SEARCH:   Advanced

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Shen, Y. Articles by Andrews, R. K. Search for Related Content PubMed PubMed Citation Articles by Shen, Y. Articles by Andrews, R. K. Related Collections Hemostasis, Thrombosis, and Vascular Biology Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 1 January 2002, Vol. 99, No. 1, pp. 145-150 HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY Functional analysis of the C-terminal flanking sequence of platelet glycoprotein Ib using canine-human chimeras Yang Shen, Jing-fei Dong, Gabriel M. Romo, Wendy Arceneaux, Andrea Aprico, Elizabeth E. Gardiner, José A. López, Michael C. Berndt, and Robert K. Andrews From the Hazel and Pip Appel Vascular Biology Laboratory, Baker Medical Research Institute, Melbourne, Australia, and the Departments of Medicine and of Molecular and Human Genetics, Baylor College of Medicine and Veterans Affairs Medical Center, Houston, TX.     Abstract Top Abstract Introduction Materials and methods Results Discussion References Platelet glycoprotein Ib-IX-V (GPIb-IX-V) mediates adhesion to von Willebrand factor (vWF) in (patho)physiological thrombus formation. vWF binds the N-terminal 282 residues of GPIb, consisting of an N-terminal flank (His1-Ile35), 7 leucine-rich repeats (Leu36-Ala200), a C-terminal flank (Phe201-Gly268), and a sulfated tyrosine sequence (Asp269-Glu282). By expressing canine-human chimeras of GPIb on Chinese hamster ovary cells, binding sites for functional anti-GPIb antibodies to individual domains were previously mapped, and it was shown that leucine-rich repeats 2 to 4 were required for optimal vWF recognition under static or flow conditions. Using novel canine-human chimeras dissecting the C-terminal flank, it is now demonstrated that (1) Phe201-Glu225 contains the epitope for AP1, an anti-GPIb monoclonal antibody that inhibits both ristocetin- and botrocetin-dependent vWF binding; (2) VM16d, an antibody that preferentially inhibits botrocetin-dependent vWF binding, recognizes the sequence Val226-Gly268, surrounding Cys248, which forms a disulfide-bond with Cys209; (3) vWF binding to chimeric GPIb is comparable to wild-type in 2 chimeras in which the sixth leucine-rich repeat was of the same species as the first disulfide loop (Phe201-Cys248) of the C-terminal flank, suggesting an interaction between these domains may be important for optimal vWF binding; and (4) replacing the C-terminal flank second disulfide loop (Asp249-Gly268) in human GPIb with the corresponding canine sequence enhanced vWF binding under static and flow conditions, providing the first evidence for a gain-of-function phenotype associated with the second loop of the C-terminal flank. (Blood. 2002;99:145-150) © 2002 by The American Society of Hematology.     Introduction Top Abstract Introduction Materials and methods Results Discussion References The platelet membrane glycoprotein Ib-IX-V (GPIb-IX-V) complex plays a central role in vascular biology. The interaction of GPIb with its adhesive ligand, von Willebrand factor (vWF), in the subendothelial matrix mediates platelet adhesion at high shear in the hemostatic response to vessel wall injury.1,2 In thrombosis, pathologic shear stress induces binding of platelet GPIb-IX-V to plasma vWF, initiating platelet aggregation.3 More recently, GPIb has been identified as a counter-receptor for P-selectin expressed on activated endothelial cells4,5 and for the leukocyte M2 integrin, Mac-1.6 These interactions potentially mediate platelet-endothelium or platelet-leukocyte adhesion in thrombosis, inflammation, and atherogenesis. GPIb-IX-V also constitutes a binding site, and a substrate for, -thrombin.2 Recent evidence suggests that GPIb acts as a cofactor for thrombin-dependent cleavage of the 7-transmembrane protease-activated receptor, PAR-1, on platelets7 and that GPIb is itself a thrombin receptor following thrombin-dependent cleavage of GPV.8 Other GPIb ligandsfactor XII and high-molecular-weight kininogenare inhibitors of thrombin binding, and they down-regulate thrombin-dependent platelet activation.9,10 All these ligands of GPIbvWF, P-selectin, Mac-1, thrombin, factor XII, and high-molecular-weight kininogenrecognize the N-terminal 282 residues of GPIb.2 The binding site for vWF comprises elements from 4 structural regions of GPIb: the N-terminal flank (His1-Ile35), 7 leucine-rich repeats (Leu36-Ala200), a C-terminal flank (Phe201-Gly268), and a sulfated tyrosine sequence (Asp269-Glu282).2,11 Previous studies with the metalloproteinase mocarhagin, which cleaves between Glu282 and Asp283 of GPIb, suggest that the vWF binding site is within residues 1 to 282.11 We have recently expressed on Chinese hamster ovary (CHO) cells canine-human and human-canine chimeras of GPIb, corresponding to boundaries between these structural domains. Murine monoclonal antibodies against the N-terminal domain of human GPIb are species-specific and do not bind canine GPIb; canine and human amino acid sequences are only 65.2% identical in the region His-1-Glu-282.12,13 The vWF activator, ristocetin, induces binding of human vWF only to human GPIb, not to the canine receptor.11,14 In contrast, the snake venom modulator, botrocetin, does not discriminate between the 2 species, inducing the binding of human vWF to human and canine GPIb.11 Botrocetin-dependent vWF binding to all the GPIb chimeras provided evidence that they retained a functional conformation. These combined studies allow detailed mapping of binding sites for anti-GPIb monoclonal antibodies and vWF, and they suggest leucine-rich repeats 2, 3, and 4 of GPIb are critical for vWF binding to GPIb-IX-V induced by ristocetin or under hydrodynamic flow.11 The C-terminal flank domain (Phe201-Gly268) contains 2 loops by virtue of disulfide bonds between Cys209-Cys248 and Cys211-Cys264. Several lines of evidence implicate the C-terminal flank as a critical factor in regulating vWF binding. Congenital gain-of-function mutations within the GPIb gene (platelet-type von Willebrand disease) result in single amino acid substitutions, Gly233 to valine or Met239 to valine, within the first of the 2 disulfide loops comprising the C-terminal flank domain.15-17 Mutation of Gly233 or Met239 to valine in recombinant GPIb, in addition to the artificial mutation of Asp235 or Lys237 to valine, also results in a gain-of-function phenotype.18,19 In contrast, mutation of Ala238 to valine results in partial loss of function.18 The anti-GPIb monoclonal antibodies, AP1 and VM16d, which map into the C-terminal flank, also inhibit vWF binding. AP1 blocks both ristocetin- and botrocetin-induced binding and rolling of GPIb-IX-expressing cells on vWF, whereas VM16d preferentially inhibits botrocetin-dependent binding11,20 but not ristocetin-dependent binding or GPIb-IX-dependent rolling on vWF.21 In addition, both AP1 and VM16d inhibit Mac-1 binding to GPIb.6 In this study, we have analyzed a series of canine-human chimeras of GPIb corresponding to the replacement of specific sequences involving the C-terminal flank. These chimeras, expressed on CHO cells, enabled more precise mapping of binding sites for the functional anti-GPIb monoclonal antibodies AP1 and VM16d, and they allowed the functional importance of each disulfide loop to be assessed regarding vWF binding to GPIb. Combined results infer a possible interaction between the sixth leucine-rich repeat and the first disulfide loop (Phe201-Cys248), and they provide the novel observation that alterations to the second loop (Asp249-Gly268) of the C-terminal flank of GPIb result in enhanced vWF binding.     Materials and methods Top Abstract Introduction Materials and methods Results Discussion References Cell lines, antibodies, and reagents Sodium iodide I 125 was purchased from Amersham (Castle Hill, Australia). Ristocetin was purchased from Boehringer-Mannheim (Mannheim, Germany). Oligonucleotides (20-25 base pair [bp]) based on canine or human cDNA sequences12,13 were obtained from Beckman (Melbourne, Australia). Human factor VIII concentrate was a gift of the Commonwealth Serum Laboratories (Melbourne, Australia). vWF was purified from human factor VIII concentrate and radio-iodinated where appropriate using the chloramine T method, as previously described.22-24 CHO cells stably transfected with GPIb and GPIX (CHO IX cells) were prepared as reported.25-27 Murine monoclonal antibodies AP123,28 and VM16d,20,29 directed against the N-terminal vWF-binding domain of GPIb, and WM23, directed against an epitope within the extracellular macroglycopeptide region of GPIb, were purified as described in detail elsewhere.23 Botrocetin was purified as described elsewhere.11 Preparation of expression vectors for canine-human chimeras of GPIb Canine-human chimeras were generated using cDNA of canine13 and human12 GPIb. The expression vector consisting of the full-length GPIb cDNA inserted at an EcoRI site of the plasmid pDX has been described.25-27,30 The strategy for generating chimeric constructs involved polymerase chain reaction amplification of the appropriate GPIb constructs using plaque-forming unit DNA polymerase to avoid 3' T extension on the amplification products, blunt-end ligation of the canine and human DNA at domain junctions, and subcloning back into the human GPIb expression plasmid using appropriate restriction sites as previously described.11 Each construct was verified by sequencing. Expression of canine-human chimeras in Chinese hamster ovary IX cells Canine-human GPIb expression vectors (1-3 µg) were stably transfected into CHO IX cells using Lipofectamine (Gibco BRL, Gaithersburg, MD) by established methods.11,25-27 CHO cells contain no endogenous GPIb, GPIb, GPIX, or GPV, but cotransfection with GPIb and GPIX facilitates stable surface expression of GPIb; GPV is not necessary for functional GPIb expression.27,30 Cells expressing surface GPIb were selected using the anti-GPIb monoclonal antibody WM23 conjugated to magnetic beads (Dynal, Oslo, Norway) according to the manufacturer's instructions. WM23 has an epitope downstream of Glu28223 present in all the chimeras. Binding of von Willebrand factor and monoclonal antibodies to Chinese hamster ovary cells CHO IX cells lacking GPIb or CHO /IX cells cotransfected with wild-type human GPIb or canine-human chimeras of GPIb were assessed for the ability to bind human vWF and monoclonal antibodies against human GPIb using previously described methods.11,25-27,30 Monoclonal antibody binding to transfected CHO cells was analyzed using fluorescein isothiocyanate-labeled secondary antibody and flow cytometry. Cells were harvested by 10-minute treatment of cultures with EDTA (0.53 mM), washed by centrifugation (10 minutes, 700g), and resuspended in 0.01 M sodium phosphate, 0.15 M sodium chloride, pH 7.4, containing 0.1% (wt/vol) bovine serum albumin (BSA) (phosphate-buffered saline [PBS]-BSA buffer). Cells were then incubated with monoclonal antibodies (5 µg/mL) for 40 minutes at 22°C, washed twice, incubated in the presence of fluorescein isothiocyanate-labeled rabbit antimouse antibody for a further 40 minutes, washed, and resuspended in PBS-BSA buffer. The geometric mean fluorescence of each sample was analyzed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA), fitted with an argon-ion laser that stimulates at 488 nm and records fluorescence above 530 nm, and Cellquest software (Becton Dickinson). For measuring vWF binding, CHO IX cells or CHO IX cells expressing wild-type GPIb or chimeras were first suspended in PBS-BSA buffer to a final concentration of 5 × 106/mL. Cells were then incubated with purified 125I-labeled human vWF (1 µg/mL, final concentration) and either ristocetin (0.1-2.0 mg/mL, final concentration) or botrocetin (0.1-10 µg/mL, final concentration). After 30 minutes at 22°C, cells were centrifuged through 20% (wt/vol) sucrose in PBS-BSA buffer (4 minutes, 8750g), and label associated with the pellet was counted in a -counter.11,27 Nonspecific binding was measured in the absence of ristocetin or botrocetin in a parallel assay. Binding of 125I-labeled monoclonal antibodies (1 µg/mL, final concentration) to cells (5 × 106/mL) was carried out using the same method as described for vWF binding. Nonspecific binding was determined by including 100 µg/mL unlabeled antibody in a parallel assay. Adhesion of Chinese hamster ovary cells to von Willebrand factor under flow Cells in the flow chamber were visualized by phase-contrast videomicroscopy and were analyzed by digital image processing as previously described.11,21,31 Cells in PBS (5 × 106/mL) were passed over a glass slide coated with vWF (50 µg/mL) at a shear stress of 10 or 15 dynes/cm2. The velocity of rolling cells was calculated by overlapping sequential images snapped at 30 frames/s and determining the distance the cells rolled during the time period of the snaps. Mean velocities were determined from a minimum of 100 cells per experiment. The number of rolling cells was calculated from a single field of view and counting each cell that rolled on the vWF surface during a 4-minute flow period. Three to 5 experimental runs were performed using different populations of cells.     Results Top Abstract Introduction Materials and methods Results Discussion References Binding of monoclonal antibodies to GPIb chimeras on Chinese hamster ovary cells Previous studies have suggested the C-terminal flank region (Phe201-Gly268) is important in regulating vWF binding because gain-of-function mutations in platelet-type von Willebrand disease occur within this sequence.17 In addition, 2 anti-GPIb monoclonal antibodies, AP1 and VM16d, that mapped to this region differentially inhibit vWF binding.11,21 The first series of chimeras (Figure 1A) was designed to complement 2 previously reported canine-human chimeras, C200 and C268, and to further dissect the C-terminal flank domain of GPIb. Sequences within the pair of disulfide loops formed by the Cys209-Cys248 and Cys211-Cys264 disulfide bonds were incrementally made canine in the chimeras C225 and C248 (Figure 1A). Additional chimeras replaced human sequence with canine sequence solely within the entire C-terminal flank (C201-268), the first disulfide loop (C201-248), or the second loop (C249-268). Expression vectors for all these chimeras of GPIb were prepared from human and canine cDNA using a blunt-end ligation method to facilitate replacement of structural domains at precise boundaries (Figure 1A). The constructs were stably transfected into CHO IX cells (CHO IX cells were stably transfected with GPIb and GPIX to facilitate GPIb expression). View larger version (17K): [in this window] [in a new window]   Figure 1. Binding of monoclonal antibodies to canine-human chimeras of GPIb. (A) Canine-human chimeras of GPIb, where residue numbers correspond to amino acid sequences of human12 and canine13 GPIb. Canine sequence is represented in black. Chimeras C200 and C268 have been reported previously.11 (B) Binding of anti-GPIb monoclonal antibodies to CHO cells expressing wild-type GPIb or the chimeras indicated, as assessed by flow cytometry. , no binding; (+) approximately 50% maximal binding; +, maximal binding. Epitopes for AP1 and VM16d were evaluated by flow cytometry of wild-type GPIb- or chimeric GPIb-expressing CHO IX cells (Figure 1B). For each population of cells, binding of AP1 or VM16d was compared with binding of WM23, a monoclonal antibody whose epitope is downstream of Glu-28220,23 and is, therefore, present on all the chimeras (Figure 1).11 WM23 bound to cells expressing wild-type human GPIb and all the chimeras, but not to CHO IX cells, which lack GPIb (data not shown). A conformation-dependent monoclonal antibody, AK2, with an epitope in the first leucine-rich repeat,11 bound to all the chimeras in which this domain contained human sequence (Figure 1B). Like wild-type human and canine GPIb, all the chimeras bound vWF in the presence of the modulator botrocetin (see next section). Together, these results suggested the chimeric receptors were expressed in a functional form, without gross disruption of the conformation of their N-terminal domains.11 As the human GPIb sequence was incrementally replaced by the canine sequence from the N-terminus, AP1 bound to C200 (canine sequence from the N-terminus up to 200, human sequence from 201 on) but not to C225 (Figure 1B). AP1 also bound to the C249-268 chimera, but not to the C201-268 or the C201-248 chimera, which contained canine sequence within the entire C-terminal flank or first disulfide loop (Phe201-Cys248), respectively. The epitope for AP1 was, therefore, localized to the sequence Phe201-Glu225 because AP1 bound to all the chimeras in which this sequence was human, but to none of the chimeras in which this sequence was canine. The VM16d epitope included elements from the sequence either side of Cys248, within Asn226-Gly268. VM16d bound to C200 and C225, but not to C268 (Figure 1B). However, there was partial binding to C248. Similarly, there was partial binding to C201-248 and C249-268, but no binding to C201-268. To confirm this partial reactivity of VM16d to C248, C201-248, and C249-268 observed by flow cytometry, binding of VM16d to these chimeras was also assessed using 125I-labeled VM16d (Figure 2). Specific binding of 125I-labeled VM16d was consistent with the flow cytometry results shown in Figure 1B. View larger version (14K): [in this window] [in a new window]   Figure 2. Binding of VM16d to GPIb chimeras. Specific binding of 125I-labeled anti-GPIb monoclonal antibody, VM16d (1 µg/mL), to CHO IX cells (5 × 106/mL) co-expressing wild-type human GPIb, or canine-human chimeras of GPIb for 30 minutes at 22°C. Binding is expressed relative to specific binding of 125I-labeled WM23 for each cell line, to account for any differences in expression of GPIb between different populations of cells. Specific binding for each antibody was calculated by subtracting nonspecific binding measured in the presence of 100 µg/mL unlabeled antibody in a parallel assay. Error bars, ± SEM (n = 2). Binding of von Willebrand factor to canine-human chimeras of GPIb on Chinese hamster ovary cells Previous analysis of recombinant GPIb expressed on mammalian cells11,18,19 and mapping of functional anti-GPIb antibodies such as AP1 and VM16d (refer to preceding section) has pointed to a role for the C-terminal flank region of GPIb in regulating vWF binding. We assessed vWF binding to the canine-human chimeras involving replacement of human with canine sequence of the entire C-terminal flank (C201-268) or the first (C201-248) or second (C249-268) disulfide loops (Figure 3A). These new chimeras were compared with a series of complementary human-canine chimeras, H104, H128, H152, and H176 (incremental restoration of human sequence from canine sequence) reported previously.11 View larger version (19K): [in this window] [in a new window]   Figure 3. Functional analysis of human-canine and canine-human chimeras of GPIb. (A) Human-canine and canine-human chimeras of GPIb, where residue numbers correspond to amino acid sequences of human12 and canine13 GPIb. Canine sequence is represented in black. Chimeras H104, H128, H152, and H176 have been described previously.11 (B) Ristocetin-dependent vWF binding to GPIb chimeras. Specific binding of 125I-labeled vWF (1 µg/mL) in the presence of ristocetin (1 mg/mL) to CHO IX cells (5 × 106/mL) co-expressing wild-type human GPIb, canine GPIb, or canine-human chimeras of GPIb for 30 minutes at 22°C. Binding is expressed relative to specific binding of 125I-labeled vWF to each cell line in the presence of botrocetin (2.5 µg/mL) in parallel assays.11 Specific binding was calculated from total binding by subtracting nonspecific binding measured in the absence of modulator in parallel assays. Error bars, ± SEM (n = 3). (C) Adhesion of GPIb chimera-expressing CHO cells to vWF under flow. Grades are defined as follows: +, 1% to 10% of cells; ++, 11% to 79% of cells; +++, more than 80% of cells. (Rolling velocities are ± SEM.) Consistent with our earlier results showing leucine-rich repeats 2 to 4 were required for ristocetin-dependent vWF binding,11 all chimeras that contained human sequence within repeats 2 to 4, but not H104, which contained human sequence only to the end of the third repeat, exhibited vWF binding in the presence of ristocetin (Figure 3B). As expected, all chimeras bound vWF in the presence of botrocetin because botrocetin does not discriminate between vWF binding to human or canine GPIb.11 (Cell line, % specific binding: IX, 100%; H104, 52% ± 3; H128, 84% ± 4; H152, 71% ± 4; H176, 34% ± 9; C201-C268, 95% ± 3; C201-C248, 126% ± 6; C249-C268, 169% ± 6). We previously showed that botrocetin-dependent binding of vWF correlates, at least within a factor of 2, with levels of binding of WM23, an anti-GPIb monoclonal antibody with an epitope downstream of Glu282 and present in all the chimeras.11 Therefore, differences in botrocetin-induced binding to different populations of cells presumably reflect variations in levels of GPIb surface expression. For this reason, we have shown levels of ristocetin-dependent binding of vWF relative to botrocetin-dependent binding in Figure 3B. Like H104, there was also no ristocetin-dependent vWF binding to chimeras C225 or C248 that lacked human repeats 2 to 4 (data not shown), consistent with lack of binding to C200 or C268.11 Ristocetin-dependent vWF binding to the new chimeras, C201-268 and C201-248, containing canine sequence in the entire C-terminal flank or its first disulfide loop, respectively, was less than binding to wild-type GPIb and comparable to binding to H128 and H176 (Figure 3B). However, one of the chimeras (C249-268), containing canine sequence only in the second disulfide loop (Asp249-Gly268) of the C-terminal flank, bound vWF in the presence of ristocetin to an even greater extent than wild-type GPIb (Figure 3B). In fact, binding of vWF to chimeric GPIb-expressing cells was comparable to binding to wild-type GPIb only when the sixth leucine-rich repeat and the first loop of the C-terminal flank (Phe201-Cys248) is of the same speciesthat is, either both human (wild-type or C249-268) or both canine (H152) (Figure 3B). This implies the possibility of an interaction between sequences within the sixth leucine-rich repeat and Phe201-Cys248. Because C249-268 bound vWF to a greater extent than wild-type receptor (Figure 3B), we tested vWF binding to this chimera over a range of ristocetin concentrations. Binding was found to be consistently enhanced (Figure 4A). In contrast, botrocetin-dependent vWF binding to C249-268 was comparable to wild-type GPIb at all botrocetin concentrations tested (Figure 4B). This suggested the C249-268 chimera was a gain-of-function receptor, a result confirmed by measuring adhesion of this cell line to vWF under flow conditions in the absence of modulators. View larger version (21K): [in this window] [in a new window]   Figure 4. Effect of ristocetin or botrocetin concentration on vWF binding to GPIb. Specific binding of 125I-labeled vWF (1 µg/mL) to CHO IX cells (106/mL) expressing wild-type human GPIb or the C249-268 chimera in the presence of ristocetin (A) or botrocetin (B) at the concentrations indicated for 30 minutes at 22°C. Specific binding was calculated from total binding by subtracting nonspecific binding measured in the absence of modulator in parallel assays. Adhesion of Chinese hamster ovary cells to von Willebrand factor under flow Under flow conditions in the absence of modulators, cells expressing C249-268, containing canine sequence only in the second disulfide loop of the C-terminal flank, rolled significantly more slowly on a vWF-coated surface than cells expressing wild-type GPIb (Figure 5). This effect was observed at shear stress of 5 and 10 dynes/cm2 and was consistent with the gain-of-function found for ristocetin-dependent binding under static conditions described above. The interactions of other relevant chimera-expressing cells with vWF under flow are summarized in Figure 3C. Compared with cells expressing wild-type GPIb, only C249-268 and the previously described chimera, H152, containing human sequence to the end of the fifth repeat,11 rolled on vWF. View larger version (14K): [in this window] [in a new window]   Figure 5. Binding of chimera-expressing CHO cells to vWF under flow. Binding of CHO IX cells expressing wild-type GPIb or CHO IX cells expressing the C249-268 chimera of GPIb. Mean rolling velocity was derived from images snapped at 30 frames/s during a 4-minute period of perfusion at 5 or 10 dynes/cm2 over a glass surface coated with 50 µg/mL vWF. Error bars, ± SEM.     Discussion Top Abstract Introduction Materials and methods Results Discussion References The N-terminal 282 residues of glycoprotein Ib (GPIb) of the platelet membrane GPIb-IX-V complex contain the binding domain for vWF and for other ligands such as P-selectin, Mac-1, -thrombin, factor XII, and high-molecular-weight kininogen.1-10 In the current study, we have focused on the structure-function relationships of the leucine-rich repeats and the C-terminal flank domain of GPIb, regions previously implicated in regulating vWF binding.2,11 Based on the species specificity of binding of vWF and anti-GPIb monoclonal antibodies to human GPIb, we used a series of canine-human chimeras involving the C-terminal flank to examine vWF binding under static and flow conditions and to map epitopes for 2 functional antibodies that recognize this region of the receptor. Anti-GPIb monoclonal antibodies AP1 and VM16d were of particular interest because of their functional effect on binding of vWF and other ligands. AP1 was mapped to the sequence Phe201-Glu225, within the first disulfide loop of the C-terminal flank of GPIb, because it specifically recognized chimeras in which this sequence was human (C200, C249-268) but not in which it was canine (C225, C248, C268, C201-268, C201-248). Previous studies showed that AP1 binding to GPIb either was unaffected or was partially decreased (by 50% or less compared with wild-type GPIb) by mutagenesis of individual residues between Gly233 and Met239.18 Unlike AP1, other anti-GPIb monoclonal antibodies that completely inhibit ristocetin-dependent binding (AK2, Hip1, and 6D1) have epitopes within the first 4 leucine-rich repeats.11 AP1 also inhibits botrocetin-dependent vWF binding to GPIb and rolling of GPIb-IX-expressing cells on vWF.21 In contrast to AP1, VM16d only inhibits botrocetin-dependent, but not ristocetin-dependent, binding of vWF to GPIb12,20 or rolling of GPIb-IX-expressing cells on vWF.21 The epitope for VM16d was apparently composed of elements on either side of Cys248 within the sequence Asn226-Gly268 because it bound to all chimeras where this sequence was human but only to some (approximately 50% of wild-type) where either Asn226-Cys248 or Asp249-Gly268 was canine (C248, C201-248, C249-268). This result was confirmed by flow cytometric analysis and by binding of radiolabeled VM16d to chimera-expressing cells. There was no binding when Asn226-Cys248 and Asp249-Gly268 sequences were canine (C268, C201-268). The partial VM16d-binding sequence Asn226-Cys248 contains the congenital and artificial gain-of-function, valine substitution mutations at Gly233, Asp235, Lys237, and Met239 and the partial loss-of-function mutation, Ala238 to valine.18 The other component of the VM16d-binding site, Asp249-Gly268, constitutes the second disulfide loop of the C-terminal flank, formed by the Cys211-Cys264 and Cys209-Cys248 disulfide bridges.1 In studies of vWF binding, this Asp249-Gly268 loop was also found to lead to a gain-of-function when the human sequence was replaced by canine sequence in GPIb (C249-268). Definition of the VM16d-binding site is significant in that this antibody not only inhibits vWF binding in the presence of botrocetin, it also inhibits the interaction of GPIb and Mac-1.6 Other antibodies that strongly inhibit vWF binding to GPIb do not inhibit Mac-1 binding. VM16d also inhibits thrombin-dependent platelet aggregation.29 Extending previous studies of the vWF-GPIb interaction,2,11,18,19 the current experiments provide additional insight into the potential role of the C-terminal flank region of GPIb in regulating vWF binding. One of the chimeras (C249-268) containing canine sequence only in the second disulfide loop (Asp249-Gly268) of the C-terminal flank bound vWF to a greater extent than wild-type GPIb in the presence of ristocetin, and rolling of the cells to vWF under flow conditions in the absence of modulators was slower than in cells expressing wild-type GPIb. These interactions are unlikely to have been affected by differences in the levels of receptor expression on different cell lines because any variation in expression levels on these cells (as assessed by WM23 binding) are well above levels of receptor where the interaction of cells with vWF under flow is significantly affected.21,31 These findings represent the first example where alterations to the second loop of the C-terminal flank result in a gain-of-function. The difference between human and canine sequences within Asp249-Gly268 include an alteration to charged residues from human to canine sequence at Asp249Val, Asp252Lys, Lys253Asn, Lys258Thr, and Gly263Asp that could account for differences in function. An interesting feature of these results, combined with those from our earlier studies,11 is how the interaction with vWF under flow is recovered as canine GPIb was incrementally made more human from the N-terminus. There was recovery of function to the extent of saltatory translocation of more than 90% of cells as human sequence was restored to the end of the fourth leucine-rich repeat (H128). Saltatory translocation occurs when cells jump along the direction of flow, where there is an increased off-rate/on-rate for adhesion compared with rolling cells.11,31 Restoring human sequence to the end of the fifth repeat (H152) resulted in rolling at 126 ± 7 µm/s compared with 42 ± 1 µm/s for wild-type GPIb. However, as even more human sequence is restored, to the end of the sixth repeat (H176) there is decreased interaction, and less than 10% of cells display saltatory translocation (Figure 3C).11 Rolling was again observed when the receptor was made human to the end of the first loop of the C-terminal flank (C249-268). These results parallel the levels of ristocetin-dependent vWF binding to the chimeras, and they support previous studies showing that ristocetin-dependent vWF binding and adhesion to vWF under flow are comparable and distinct from botrocetin-dependent vWF binding.21 The simplest explanation for these results is that optimal binding of chimeric GPIb-expressing cells to vWF, comparable to wild-type, only occurs when the sixth leucine-rich repeat and the first loop of the C-terminal flank (Phe201-Cys248) is of the same species, either both human (wild-type or C249-268) or both canine (H152) (Figure 3C). Saltatory translocation (or partial ristocetin-dependent binding), however, is present when leucine-rich repeats 2 to 4 are human (H128, H176, C201-248, C201-268), consistent with previous results.11 Together, these findings raise the possibility of an interaction between sequences within the sixth leucine-rich repeat and Phe201-Cys248, an interaction that could be disrupted when respective sequences are cross-species. This may be an important consideration underlying the gain-of-function or loss-of-function mutations within the sequence spanning Gly233-Met239 within this first disulfide loop.18 In support of this supposition, alanine substitution point mutations of residues within the sixth leucine-rich repeat affected the capacity of recombinant GPIb to support vWF binding.32 Definitive evidence awaits structural determination of the leucine-rich repeat and the C-terminal flank domains of GPIb. In conclusion, the current study has further defined the epitopes for 2 functionally important anti-GPIb monoclonal antibodies, AP1 and VM16d, that inhibit interactions involving the platelet membrane GPIb-IX-V complex and vWF, Mac-1, and thrombin. In addition, analysis of canine-human chimeras of GPIb expressed on CHO cells provides further evidence for a role of the C-terminal flank domain and of leucine-rich repeats in regulating vWF binding under static or flow conditions.     Footnotes Submitted May 29, 2001; accepted August 17, 2001. Supported in part by the National Health and Medical Research Council of Australia and the National Heart Foundation of Australia. Y.S. and J.D. are co-first authors. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Robert K. Andrews, Baker Medical Research Institute, PO Box 6492, St Kilda Rd Central, Melbourne 8008, Australia; e-mail: rkandrews{at}hotmail.com.     References Top Abstract Introduction Materials and methods Results Discussion References 1. López JA. The platelet glycoprotein Ib-IX complex. Blood Coag Fibrinol. 1994;5:97-119[Medline] [Order article via Infotrieve]. 2. Berndt MC, Shen Y, Dopheide SM, Gardiner EE, Andrews RK. The vascular biology of the glycoprotein Ib-IX-V complex. Thromb Haemost. 2001;86:178-188[Medline] [Order article via Infotrieve]. 3. Kroll MH, Hellums JD, McIntyre LV, Schafer AI, Moake JL. Platelets and shear stress. Blood. 1996;88:1525-1541[Free Full Text]. 4. Romo GM, Dong J-F, Schade AJ, et al. The glycoprotein Ib-IX-V complex is a platelet counterreceptor for P-selectin. J Exp Med. 1999;190:803-814[Abstract/Free Full Text]. 5. Katayama T, Ikeda Y, Handa M, et al. Immunoneutralization of glycoprotein Ib attenuates endotoxin-induced interactions of platelets and leukocytes with rat venular endothelium in vivo. Circ Res. 2000;86:1031-1037[Abstract/Free Full Text]. 6. Simon DI, Chen Z, Xu H, et al. Platelet glycoprotein Ib is a counterreceptor for the leukocyte integrin Mac-1 (CD11b/CD18). J Exp Med. 2000;192:193-204[Abstract/Free Full Text]. 7. De Candia E, Hall SW, Rutella S, Landolfi R, Andrews RK, De Cristofaro R. Binding of thrombin to the glycoprotein Ib accelerates the hydrolysis of PAR-1 on intact platelets. J Biol Chem. 2001;276:4692-4698[Abstract/Free Full Text]. 8. Ramakrishnan V, DeGuzman F, Bao M, Hall S, Leung L, Phillips D. A thrombin receptor function for platelet glycoprotein Ib-IX unmasked by cleavage of glycoprotein V. Proc Natl Acad Sci U S A. 2001;98:1823-1828[Abstract/Free Full Text]. 9. Bradford HN, Pixley RA, Colman RW. Human factor XII binding to the glycoprotein Ib-IX-V complex inhibits thrombin-induced platelet aggregation. J Biol Chem. 2000;275:22756-22763[Abstract/Free Full Text]. 10. Bradford HN, Dela Cadena RA, Kunapuli SP, Dong JF, Lopez JA, Colman RW. Human kininogens regulate thrombin binding to platelets through the glycoprotein Ib-IX-V complex. Blood. 1997;90:1508-1515[Abstract/Free Full Text]. 11. Shen Y, Romo GM, Dong J-F, et al. Requirement of leucine-rich repeats of GP Ib for shear-dependent and static binding of von Willebrand factor to the platelet membrane GP Ib-IX-V complex. Blood. 2000;95:903-910[Abstract/Free Full Text]. 12. López JA, Chung DW, Fujikawa K, Hagen FS, Papayannopoulou T, Roth GJ. Cloning of the  chain of human platelet glycoprotein Ib: a transmembrane protein with homology to leucine-rich 2-glycoprotein. Proc Natl Acad Sci U S A. 1987;84:5615-5619[Abstract/Free Full Text]. 13. Kenny D, Morateck PA, Fahs SA, Warltier DC, Montgomery RR. Cloning and expression of canine glycoprotein Ib. Thromb Haemost. 1999;82:1327-1333[Medline] [Order article via Infotrieve]. 14. Read MS, Shermer RW, Brinkhous KM. Venom coagglutinin: an activator of platelet aggregation dependent on von Willebrand factor. Proc Natl Acad Sci U S A. 1978;83:4514-4518. 15. Miller JL, Cunningham D, Lyle VA, Finch CN. Mutation in the gene encoding the  chain of platelet glycoprotein Ib in platelet-type von Willebrand disease. Proc Natl Acad Sci U S A. 1991;88:4761-4765[Abstract/Free Full Text]. 16. Russell SD, Roth GJ. Pseudo-von Willebrand disease: a mutation in the platelet glycoprotein Ib gene associated with a hyperactive surface receptor. Blood. 1993;81:1787-1791[Abstract/Free Full Text]. 17. Miller JL. Platelet-type von Willebrand disease. Thromb Haemost. 1996;75:865-869[Medline] [Order article via Infotrieve]. 18. Dong J-F, Schade A, Romo GM, et al. Novel gain-of-function mutations of platelet glycoprotein Ib by valine mutagenesis in the Cys209-Cys248 disulfide loop: functional analysis under static and dynamic conditions. J Biol Chem. 2000;275:27663-27670[Abstract/Free Full Text]. 19. Marchese P, Saldivar E, Ware J, Ruggeri ZM. Adhesive properties of the isolated amino-terminal domain of platelet glycoprotein Ib in a flow field. Proc Natl Acad Sci U S A. 1999;96:7837-7842[Abstract/Free Full Text]. 20. Ward CM, Andrews RK, Smith AI, Berndt MC. Mocarhagin, a novel cobra venom metalloproteinase, cleaves the platelet von Willebrand factor receptor glycoprotein Ib: identification of the sulfated tyrosine/anionic sequence Tyr-276-Glu-282 of glycoprotein Ib as a binding site for von Willebrand factor and -thrombin. Biochemistry. 1996;35:4929-4938[CrossRef][Medline] [Order article via Infotrieve]. 21. Dong J-F, Berndt MC, Schade A, McIntire LV, Andrews RK, López JA. Ristocetin- but not botrocetin-dependent binding of von Willebrand factor to the platelet membrane glycoprotein Ib-IX-V complex correlates with shear-dependent interactions. Blood. 2001;97:162-168[Abstract/Free Full Text]. 22. Booth WJ, Furby FH, Berndt MC, Castaldi PA. Factor VIII/von Willebrand factor has potent lectin activity. Biochem Biophys Res Commun. 1984;118:495-501[CrossRef][Medline] [Order article via Infotrieve]. 23. Berndt MC, Du X, Booth WJ. Ristocetin-dependent reconstitution of binding of von Willebrand factor to purified human platelet membrane glycoprotein Ib-IX complex. Biochemistry. 1988;27:633-640[CrossRef][Medline] [Order article via Infotrieve]. 24. Andrews RK, Booth WJ, Gorman JJ, Castaldi PA, Berndt MC. Purification of botrocetin from Bothrops jararaca venom: analysis of the botrocetin-mediated interaction between von Willebrand factor and the human platelet membrane glycoprotein Ib-IX complex. Biochemistry. 1989;28:8317-8326[CrossRef][Medline] [Order article via Infotrieve]. 25. Dong J-F, Li CQ, López JA. Tyrosine sulfation of the glycoprotein Ib-IX complex: identification of sulfated residues and effect on ligand binding. Biochemistry. 1994;33:13946-13953[CrossRef][Medline] [Order article via Infotrieve]. 26. Dong J-F, Huyn W, López JA. Aggregation of mammalian cells expressing the platelet glycoprotein GP Ib-IX complex and the requirement for tyrosine sulfation of GP Ib. Blood. 1995;86:4175-4183[Abstract/Free Full Text]. 27. López JA, Leung B, Reynolds CC, Li CQ, Fox JEB. Efficient plasma membrane expression of a functional human glycoprotein Ib-IX complex requires the presence of its three subunits. J Biol Chem. 1992;267:12851-12859[Abstract/Free Full Text]. 28. Montgomery RR, Kunicki TJ, Taves C, Pidard D, Corcoran M. Diagnosis of Bernard-Soulier syndrome and Glanzmann's thrombasthenia with a monoclonal assay on whole blood. J Clin Invest. 1983;71:385-389. 29. Mazurov AV, Vinogradov DV, Vlasik TN, Repin VS, Booth WJ, Berndt MC. Characterization of an antiglycoprotein Ib monoclonal antibody that specifically inhibits platelet-thrombin interaction. Thromb Res. 1991;62:673-684[CrossRef][Medline] [Order article via Infotrieve]. 30. Li CQ, Dong J-F, Lanza F, Sanan DA, Sae-Tung G, López JA. Expression of glycoprotein GP V in heterologous cells and evidence for its association with GP Ib in forming a GP Ib-IX-V complex on the cell surface. J Biol Chem. 1995;270:16302-16307[Abstract/Free Full Text]. 31. Fredrickson BJ, Dong J-F, McIntyre LV, López JA. Shear-dependent rolling on von Willebrand factor of mammalian cells expressing the platelet glycoprotein Ib-IX-V complex. Blood. 1998;92:3684-3693[Abstract/Free Full Text]. 32. Shimizu A, Matsushita T, Nakayama T, et al. Identification of the amino acid residues of the platelet GPIb essential for the von Willebrand factor binding by the charged-to alanine scanning mutagenesis [abstract]. Blood. 2000;96:623. © 2002 by The American Society of Hematology.   CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: Y. Shen, S. L. Cranmer, A. Aprico, J. C. Whisstock, S. P. Jackson, M. C. Berndt, and R. K. Andrews Leucine-rich Repeats 2-4 (Leu60-Glu128) of Platelet Glycoprotein Ib{alpha} Regulate Shear-dependent Cell Adhesion to von Willebrand Factor J. Biol. Chem., September 8, 2006; 281(36): 26419 - 26423. [Abstract] [Full Text] [PDF] M. Othman, C. Notley, F. L. Lavender, H. White, C. D. Byrne, D. Lillicrap, and D. F. O'Shaughnessy Identification and functional characterization of a novel 27-bp deletion in the macroglycopeptide-coding region of the GPIBA gene resulting in platelet-type von Willebrand disease Blood, June 1, 2005; 105(11): 4330 - 4336. [Abstract] [Full Text] [PDF] Y. Peng, M. C. Berndt, M. A. Cruz, and J. A. Lopez The {alpha}1 helix-{beta}13 strand spanning Leu214 to Val229 of platelet glycoprotein Ib{alpha} facilitates the interaction with von Willebrand factor: evidence from characterization of the epitope of monoclonal antibody AP1 Blood, December 15, 2004; 104(13): 3971 - 3978. [Abstract] [Full Text] [PDF] S. W. Kerrigan, I. Douglas, A. Wray, J. Heath, M. F. Byrne, D. Fitzgerald, and D. Cox A role for glycoprotein Ib in Streptococcus sanguis-induced platelet aggregation Blood, June 28, 2002; 100(2): 509 - 516. [Abstract] [Full Text] [PDF] This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Shen, Y. Articles by Andrews, R. K. Search for Related Content PubMed PubMed Citation Articles by Shen, Y. Articles by Andrews, R. K. Related Collections Hemostasis, Thrombosis, and Vascular Biology Social Bookmarking                   What's this?

	Copyright © 2002 by American Society of Hematology         Online ISSN: 1528-0020