Page 167 - Haematologica-5

haematologica | 2018; 103(5)
Fibrinogen, GPVI and platelet activation
and fibrin but is only involved in post-adhesive events on laminin.6,11 Determining the importance of the interaction of GPVI with each ligand in mediating platelet activation in vivo will require development of selective inhibitors.
GPVI is involved in arterial thrombosis and in several of the more recently discovered roles of platelets, including maintenance of vascular integrity at sites of inflammatory challenge.12 We and others have reported that the absence of GPVI reduces experimental thrombosis in mouse mod- els of atherosclerotic plaque rupture13,14 and abolishes occlusive thrombus formation following FeCl3 injury.15 In contrast, the absence or blockade of GPVI has a relatively minor impact on hemostasis in mice16 and patients defi- cient in GPVI have a relatively mild bleeding diathesis.17–19 These results highlight that GPVI is a promising anti- thrombotic target with inhibitors predicted to have a minor effect on hemostasis.20
Following ligand binding, GPVI stimulates signals that convert integrin aIIbβ3 from a low to a high affinity state for fibrinogen and other physiological ligands.21 Ligand engagement of integrin aIIbβ3 has been reported to gen- erate outside-in signals that are similar to those of GPVI, including activation of Src and Syk kinases, PLCγ2 and Ca2+ mobilization.22–24 Paradoxically, however, human but not mouse platelets generate extensive lamellipodial sheets and stress fibers on fibrinogen whereas on colla- gen, which stimulates similar signals, full spreading of platelets is seen in both species.25 One explanation for this difference is the presence of the low affinity immune receptor, FcγRIIA, in the human but not the rodent genome, as FcγRIIA-transfected transgenic mouse platelets exhibit increased spreading and Syk activation upon adhesion to fibrinogen, although the increase in spreading is only partial.26–28 Outside-in signaling by aIIbβ3 is also mediated by two conserved tyrosines pres- ent in a NxxY motif in the integrin β3 intracytoplasmic domain independent of Src and Syk activation. Mutation of these two tyrosine residues to phenylalanine leads to a re-bleeding diathesis that has been attributed to a defect in clot retraction.29 This shows that engagement of integrin aIIbβ3 leads to activation of multiple signaling pathways.
In the present study, we showed that full spreading of human platelets on fibrinogen is abolished in patients deficient in GPVI and that transgenic mouse platelets expressing human GPVI, in contrast to wild-type platelets, spread fully on fibrinogen. Direct binding of fib- rinogen to human GPVI was demonstrated using human GPVI-transfected cell lines and recombinant GPVI. Inhibiting the binding of fibrinogen to GPVI limited platelet aggregation under conditions that excluded involvement of collagen and fibrin.
Methods
Patients
Family 1 and family 2 are two families who are heterozygous or homozygous for an adenine insertion in exon 6 of GP6 that gen- erates a premature stop codon in position 242 of the protein.17 They have been described previously.30 Patient 3 is a 10-year old boy suffering from an autoimmune disease with anti-GPVI anti- bodies. The platelets of this patient do not aggregate to collagen and GPVI is not detected at the platelet surface using flow cytom- etry and western blot (data not shown).
Mice
Wild-type mice were generated from breeding of heterozy- gotes or purchased from Harlan Laboratories (Hillcrest, UK) or Charles River (Lyon, France). GPVI-/- mice were provided by Dr Jerry Ware.31 Mouse platelets expressing human GPVI have been described and characterized previously.32 Syk chimera mice have been described previously.27 Ethical approval for ani- mal experimentation was obtained from the French Ministry of Research and UK Home Office in accordance with the European Union guidelines, the Guide for the Care and Use of Laboratory Animals.
Reagents
PRT-060318 was obtained from Caltag Medsystems (Buckingham, UK), REOPRO from E. Lilly (Indianapolis, IN, USA). Recombinant GPVI was made as described elsewhere.33 Fibrinogen was from Kabi (Bad Homburg, Germany) or from ERL (South Bend, IN, USA). RAM.1 (anti-GPIbβ) was generated in U949.34 The blocking Fab fragment of monoclonal antibody directed against human GPVI, 9O12.2, and its humanized ver- sion are referred to as 9O12 in this manuscript.35,36 All other reagents were from previously described sources.11,37 The anti- fibrin antibody 59D8 was obtained from CT Esmon (Oklahoma Medical Research Foundation, OK, USA).38
Generation and characterization of RBL-2H3 cells
The cDNA of WT human GPVI
between the 5’-XhoI and 3’-BamHI restriction sites. Rat basophilic leukemia cells, RBL-2H3, were cultured in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum albumin and stably transfected with 1 μg of DNA corre- sponding to the empty vector or WThGPVI vector mixed with FuGENE6 (Roche, Boulogne-Billancourt, France) and selected in growth medium containing G418 0.7%; 1 mg/mL geneticin (GibcoBRL, Invitrogen, Cergy Pontoise). Cell surface expression of recombinant GPVI and constitutively expressed integrin aIIbβ3 was confirmed by flow cytometry and immunoblot (data not shown).
Cell adhesion to fibrinogen
LAB TEK 4 wells were coated with 400 μL/well of collagen (50 μg/mL) or fibrinogen (100 μg/mL) overnight at 37°C. Wells were saturated with human serum albumin (10 mg/mL) for 1 h at 37°C. Trypsinised RBL cells (3x105 cells/mL) were incubated with phosphate-buffered saline or 9O12 (50 μg/mL) and/or REOPRO (40 μg/mL) for 15 min at 37°C. Subsequently, 100,000 cells were added to the wells (300 μL) for 1 h at 37°C. After three washing steps, cells were fixed with 400 μL paraformalde- hyde 4% for 20 min. Pictures were taken with an EVOS optic microscope (x10). Actin was stained with Alexa-488-phalloidin and the nucleus with DAPI.
Washed platelets
Human blood was taken from patients or from healthy donors using 3.8% (v/v) sodium citrate (1:9) as the anticoagu- lant. Human and mouse washed platelets were obtained by centrifugation using prostacyclin (2.8 μmol/L) and resuspended in modified Tyrode-HEPES buffer as described elsewhere.37,39
Platelet spreading
Platelet adhesion to immobilized fibrinogen was achieved as described previously.37 Images of the platelets were obtained with a Zeiss Axiovert 200 mol/L microscope or with a Leica DMI400 microscope. Platelet surface area was analyzed using ImageJ software (NIH, Bethesda, USA).
33
was inserted in pSRαNeo
899