An important role of the SRC family kinase Lyn in stimulating platelet granule secretion

Abstract

The Src family kinases (SFKs) have been proposed to play stimulatory and inhibitory roles in platelet activation. The mechanisms for these apparently contradictory roles are unclear. Here we show that SFK, mainly Lyn, is important in stimulating a common signaling pathway leading to secretion of platelet granules. Lyn knock-out or an isoform-nonselective SFK inhibitor, PP2, inhibited platelet secretion of both dense and alpha granules and the secretion-dependent platelet aggregation induced by thrombin, collagen, and thromboxane A(2). The inhibitory effect of Lyn knock-out on platelet aggregation was reversed by supplementing granule content ADP, indicating that the primary role of Lyn is to stimulate granule secretion. Inhibitory effect of PP2 on platelet aggregation induced by thrombin and thromboxane A(2) were also reversed by supplementing ADP. Furthermore, PP2 treatment or Lyn knock-out diminished agonist-induced Akt activation and cyclic GMP production. The inhibitory effect of PP2 or Lyn knock-out on platelet response can be corrected by supplementing cyclic GMP. These data indicate that Lyn stimulates platelet secretion by activating the phosphoinositide 3-kinase-Akt-nitric oxide (NO)-cyclic GMP pathway and also provide an explanation why Lyn can both stimulate and inhibit platelet activation.

FIGURE 1.

Effects of the SFK inhibitor PP2 on GPCR-induced ATP secretion and platelet aggregation. A–D, washed human platelets in modified Tyrode's buffer (3 × 10⁸/ml) were preincubated with PP2 (10 μm) or DMSO control (0.1%) at 37 °C for 2 min and then exposed to various concentrations of U46619 (A and B) or thrombin (C and D) and simultaneously recorded for ATP secretion and aggregation. The aggregation and ATP release traces are representatives of at least three different experiments. E and F, aggregation and secretion results in the experiments described in A–D were quantitated. Secretion values were normalized with respect to platelets pretreated with DMSO and stimulated with the lowest concentration of U46619 (U) (E) or thrombin (Throm) (F).

FIGURE 2.

Specificity of the effect of PP2 on platelet secretion and aggregation and restoration of the platelet aggregation by exogenous ADP in the PP2-treated platelets. A, washed human platelets in modified Tyrode's buffer (3 × 10⁸/ml) were preincubated with DMSO (0.1%), PP2 (10 μm), or an analog control for PP2, PP3 (10 μm), which does not inhibit SFK at 37 °C for 2 min, and then exposed to U46619 (500 nm). B, washed human platelets were preincubated with DMSO or PP2 (10 μm) (PP2) for 2 min at 37 °C and then stimulated with thrombin (0.025 unit/ml). PP2-treated platelets were also stimulated with thrombin followed by 0.5 μm ADP (PP2+ADP). Platelets were also stimulated with ADP alone (0.5 μm) (ADP). C, quantitative data (mean ± S.D.) from four experiments as described in B. D, washed human platelets were preincubated with DMSO or PP2 (10 μm) for 2 min at 37 °C and then stimulated with U46619 (250 nm). PP2-treated platelets were also stimulated with U46619 followed by 0.5 μm ADP. Platelets were also stimulated with 0.5 μm ADP alone.

FIGURE 3.

The inhibitory effect of PP2 on P-selectin expression. A and B, washed human platelets in modified Tyrode's buffer were preincubated with PP2 (10 μm) or DMSO for 2 min at 37 °C and then stimulated with U46619 (U) (250 nm) or thrombin (T) (0.025 unit/ml) for 5 min at 37 °C with stirring and subsequently fixed with paraformaldehyde. Fixed platelets were incubated with a monoclonal anti-human P-selectin antibody, SZ51, or negative control mouse IgG (M IgG) for 30 min at 22 °C. After washing once with PBS, platelets were further incubated with a fluorescein isothiocyanate-conjugated rabbit anti-mouse Ig antibody. Surface expression of P-selectin was analyzed using flow cytometry. Data from a representative experiment are shown in A. Quantitative results from three experiments are expressed as the P-selectin expression index (fluorescence intensity of platelets stimulated with an agonist/fluorescence intensity of unstimulated platelets) (B).

FIGURE 4.

The inhibitory effect of Lyn deficiency on platelet aggregation and secretion. A, washed platelets from Lyn^+/+ or Lyn^−/− mice were stimulated with thrombin (0.025 units/ml) in a lumiaggregometer at 37 °C. Real-time ATP secretion and platelet aggregation were simultaneously recorded. B, aggregation and secretion results for the experiments described in A were quantitated (n = 3). Secretion values were normalized with respect to platelets from Lyn^+/+ mice. C and D, washed platelets from Lyn^+/+ (C) or Lyn^−/− mice (D) were incubated with various concentrations of thrombin for 5 min at 37 °C with stirring and then fixed with paraformaldehyde. Fixed platelets were incubated with a fluorescein isothiocyanate-labeled monoclonal anti-mouse P-selectin antibody, SZ51, for 30 min at 22 °C, and analyzed using flow cytometry. The P-selectin expression index (median of fluorescence intensity with a certain concentration of thrombin/median of fluorescence intensity of unstimulated platelets (control)) is also shown. E, aggregation traces of platelets from either Lyn knock-out mice (Lyn^−/−) or wild type controls (Lyn^+/+) stimulated with thrombin or thrombin plus 0.5 μm ADP (Lyn^−/−+ADP). F, aggregation results for the experiments described in E were quantitated (n = 3). WT, wild type.

FIGURE 5.

Effects of PP2 and Lyn knock-out on collagen-induced platelet secretion and aggregation. A, washed human platelets were preincubated with PP2 or DMSO for 2 min and then exposed to collagen in a lumiaggregometer at 37 °C. Real-time ATP secretion and platelet aggregation were simultaneously recorded. B, washed human platelets were preincubated with PP2 or DMSO for 2 min and then stimulated with collagen (Coll) (0.5 μg/ml) for 5 min at 37 °C with stirring and then analyzed for P-selectin expression by flow cytometric analysis of the binding of a monoclonal anti-human P-selectin antibody, SZ51. C, aggregation and ATP secretion traces of washed platelets from Lyn^+/+ or Lyn^−/− mice stimulated with collagen (0.5 μg/ml) in a lumiaggregometer at 37 °C. D, aggregation traces of Lyn^−/− or Lyn^+/+ mouse platelets stimulated with collagen (0.5 μg/ml) or collagen plus 0.5 μm ADP (Lyn^−/−+ADP). E, quantitative data (mean ± S.D.) from three experiments as described in D. WT, wild type.

FIGURE 6.

The effect of Fyn deficiency on platelet aggregation and secretion. A, washed platelets from Fyn^+/+ or Fyn^−/− mice were stimulated with collagen (0.5 μg/ml) in a lumiaggregometer at 37 °C. Real-time ATP secretion and platelet aggregation were simultaneously recorded. B, washed platelets from Fyn^+/+ or Fyn^−/− mice were stimulated with U46619 (250 nm) in a lumiaggregometer at 37 °C. Real-time ATP secretion and platelet aggregation were simultaneously recorded. C and D, aggregation (C) and secretion (D) results for the experiments described A and B were quantitated (n = 3). Secretion values were normalized with respect to wild type platelets stimulated with collagen. U, U46616; Coll, collagen.

FIGURE 7.

The effect of PP2 on Akt phosphorylation and cGMP production. A, washed human platelets were preincubated with PP2 (10 μm) or DMSO for 2 min at 37 °C. Platelets were then stimulated with collagen (Coll) in the aggregometer for 5 min at 37 °C and solubilized with SDS-PAGE sample buffer. Phosphorylation of Akt (p-Akt) was detected by Western blotting with an antibody specifically recognizing the phosphorylated Ser⁴⁷³ site in Akt. Equal sample loading was assessed by Western blotting using an anti-Akt antibody. B, densitometry measurements from results in A. Values were normalized with respect to sample with stimulation for each immunoblot and are expressed as relative phosphorylation (mean ± S.D. from three separate experiments). Statistical significance was determined using Student's t test. C, washed human platelets were preincubated with PP2 (10 μm) or DMSO (0.1%) for 2 min at 37 °C. Platelets were then stimulated with thrombin in the aggregometer for 5 min at 37 °C and solubilized with SDS-PAGE sample buffer. Phosphorylation of Akt was detected by Western blotting. D, densitometry measurements from the results in C. Values were normalized with the value obtained with stimulated platelets for each immunoblot and are expressed as relative phosphorylation. E and F, washed human platelets were preincubated for 2 min at 37 °C with DMSO or PP2 (10 μm). The platelets were then stimulated with collagen (0.5 μg/ml) (E) or thrombin (0.05 units/ml) (F) in a platelet aggregometer for 5 min. cGMP concentrations were determined using a cGMP enzyme immunoassay kit. Results are expressed as mean ± S.D. (n = 3) (p < 0.01).

FIGURE 8.

The effect of Lyn deficiency on Akt phosphorylation and cGMP production. A, washed platelets from Lyn^+/+ or Lyn^−/− mice were stimulated with collagen (0.5 μg/ml), thrombin (0.05 units/ml), or U46619 (500 nm) in a platelet aggregometer at 37 °C for 5 min. cGMP concentrations were determined using a cGMP enzyme immunoassay kit. Results are expressed as mean ± S.D. (n = 3). B and C, washed platelets from Lyn^+/+ or Lyn^−/− mice were stimulated with collagen (B) or thrombin (C) in the aggregometer for 5 min at 37 °C and solubilized with SDS-PAGE sample buffer. Phosphorylation of Akt was detected by Western blotting. Values were normalized with the value of stimulated wild type platelets for each immunoblot and are expressed as relative phosphorylation (mean ± S.D. from three separate experiments).

FIGURE 9.

8-Bromo-cGMP rescues platelet aggregation and secretion in PP2-treated platelets. A, washed human platelets in modified Tyrode's buffer (3 × 10⁸/ml) were preincubated with DMSO (control) or PP2 (10 μm) for 2 min at 37 °C and then stimulated with thrombin (0.025 units/ml), followed by the addition of 8-bromo-cGMP (5 μm) (PP2+cGMP) or buffer (PP2). B, quantitative data (mean ± S.D.) on the percentage of aggregation (light transmission) from three experiments as described in A. C, 6 min after the addition of thrombin, luciferase/luciferin reagent was added to the platelet suspension to measure the total amount of ATP released. The data represent the mean ± S.D. of ATP release from three experiments. D, platelets from Lyn knock-out mice were stimulated with thrombin (Lyn^−/−) or thrombin followed by the addition of 8-bromo-cGMP (Lyn^−/−+Br-cGMP). Platelets from wild type controls were also stimulated with thrombin (Lyn^+/+).

FIGURE 10.

Exogenous ADP rescues cGMP in Lyn-deficient platelets. A and B, washed platelets (3 × 10⁸/ml) from Lyn^+/+ or Lyn^−/− mice were stimulated with collagen in the aggregometer for 5 min at 37 °C. They were stimulated with collagen (0.5 μg/ml) (A) or thrombin (0.025 unit/ml) (B) with or without the addition of ADP (0.5 μm). cGMP concentrations were determined using a cGMP enzyme immunoassay kit. Results are expressed as mean ± S.D. (n = 3). C, a positive feedback loop regulating platelet secretion. Platelet agonists, thrombin or collagen, bind to their receptors, which, by activating the SFK pathway, induce the activation of the PI3K/Akt pathway. PI3K/Akt induces platelet secretion via PKG. Secreted ADP activates the PI3K/Akt pathway through its G_i-coupled P2Y12 receptor, which further induces cGMP elevation.