Canonical Wnt signaling negatively regulates platelet function

Abstract

Wnts regulate important intracellular signaling events, and dysregulation of the Wnt pathway has been linked to human disease. Here, we uncover numerous Wnt canonical effectors in human platelets where Wnts, their receptors, and downstream signaling components have not been previously described. We demonstrate that the Wnt3a ligand inhibits platelet adhesion, activation, dense granule secretion, and aggregation. Wnt3a also altered platelet shape change and inhibited the activation of the small GTPase RhoA. In addition, we found the Wnt-beta-catenin signaling pathway to be functional in platelets. Finally, disruption of the Wnt Frizzled 6 receptor in the mouse resulted in a hyperactivatory platelet phenotype and a reduced sensitivity to Wnt3a. Taken together our studies reveal a novel functional role for Wnt signaling in regulating anucleate platelet function and may provide a tractable target for future antiplatelet therapy.

Fig. 1.

The canonical Wnt-β-catenin pathway is present in platelets. (A) Wnt binds to a surface receptor complex comprising of the Fzd and LRP5/6 receptors. In the absence of Wnt, β-cat is phosphorylated by a destruction complex containing CK1, GSK3β, Axin-1, FRAT-1, and APC, which target it for proteosomal degradation. In the presence of Wnt, β-cat is not phosphorylated and accumulates in the cytosol. Activatory signals are denoted by normal arrows, inhibitory signals by flat-headed arrows. (B) Positive control lysates (Ctrl), Resting (R) and 5 μM TRAP-activated (A) platelets were resolved by SDS/PAGE and immunoblotted with antibodies to (i) Fzd isoforms 1–9, (ii) LRP5/6, (iii) Dvl-2, (iv) Axin-1, (v) APC, (vi) FRAT-1, (vii) CK1α, (viii) GSK-3β and (ix) β-cat. Representative blots are shown from 3 replicates.

Fig. 2.

Wnt3a inhibits platelet aggregation. (A) A representative aggregation trace showing Wnt3a to decrease aggregation in a concentration-dependant manner in 1.5 μM TRAP-stimulated platelets. (B) The mean result of 3 independent TRAP (1.5 μM)-induced aggregation experiments in the presence of increasing doses of Wnt3a are shown (IC₅₀ = 32 nM). (C) Aggregation was measured in response to numerous platelet agonists in the absence (black bars) and presence (gray bars) of 32 nM Wnt3a. All data shown are representative of 3 or more independent platelet preparations ± SEM. (*, P < 0.05)

Fig. 3.

Effects of Wnt3a on granule secretion. (A) (i) CD62P expression was measured using flow cytometry in the absence (black bars) and presence of increasing concentrations of Wnt3a (32 nM is denoted by dark gray bars and 50 nM by light gray bars). No treatment or EDTA-treated samples served as controls. (ii) A representative histogram trace detailing CD62P expression in resting (gray shaded curve) and TRAP-activated platelets in the absence (black dotted line) and presence (black solid line) of Wnt3a. (B) ATP release from 1.5 μM and 5 μM TRAP stimulated platelets in the absence (black bars) and presence (light gray bars) of 50 nM Wnt3a measured in a luminescent aggregometer. All results represented as a mean value ± SEM from 3 independent platelet preparations (*, P < 0.05).

Fig. 4.

Wnt3a inhibits integrin αIIbβ3 activation. (A) (i) A FITC conjugated antibody for PAC-1 binding was used in flow cytometry to measure αIIbβ3 activation to increasing concentrations of TRAP in the absence (black bars) and presence of 32 nM (dark gray bars) and 50 nM (light gray bars) Wnt3a. (ii) A representative histogram trace shows resting (solid gray curve) and 2.5 μM TRAP stimulated platelets in the absence (black dotted line) and presence of 50 nM Wnt3a (black solid line). (B) A static adhesion assay was performed to quantify the number of platelets adhering to Fb in the presence of increasing concentration of Wnt3a. Data are expressed as a % of the positive control (platelet-only sample, containing no Wnt3a). Abciximab (10 μg/ml) was used as a negative control. All results represented as a mean value ± SEM from 3 independent platelet preparations (*, P < 0.05).

Fig. 5.

Wnt3a inhibits RhoA activation and the rho-kinase dependant pathway of platelet shape change. (A) Inhibition of RhoA activation when 50 nM Wnt3a was added to platelets before thrombin activation (0–0.1U/ml). Anti-RhoA blot shows total RhoA in lysed samples. 5 μM ADP-induced platelet shape change in response to a variety of conditions was monitored by Optical Density (OD) in a ChronoLog aggregometer by measuring (B) maximum % increase in OD and (C) % increase in OD 3 min after stimulation, both versus vehicle-treated only. Data shown are representative of 3 separate donors and are presented as mean ± SEM (*, P < 0.05).

Fig. 6.

Modification of Wnt canonical pathway events in platelets. (A) A phospho-GSK3β (S9) ELISA was carried out using resting and 1.5 μM TRAP-activated washed platelet samples in the presence/absence of Wnt3a. The phospho-GSK3 concentration (pg/ml) of each sample is represented as a mean value ± SEM from at least 3 separate platelet preparations. (B) Resting (R) and 1.5 μM TRAP-activated (A) platelet lysates, pretreated with 50 nM Wnt3a (50W and 50W/A respectively) were immunoblotted for phosphorylated (S37) β-cat. Blots were stripped and reprobed with anti-β-cat to demonstrate equal loading. Data shown are representative of 3 replicate experiments.

Fig. 7.

Fzd4 and 6 are present in platelets and Fzd6 −/− platelets have an increased activatory phenotype. (A) RT-PCR of platelet RNA using primers specific for Fzd4, 6 and GAPDH demonstrated their expression in platelets (predicted product sizes 211, 222, and 288 bp, respectively. (B) Fzd6−/− mice have increased platelet activation. Platelets were isolated from Fzd6+/− and Fzd6−/− mice and activated with thrombin (n = 4± SD.; P < 0.01 vs. Fz6+/−). (C) Wnt3a does not antagonize Fzd6−/− platelet activation. Fzd6+/− (Left) and Fzd6−/− (Right) platelets were incubated in the absence/presence of Wnt3a and stimulated with thrombin (0.25 U/ml).