Blood, Vol. 114, Issue 9, 1893-1899, August 27, 2009

The low-frequency isoform of platelet glycoprotein VIb attenuates ligand-mediated signal transduction but not receptor expression or ligand binding
Blood Trifiro et al. 114: 1893

Supplemental materials for: Trifiro et al

Recombinant soluble (rs) GPVIa and GPVIb ectodomain dimers (residues 1 to 269). Platelet mRNA from homozygous GP6aa or GP6bb donors served as template to amplify cDNA using the Superscript one step RT-PCR kit (Gibco BRL, Gaithersburg, MD) and primers GP6F (5′-CTC AGG ACA GGG CTG AGG AA -3′, nucleotide nt 4–23) and GP6R (5′-CCA TGA TCC CTC CCT TGG AT -3′, nt 1089–1070). cDNA was subcloned into pGEM-T (Promega, Madison, WI) and sequenced.

Tandem cysteine residues were inserted upstream from FLAG in p3XFLAG-CMV™-14 (Sigma Chemical Corp., St Louis, MO) using QuikChange®II Site-Directed Mutagenesis Kit (Stratagene) and primers:
forward = GTACCAGTCGACTGTAGAGGATGCCGGGCTGACTAC; and
reverse = GTAGTCAGCCCGGCATCCTCTACAGTCGACTGGTAC,
to create cys-p3XFLAG. GPVIa or GPVIb cDNA were amplified using the full-length GP6a and GP6b cloned in pGEM-T as templates. The forward primer included a 5′ Hind III restriction site (5′-GACCCAAGCTTTCAGGACAG-3′); and the reverse primer, a 3′ Bgl II restriction site (5′-GAAGATCTTCGTTGCCCTTGGTGTAGTACT-3′). A Hind III / Bgl II segment was excised and subcloned into cys-p3XFLAG to create pGPVI-Cys-3XFLAG (Figure S1).

HEK293T cells were transiently transfected with the pGPVIa-Cys-3XFLAG or pGPVIb-Cys-3XFLAG, using the Effectene protocol (Qiagen; Valencia, CA). After 48 hours, rsGPVI was affinity purified from the media using Anti-FLAG®M2 Affinity Gel (Sigma), according to the manufacturer’s instructions, but in the presence of 1mM N-Ethylmaleimide (NEM) (Sigma). rsGPVIa and rsGPVIb were monitored by SDS-PAGE and Western blotting, using biotinylated convulxin (biotin-CVX) or murine monoclonal anti-human GPVI antibody LJ6.5.1 rsGPVI dimers have an MWApp of 110 kDa under nonreduced conditions; monomers, an MWApp of 60 kDa under nonreduced conditions.

Dimers were separated from monomers using a Superdex 200 10/300 GL column (Amersham Biosciences; Piscataway, NJ) with a bed volume of 24 mL. The column was pre-equilibrated in PBS (pH 7.4) containing 1 mM N-ethylmaleimide (NEM) (Sigma-Aldrich, St Louis, MO, USA) to prevent disulfide exchange, and 0.1 mg of rsGPVIa or rsGPVIb was loaded in 0.2 mL of PBS with 1 mM NEM and run at 0.2 mL per minute. Identification of protein-enriched eluted fractions was monitored by absorbance at 280 nm. An analysis of peak fractions yielded two partially overlapping peaks, the first with an average retention time corresponding to an MWApp of 100 kDa, and the second slower peak corresponding to an MWApp of 50 kDa. These experimentally derived MWApp values correspond well to the MWApp expected of GPVI dimers and monomers, respectively. The protein composition of pooled peak fractions was then analyzed by western blot.

Binding of soluble GPVIa or GPVIb ectodomains to ligands. Aliquots (100 µl) of type I collagen (20 µg/ml), CVX (5 µg/ml) or CRP (1 µg/ml) were added to the wells of 96-well microtiter plates and incubated overnight at 4°C. The wells were rinsed twice with 150 µl of TBS and incubated a further 1 hour with 150 µl of 2% (w/v) bovine serum albumin (BSA) to block any remaining reactive sites. Following two additional rinses with TBS, 100 µl of soluble rsGPVIa or rsGPVIb (0.5, 1, 2, 5 or 10 µg per ml) were added to separate wells, each in the presence of 1mM of NEM. The plates were incubated at 37°C for 90 min, then the protein suspensions were removed by aspiration, and the wells were rinsed three times with 150 µl TBS. Bound GPVI was detected using, as primary antibody, murine monoclonal anti-FLAG (1:50000 dilution in TBS) (Upstate Group, Charlottesville, VA), and as secondary antibody, horseradish peroxidase conjugated-Goat anti Mouse IgG (Upstate). Color was developed using the ABTS peroxidase substrate (KPL, Inc., Gaithersburg, MD) according to the manufacturer's instructions.

Stable transfected Dami cell lines. Full-length GPVIa or GPVIb cDNA was subcloned into the mammalian expression vector pcDNA3 (Invitrogen, Carlsbad, CA) and separately transfected into Dami cells using the Effectene transfection system (Qiagen). Selection was carried out for 2 weeks in Iscove modified Dulbecco medium (IMDM) supplemented with 0.5 mg/mL geneticin (GibcoBRL). The surviving cells were cloned to establish stable cell lines that could be screened for adhesion to collagen, convulxin or CRP using a colorimetric assay.2

Co-precipitation of FcRγ with GPVIa or GPVIb. Stable Dami cell lines expressing recombinant (r) GPVIa or GPVIb were solubilized in 1% (wt/vol) digitonin at a cell density of 107/mL. Soluble protein was incubated with rabbit anti-FcRγ IgG or LJ6.5 for 2 hours at ambient temperature, and protein G–Sepharose (Pierce Chemical, Rockford, IL) was then added. After a 1-hour incubation, the beads were washed 4 times in Tris (tris(hydroxymethyl)aminomethane)–buffered saline (TBS) containing 0.1%Tween 20 (Promega), resuspended in NuPAGE loading buffer (Invitrogen) and incubated at 70°C for 10 minutes. The proteins thus eluted were separated by NuPAGE using a 4% to 12% gradient slab gel in MOPS (3-N-morpholino)-propane sulfonic acid)–SDS buffer. Proteins separated in the polyacrylamide gel were transferred electrophoretically to a polyvinylidenedifluoride (PVDF) membrane, and the membrane was then probed with either rabbit anti-FcRγ IgG or LJ6.5. Bound probes were visualized by enhanced chemiluminescence (ECL; Amersham) using horseradish peroxidase (HRP)–conjugated secondary reagents.

Calmodulin (CaM) binding to GPVI cytoplasmic tails. Four maltose-binding protein (MBP) fusion constructs were synthesized, following the protocol of Suzuki-Inoue et al.,3 in which the entire 51 amino acid GPVI cytoplasmic domain (residues 289–339) was inserted downstream from MBP. These constructs are designated by the amino acid substitution at position 317 and/or 322 and are named QH (representing GPVIa); QN, LH and LN (representing GPVIb).

Washed platelets, as the source of CaM, were lysed in 2% v/v Triton X-100 in the presence of 1mM PMSF, 10ug/ml leupeptin, 10ug/ml aprotonin, 1ug/ml pepstatin A, and 10 mM EGTA, and the soluble protein was separated by high speed centrifugation. An aliquot of the platelet lysate was pre-cleared by adsorption with Protein G Sepharose beads (Amersham, Piscataway, NJ) that had been washed in lysis buffer. Equal protein (µg) from each of the recombinant MBP-GPVI constructs was incubated with an aliquot of the soluble platelet protein for 1 hour at 4ºC, then mouse monoclonal anti-calmodulin (Upstate) was added, and the mixture was adsorbed with pre-washed protein G beads. The beads were pelleted, rinsed, and resuspended in Laemmli buffer containing SDS, and incubated at 80°C for 15 minutes. The beads were pelleted once again, and the soluble proteins contained in the supernatant were separated by SDS-PAGE on a 4–12% polyacrylamide gel.

The relative amount of MBP-GPVI in each anti-CaM immunoprecipitate was determined by western blot using alkaline phosphatase conjugated anti-MBP (Sigma). The relative amount of precipitated CaM was determined by western blot of the same membranes with alkaline phosphatase conjugated anti-calmodulin (Upstate).

CaM binding to full-length GPVIa or GPVIb isolated from transfected Dami cells. Dami cell lines stably transfected with GPVIa or GPVIb were washed twice in PBS, resuspended in 40 mM Tris-HCl, 10mM EGTA, pH7.3, containing 1 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 µg/ml pepstatin, and 2% v/v Triton X100 (lysis buffer) and incubated for 1 hour on wet ice. Insoluble material was then pelleted by centrifugation at 16,000 × g for 1 min at room temperature, and the supernatant was aspirated and maintained at 4°C (wet ice). Lysate proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane, and the relative amount of GPVI in the lysis supernatant from each cell line was determined by a western blot using biotin-conjugated LJ6.5 followed by peroxidase-conjugated streptavidin (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). An appropriate dilution of each cell lysate that contained an equivalent amount of GPVI protein was precleared by mixing with 50 µl of a 1:1 slurry of washed Protein G agarose beads (Amersham, Uppsala, Sweden) in lysis buffer for 1 hr at 4°C. The beads were removed by centrifugation at 16,000 × g for 1 min at 4°C, and the lysate was then incubated overnight (12–18 hr) at 4°C with 4 µg of monoclonal anti-calmodulin (Upstate). The mixture was then added to 50 µl of a 1:1 slurry of a new batch of washed Protein G agarose beads and incubated for 2 hr at 4°C. The agarose beads were pelleted by centrifugation at 2000 × g for 5 min at 4°C, then resuspended and pelleted an additional three times in ice cold lysis buffer to rinse the beads, and finally resuspended in 25 µl of 2× Laemmli sample buffer (Invitrogen). The mixture was incubated in a boiling water bath for 5 minutes, and the beads were pelleted by centrifugation at 16,000 × g for 1min at room temperature. The samples were subjected to SDS-PAGE, transferred to a nitrocellulose membrane, and the relative amount of GPVI recovered was determined by western blot. The amount of calmodulin present in each sample was also determined by western blot of the same membranes with monoclonal anti calmodulin antibody.

Monoclonal LJ6.5 IgG was conjugated with biotin using EZ-Link Sulfo-NHS-Biotin Reagent (Pierce, Rockford, IL, USA), according to the manufacturer’s instructions.

A relative quantification of the protein bands in photographic films derived from western blots was performed by optical densitometry using the 2(-Delta Delta C(T)) method, as described by Livak and Schmittgen.4

Fyn/Lyn binding to GPVI cytoplasmic tails. We employed an ELISA, described by Suzuki-Inoue et al.,3 in which we measure the binding of maltose binding protein-GPVI cytoplasmic domain fusion proteins (MBP-GPVI) to the SH3 domains of the Src kinases Fyn and Lyn. For this ELISA, plasmids expressing glutathione sulfonyl transferase (GST)-SH3 fusion proteins were generously provided by Dr. Steve Watson (University of Birmingham, Birmingham, UK).

Briefly, we measured the binding of MBP-GPVI to GST-Fyn-SH3, GST-Lyn-SH3 and GST-Btk-SH3, as a negative control, that were attached to pre-blocked glutathione coated microtiter plates (Sigma, St. Louis, MO.). Firstly, conditions to saturate microtiter wells with equivalent amounts of GST-SH3 constructs were established in a separate ELISA measuring the binding of alkaline-phosphatase (AP)-conjugated anti-GST. Using these optimized conditions, MBP-GPVI constructs were added to the GST-SH3 coated wells for 60 minutes in the absence or presence of 0.3 µM or 1 µM calcium chloride, the plates were washed, and the amount of MBP-GPVI construct bound was detected with alkaline phosphatase-conjugated mouse monoclonal anti-MBP (Sigma). These concentrations of calcium represent the high end of the experimentally determined range of platelet cytosolic calcium that is produced upon engagement of GPVI by agonists.3

Quantitation of total platelet GPVI by western blot. This is a modification of the method that we initially reported.5 Nine volumes of blood were mixed with one volume of 10 mM EDTA, and platelet -rich plasma was isolated, as described.5 Platelets were pelleted by centrifugation, resuspended in 15 mmol/L Tris-HCl, pH 7.4, containing 145 mmol/L NaCl (TBS) plus 1 mmol/L EDTA (TBS-EDTA), and washed by three successive centrifugations and resuspensions in TBS-EDTA. Platelets were then resuspended in TBS-EDTA containing 2 mM N-ethylmaleimide, and the platelet count was adjusted to 5 × 10E6 per µl. One volume of the platelet suspension was mixed with an equal volume of sample loading buffer containing 2% (v/v) sodium dodecyl sulfate (SDS). The mixture was incubated in a boiling water bath for 10 minutes to affect complete lysis of platelets. The anticoagulation of blood in EDTA, the subsequent platelet isolation and washing in the presence of EDTA and the lysis of platelets by SDS in the presence of EDTA minimizes and/or eliminates ADAM-10 cleavage of GPVI.6

Platelet lysate proteins were separated by SDS-PAGE on a 12% slab gel under nonreduced conditions (lysis buffer included 1 mM NEM), as previously described.5 The separated proteins were transferred to a polyvinylidine difluoride (PVDF) membrane at 250 mA overnight, and the membrane was then blocked by incubation for 90 minutes in a solution of 5% fat-free milk in 10 mmol/L Tris-HCl, pH 7.4, containing 145 mmol/L NaCl (TBS) plus 0.05% (vol/vol) Tween 20 (TBS-T). The murine monoclonal antibody LJ6.5 was used as ligand, replacing CVX,7 and with biotin using EZ-Link Sulfo-NHS-Biotin Reagent (Pierce, Rockford, IL, USA), according to the manufacturer’s instructions. The membrane was incubated for 120 minutes at ambient temperature in 2 µg/ml (final) biotin-conjugated murine monoclonal antibody LJ6.5 diluted in TBS-T. The membrane was then incubated for 10 min with gentle agitation in four successive 50 ml volumes of TBS-T and then at ambient temperature for 30 minutes in 20 ml of a 1:5000 dilution of horseradish peroxidase–conjugated streptavidin (Zymed Laboratories) in TBS-T. Four additional rinses followed, and then GPVI was visualized by enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech). From the developed image, the density and area of bands corresponding to GPVI were optically scanned and quantified by using Scion Image software (Scion Corp).

All platelet samples were normalized to a standard platelet sample, as described.5 Four aliquots (5, 10, 20, and 40 µg total protein per well) of the stock standard sample generated a linear relationship. To assure that limiting amounts of test GPVI were analyzed in each test sample, duplicate aliquots were analyzed at 40, 20 and 10 µg total protein per well. The amount of GPVI per 20 µg of platelet protein was expressed in arbitrary units relative to the standard sample, where the amount of GPVI in the standard sample per 20 µg of platelet protein = 10.

Comparison of total platelet GPVI level and surface GPVI expression. Previous studies by Kahn and coworkers8,9 have established that the surface expression of platelet GPVI varies several-fold among normal subjects or patients with prior myocardial infarction (MI) and patients with prior MI exhibit a significant increase in mean platelet surface GPVI. Since we had conducted comparisons of total platelet GPVI levels among normal donors for several years, we sought to confirm that the level of surface GPVI varies proportionally to the level of total GPVI. In a subset of 22 of the 132 donors analyzed in this study, we compared total GPVI, measured by western blot, and surface GPVI, measured by the binding of biotinylated-HY101 in flow cytometry (Figure S2). Surface HY101 binding was measured using a whole blood assay, as previously described,10 and the results are expressed as geometric mean fluorescence intensity (GMFI). To minimize platelet activation and GPVI shedding, blood was anticoagulated in EDTA, and processed within 30 minutes. A direct correlation between total GPVI and surface GPVI was observed by linear regression analysis (R2 = 0.8574; p

Recombinant soluble GPVI isoforms rsGPVIa and rs GPVIb. By western blot with LJ6.5 (Figure S3), purified recombinant rsGPVIa and rsGPVIb each consisted of dimers and monomers (approximate proportion of 1:2) with apparent molecular weights of 110 kDa and 60 kDa. Purified rsGPVIa and rsGPVIb were subjected to size exclusion chromatography using a Superdex 200 10/300 GL column in the presence of NEM (non-reduced). By western blot with LJ6.5 (Figure S3), the pooled peak fractions with faster retention times were found to be enriched in rsGPVIa or rsGPVIb dimers. By optical densitometry, the fraction of rsGPVIa or rsGPVIb dimers in each of three separate preparations ranged from 88–92%.

Files in this Data Supplement:

  • Table S1. Relative content of total platelet GPVI as a function of GP6 genotype (PDF, 13.5 KB)

  • Table S2. Cloned, stably-transfected Dami cell lines (PDF, 13.6 KB)

  • Figure S1. Soluble, recombinant human GPVIa and GPVIb ectodomains (PDF, 38.8 KB) -
    (A) pGPVI-Cys-3XFLAG includes a 5′ uncoded sequence containing a HindIII restriction site followed downstream by the GPVI sequence (Methionine-1 through Asparagine-269), a 15-amino acid linker sequence (black highlighted) that includes a BglII restriction site and two cysteine codons, a 22-amino acid, triple FLAG sequence (grey highlighted) and tandem stop codons. (B) The corresponding amino acid sequence. The two cysteine residues (C) in the linker sequence are underlined.





  • Figure S2. Comparison of total and surface GPVI (JPG, 17.6 KB) -





  • Figure S3. Soluble, recombinant human GPVIa and GPVIb (JPG, 7.69 KB) -
    HEK293 cells were transfected with either pGPVIa-Cys-3XFLAG or pGPVIb-Cys-3XFLAG. After 48 hours in culture, the media were removed, and soluble GPVI was purified by adsorption to anti-FLAG agarose and elution. The eluted proteins were separated by SDS-PAGE under nonreduced conditions (in the presence of 1 mM NEM) and transferred to a nitrocellulose membrane. GPVI monomers (60 kDa) and dimers (110 kDa) were detected by western blot using LJ6.5 anti-human GPVI monoclonal antibody in both the purified GPVIa (lane 1) and GPVIb (lane 2) preparations. Comparisons can be made to dimer-enriched preparations of purified GPVIa (lane 3) or GPVIb (lane 4) prepared by Superdex 200 10/300 GL column chromatography.





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