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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY

Functional analysis of the C-terminal flanking sequence of platelet ␣ using canineÐhuman chimeras

Yang Shen, Jing-fei Dong, Gabriel M. Romo, Wendy Arceneaux, Andrea Aprico, Elizabeth E. Gardiner, Jose´A.Lo´pez, Michael C. Berndt, and Robert K. Andrews

Platelet glycoprotein Ib-IX-V (GPIb-IX-V) quired for optimal vWF recognition under the same species as the first disulfide loop mediates adhesion to von Willebrand fac- static or flow conditions. Using novel ca- (Phe201-Cys248) of the C-terminal flank, tor (vWF) in (patho)physiological throm- nineÐhuman chimeras dissecting the C- suggesting an interaction between these bus formation. vWF binds the N-terminal terminal flank, it is now demonstrated that domains may be important for optimal vWF 282 residues of GPIb␣, consisting of an (1) Phe201-Glu225 contains the epitope for binding; and (4) replacing the C-terminal N-terminal flank (His1ÐIle35), 7 leucine- AP1, an anti-GPIb␣ monoclonal antibody flank second disulfide loop (Asp249-Gly268) rich repeats (Leu36ÐAla200), a C-terminal that inhibits both ristocetin- and botrocetin- in human GPIb␣ with the corresponding flank (Phe201ÐGly268), and a sulfated ty- dependent vWF binding; (2) VM16d, an anti- canine sequence enhanced vWF binding rosine sequence (Asp269ÐGlu282). By ex- body that preferentially inhibits botrocetin- under static and flow conditions, providing pressing canineÐhuman chimeras of dependent vWF binding, recognizes the the first evidence for a gain-of-function phe- GPIb␣ on Chinese hamster ovary cells, sequence Val226-Gly268, surrounding notype associated with the second loop of binding sites for functional anti-GPIb␣ Cys248, which forms a disulfide-bond with the C-terminal flank. (Blood. 2002;99: antibodies to individual domains were Cys209; (3) vWF binding to chimeric GPIb␣ 145-150) previously mapped, and it was shown is comparable to wild-type in 2 chimeras in that leucine-rich repeats 2 to 4 were re- which the sixth leucine-rich repeat was of © 2002 by The American Society of Hematology Introduction

The platelet membrane glycoprotein Ib-IX-V (GPIb-IX-V) com- Ala200), a C-terminal flank (Phe201ÐGly268), and a sulfated plex plays a central role in vascular biology. The interaction of tyrosine sequence (Asp269ÐGlu282).2,11 Previous studies with the GPIb␣ with its adhesive ligand, (vWF), in metalloproteinase mocarhagin, which cleaves between Glu282 and the subendothelial matrix mediates platelet adhesion at high shear Asp283 of GPIb␣, suggest that the vWF binding site is within in the hemostatic response to vessel wall injury.1,2 In thrombosis, residues 1 to 282.11 We have recently expressed on Chinese hamster pathologic shear stress induces binding of platelet GPIb-IX-V to ovary (CHO) cells canineÐhuman and humanÐcanine chimeras of plasma vWF, initiating platelet aggregation.3 More recently, GPIb␣ GPIb␣, corresponding to boundaries between these structural has been identified as a counter-receptor for P-selectin expressed domains. Murine monoclonal antibodies against the N-terminal on activated endothelial cells4,5 and for the leukocyte ␣M␤2 domain of human GPIb␣ are species-specific and do not bind integrin, Mac-1.6 These interactions potentially mediate platelet- canine GPIb␣; canine and human amino acid sequences are only endothelium or platelet-leukocyte adhesion in thrombosis, inflam- 65.2% identical in the region His-1ÐGlu-282.12,13 The vWF activator, mation, and atherogenesis. GPIb-IX-V also constitutes a binding ristocetin, induces binding of human vWF only to human GPIb␣, not to site, and a substrate for, ␣-.2 Recent evidence suggests that the canine receptor.11,14 In contrast, the snake venom modulator, GPIb␣ acts as a cofactor for thrombin-dependent cleavage of the botrocetin, does not discriminate between the 2 species, inducing the 7-transmembrane protease-activated receptor, PAR-1, on platelets7 and binding of human vWF to human and canine GPIb␣.11 Botrocetin- that GPIb␣ is itself a thrombin receptor following thrombin-dependent dependent vWF binding to all the GPIb␣ chimeras provided evidence cleavage of GPV.8 Other GPIb␣ ligands—factor XII and high-molecularÐ that they retained a functional conformation. These combined studies weight —are inhibitors of thrombin binding, and they down- allow detailed mapping of binding sites for anti-GPIb␣ monoclonal regulate thrombin-dependent platelet activation.9,10 antibodies and vWF, and they suggest leucine-rich repeats 2, 3, and 4 of All these ligands of GPIb␣—vWF, P-selectin, Mac-1, thrombin, GPIb␣ are critical for vWF binding to GPIb-IX-V induced by ristocetin factor XII, and high-molecularÐweight kininogen—recognize the or under hydrodynamic flow.11 N-terminal 282 residues of GPIb␣.2 The binding site for vWF The C-terminal flank domain (Phe201-Gly268) contains 2 loops comprises elements from 4 structural regions of GPIb␣: the by virtue of disulfide bonds between Cys209-Cys248 and Cys211- N-terminal flank (His1ÐIle35), 7 leucine-rich repeats (Leu36Ð Cys264. Several lines of evidence implicate the C-terminal flank as

From the Hazel and Pip Appel Vascular Biology Laboratory, Baker Medical Y.S. and J.D. are co-first authors. Research Institute, Melbourne, Australia, and the Departments of Medicine and Reprints: Robert K. Andrews, Baker Medical Research Institute, PO Box 6492, of Molecular and Human Genetics, Baylor College of Medicine and Veterans St Kilda Rd Central, Melbourne 8008, Australia; e-mail: [email protected]. Affairs Medical Center, Houston, TX. The publication costs of this article were defrayed in part by page charge Submitted May 29, 2001; accepted August 17, 2001. payment. Therefore, and solely to indicate this fact, this article is hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734. Supported in part by the National Health and Medical Research Council of Australia and the National Heart Foundation of Australia. © 2002 by The American Society of Hematology

BLOOD, 1 JANUARY 2002 ⅐ VOLUME 99, NUMBER 1 145 From www.bloodjournal.org by guest on September 11, 2016. For personal use only.

146 SHEN et al BLOOD, 1 JANUARY 2002 ⅐ VOLUME 99, NUMBER 1 a critical factor in regulating vWF binding. Congenital gain-of- by established methods.11,25-27 CHO cells contain no endogenous GPIb␣, function mutations within the GPIb␣ (platelet-type von GPIb␤, GPIX, or GPV, but cotransfection with GPIb␤ and GPIX facilitates Willebrand disease) result in single amino acid substitutions, stable surface expression of GPIb␣; GPV is not necessary for functional ␣ 27,30 ␣ Gly233 to valine or Met239 to valine, within the first of the 2 GPIb expression. Cells expressing surface GPIb were selected using the anti-GPIb␣ monoclonal antibody WM23 conjugated to magnetic beads disulfide loops comprising the C-terminal flank domain.15-17 Muta- ␣ (Dynal, Oslo, Norway) according to the manufacturer’s instructions. tion of Gly233 or Met239 to valine in recombinant GPIb ,in WM23 has an epitope downstream of Glu28223 present in all the chimeras. addition to the artificial mutation of Asp235 or Lys237 to valine, also results in a gain-of-function phenotype.18,19 In contrast, Binding of von Willebrand factor and monoclonal antibodies mutation of Ala238 to valine results in partial loss of function.18 to Chinese hamster ovary cells The anti-GPIb␣ monoclonal antibodies, AP1 and VM16d, which map into the C-terminal flank, also inhibit vWF binding. AP1 CHO ␤IX cells lacking GPIb␣ or CHO ␤/IX cells cotransfected with blocks both ristocetin- and botrocetin-induced binding and rolling wild-type human GPIb␣ or canineÐhuman chimeras of GPIb␣ were of GPIb-IXÐexpressing cells on vWF, whereas VM16d preferen- assessed for the ability to bind human vWF and monoclonal antibodies ␣ 11,25-27,30 tially inhibits botrocetin-dependent binding11,20 but not ristocetin- against human GPIb using previously described methods. Mono- clonal antibody binding to transfected CHO cells was analyzed using dependent binding or GPIb-IXÐdependent rolling on vWF.21 In fluorescein isothiocyanateÐlabeled secondary antibody and flow cytometry. ␣ 6 addition, both AP1 and VM16d inhibit Mac-1 binding to GPIb . Cells were harvested by 10-minute treatment of cultures with EDTA (0.53 In this study, we have analyzed a series of canineÐhuman mM), washed by centrifugation (10 minutes, 700g), and resuspended in chimeras of GPIb␣ corresponding to the replacement of specific 0.01 M sodium phosphate, 0.15 M sodium chloride, pH 7.4, containing sequences involving the C-terminal flank. These chimeras, ex- 0.1% (wt/vol) bovine serum albumin (BSA) (phosphate-buffered saline pressed on CHO cells, enabled more precise mapping of binding [PBS]ÐBSA buffer). Cells were then incubated with monoclonal antibodies sites for the functional anti-GPIb␣ monoclonal antibodies AP1 and (5 ␮g/mL) for 40 minutes at 22¡C, washed twice, incubated in the presence VM16d, and they allowed the functional importance of each of fluorescein isothiocyanateÐlabeled rabbit antimouse antibody for a disulfide loop to be assessed regarding vWF binding to GPIb␣. further 40 minutes, washed, and resuspended in PBSÐBSA buffer. The Combined results infer a possible interaction between the sixth geometric mean fluorescence of each sample was analyzed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA), fitted with an leucine-rich repeat and the first disulfide loop (Phe201-Cys248), argon-ion laser that stimulates at 488 nm and records fluorescence above and they provide the novel observation that alterations to the 530 nm, and Cellquest software (Becton Dickinson). ␣ second loop (Asp249-Gly268) of the C-terminal flank of GPIb For measuring vWF binding, CHO ␤IX cells or CHO ␤IX cells expressing result in enhanced vWF binding. wild-type GPIb␣ or chimeras were first suspended in PBSÐBSA buffer to a final concentration of 5 ϫ 106/mL. Cells were then incubated with purified 125I-labeled human vWF (1 ␮g/mL, final concentration) and either ristocetin Materials and methods (0.1-2.0 mg/mL, final concentration) or botrocetin (0.1-10 ␮g/mL, final concen- tration). After 30 minutes at 22¡C, cells were centrifuged through 20% (wt/vol) Cell lines, antibodies, and reagents sucrose in PBSÐBSA buffer (4 minutes, 8750g), and label associated with the pellet was counted in a ␥-counter.11,27 Nonspecific binding was measured in the Sodium iodide I 125 was purchased from Amersham (Castle Hill, Australia). absence of ristocetin or botrocetin in a parallel assay. Ristocetin was purchased from Boehringer-Mannheim (Mannheim, Germany). Binding of 125I-labeled monoclonal antibodies (1 ␮g/mL, final concen- Oligonucleotides (20-25 [bp]) based on canine or human cDNA tration) to cells (5 ϫ 106/mL) was carried out using the same method as sequences12,13 were obtained from Beckman (Melbourne, Australia). Human described for vWF binding. Nonspecific binding was determined by factor VIII concentrate was a gift of the Commonwealth Serum Laboratories including 100 ␮g/mL unlabeled antibody in a parallel assay. (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 Adhesion of Chinese hamster ovary cells to von Willebrand (CHO ␤IX cells) were prepared as reported.25-27 Murine monoclonal antibodies factor under flow 23,28 20,29 AP1 and VM16d, directed against the N-terminal vWF-binding domain Cells in the flow chamber were visualized by phase-contrast videomicros- ␣ of GPIb , and WM23, directed against an epitope within the extracellular copy and were analyzed by digital image processing as previously ␣ macroglycopeptide region of GPIb , were purified as described in detail described.11,21,31 Cells in PBS (5 ϫ 106/mL) were passed over a glass slide 23 11 elsewhere. Botrocetin was purified as described elsewhere. coated with vWF (50 ␮g/mL) at a shear stress of 10 or 15 dynes/cm2. The velocity of rolling cells was calculated by overlapping sequential images Preparation of expression vectors for canineÐhuman chimeras snapped at 30 frames/s and determining the distance the cells rolled during ␣ of GPIb the time period of the snaps. Mean velocities were determined from a CanineÐhuman chimeras were generated using cDNA of canine13 and minimum of 100 cells per experiment. The number of rolling cells was human12 GPIb␣. The expression vector consisting of the full-length calculated from a single field of view and counting each cell that rolled on GPIb␣ cDNA inserted at an EcoRI site of the plasmid pDX has been the vWF surface during a 4-minute flow period. Three to 5 experimental described.25-27,30 The strategy for generating chimeric constructs involved runs were performed using different populations of cells. polymerase chain reaction amplification of the appropriate GPIb␣ con- structs using plaque-forming unit DNA polymerase to avoid 3Ј 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␣ Results expression plasmid using appropriate restriction sites as previously de- Binding of monoclonal antibodies to GPIb␣ chimeras on scribed.11 Each construct was verified by sequencing. Chinese hamster ovary cells

Expression of canineÐhuman chimeras in Chinese hamster Previous studies have suggested the C-terminal flank region ␤ ovary IX cells (Phe201-Gly268) is important in regulating vWF binding because CanineÐhuman GPIb␣ expression vectors (1-3 ␮g) were stably transfected gain-of-function mutations in platelet-type von Willebrand disease into CHO ␤IX cells using Lipofectamine (Gibco BRL, Gaithersburg, MD) occur within this sequence.17 In addition, 2 anti-GPIb␣ monoclonal From www.bloodjournal.org by guest on September 11, 2016. For personal use only.

BLOOD, 1 JANUARY 2002 ⅐ VOLUME 99, NUMBER 1 FUNCTIONAL ANALYSIS OF GPIb␣ 147 antibodies, AP1 and VM16d, that mapped to this region differen- tially inhibit vWF binding.11,21 The first series of chimeras (Figure 1A) was designed to complement 2 previously reported canineÐ 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 Figure 2. Binding of VM16d to GPIb␣ chimeras. Specific binding of 125I-labeled flank (C201-268), the first disulfide loop (C201-248), or the second anti-GPIb␣ monoclonal antibody, VM16d (1 ␮g/mL), to CHO ␤IX cells (5 ϫ 106/mL) loop (C249-268). Expression vectors for all these chimeras of co-expressing wild-type human GPIb␣, or canine–human chimeras of GPIb␣ for 30 125 GPIb␣ were prepared from human and canine cDNA using a minutes at 22°C. Binding is expressed relative to specific binding of I-labeled WM23 for each cell line, to account for any differences in expression blunt-end ligation method to facilitate replacement of structural of GPIb␣ between different populations of cells. Specific binding for each antibody domains at precise boundaries (Figure 1A). The constructs were was calculated by subtracting nonspecific binding measured in the presence of 100 stably transfected into CHO ␤IX cells (CHO ␤IX cells were stably ␮g/mL unlabeled antibody in a parallel assay. Error bars, Ϯ SEM (n ϭ 2). transfected with GPIb␤ and GPIX to facilitate GPIb␣ expression). Epitopes for AP1 and VM16d were evaluated by flow cytometry and C225, but not to C268 (Figure 1B). However, there was partial of wild-type GPIb␣- or chimeric GPIb␣-expressing CHO ␤IX cells binding to C248. Similarly, there was partial binding to C201-248 (Figure 1B). For each population of cells, binding of AP1 or and C249-268, but no binding to C201-268. To confirm this partial VM16d was compared with binding of WM23, a monoclonal reactivity of VM16d to C248, C201-248, and C249-268 observed antibody whose epitope is downstream of Glu-28220,23 and is, by flow cytometry, binding of VM16d to these chimeras was also therefore, present on all the chimeras (Figure 1).11 WM23 bound to assessed using 125I-labeled VM16d (Figure 2). Specific binding of cells expressing wild-type human GPIb␣ and all the chimeras, but 125I-labeled VM16d was consistent with the flow cytometry results not to CHO ␤IX cells, which lack GPIb␣ (data not shown). A shown in Figure 1B. conformation-dependent monoclonal antibody, AK2, with an epitope in the first leucine-rich repeat,11 bound to all the chimeras in which Binding of von Willebrand factor to canineÐhuman chimeras ␣ this domain contained human sequence (Figure 1B). Like wild-type of GPIb on Chinese hamster ovary cells ␣ human and canine GPIb , all the chimeras bound vWF in the Previous analysis of recombinant GPIb␣ expressed on mammalian presence of the modulator botrocetin (see next section). Together, cells11,18,19 and mapping of functional anti-GPIb␣ antibodies such these results suggested the chimeric receptors were expressed in a as AP1 and VM16d (refer to preceding section) has pointed to a functional form, without gross disruption of the conformation of role for the C-terminal flank region of GPIb␣ in regulating vWF 11 their N-terminal domains. binding. We assessed vWF binding to the canineÐhuman chimeras ␣ As the human GPIb sequence was incrementally replaced by involving replacement of human with canine sequence of the entire the canine sequence from the N-terminus, AP1 bound to C200 C-terminal flank (C201-268) or the first (C201-248) or second (canine sequence from the N-terminus up to 200, human sequence (C249-268) disulfide loops (Figure 3A). These new chimeras were from 201 on) but not to C225 (Figure 1B). AP1 also bound to the compared with a series of complementary humanÐcanine chimeras, C249-268 chimera, but not to the C201-268 or the C201-248 H104, H128, H152, and H176 (incremental restoration of human chimera, which contained canine sequence within the entire sequence from canine sequence) reported previously.11 C-terminal flank or first disulfide loop (Phe201-Cys248), respec- Consistent with our earlier results showing leucine-rich repeats tively. The epitope for AP1 was, therefore, localized to the 2 to 4 were required for ristocetin-dependent vWF binding,11 all sequence Phe201-Glu225 because AP1 bound to all the chimeras in chimeras that contained human sequence within repeats 2 to 4, but which this sequence was human, but to none of the chimeras in not H104, which contained human sequence only to the end of the which this sequence was canine. third repeat, exhibited vWF binding in the presence of ristocetin The VM16d epitope included elements from the sequence either (Figure 3B). As expected, all chimeras bound vWF in the presence side of Cys248, within Asn226-Gly268. VM16d bound to C200 of botrocetin because botrocetin does not discriminate between vWF binding to human or canine GPIb␣.11 (Cell line, % specific binding: ␣␤IX, 100%; H104, 52% Ϯ 3; H128, 84% Ϯ 4; H152, 71% Ϯ 4; H176, 34% Ϯ 9; C201-C268, 95% Ϯ 3; C201-C248, 126% Ϯ 6; C249-C268, 169% Ϯ 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, Figure 1. Binding of monoclonal antibodies to canineÐhuman chimeras of GPIb␣. (A) Canine–human chimeras of GPIb␣, where residue numbers correspond there was also no ristocetin-dependent vWF binding to chimeras to amino acid sequences of human12 and canine13 GPIb␣. Canine sequence is C225 or C248 that lacked human repeats 2 to 4 (data not shown), represented in black. Chimeras C200 and C268 have been reported previously.11 (B) consistent with lack of binding to C200 or C268.11 Binding of anti-GPIb␣ monoclonal antibodies to CHO cells expressing wild-type GPIb␣ or the chimeras indicated, as assessed by flow cytometry. Ϫ, no binding; (ϩ) Ristocetin-dependent vWF binding to the new chimeras, C201- approximately 50% maximal binding; ϩ, maximal binding. 268 and C201-248, containing canine sequence in the entire From www.bloodjournal.org by guest on September 11, 2016. For personal use only.

148 SHEN et al BLOOD, 1 JANUARY 2002 ⅐ VOLUME 99, NUMBER 1

Figure 3. Functional analysis of humanÐcanine and canineÐhuman 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 ␮g/mL) in the presence of ristocetin (1 mg/mL) to CHO ␤IX cells (5 ϫ 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 ␮g/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, Ϯ SEM (n ϭ 3). (C) Adhesion of GPIb␣ chimera-expressing CHO cells to vWF under flow. Grades are defined as follows: ϩ, 1% to 10% of cells; ϩϩ, 11% to 79% of cells; ϩϩϩ, more than 80% of cells. (Rolling velocities are Ϯ SEM.)

C-terminal flank or its first disulfide loop, respectively, was less Adhesion of Chinese hamster ovary cells to von Willebrand than binding to wild-type GPIb␣ and comparable to binding to factor under flow H128 and H176 (Figure 3B). However, one of the chimeras (C249-268), containing canine sequence only in the second disul- Under flow conditions in the absence of modulators, cells express- fide loop (Asp249-Gly268) of the C-terminal flank, bound vWF in ing C249-268, containing canine sequence only in the second the presence of ristocetin to an even greater extent than wild-type disulfide loop of the C-terminal flank, rolled significantly more GPIb␣ (Figure 3B). In fact, binding of vWF to chimeric GPIb␣- slowly on a vWF-coated surface than cells expressing wild-type ␣ expressing cells was comparable to binding to wild-type GPIb␣ GPIb (Figure 5). This effect was observed at shear stress of 5 and 2 only when the sixth leucine-rich repeat and the first loop of the 10 dynes/cm and was consistent with the gain-of-function found C-terminal flank (Phe201-Cys248) is of the same species—that is, for ristocetin-dependent binding under static conditions described either both human (wild-type or C249-268) or both canine (H152) above. The interactions of other relevant chimera-expressing cells (Figure 3B). This implies the possibility of an interaction between with vWF under flow are summarized in Figure 3C. Compared ␣ sequences within the sixth leucine-rich repeat and Phe201-Cys248. with cells expressing wild-type GPIb , only C249-268 and the Because C249-268 bound vWF to a greater extent than wild- previously described chimera, H152, containing human sequence 11 type receptor (Figure 3B), we tested vWF binding to this chimera to the end of the fifth repeat, rolled on vWF. 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 Discussion GPIb␣ at all botrocetin concentrations tested (Figure 4B). This ␣ ␣ suggested the C249-268 chimera was a gain-of-function receptor, a The N-terminal 282 residues of glycoprotein Ib (GPIb ) of the platelet result confirmed by measuring adhesion of this cell line to vWF membrane GPIb-IX-V complex contain the binding domain for vWF ␣ under flow conditions in the absence of modulators. and for other ligands such as P-selectin, Mac-1, -thrombin, factor XII, and high-molecularÐweight kininogen.1-10 In the current study, we have focused on the structureÐfunction 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 specific- ity of binding of vWF and anti-GPIb␣ monoclonal antibodies to human GPIb␣, we used a series of canineÐhuman chimeras involving the C-terminal flank to examine vWF binding under static and flow

Figure 4. Effect of ristocetin or botrocetin concentration on vWF binding to GPIb␣. Specific binding of 125I-labeled vWF (1 ␮g/mL) to CHO ␤IX cells (106/mL) Figure 5. Binding of chimera-expressing CHO cells to vWF under flow. Binding expressing wild-type human GPIb␣ or the C249-268 chimera in the presence of of CHO ␤IX cells expressing wild-type GPIb␣ or CHO ␤IX cells expressing the ristocetin (A) or botrocetin (B) at the concentrations indicated for 30 minutes at 22°C. C249-268 chimera of GPIb␣. Mean rolling velocity was derived from images snapped Specific binding was calculated from total binding by subtracting nonspecific binding at 30 frames/s during a 4-minute period of perfusion at 5 or 10 dynes/cm2 over a glass measured in the absence of modulator in parallel assays. surface coated with 50 ␮g/mL vWF. Error bars, Ϯ SEM. From www.bloodjournal.org by guest on September 11, 2016. For personal use only.

BLOOD, 1 JANUARY 2002 ⅐ VOLUME 99, NUMBER 1 FUNCTIONAL ANALYSIS OF GPIb␣ 149 conditions and to map epitopes for 2 functional antibodies that recognize cells with vWF under flow is significantly affected.21,31 These this region of the receptor. findings represent the first example where alterations to the second Anti-GPIb␣ monoclonal antibodies AP1 and VM16d were of loop of the C-terminal flank result in a gain-of-function. The particular interest because of their functional effect on binding of vWF difference between human and canine sequences within Asp249- and other ligands. AP1 was mapped to the sequence Phe201-Glu225, Gly268 include an alteration to charged residues from human to within the first disulfide loop of the C-terminal flank of GPIb␣, because canine sequence at Asp249Val,Asp252Lys, Lys253Asn, Lys258Thr, it specifically recognized chimeras in which this sequence was human and Gly263Asp that could account for differences in function. (C200, C249-268) but not in which it was canine (C225, C248, C268, An interesting feature of these results, combined with those C201-268, C201-248). Previous studies showed that AP1 binding to from our earlier studies,11 is how the interaction with vWF under GPIb␣ either was unaffected or was partially decreased (by 50% or less flow is recovered as canine GPIb␣ was incrementally made more compared with wild-type GPIb␣) by mutagenesis of individual residues human from the N-terminus. There was recovery of function to the between Gly233 and Met239.18 UnlikeAP1, other anti-GPIb␣ monoclo- extent of saltatory translocation of more than 90% of cells as nal antibodies that completely inhibit ristocetin-dependent binding human sequence was restored to the end of the fourth leucine-rich (AK2, Hip1, and 6D1) have epitopes within the first 4 leucine-rich repeat (H128). Saltatory translocation occurs when cells jump repeats.11 AP1 also inhibits botrocetin-dependent vWF binding to along the direction of flow, where there is an increased off-rate/on- GPIb␣ and rolling of GPIb-IX-expressing cells on vWF.21 rate for adhesion compared with rolling cells.11,31 Restoring human In contrast to AP1, VM16d only inhibits botrocetin-dependent, but sequence to the end of the fifth repeat (H152) resulted in rolling at not ristocetin-dependent, binding of vWF to GPIb␣12,20 or rolling of 126 Ϯ 7 ␮m/s compared with 42 Ϯ 1 ␮m/s for wild-type GPIb␣. GPIb-IXÐexpressing cells on vWF.21 The epitope for VM16d was However, as even more human sequence is restored, to the end of apparently composed of elements on either side of Cys248 within the the sixth repeat (H176) there is decreased interaction, and less than sequence Asn226-Gly268 because it bound to all chimeras where this 10% of cells display saltatory translocation (Figure 3C).11 Rolling sequence was human but only to some (approximately 50% of was again observed when the receptor was made human to the end wild-type) where either Asn226-Cys248 or Asp249-Gly268 was canine of the first loop of the C-terminal flank (C249-268). These results (C248, C201-248, C249-268). This result was confirmed by flow parallel the levels of ristocetin-dependent vWF binding to the cytometric analysis and by binding of radiolabeled VM16d to chimera- chimeras, and they support previous studies showing that ristocetin- expressing cells. There was no binding when Asn226-Cys248 and dependent vWF binding and adhesion to vWF under flow are Asp249-Gly268 sequences were canine (C268, C201-268). The partial comparable and distinct from botrocetin-dependent vWF binding.21 VM16d-binding sequence Asn226-Cys248 contains the congenital and The simplest explanation for these results is that optimal binding of artificial gain-of-function, valine substitution mutations at Gly233, chimeric GPIb␣-expressing cells to vWF, comparable to wild-type, Asp235, Lys237, and Met239 and the partial loss-of-function mutation, only occurs when the sixth leucine-rich repeat and the first loop of Ala238 to valine.18 The other component of the VM16d-binding site, the C-terminal flank (Phe201-Cys248) is of the same species, either Asp249-Gly268, constitutes the second disulfide loop of the C-terminal both human (wild-type or C249-268) or both canine (H152) flank, formed by the Cys211-Cys264 and Cys209-Cys248 disulfide (Figure 3C). Saltatory translocation (or partial ristocetin-dependent bridges.1 In studies of vWF binding, this Asp249-Gly268 loop was also binding), however, is present when leucine-rich repeats 2 to 4 are found to lead to a gain-of-function when the human sequence was human (H128, H176, C201-248, C201-268), consistent with previous replaced by canine sequence in GPIb␣ (C249-268). Definition of the results.11 Together, these findings raise the possibility of an interaction VM16d-binding site is significant in that this antibody not only inhibits between sequences within the sixth leucine-rich repeat and Phe201- vWF binding in the presence of botrocetin, it also inhibits the interaction Cys248, an interaction that could be disrupted when respective se- of GPIb␣ and Mac-1.6 Other antibodies that strongly inhibit vWF quences are cross-species. This may be an important consideration binding to GPIb␣ do not inhibit Mac-1 binding. VM16d also inhibits underlying the gain-of-function or loss-of-function mutations within the thrombin-dependent platelet aggregation.29 sequence spanning Gly233-Met239 within this first disulfide loop.18 In Extending previous studies of the vWF-GPIb␣ interac- support of this supposition, alanine substitution point mutations of tion,2,11,18,19 the current experiments provide additional insight into residues within the sixth leucine-rich repeat affected the capacity of the potential role of the C-terminal flank region of GPIb␣ in recombinant GPIb␣ to support vWF binding.32 Definitive evidence regulating vWF binding. One of the chimeras (C249-268) contain- awaits structural determination of the leucine-rich repeat and the ing canine sequence only in the second disulfide loop (Asp249- C-terminal flank domains of GPIb␣. Gly268) of the C-terminal flank bound vWF to a greater extent than In conclusion, the current study has further defined the epitopes wild-type GPIb␣ in the presence of ristocetin, and rolling of the for 2 functionally important anti-GPIb␣ monoclonal antibodies, cells to vWF under flow conditions in the absence of modulators AP1 and VM16d, that inhibit interactions involving the platelet was slower than in cells expressing wild-type GPIb␣. These membrane GPIb-IX-V complex and vWF, Mac-1, and thrombin. In interactions are unlikely to have been affected by differences in the addition, analysis of canineÐhuman chimeras of GPIb␣ expressed levels of receptor expression on different cell lines because any on CHO cells provides further evidence for a role of the C-terminal variation in expression levels on these cells (as assessed by WM23 flank domain and of leucine-rich repeats in regulating vWF binding binding) are well above levels of receptor where the interaction of under static or flow conditions. References

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2002 99: 145-150 doi:10.1182/blood.V99.1.145

Functional analysis of the C-terminal flanking sequence of platelet glycoprotein Ib α 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

Updated information and services can be found at: http://www.bloodjournal.org/content/99/1/145.full.html Articles on similar topics can be found in the following Blood collections Hemostasis, Thrombosis, and Vascular Biology (2485 articles)

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