Uncoupling ITIM receptor G6b-B from tyrosine phosphatases Shp1 and Shp2 disrupts murine platelet homeostasis

Abstract

The immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptor G6b-B has emerged as a key regulator of platelet homeostasis. However, it remains unclear how it mediates its effects. Tyrosine phosphorylation of ITIM and immunoreceptor tyrosine-based switch motif (ITSM) within the cytoplasmic tail of G6b-B provides a docking site for Src homology 2 domain-containing protein-tyrosine phosphatases Shp1 and Shp2, which are also critical regulators of platelet production and function. In this study, we investigate the physiological consequences of uncoupling G6b-B from Shp1 and Shp2. To address this, we generated a transgenic mouse model expressing a mutant form of G6b-B in which tyrosine residues 212 and 238 within ITIM and ITSM were mutated to phenylalanine. Mice homozygous for the mutation (G6b-B diY/F) were macrothrombocytopenic, as a result of the reduction in platelet production, and had large clusters of megakaryocytes and myelofibrosis at sites of hematopoiesis, similar to those observed in G6b-deficient mice and patients. Platelets from G6b-B diY/F mice were hyporesponsive to collagen, as a result of the significant reduction in the expression of the immunoreceptor tyrosine-based activation motif (ITAM)-containing collagen receptor complex GPVI-FcR γ-chain, as well as thrombin, which could be partially rescued by costimulating the platelets with adenosine diphosphate. In contrast, platelets from G6b-B diY/F, G6b KO, and megakaryocyte-specific Shp2 KO mice were hyperresponsive to antibody-mediated cross-linking of the hemi-ITAM-containing podoplanin receptor CLEC-2, suggesting that G6b-B inhibits CLEC-2-mediated platelet activation through Shp2. Findings from this study demonstrate that G6b-B must engage with Shp1 and Shp2 to mediate its regulatory effects on platelet homeostasis.

Graphical abstract

Figure 1.

Loss of ITIM/ITSM phosphorylation prevents G6b-B–Shp1–Shp2 interaction. Flow cytometric (A) and western blot (B) analysis of G6b-B expression in platelets from WT and G6b-B diY/F mice. Mean ± SEM (n = 6) of G6b-FITC median fluorescence intensity (MFI), with rat IgG_2A subtracted (Ai) and representative histogram (Aii). Representative blots (Bi) and quantification, normalized to tubulin reblots (mean ± SEM, n = 3) (Bii). (C) Lysates were prepared using washed platelets (5 × 10⁸/mL) under basal conditions and 30 μg/mL collagen–stimulated conditions (90 seconds, 37°C, stirring at 1200 rpm) from WT and G6b-B diY/F mice. Coimmunoprecipitation of Shp1 and Shp2 was investigated by western blotting, following immunoprecipitation using anti–G6b-B or nonimmune rabbit polyclonal antibodies.

Figure 2.

Uncoupling of G6b-B–Shp1–Shp2 disrupts platelet production. (A) Percentage of reticulated platelets following staining with Retic-Count (mean ± SEM, n = 6). (B) Clearance of platelets in WT and G6b-B diY/F mice, following labeling with IV NHS-biotin. (Bi) Biotin-labeled platelets measured by streptavidin-PE binding in tail vein–sampled whole blood (mean ± SEM, n = 5 or 6 per data point). (Bii) Rate of platelet elimination calculated from slope of loss of biotinylated platelets (mean ± SEM, n = 6). (Ci) Platelet recovery following anti-GPIbα antibody–mediated platelet depletion in WT and G6b-B diY/F mice (n = 8-20 per time point). (Cii) Platelet-recovery rate calculated from recovery data for WT and G6b-B diY/F mice between days 3 and 7 (mean ± SEM, n = 8-11). Representative images (Di) and quantification of the number of MKs (Dii) in hematoxylin and eosin (H&E)–stained spleen and femur sections from WT and G6b-B diY/F mice (mean ± SEM, n = 6 mice, 5 images per mouse). Arrowheads indicate MKs. Representative images of reticulin staining showing myelofibrosis of WT and G6b-B diY/F spleens and femurs (scale bars, 50 μm). (Diii) Spleen/body weight ratio in WT, G6b-B diY/F, and G6b KO mice (mean ± SEM, n = 22). ***P < .001.

Figure 3.

Aberrant platelet activation in G6b-B diY/F mice. (A) Blood loss/body weight ratio following excision of 5 mm of tail tip in isoflurane-anesthetized mice of the indicated genotypes. Data were analyzed using a bimodal function, comprising a gamma and normal distribution for low- and high-bleeding tendency, respectively. Likelihood ratio tests were performed to determine whether the probability of bleeding (P_bleed) differed between genotypes. (B-D) Flow cytometric measurement of P-selectin exposure and fibrinogen binding of WT and G6b-B diY/F platelets in whole blood using ITAM-activating (Bi-ii) and G protein–coupled receptor–activating (Ci-ii) agonists (mean ± SEM, n = 5 or 6 per stimulation). Thrombin stimulation was in the presence of 10 μM glycine-proline-arginine-proline to prevent blood clotting. (Di-ii) Rescue of activating PAR4 peptide (PAR4p)-stimulated fibrinogen binding in the presence of 10 μM ADP (mean ± SEM, n = 5 or 6). Platelets were gated using forward and side scatter, and 10 000 events were collected. *P < .05; **P < .01; ***P < .001.

Figure 4.

Aberrant platelet function in G6b-B diY/F mice. (A) Mean aggregation and ATP release traces in washed platelets (2 × 10⁸/mL). For ADP aggregations, platelets were washed in the presence of 0.02 U/mL apyrase and supplemented with 1 μM CaCl₂ and 50 μg/mL fibrinogen (mean ± SEM, n = 4 or 5). (Bi) Representative images of WT and G6b-B diY/F washed platelet (2 × 10⁷/mL) spreading on fibrinogen under basal conditions and 0.1 U/mL thrombin–preactivated conditions (scale bars, 5 μm). Quantification of surface area coverage (Bii), platelet perimeter (Biii), total platelets per image (Biv), and 4 stages of spreading in the presence of thrombin (Bv) (mean ± SEM, n = 5 or 6). ***P < .001.

Figure 5.

Reduced platelet adhesion and activation under flow conditions. PPACK, heparin, and fragmin anticoagulated whole blood from WT, G6b-B diY/F and G6b KO mice was flowed for 3.5 minutes (1000 s⁻¹ shear) over coverslips coated with collagen, vWF-BP + laminin, and vWF-BP + laminin + rhodocytin. Representative brightfield images (A) and representative fluorescence images (B) following staining with Alexa Fluor 647–conjugated annexin V, P-selectin–FITC, and JON/A-PE antibodies to measure phosphatidylserine-positive platelets, α-granule release, and αIIbβ3 integrin activation (αIIbβ3^act), respectively (scale bars, 10 μm). (C) Heat map showing the effect size of morphology scores and surface area coverage (SAC) of the indicated parameters, normalized to WT (n = 5 or 6).

Figure 6.

G6b-B–Shp2 regulates CLEC-2 signaling. Washed platelets (4 × 10⁸/mL) from G6b KO (A), G6b-B diY/F (B), Shp1 KO (C), and Shp2 KO (D) mice and litter-matched WT controls were activated with 10 μg/mL activating CLEC-2 antibody in the presence of 10 μM lotrafiban, followed by lysing and investigation of named phospho-tyrosine sites by western blotting. Representative western blots from 3 independent experiments. *P < .05; **P < .01.

Figure 7.

Structural basis of G6b-B recognition by SHP2. The specific interactions are shown by comparison of N-SH2 apo (black) and N-SH2 + 1 equivalent p-ITIM (sky blue) (A), C-SH2 apo (black) and C-SH2 + 1 equivalent p-ITSM (red) (B), and tandem SH2 apo (teal) and tandem SH2 + 1 equivalent p-ITIM+p-ITSM (red) (C). Selected chemical shift perturbations in SOFAST-HMQC NMR spectra are labeled. (D) Lowest-energy model of tandem SH2 bound to p-ITIM+p-ITSM generated by HADDOCK based on restraints generated from the NMR data. Residues showing large chemical shift perturbations are shown in blue and are defined as active residues directly involved in interaction with the peptide. Residues showing small, yet significant, chemical shift perturbations are shown in cyan and are defined as passively involved residues.