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Western blotting
For stimulation on fibrinogen, washed platelets were pre-treat- ed with 10 μmol/L indomethacin and 2 U/mL apyrase. Platelets (1.5 mL containing 5x108/mL) were allowed to adhere to 10 cm dishes coated with 100 μg/mL fibrinogen or heat-inactivated bovine serum albumin for 45 min at 37°C. Non-adherent platelets were removed and lysed by addition of 2X lysis buffer (150 mmol/L NaCl, 10 mmol/L Tris, 1 mmol/L EGTA, 1 mmol/L EDTA, 1% NP-40; pH 7.4, plus 1.25 mmol/L Na3VO4, 50 μg/mL AEBSF, 2.5 μg/mL leupeptin, 2.5 μg/mL aprotinin and 0.25 μg/mL pep- statin). Adherent platelets were washed twice with Tyrode buffer then lysed with 1X lysis buffer on ice for 15 min before scraping. Proteins were immunoprecipitated with a-Syk antibody and pro- tein A-sepharose beads for 2 h. The beads were washed, proteins eluted in sodium dodecyl sulfate (SDS) sample buffer, separated by SDS-polyacrylamide gel electrophoresis (PAGE), electro-trans- ferred, and western blotted with the stated antibodies. For whole platelet lysates, washed platelets (5x108/mL) were lysed directly with an equal volume of 2xSDS sample buffer, separated by SDS- PAGE, electro-transferred, and western blotted with the stated antibodies.
Ca2+ assay and in vitro perfusion assay
Intraplatelet Ca2+ concentrations following platelet adhesion to
fibrinogen were measured using a dual-dye ratiometric method and hirudinated blood perfusion was performed as previously described.40 Three-dimensionsal reconstructed images were obtained using the 3D module of Leica LAS X software.
Solid-based binding assay
Binding studies were performed with the recombinant proteins, GPVI-Fc fusion (dimer) and GPVI-His tagged (monomer). Cover slips were coated with collagen or fibrinogen overnight at 4°C. The plates were blocked with 3% bovine serum albumin – phos- phate-buffered saline for 1 h and washed prior to addition of monomeric or dimeric GPVI at a concentration of 100 nmol/L for 1 h. After washing, 4 μg/mL of secondary antibodies, horseradish peroxidase (HRP)-conjugated goat anti-human IgG Fc or HRP-con- jugated anti-His Tag, were added for 1 h. GPVI binding was detected using 3,3′,5,5′-tetramethylbenzidine. The reaction was stopped with H2SO4 (2 mol/L) and absorbance was measured at 450 nm with a spectrofluorometer.
Surface plasmon resonance
Surface plasmon resonance was performed on a Pioneer plat- form from PALL® FortéBio® (Portsmouth, UK). IF-1 purified fib- rinogen was diluted to 100 μg/mL using 10 mmol/L NaAc pH 5.0. IF-1 fibrinogen was adsorbed to the chip surface via amine cou- pling to a level of 3825 resonance units (RU) at flow-cell 1 and 3423 RU on flow-cell 3. Flow-cell 2 was activated using amine coupling and blocked using 1 mol/L ethanolamine and was the designated reference channel. GPVI analytes were dialysed and diluted to 1 μmol/L using the same batch of running buffer as used for the blanks. Analytes were injected using the OneStep® titration function at a flow rate of 30 μL/min with a 100% loop-inject and 400 s dissociation. The chip surface was regenerated by flushing with 1 mol/L NaCl at 60 μL/min for 10 s, followed by a further 400 s dissociation. Qdat data analysis software (PALL® FortéBio®, UK) was used to analyze the data. Binding data were fitted using a one site KA/KD model and analyte aggregation parameters adjusted per binding curve according to goodness of fit and curve type.
Statistics
The statistical analyses were performed using the GraphPad Prism program, version 5.0 (Prism, GraphPad, LaJolla, CA, USA).
The values are indicated as mean ± standard error of the mean (SEM). The statistical analysis is described in the Figure legends.
Results
Abolition of spreading on fibrinogen in glycoprotein VI-deficient human platelets
Human platelets undergo robust spreading on immobi- lized fibrinogen, generating lamellipodial sheets and stress fibers.41 This is illustrated in Figure 1A with over 90% of platelets from a control donor undergoing full spreading on fibrinogen over 30 min; the small number of partially- spread platelets most likely represent newly adhered cells. In 2013, Matus et al. described four unrelated families with index cases who are homozygous for an adenine insertion in exon 6 of human GP6, which leads to a premature ‘stop codon’ in position 242 prior to the transmembrane domain.17 All four homozygous patients lack expression of GPVI on their platelets and heterozygous relatives express approximately 50% of the receptor. Since then, two fur- ther unrelated families with the same mutation have been identified by the same group and also been shown to lack surface expression of GPVI with absent platelet aggrega- tion to collagen.30 Unexpectedly, in studying platelets from two unrelated index cases in these families, we observed reduced adhesion on immobilized fibrinogen and a failure to form lamellipodial sheets and stress fibers (Figure 1A). The absence of GPVI was confirmed by flow cytometry and by abolition of aggregation to collagen but not to other agonists in both cases30 (data not shown). In contrast, spreading and adhesion of platelets from heterozygote carriers from each family and platelets from a control were similar (Figure 1A). The same result was also seen in a patient with an auto-immune thrombocytopenia associat- ed with the absence of GPVI expression (Figure 1Bi). Adhesion of platelets was blocked by the aIIbβ3 receptor antagonist, REOPRO (Figure 1B), as previously shown in controls. These results demonstrate that adhesion of human platelets on fibrinogen is critically dependent on integrin aIIbβ3 with a minor contribution from GPVI, but that full spreading requires GPVI.
Mouse platelets expressing human glycoprotein VI undergo full spreading on fibrinogen
Mouse platelets adhere and undergo limited spreading on human or mouse fibrinogen, forming filopodia and limited lamellipodia but not stress fibers (Figure 2A). A similar response is seen in platelets deficient in GPVI (Figure 2A), whereas adhesion is abolished in the absence of the integrin β3-subunit (Figure 2B). A similar level of adhesion is seen in human GPVI transgenic mouse platelets but is associated with the formation of lamellipo- dial sheets and stress fibers (Figure 2C). These results demonstrate that full spreading but not adhesion of mouse platelets is dependent on human GPVI and not mouse GPVI. One potential explanation for these results is that human but not mouse GPVI is able to bind to fibrinogen and mediate platelet activation.
Fibrinogen binds to monomeric human glycoprotein VI
To test whether fibrinogen is able to bind to GPVI, increasing concentrations of recombinant soluble GPVI extracellular domain, expressed either as a monomer (GPVI-ex) or dimer (GPVI-Fc), was flowed over immobi-
haematologica | 2018; 103(5)