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B
post-hoc test,
****P<0.0001). (B). Washed platelets from a control or a patient with an immune thrombocytopenic purpura presenting with undetectable levels of GPVI on platelets (Patient 3) were allowed to adhere to fibrinogen (100 μg/mL). (B)(i). Representative epifluo- rescence images of washed platelets from patient 3 adhering to immobilized fibrinogen for 30 min, in the presence or absence of REOPRO (40 μg/mL). Scale bars represent 10 μm. (B)(ii). Bar graph represents the number of platelets adhering to fibrinogen per mm2. Adhesion is expressed as mean±SEM in eight random fields, in two separate experiments.
haematologica | 2018; 103(5)
Fibrinogen, GPVI and platelet activation
lized fibrinogen and binding monitored by surface plas- mon resonance. As shown in Figure 3A, clear binding of monomeric GPVI (ka = 1.17 ± 0.01 x 104 M-1s-1) was observed with a kd of 3.94 ± 0.01 x 10-3 s-1. Binding was fit- ted to a single site with an equilibrium dissociation con- stant (KD) of 336 ± 1 nmol/L. In contrast, binding of dimer- ic GPVI to fibrinogen was not detected at concentrations up to 1 μmol/L (Figure 3Ai). In a second approach, fibrino- gen was immobilized on a plastic surface and a solid phase binding assay was performed. There was increased binding of monomeric GPVI, but not dimeric GPVI, which was inhibited by D-dimer (Figure 3Aii) where the binding motif in fibrin resides.30 To further investigate the ability of GPVI to bind to fibrinogen, we transfected rat RBL-2H3 basophilic cells, which constitutively express integrin aIIbβ3 at low levels, with human GPVI and studied adhe- sion to immobilized fibrinogen. We observed a 3-fold increase in adhesion of GPVI-transfected cells to fibrino- gen and to collagen relative to the adhesion of mock-trans- fected control cells (Figure 3Bi, ii). RBL-2H3 cells express- ing human GPVI also formed stress fibers upon adhesion to fibrinogen. The human GPVI-blocking monoclonal
A
antibody 9012 Fab blocked the increase in adhesion. Blocking the integrin aIIbβ3 with REOPRO reduced cell adhesion to immobilized fibrinogen to the same level as 9O12 Fab, with no further inhibition in the presence of both inhibitors (data not shown), indicating the presence of additional binding proteins for fibrinogen in the adherent cell line although binding to these was not sufficient to induce spreading (Figure 3Biii and not shown). These results demonstrate that GPVI binds to immobilized fib- rinogen and is able to contribute to cell adhesion.
Spreading of human platelets but not mouse platelets is dependent on Syk
The formation of lamellipodial sheets and stress fibers in human platelets on fibrinogen and collagen is blocked by the inhibitors of Src and Syk tyrosine kinases, PP2 and PRT060318, respectively (Figure 4Ai & ii). Adhesion of human platelets to fibrinogen induces phosphorylation of Syk which co-precipitates with the phosphorylated FcR γ- chain (Figure 4Aiii). These results provide further evidence of GPVI activation in human platelets by immobilized fib- rinogen. In contrast, the morphological modifications of
Figure 1. Glycoprotein VI supports platelet adhesion and spreading on immobilized fibrinogen. (A, B) Washed human platelets were allowed to adhere to immobilized fibrinogen (10 μg/mL) for 30 min. (A)(i). Representative epifluorescence images of fibrinogen-adherent platelets from healthy donors (Control) or members of a family with a mutation in the GP6 gene (Heterozygotes: Family 1 +/-; homozygotes: Family 1 -/-). Scale bars represent 10 μm. (A)(ii). Bar graph representing the percentage of platelets spreading on fibrinogen. Spreading is expressed as the mean±SEM in five random fields, in two separate experiments (one-way ANOVA, Kruskal-Wallis post-hoc test, ***P<0.0002; ****P<0.0001). (A)(iii). Bar graph representing the number of platelets adhering to immo- bilized fibrinogen per mm2 of a control (Control) and two families with a muta- tion in the GP6 gene. Adhesion is expressed as mean±SEM in five ran- dom fields, in two separate experi- ments (one-way ANOVA, Kruskal-Wallis
**P<0.002;
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