jackson S.P. Biochem. J. 2005; 387: 849-858Crossref PubMed Scopus (39) Google Scholar) using glass microslides (VitroCom) coated with 150 μg/ml human. Cells in Tyrode's/EDTA buffer (12 mm NaHCO3, 10 mm HEPES, 137 mm NaCl, 2.7 mm KCl, 5.5 mm glucose, pH 7.5) containing 2 mm EDTA were perfused at 50 s-1 followed by incremental increases in shear rate every 30 s. In this assay, cells interact with VWF in a strictly GPIbα-dependent manner (14Cranmer S.L. Ulsemer P. Cooke B.M. Salem H.H. de la Salle C. Lanza F. Jackson S.P. J. Biol. Chem. 1999; 274: 6097-6106Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 15Williamson D. Pikovski I. Cranmer S.L. Mangin P. Mistry N. Domagala T. Chehab S. Lanza F. Salem H.H. Jackson S.P. J. Biol. Chem. 2002; 277: 2151-2159Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 16Cranmer S.L. Pikovski I. Mangin P. Thompson P.E. Domagala T. Frazzetto M. Salem H.H. Jackson S.P. Biochem. J. 2005; 387: 849-858Crossref PubMed Scopus (39) Google Scholar) and roll rather than stably adhering to the VWF-coated surface, because the presence of 2 mm EDTA precludes any stationary integrin-dependent adhesion. Once cells have tethered, they continue rolling and do not become stationary. The term "adherent" is used to describe any cell that has tethered to surface-coated VWF from the bulk flow. Cell rolling velocity was analyzed for 25 cells over five separate fields for 30 s at each shear rate. The shear rates used for GPIbα-expressing CHO cells are based on previous studies and discussed elsewhere (14Cranmer S.L. Ulsemer P. Cooke B.M. Salem H.H. de la Salle C. Lanza F. Jackson S.P. J. Biol. Chem. 1999; 274: 6097-6106Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 15Williamson D. Pikovski I. Cranmer S.L. Mangin P. Mistry N. Domagala T. Chehab S. Lanza F. Salem H.H. Jackson S.P. J. Biol. Chem. 2002; 277: 2151-2159Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 16Cranmer S.L. Pikovski I. Mangin P. Thompson P.E. Domagala T. Frazzetto M. Salem H.H. Jackson S.P. Biochem. J. 2005; 387: 849-858Crossref PubMed Scopus (39) Google Scholar). Statistical analyses using one-way analysis of variance and Student's unpaired t test, with p < 0.05 considered significant, were performed using Prism software (Graphpad, San Diego, CA). To establish the functional role of Leu60-Glu128 of GPIbα, especially the prominent negative charge patch that this region contributes to the concave face of the repeats (complementary to a positive charge patch on the interacting face of VWF-A1) (1Andrews R.K. Gardiner E.E. Shen Y. Whisstock J.C. Berndt M.C. Int. J. Biochem. Cell Biol. 2003; 35: 1170-1174Crossref PubMed Scopus (168) Google Scholar, 9Huizinga 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 (503) Google Scholar, 10Dumas 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, 11Whisstock J.C. Shen Y. López J.A. Andrews R.K. Berndt M.C. Thromb. Haemostasis. 2002; 87: 329-333Crossref PubMed Scopus (13) Google Scholar), we expressed human/canine homology swaps of GPIbα (Fig. 1A). This approach enables functional analysis of the N-terminal domain of GPIbα that is conformationally sensitive and not amenable to analysis by short peptides or scanning mutagenesis (6Shen Y. Romo G.M. Dong J.-F. Schade A. McIntire L.V. Kenny D. Whisstock J.C. Berndt M.C. López J.A. Andrews R.K. Blood. 2000; 95: 903-910Crossref PubMed Google Scholar, 7Shen Y. Dong J.-F. Romo G.M. Arceneaux W. Aprico A. Gardiner E.E. López J.A. Berndt M.C. Andrews R.K. Blood. 2002; 99: 145-150Crossref PubMed Scopus (27) Google Scholar). Furthermore, expressing mutants on CHO cells enables antibody binding and adhesion to VWF under flow to be evaluated. Several lines of evidence confirm that GPIbα homology swaps are expressed in a functional form without significant conformational disruption. First, modeling of constructs as described previously (6Shen Y. Romo G.M. Dong J.-F. Schade A. McIntire L.V. Kenny D. Whisstock J.C. Berndt M.C. López J.A. Andrews R.K. Blood. 2000; 95: 903-910Crossref PubMed Google Scholar, 11Whisstock J.C. Shen Y. López J.A. Andrews R.K. Berndt M.C. Thromb. Haemostasis. 2002; 87: 329-333Crossref PubMed Scopus (13) Google Scholar) suggests no significant structural disorder because all core-structural residues are conserved across the species. The models also illustrate conspicuous electrostatic differences in surface charge between human and canine GPIbα (Fig. 1B). A negative patch on human GPIbα is predominantly centered on Asp63 and other residues comprising this surface. In canine GPIbα, Asp63 is substituted by Arg, which together with His61 results in loss of the negative patch. There is no direct contact between the human GPIbα sequence, Leu60-Glu128 (LRR2-4), and VWF-A1 (Fig. 1B), although the co-crystal structure (10Dumas 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) reveals a single water-mediated contact between Asp63 and Arg571 of VWF-A1 (an interaction that is predicted to be abolished in the HUMCAN60-128 swap). Second, GPIbα homology swaps expressed as a GPIb-IX complex on CHO cells are recognized by conformation-sensitive antibodies (6Shen Y. Romo G.M. Dong J.-F. Schade A. McIntire L.V. Kenny D. Whisstock J.C. Berndt M.C. López J.A. Andrews R.K. Blood. 2000; 95: 903-910Crossref PubMed Google Scholar, 7Shen Y. Dong J.-F. Romo G.M. Arceneaux W. Aprico A. Gardiner E.E. López J.A. Berndt M.C. Andrews R.K. Blood. 2002; 99: 145-150Crossref PubMed Scopus (27) Google Scholar, 11Whisstock J.C. Shen Y. López J.A. Andrews R.K. Berndt M.C. Thromb. Haemostasis. 2002; 87: 329-333Crossref PubMed Scopus (13) Google Scholar): epitopes for AN51 mapping to the N-terminal sequence; His1-Ile35 and VM16d mapping to the C-terminal flank sequence; Val226-Gly268 are both present in WT-GPIbα and HUMCAN60-128; and 6D1 maps to Leu104-Glu128, present in WT-GPIbα and CANHUM60-128 (Fig. 2, cf. Fig. 1A). WM23, with an epitope C-terminal of Glu282, recognizes WT-GPIbα, HUMCAN60-128, and CANHUM60-128 confirming equivalent expression on cells used for functional analysis. Third, all of the mutants bind human VWF in the presence of the modulator botrocetin, which does not discriminate between species (6Shen Y. Romo G.M. Dong J.-F. Schade A. McIntire L.V. Kenny D. Whisstock J.C. Berndt M.C. López J.A. Andrews R.K. Blood. 2000; 95: 903-910Crossref PubMed Google Scholar), suggesting that the receptors are expressed in a functional form competent to bind ligand (Fig. 3). HUMCAN60-128 retaining the VWF-contacting residues also retains the capacity to mediate VWF-dependent adhesion at low shear (Fig. 4).FIGURE 2Binding of anti-GPIbα monoclonal antibodies to wild-type or mutant GPIbα. Flow cytometry of CHO cell lines expressing human WT-GPIbα, HUMCAN60-128, or CANHUM60-128, probed with anti-GPIbα monoclonal antibodies (WM23, AN51, 6D1, or VM16d) as indicated or the control antibody, CR1 (gray histogram shown in all panels). Epitopes for the anti-GPIbα antibodies are as described in the legend to Fig. 1A.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3Botrocetin-dependent binding of VWF to GPIbα-expressing CHO cells. Binding of fluorescein isothiocyanate-labeled human VWF (5 μg/ml, final concentration) to CHOβIX cells or CHOαβIX cells expressing WT or mutant GPIbα (106/ml) measured by flow cytometry using a method similar to that reported previously for platelets (18Goto S. Salomon D.R. Ikeda Y. Ruggeri Z.M. J. Biol. Chem. 1995; 270: 23352-23361Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar) in the absence (gray histograms) or presence (red histograms) of botrocetin (6Shen Y. Romo G.M. Dong J.-F. Schade A. McIntire L.V. Kenny D. Whisstock J.C. Berndt M.C. López J.A. Andrews R.K. Blood. 2000; 95: 903-910Crossref PubMed Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4Adhesion of GPIbα-expressing CHO cells to VWF. A, adhesion of WT-GPIbα-expressing CHO cells or CHO cells expressing HUMCAN60-128 or CANHUM60-128 mutant GPIbα to immobilized human VWF with increasing shear rate (s-1). B, rolling velocity of adherent cells from A. C, ratio of the number of adherent HUMCAN60-128 and WT-GPIbα cells from A. D, ratio of rolling velocities of HUMCAN60-128 and WT-GPIbα cells from B.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Adhesion of GPIbα-expressing cells to VWF in a flow chamber, mimicking pathophysiological shear (14Cranmer S.L. Ulsemer P. Cooke B.M. Salem H.H. de la Salle C. Lanza F. Jackson S.P. J. Biol. Chem. 1999; 274: 6097-6106Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 15Williamson D. Pikovski I. Cranmer S.L. Mangin P. Mistry N. Domagala T. Chehab S. Lanza F. Salem H.H. Jackson S.P. J. Biol. Chem. 2002; 277: 2151-2159Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 16Cranmer S.L. Pikovski I. Mangin P. Thompson P.E. Domagala T. Frazzetto M. Salem H.H. Jackson S.P. Biochem. J. 2005; 387: 849-858Crossref PubMed Scopus (39) Google Scholar), confirms that CHO cells expressing WT-GPIbα adhere to human VWF (Fig. 3), whereas CHOβIX cells or cells expressing canine GPIbα do not bind human VWF (6Shen Y. Romo G.M. Dong J.-F. Schade A. McIntire L.V. Kenny D. Whisstock J.C. Berndt M.C. López J.A. Andrews R.K. Blood. 2000; 95: 903-910Crossref PubMed Google Scholar, 7Shen Y. Dong J.-F. Romo G.M. Arceneaux W. Aprico A. Gardiner E.E. López J.A. Berndt M.C. Andrews R.K. Blood. 2002; 99: 145-150Crossref PubMed Scopus (27) Google Scholar, 14Cranmer S.L. Ulsemer P. Cooke B.M. Salem H.H. de la Salle C. Lanza F. Jackson S.P. J. Biol. Chem. 1999; 274: 6097-6106Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 15Williamson D. Pikovski I. Cranmer S.L. Mangin P. Mistry N. Domagala T. Chehab S. Lanza F. Salem H.H. Jackson S.P. J. Biol. Chem. 2002; 277: 2151-2159Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 16Cranmer S.L. Pikovski I. Mangin P. Thompson P.E. Domagala T. Frazzetto M. Salem H.H. Jackson S.P. Biochem. J. 2005; 387: 849-858Crossref PubMed Scopus (39) Google Scholar). In contrast, the HUMCAN60-128 swap (Leu60-Glu128 replaced by canine sequence) showed a substantial decrease in the number of cells adhering from flow at 50 s-1 and a significantly reduced ability to maintain adhesion with increasing shear (Fig. 4, A and C). This is despite the fact that the two major contact sites for VWF-A1 predicted by crystal structures were preserved. The interaction at low shear suggests that although HUMCAN60-128 was competent to bind, shear-dependent adhesion was severely compromised without human Leu60-Glu128 (and almost non-existent at high shear, where WT-GPIbα still remained functional). HUMCAN60-128 cells that did roll on VWF rolled 2.5-6-fold faster than WT-GPIbα; this difference became more pronounced with increasing shear rate (Fig. 4, C and D). The lack of binding of CANHUM60-128 (Fig. 4A) suggests human Leu60-Glu128 alone is not sufficient to support adhesion to VWF. In this regard, the co-crystal structures of GPIbα N-terminal domain and VWF-A1 fragments (9Huizinga 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 (503) Google Scholar, 10Dumas 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) demonstrate that elements flanking LRR2-4 make contact under the static conditions used for crystallography, and these regions are evidently required for optimal VWF recognition at low or high shear. However, the combined functional data show that the relative functional importance of specific structural elements within Leu60-Glu128 increases as the shear force increases. The aim of this study was to reconcile discrepancies between structural and functional analyses of binding of platelet GPIbα (the major ligand-binding subunit of GPIb-IX-V) to VWF, an interaction that initiates pathophysiological thrombus formation under shear stress (1Andrews R.K. Gardiner E.E. Shen Y. Whisstock J.C. Berndt M.C. Int. J. Biochem. Cell Biol. 2003; 35: 1170-1174Crossref PubMed Scopus (168) Google Scholar, 2Kroll M.H. Hellums J.D. McIntire L.V. Schafer A.I. Moake J.L. Blood. 1996; 88: 1525-1541Crossref PubMed Google Scholar, 3Ozaki Y. Asazuma N. Suziki-Inoue K. Berndt M.C. J. Thromb. Haemost. 2005; 3: 1745-1751Crossref PubMed Scopus (109) Google Scholar, 4Bhatt D.L. Topol E.J. Nat. Rev. Drug Discov. 2003; 2: 15-28Crossref PubMed Scopus (402) Google Scholar, 5Phillips D.R. Conley P.B. Sinha U. Andre P. J. Thromb. Haemost. 2005; 3: 1577-1589Crossref PubMed Scopus (73) Google Scholar). Our previous studies using human/canine chimeras of GPIbα suggested that the LRR2-4 sequence, Leu60-Glu128, is required for GPIbα-dependent adhesion to VWF under flow conditions (6Shen Y. Romo G.M. Dong J.-F. Schade A. McIntire L.V. Kenny D. Whisstock J.C. Berndt M.C. López J.A. Andrews R.K. Blood. 2000; 95: 903-910Crossref PubMed Google Scholar), whereas co-crystal structures of GPIbα and VWF fragments under static conditions reveal interactive sites, predominantly N- and C-terminal to LRR2-4, and a bidentate mode of GPIbα binding to ligand (8Sadler J.E. Science. 2002; 297: 1128-1129Crossref PubMed Scopus (40) Google Scholar, 9Huizinga 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 (503) Google Scholar, 10Dumas 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). All of the chimeras showing impaired VWF binding, however, not only lack human Leu60-Glu128 sequence (6Shen Y. Romo G.M. Dong J.-F. Schade A. McIntire L.V. Kenny D. Whisstock J.C. Berndt M.C. López J.A. Andrews R.K. Blood. 2000; 95: 903-910Crossref PubMed Google Scholar) but also lack either N- or C-terminal contact sites (8Sadler J.E. Science. 2002; 297: 1128-1129Crossref PubMed Scopus (40) Google Scholar, 9Huizinga 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 (503) Google Scholar). To establish the functional role of Leu60-Glu128, we expressed human/canine homology swaps of GPIbα on CHO cells. Three lines of evidence (molecular modeling, conformation-specific antibody binding, and botrocetin-dependent VWF binding) support the correct folding of the mutant receptor as published previously (6Shen Y. Romo G.M. Dong J.-F. Schade A. McIntire L.V. Kenny D. Whisstock J.C. Berndt M.C. López J.A. Andrews R.K. Blood. 2000; 95: 903-910Crossref PubMed Google Scholar, 7Shen Y. Dong J.-F. Romo G.M. Arceneaux W. Aprico A. Gardiner E.E. López J.A. Berndt M.C. Andrews R.K. Blood. 2002; 99: 145-150Crossref PubMed Scopus (27) Google Scholar); in addition, the HUMCAN60-128 mutant still supports adhesion to VWF at low shear rates (see above). These functional data, on adhesion of GPIbα-expressing cells to VWF in a flow chamber mimicking pathophysiological shear (14Cranmer S.L. Ulsemer P. Cooke B.M. Salem H.H. de la Salle C. Lanza F. Jackson S.P. J. Biol. Chem. 1999; 274: 6097-6106Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 15Williamson D. Pikovski I. Cranmer S.L. Mangin P. Mistry N. Domagala T. Chehab S. Lanza F. Salem H.H. Jackson S.P. J. Biol. Chem. 2002; 277: 2151-2159Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 16Cranmer S.L. Pikovski I. Mangin P. Thompson P.E. Domagala T. Frazzetto M. Salem H.H. Jackson S.P. Biochem. J. 2005; 387: 849-858Crossref PubMed Scopus (39) Google Scholar), however, show how the relative functional importance of elements within Leu60-Glu128 increases as the shear force increases. This is not to imply that regions outside LRR2-4 are not important for the interaction of GPIbα with VWF but rather that the elements within LRR2-4 become increasingly critical for binding as shear rate increases. The lack of binding of CANHUM60-128 to VWF suggests that human Leu60-Glu128 alone is not sufficient to support adhesion to VWF at low or high shear. The structure of the N-terminal domain of GPIbα has been described as a cupped human hand with LRR domains as the palm and fingers, the extended loop of the C-terminal flanking sequence as the thumb, and the anionic/sulfated sequence as the wrist (17Uff S. Clemetson J.M. Harrison T. Clemetson K.J. Emsley J. J. Biol. Chem. 2002; 277: 35657-35663Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). The dimensions of VWF-A1 preclude contact with a ligand-binding surface at the palm of GPIbα because of steric hindrance from the thumb, but Uff et al. (17Uff S. Clemetson J.M. Harrison T. Clemetson K.J. Emsley J. J. Biol. Chem. 2002; 277: 35657-35663Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar) propose that "the GPIbα crystal structure [may represent] the low affinity or "closed" form of the receptor and that a conformational change in the thumb [may be] required to unmask the A1 domain binding site." In contrast, the GPIbα·VWF-A1 complex crystal structure, with only the N- and C-terminal projections (but not the concave surface) in direct contact (8Sadler J.E. Science. 2002; 297: 1128-1129Crossref PubMed Scopus (40) Google Scholar), would appear more consistent with a low-affinity structure postulated by Uff et al. (17Uff S. Clemetson J.M. Harrison T. Clemetson K.J. Emsley J. J. Biol. Chem. 2002; 277: 35657-35663Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). However, the VWF-A1 domain could be satisfactorily docked against the concave surface of GPIbα if the "thumb" is moved (17Uff S. Clemetson J.M. Harrison T. Clemetson K.J. Emsley J. J. Biol. Chem. 2002; 277: 35657-35663Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). In the absence of VWF modulators ristocetin and botrocetin, shear stress may provide the necessary impetus to open the structure sufficiently to allow interaction between VWF-A1 and the concave surface of the LRR domain, mediated predominantly by complementary electrostatic patches on GPIbα and VWF-A1 (9Huizinga 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 (503) Google Scholar, 10Dumas 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, 11Whisstock J.C. Shen Y. López J.A. Andrews R.K. Berndt M.C. Thromb. Haemostasis. 2002; 87: 329-333Crossref PubMed Scopus (13) Google Scholar). This model would predict that although the contact sites may confer specificity, there is an increasing requirement for the electronegative surface encompassing Leu60-Glu128 as the shear rate increases, consistent with the functional data (6Shen Y. Romo G.M. Dong J.-F. Schade A. McIntire L.V. Kenny D. Whisstock J.C. Berndt M.C. López J.A. Andrews R.K. Blood. 2000; 95: 903-910Crossref PubMed Google Scholar) (Fig. 4). Definitive resolution of the contact surface between receptor and ligand will undoubtedly require further structural information. However, in the context of shear this will be almost impossible to achieve using current technologies. Instead, the functional analysis of homology domain swaps of GPIbα identifies shear-dependent regions of the receptor involved in binding VWF.
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