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• W1856218379 abstract "Platelet-type von Willebrand disease is a bleeding disorder resulting from gain-of-function mutations of glycoprotein (GP) Ibα that increase its affinity for von Willebrand factor (vWf). The two known naturally occurring mutations, G233V and M239V, both enrich the valine content of an already valine-rich region within the Cys209–Cys248 disulfide loop. We tested the effect of converting other non-valine residues in this region to valine. Of 10 mutants expressed in CHO cells as components of GP Ib-IX complexes, four displayed a gain-of-function phenotype (G233V, D235V, K237V, and M239V) based on 125I-vWf binding and adhesion to immobilized vWf. The remainder displayed loss-of-function phenotypes. The gain-of-function mutants bound vWf spontaneously and had a heightened response to low concentrations of ristocetin or botrocetin, whereas the loss-of-function mutants bound vWf more poorly than wild-type GP Ibα. No distinct gain- or loss-of-function conformations were identified with conformation-sensitive antibodies. Compared with cells expressing wild-type GP Ibα, cells expressing the gain-of-function mutants rolled significantly more slowly over immobilized vWf under flow than wild-type cells and were able to adhere to vWf coated at lower densities. In aggregate, these data indicate that the region of GP Ibα bounded by Asn226 and Ala244 regulates the affinity for vWf. Platelet-type von Willebrand disease is a bleeding disorder resulting from gain-of-function mutations of glycoprotein (GP) Ibα that increase its affinity for von Willebrand factor (vWf). The two known naturally occurring mutations, G233V and M239V, both enrich the valine content of an already valine-rich region within the Cys209–Cys248 disulfide loop. We tested the effect of converting other non-valine residues in this region to valine. Of 10 mutants expressed in CHO cells as components of GP Ib-IX complexes, four displayed a gain-of-function phenotype (G233V, D235V, K237V, and M239V) based on 125I-vWf binding and adhesion to immobilized vWf. The remainder displayed loss-of-function phenotypes. The gain-of-function mutants bound vWf spontaneously and had a heightened response to low concentrations of ristocetin or botrocetin, whereas the loss-of-function mutants bound vWf more poorly than wild-type GP Ibα. No distinct gain- or loss-of-function conformations were identified with conformation-sensitive antibodies. Compared with cells expressing wild-type GP Ibα, cells expressing the gain-of-function mutants rolled significantly more slowly over immobilized vWf under flow than wild-type cells and were able to adhere to vWf coated at lower densities. In aggregate, these data indicate that the region of GP Ibα bounded by Asn226 and Ala244 regulates the affinity for vWf. glycoprotein phosphate-buffered saline von Willebrand factor von Willebrand disease Chinese hamster ovary wild type Injuries to blood vessels must be sealed rapidly and efficiently to prevent blood loss. The process of sealing these defects begins when platelets adhere to subendothelial matrix exposed when the overlying endothelial layer is removed. This process sets off a sequence of events that includes the activation of the platelets, their spread along the exposed matrix, release of their granule contents, and their aggregation to form a hemostatic plug. The entire process is initiated by an interaction between the glycoprotein (GP)1 Ib-IX-V complex on the platelet surface and von Willebrand factor (vWf) exposed on the subendothelium. von Willebrand factor also circulates in the plasma but does not, under normal circumstances, interact in the fluid phase with the platelet complex. This interaction requires an activation stimulus: either very high fluid shear forces, such as may be found in regions of arterial stenosis, the presence of the nonphysiological modulators ristocetin (a peptide antibiotic) or botrocetin (a family of snake venom proteins), or immobilization of vWf on a surface (1Andrews R.K. López J.A. Berndt M.C. Int. J. Biochem. Cell Biol. 1997; 29: 91-105Crossref PubMed Scopus (177) Google Scholar). The GP Ib-IX-V complex comprises four transmembrane polypeptide chains, GP Ibα, GP Ibβ, GP IX, and GP V, but only the largest of these, GP Ibα, has been shown to interact with vWf (1Andrews R.K. López J.A. Berndt M.C. Int. J. Biochem. Cell Biol. 1997; 29: 91-105Crossref PubMed Scopus (177) Google Scholar). This polypeptide is a typical type I transmembrane protein and can be subdivided into four functional domains: a 45-kDa region of approximately 300 amino acids at the amino terminus; a rigid mucin-like stalk, the macroglycopeptide, which separates the ligand-binding domain from the plasma membrane; a single transmembrane domain; and a 97-amino acid cytoplasmic domain that connects the entire complex to the platelet cytoskeleton (2López J.A. Dong J.F. Curr. Opin. Hematol. 1997; 4: 323-329Crossref PubMed Scopus (93) Google Scholar). The vWf-binding region is contained in the 45-kDa N-terminal domain. In this region, three sites have been implicated as important for the interaction with vWf (Fig. 1 A): the N-terminal disulfide loop and adjacent leucine-rich repeats (His1–Ala200), the C-terminal disulfide loop region (Phe201–Gly268), and an anionic, tyrosine-sulfated sequence, Asp269–Glu282. Ample evidence supports the involvement of each region in vWf binding. First, mutations from patients with the deficiency disorder of the complex, Bernard-Soulier syndrome, implicate the region spanning residues His1 to Ala200 that contains seven tandem repeats of a conserved 24-amino acid leucine-rich sequence. These repeats assign GP Ibα to a large family with similar motifs, a family to which GP Ibβ, GP IX, and GP V also belong (3López J.A. Blood Coagul. Fibrinolysis. 1994; 5: 97-119Crossref PubMed Scopus (291) Google Scholar). Mutagenesis and peptide studies also implicate the highly acidic region spanning residues Glu269–Glu287 as being involved in vWf binding (4Murata M. Ware J. Ruggeri Z.M. J. Biol. Chem. 1991; 266: 15474-15480Abstract Full Text PDF PubMed Google Scholar). This region has been shown to be postranslationally sulfated on three tyrosines (5Dong J.-F. Li C.Q. López J.A. Biochemistry. 1994; 33: 13946-13953Crossref PubMed Scopus (107) Google Scholar, 6Ward C.M. Andrews R.K. Smith A.I. Berndt M.C. Biochemistry. 1996; 35: 4929-4938Crossref PubMed Scopus (181) Google Scholar), a requirement for the optimal binding of vWf, whether induced by modulator (5Dong J.-F. Li C.Q. López J.A. Biochemistry. 1994; 33: 13946-13953Crossref PubMed Scopus (107) Google Scholar, 6Ward C.M. Andrews R.K. Smith A.I. Berndt M.C. Biochemistry. 1996; 35: 4929-4938Crossref PubMed Scopus (181) Google Scholar, 7Marchese P. Murata M. Mazzucato M. Pradella P. De Marco L. Ware J. Ruggeri Z.M. J. Biol. Chem. 1995; 270: 9571-9578Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar) or under flow conditions with vWf immobilized on a surface (8Fredrickson B.J. Dong J.F. McIntire L.V. López J.A. Blood. 1998; 92: 3684-3693Crossref PubMed Google Scholar). Between these two regions lie two disulfide loops formed by bonds between Cys209 and Cys248 and between Cys211 and Cys264 (Fig. 1 B). Synthetic peptide blocking studies have suggested a role for these loops in vWf binding (9Katagiri Y. Hayashi Y. Yamamoto K. Tanoue K. Kosaki G. Yamazaki H. Thromb. Haemostasis. 1990; 63: 122-126Crossref PubMed Scopus (78) Google Scholar, 10Cruz M.A. Petersen E. Turci S.M. Handin R.I. J. Biol. Chem. 1992; 267: 1303-1309Abstract Full Text PDF PubMed Google Scholar), as have studies of the rare bleeding disorder platelet-type (pseudo) von Willebrand disease (vWD) (11Miller J.L. Cunningham D. Lyle V.A. Finch C.N. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4761-4765Crossref PubMed Scopus (141) Google Scholar, 12Russell S.D. Roth G.J. Blood. 1993; 81: 1787-1791Crossref PubMed Google Scholar, 13Takahashi H. Murata M. Moriki T. Anbo H. Furukawa T. Nikkuni K. Shibata A. Handa M. Kawai Y. Watanabe K. Ikeda Y. Blood. 1995; 85: 727-733Crossref PubMed Google Scholar). These findings are supported by functional analysis of anti-GP Ibα monoclonal antibodies. von Willebrand factor binding to GP Ibα induced by ristocetin or botrocetin is strongly inhibited by antibodies that recognize epitopes within the N-terminal 128 residues (AK2, Hip1, C-34, 6D1), or the C-terminal disulfide loop region between Phe201 and Gly268 (AP1) (14Shen Y. Romo G.M. Dong J. 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). Other antibodies that preferentially inhibit botrocetin-induced vWf binding map to Phe201–Gly268 (VM16d) or to the sulfated sequence (SZ2). Paradoxically, platelet-type vWD is caused by mutations of GP Ibα that increase its affinity for vWf (15Miller J.L. Castella A. Blood. 1982; 60: 790-794Crossref PubMed Google Scholar). The bleeding diathesis is thought to result from rapid clearance from the plasma of the highest molecular weight multimers of vWf (16Miller J.L. Thromb. Haemostasis. 1996; 75: 865-869Crossref PubMed Scopus (64) Google Scholar), which are the most effective multimers in mediating platelet adhesion (17Federici A.B. Bader R. Pagani S. Colibretti M.L. De Marco L. Mannucci P.M. Br. J. Haematol. 1989; 73: 93-99Crossref PubMed Scopus (131) Google Scholar). The platelets of patients with this disorder either bind vWf in the absence of modulators or require much lower ristocetin concentrations to induce the interaction (15Miller J.L. Castella A. Blood. 1982; 60: 790-794Crossref PubMed Google Scholar, 18Weiss H.J. Meyer D. Rabinowitz R. Pietu G. Girma J.-P. Vicic W.J. Rogers J. N. Engl. J. Med. 1982; 306: 326-333Crossref PubMed Scopus (177) Google Scholar). Miller and co-workers showed that one case of platelet-type vWD resulted from a Val for Gly substitution at residue 233 (11Miller J.L. Cunningham D. Lyle V.A. Finch C.N. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4761-4765Crossref PubMed Scopus (141) Google Scholar); Russell and Roth described a substitution of Val for Met at residue 239 in another kindred with the disorder (12Russell S.D. Roth G.J. Blood. 1993; 81: 1787-1791Crossref PubMed Google Scholar). The latter mutation was subsequently described in an unrelated Japanese kindred (13Takahashi H. Murata M. Moriki T. Anbo H. Furukawa T. Nikkuni K. Shibata A. Handa M. Kawai Y. Watanabe K. Ikeda Y. Blood. 1995; 85: 727-733Crossref PubMed Google Scholar) and as occurring de novo in another patient (19Kunishima S. Heaton D.C. Naoe T. Hickton C. Mizuno S. Saito H. Kamiya T. Blood Coagul. Fibrinolysis. 1997; 8: 311-315Crossref PubMed Scopus (24) Google Scholar). Both mutations are close to each other in the primary structure of the polypeptide within the Cys209–Cys248 disulfide loop, implicating this loop either as being directly involved in vWf binding or as a regulatory region that under normal circumstances directly or indirectly prevents the exposure of the ligand-binding regions. Of interest, not only do these mutations affect residues in close proximity to each other in the linear sequence of GP Ibα, both mutations also convert existing non-valine amino acids to valine. Because of these common features, we examined the effect of converting other non-valine residues in the region to valine. We compared the mutants with wild-type GP Ibα and with the known platelet-type vWD mutants expressed as part of recombinant GP Ib-IX complexes in Chinese hamster ovary (CHO) cells in several ways; we examined the effect of the mutations on expression of the complex, on its ability to support modulator-induced vWf binding, and on the ability of GP Ib-IX complex-expressing cells to adhere to and roll on a matrix of vWf under conditions of flow. Mutagenesis was performed directly on the GP Ibα cDNA cloned into the EcoRI site of the mammalian expression vector pDX (20López J.A. Leung B. Reynolds C.C. Li C.Q. Fox J.E.B. J. Biol. Chem. 1992; 267: 12851-12859Abstract Full Text PDF PubMed Google Scholar) using a commercial polymerase chain reaction-based mutagenesis kit (QuikChangeTM; Stratagene, La Jolla, CA). Eleven mutations were created in which each codon for non-valine amino acid residues within a region spanning Asn226–Ala244 was converted to a valine codon. Mutated GP Ibα cDNAs were sequenced completely to verify the mutations and to eliminate those mutants that carried unwanted mutations. The sequencing reactions were performed using the ABI Dye terminator kit, and the sequencing gels were run on an ABI model 373A automated sequencer (ABI, San Leandro, CA). To develop stable cell lines expressing mutant GP Ibα polypeptides, the mutated GP Ibα cDNAs were transfected into CHO βIX cells (which stably express GP Ibβ and GP IX) (21López J.A. Weisman S. Sanan D.A. Sih T. Chambers M. Li C.Q. J. Biol. Chem. 1994; 269: 23716-23721Abstract Full Text PDF PubMed Google Scholar) using a commercial kit (LipofectAMINE, Life Technologies Inc.) (5Dong J.-F. Li C.Q. López J.A. Biochemistry. 1994; 33: 13946-13953Crossref PubMed Scopus (107) Google Scholar, 22Li C.Q. Dong J. Lanza F. Sanan D.A. Sae-Tung G. López J.A. J. Biol. Chem. 1995; 270: 16302-16307Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The plasmid pREP4 (Invitrogen, Carlsbad, CA), which carries the hygromycin resistance gene, was cotransfected with each of the GP Ibα mutant cDNAs. Transfected cells were grown for 3 days in α-minimal essential medium (Life Technologies) containing 10% heat-inactivated fetal bovine serum and then in the same medium containing increasing doses of hygromycin for selection. The final hygromycin concentration was 500 μg/ml. The cells were then repeatedly sorted for GP Ib-IX expression with antibody-coupled magnetic beads. For this, cells were detached with 0.53 mmEDTA and resuspended in phosphate-buffered saline (PBS) containing 1% bovine serum albumin. The cells were incubated first with the monoclonal GP Ibα antibody WM23 (kindly provided by Dr. Michael C. Berndt, Baker Medical Research Institute, Melbourne, Australia) for 30 min at room temperature and then with magnetic beads coated with sheep anti-mouse IgG (Dynabeads, Dynal, Inc., Lake Success, NY). Bead-bound cells were separated from unbound cells on a magnetic apparatus (Dynal). To determine the effect of the mutations on the surface expression of GP Ibα, we transiently expressed the wild-type or mutant cDNAs by the method described above but without drug selection and studied expression of the complex by flow cytometry 3 days after transfection. Cells expressing the wild-type or mutant polypeptides were labeled with WM23. This antibody binds in a domain distinct from the mutated region; its binding is therefore unlikely to be affected by the mutations (23Berndt M.C. Du X. Booth W.J. Biochemistry. 1988; 27: 633-640Crossref PubMed Scopus (130) Google Scholar, 24Andrews R.K. Booth W.J. Gorman J.J. Castaldi P.A. Berndt M.C. Biochemistry. 1989; 28: 8317-8326Crossref PubMed Scopus (155) Google Scholar). The procedure for performing flow cytometry was described previously (25Dong J. Li C.Q. Sae-Tung G. Hyun W. Afshar-Kharghan V. López J.A. Biochemistry. 1997; 36: 12421-12427Crossref PubMed Scopus (47) Google Scholar). Briefly, cells grown in a monolayer were detached with 0.53 mm EDTA, washed with PBS, and incubated with 1 μg/ml WM23 in PBS containing 1% bovine serum albumin for 60 min at room temperature. The cells were then washed to remove unbound antibody and incubated with fluorescein isothiocyanate-conjugated rabbit anti-mouse IgG (Zymed Laboratories Inc., South San Francisco, CA) for 30 min at room temperature. The cells were then analyzed by flow cytometry on a FACScan flow cytometer (Becton Dickinson, San Jose, CA), stimulating with 488-nm laser light and collecting light emitted at >530 nm. The binding of several monoclonal antibodies with epitopes in the GP Ibα ligand-binding domain was also analyzed by flow cytometry to determine if the mutations affected the global conformation of this region. These antibodies have previously been shown to block the vWf-GP Ib-IX-V complex interaction (5Dong J.-F. Li C.Q. López J.A. Biochemistry. 1994; 33: 13946-13953Crossref PubMed Scopus (107) Google Scholar, 6Ward C.M. Andrews R.K. Smith A.I. Berndt M.C. Biochemistry. 1996; 35: 4929-4938Crossref PubMed Scopus (181) Google Scholar, 26Ruan C. Tobelem G. McMichael A.J. Drouet L. Legrand Y. Degos L. Kieffer N. Lee H. Caen J.P. Br. J. Haematol. 1981; 49: 511-519Crossref PubMed Scopus (70) Google Scholar). The antibodies tested were AN51 (DAKO, Carpinteria, CA), AK2 (RDI Research Diagnostic Inc., Flanders, NJ), AP1 (a gift from Dr. Robert Montgomery, the Blood Research Institute, Milwaukee, WI), C-34 (obtained from the 5th International Workshop on Leukocyte Antigens), and SZ2 (RDI Research Diagnostics, Inc.). Purified human vWf (a gift from Dr. Michael Berndt) was iodinated with Na125I as described previously (5Dong J.-F. Li C.Q. López J.A. Biochemistry. 1994; 33: 13946-13953Crossref PubMed Scopus (107) Google Scholar). Free iodine was separated from labeled vWf by passing the iodination reaction solution through a Sephadex G25 column (PD10 column; Amersham Pharmacia Biotech). The specific activity of the 125I-labeled vWf was 0.27 mCi/mg of protein. Modulator-induced vWf binding to the cells expressing the GP Ibα mutants was assessed by methods described previously (5Dong J.-F. Li C.Q. López J.A. Biochemistry. 1994; 33: 13946-13953Crossref PubMed Scopus (107) Google Scholar, 20López J.A. Leung B. Reynolds C.C. Li C.Q. Fox J.E.B. J. Biol. Chem. 1992; 267: 12851-12859Abstract Full Text PDF PubMed Google Scholar). Briefly, CHO cells expressing the mutants were detached with 0.53 mmEDTA and washed with Ca2+- and Mg2+-free Tyrode's buffer (138 mm NaCl, 5.5 mm glucose, 12 mm NaHCO3, 0.36 mmNaH2PO4, 2.9 mm KCl, pH 7.4). Washed cells were resuspended in Tyrode's buffer containing 1% bovine serum albumin to a final concentration of 4 × 106cells/ml. An aliquot of each cell suspension was used to determine the cell surface levels of GP Ibα by flow cytometry. To assess vWf binding, 25-μl aliquots of the cell suspension were mixed with either 1.0 mg/ml ristocetin (Sigma) or 20 μg/ml of purified botrocetin (24Andrews R.K. Booth W.J. Gorman J.J. Castaldi P.A. Berndt M.C. Biochemistry. 1989; 28: 8317-8326Crossref PubMed Scopus (155) Google Scholar) and increasing quantities of 125I-vWf. The final volume of the reaction mixture was adjusted to 100 μl with Tyrode's buffer. The mixture was incubated for 30 min at room temperature and then loaded onto a minicolumn containing 20% sucrose in Tyrode's buffer. Cell-bound vWf was separated from vWf in solution by centrifuging the minicolumn for 5 min at 10,000 × g at room temperature. The capillary tips containing the cell pellets were excised, and the radioactivity was counted in a γ-counter. Specific binding was first determined by subtracting background counts (binding to CHO βIX cells) and then corrected for the surface receptor levels as determined by flow cytometry. The same procedure was used to examine the binding of vWf to the mutants at low concentrations of the modulators. For ristocetin, the cells were incubated at ristocetin concentrations of 0, 0.2, and 0.4 mg/ml, and for botrocetin, they were incubated at concentrations of 0, 0.1, and 0.2 μg/ml, both in the presence of 1.6 μg/ml125I-vWf. Human vWf was purified from blood cryoprecipitate by glycine and NaCl precipitation and subsequent separation on a 2.5 × 50-cm Sepharose 4B column (bed volume of 3000 ml; Amersham Pharmacia Biotech) as described previously (8Fredrickson B.J. Dong J.F. McIntire L.V. López J.A. Blood. 1998; 92: 3684-3693Crossref PubMed Google Scholar). A commercial kit was used to determine the concentration of vWf according to the manufacturer's instructions (Ramo Laboratories, Inc., Houston, TX). Purified vWf was immobilized onto 11 × 22-mm2 glass coverslips by incubating the coverslips with 200 μl of PBS containing the specified vWf concentration for 45 min at room temperature. Unbound vWf was removed by washing the coverslips with 0.9% NaCl. Cell rolling was induced in a parallel plate flow chamber and observed by phase-contrast video microscopy. The parallel plate flow chamber is composed of a polycarbonate slab, a silicon gasket, and a vWf-coated glass coverslip. The three components were held together by vacuum in such a way that the coverslip formed the bottom of the chamber. A syringe pump connected to the outlet port draws PBS through the chamber at defined flow rates to generate desired wall shear stresses, which are determined by the height of the gap (the thickness of the silicon gasket), the width of the chamber, the fluid viscosity, and the flow rate (27Slack S.M. Turitto V.T. Thromb. Haemostasis. 1994; 72: 777-781Crossref PubMed Scopus (36) Google Scholar). The assembled parallel plate flow chamber was mounted onto an inverted stage microscope (DIAPHOT-TMD; Nikon, Garden City, NY) equipped with a silicon-intensified target video camera (model C2400; Hammatus, Waltman, MA) connected to a video cassette recorder. The parallel plate flow chamber was maintained at 37 °C by a thermostatic air bath during the experiments. The cell suspension either was allowed to preincubate in the chamber for 1 min (0.6 ml of a suspension containing 500,000 cells/ml) before perfusing the chamber with PBS or was perfused directly through the chamber (100,000 cells/ml) without prior incubation. Cell rolling in a single view field was recorded in real time for 3–5 min on videotape. The video data were analyzed off-line using Inovision imaging software (IC-300 Modular Image Processing; Workstation Inovision Corp., Durham, NC) to quantify the number and velocity of rolling cells (28Jones D.A. Abbassi O. McIntire L.V. McEver R.P. Smith C.W. Biophys. J. 1993; 65: 1560-1569Abstract Full Text PDF PubMed Scopus (209) Google Scholar, 29Kukreti S. Konstantopoulos K. Smith C.W. McIntire L.V. Blood. 1997; 89: 4104-4111Crossref PubMed Google Scholar). A rolling cell was defined as a cell that translocated in the direction of fluid flow while maintaining constant contact with the vWf matrix. The rolling velocity was defined as the distance traveled by a single cell during a defined period (1–5 s). The data were analyzed using Student'st test. A p value of <0.05 was considered statistically significant. The region of GP Ibα affected by the platelet-type vWD gain-of-function mutations, which lies within the Cys209–Cys248 disulfide loop, is rich in valine residues (Fig. 1 B). In addition, each of the known platelet-type vWD mutations changes a non-valine residue to valine. To evaluate further the role of this region in regulating the binding of vWf, we converted all non-valine residues to valine within a region encompassed by Asn226and Ala244. The mutations were introduced individually into the human GP Ibα cDNA as single or double nucleotide substitutions to convert the existing codon to a valine codon (TableI). The resulting mutants were transfected into CHO βIX cells, which stably express GP Ibβ and GP IX, both of which are necessary for optimal surface expression of a functional vWf-binding complex (20López J.A. Leung B. Reynolds C.C. Li C.Q. Fox J.E.B. J. Biol. Chem. 1992; 267: 12851-12859Abstract Full Text PDF PubMed Google Scholar, 30Dong J. Gao S. López J.A. J. Biol. Chem. 1998; 273: 31449-31454Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Transient expression studies showed that 10 of the mutants (N226V, K231V, Q232V, G233V, D235V, K237V, A238V, M239V, T240V, and A244V) were expressed on the CHO βIX cell surface at levels similar to that of wild-type GP Ibα (data not shown). The Y228V mutant was expressed in only trace quantities on the cell surface and was not evaluated further. We then established stable cell lines expressing each of the mutants by cotransfecting into CHO βIX cells each mutant GP Ibα plasmid with pREP-4, a plasmid containing a gene encoding hygromycin resistance. The stable cell lines were used in all subsequent experiments.Table ISummary of the valine mutations of GP IbαMutantsCodon changesAmino acid changesN266VAAT to GTTAsn266 to ValY288VATC to GTCTyr288to ValK231VAAG to GTGLys231 to ValQ232VCAA to GTAGln232 to ValG233V1-aMutation reported in platelet-type von Willebrand disease.GGT to GTTGly233 to ValD235VGAC to GTCAsp235to ValK237VAAG to GTCLys237 to ValA238VGCC to GTCAla238 to ValM239V1-aMutation reported in platelet-type von Willebrand disease.ATG to GTGMet239 to ValT240VACC to GTCThr240to ValA244VGCC to GTCAla244 to Val1-a Mutation reported in platelet-type von Willebrand disease. Open table in a new tab To determine the effect of the valine mutations on vWf binding, we first evaluated binding in the presence of either ristocetin or botrocetin. In the presence of ristocetin, we found two patterns of binding of the mutants compared with the binding of the wild-type complex: they either bound similar or higher amounts of vWf (G233V, D235V, K237V, and M239V) or they bound less (N226V, K231V, Q232V, A238V, T240V, and A244V) (Fig.2, A and B). Both G233V and K237V bound significantly more vWf than did the wild-type cells at all concentrations of vWf used (n = 6,p < 0.01), whereas vWf binding to D235V and M239V was higher at the lower vWf concentrations but indistinguishable from that of the wild-type cells at concentrations that were saturating for the wild-type complex (n = 6, p = 0.08 for D235V, and p = 0.10 for M239V, for a vWf concentration of 1.6 μg/ml). The loss-of-function mutants bound significantly less vWf at all concentrations (n = 3, p < 0.05) (Fig. 2 B). The mutants defined as gain-of-function mutants by their ristocetin sensitivity displayed similar botrocetin-induced vWf binding characteristics as the wild-type cells (Fig. 2 C) except for mutant M239V, for which vWf binding saturated at lower vWf concentrations (n = 8, p < 0.02 at 0.2 μg/ml and n = 8, p < 0.01 at 0.4 μg/ml). Of the mutants defined as loss-of-function by defective ristocetin-induced binding, only two, N226V and K231V, demonstrated lower botrocetin-induced binding than did CHO αβIX cells (n = 3, p < 0.05) (Fig.2 D). The rest of the loss-of-function mutants bound vWf in a manner similar to the binding of CHO αβIX cells, with the exception of the cells expressing the T240V mutant, which bound more vWf than the wild-type at the higher vWf concentrations. One defining characteristic of the platelet-type vWD phenotype is the ability of the platelets to bind vWf at much lower ristocetin concentrations than those that support vWf binding to normal platelets (11Miller J.L. Cunningham D. Lyle V.A. Finch C.N. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4761-4765Crossref PubMed Scopus (141) Google Scholar, 12Russell S.D. Roth G.J. Blood. 1993; 81: 1787-1791Crossref PubMed Google Scholar, 13Takahashi H. Murata M. Moriki T. Anbo H. Furukawa T. Nikkuni K. Shibata A. Handa M. Kawai Y. Watanabe K. Ikeda Y. Blood. 1995; 85: 727-733Crossref PubMed Google Scholar). The same increased sensitivity to ristocetin has also been observed for recombinant GP Ibα fragments containing the mutations (31Murata M. Russell S.R. Ruggeri Z.M. Ware J. J. Clin. Invest. 1993; 91: 2133-2137Crossref PubMed Scopus (37) Google Scholar, 32Moriki T. Murata M. Kitaguchi T. Anbo H. Handa M. Watanabe K. Takahashi H. Ikeda Y. Blood. 1997; 90: 698-705Crossref PubMed Google Scholar). We therefore examined the ristocetin sensitivity of the four mutants with levels of vWf binding similar to or higher than that of wild-type GP Ibα. Two features of the mutants immediately became apparent (Fig. 3,top panel). First, they all bound significantly more vWf than wild-type cells in the absence of ristocetin (n = 4, p < 0.05). Second, each mutant also displayed a heightened sensitivity to low ristocetin concentrations compared with cells expressing the wild-type complex. At ristocetin concentrations of both 0.2 mg/ml and 0.4 mg/ml, the four mutants bound substantially more vWf than did the wild-type cells (n = 4, p < 0.01). Earlier studies have suggested that gain-of-function mutants associated with platelet-type vWD also display enhanced sensitivity to botrocetin (33Takahashi H. Nagayama R. Hattori A. Shibata A. Am. J. Hematol. 1985; 18: 179-189Crossref PubMed Scopus (7) Google Scholar). In contrast to the findings at higher concentrations of botrocetin, which showed no differences between the gain-of-function mutants and wild-type GP Ibα in vWf binding at saturating vWf concentrations (Fig. 2 C), the gain-of-function mutants were significantly more sensitive to low concentrations of botrocetin than was wild-type GP Ibα (Fig. 3, bottom panel). Only two of the mutants, G233V and K237V, showed enhanced sensitivity to botrocetin at 0.1 μg/ml, but botrocetin at 0.2 μg/ml enhanced vWf binding to all four mutants. Neither concentration of botrocetin stimulated vWf binding to wild-type GP Ibα. It has been proposed that the mutations in platelet-type vWD favor an active GP Ibα conformation that may be detectable by differences in monoclonal antibody binding characteristics between the mutant and wild-type polypeptides. We therefore examined the binding to the mutants of several GP Ibα monoclonal antibodies with epitopes in the ligand-binding domain (Fig. 4). Binding was normalized to the binding of WM23 to adjust for any potential differences in the surface expression of GP Ibα between cell lines. With the exception of SZ2, the antibodies bound at slightly lower levels to the mutants than to cells expressing wild-type GP Ibα. SZ2 binding was essentially equivalent for the mutants and wild-type cells. The binding patterns of four antibodies, AK2, AN51, AP1, and C-34, were similar. Of the mutants, only G2" @default.
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