Physical and functional association of the Src family kinases Fyn and Lyn with the collagen receptor glycoprotein VI-Fc receptor gamma chain complex on human platelets

Abstract

We have previously shown that uncharacterized glycoprotein VI (GPVI), which is constitutively associated and coexpressed with Fc receptor gamma chain (FcRgamma) in human platelets, is essential for collagen-stimulated tyrosine phosphorylation of FcRgamma, Syk, and phospholipase Cgamma2 (PLCgamma2), leading to platelet activation. Here we investigated involvement of the Src family in the proximal signals through the GPVI-FcRgamma complex, using the snake venom convulxin from Crotalus durissus terrificus, which specifically recognizes GPVI and activates platelets through cross-linking GPVI. Convulxin-coupled beads precipitated the GPVI-FcRgamma complex from platelet lysates. Collagen and convulxin induced tyrosine phosphorylation of FcRgamma, Syk, and PLCgamma2 and recruited tyrosine-phosphorylated Syk to the GPVI-FcRgamma complex. Using coprecipitation methods with convulxin-coupled beads and antibodies against FcRgamma and the Src family, we showed that Fyn and Lyn, but not Yes, Src, Fgr, Hck, and Lck, were physically associated with the GPVI-FcRgamma complex irrespective of stimulation. Furthermore, Fyn was rapidly activated by collagen or cross-linking GPVI. The Src family-specific inhibitor PP1 dose-dependently inhibited collagen- or convulxin-induced tyrosine phosphorylation of proteins including FcRgamma, Syk, and PLCgamma2, accompanied by a loss of aggregation and ATP release reaction. These results indicate that the Src family plays a critical role in platelet activation via the collagen receptor GPVI-FcRgamma complex.

Figure 1

Affinity precipitation of the GPVI–FcRγ complex with convulxin-coupled Sepharose 4B from platelet lysates. Unstimulated gel-filtered platelets were lysed directly in SDS sample buffer or in Triton X-100 lysis buffer. Whole lysates (lane 1) and proteins precipitated by affinity with convulxin-coupled Sepharose 4B (lane 2) or Sepharose 4B alone (lane 3) were resolved by 10% (A) or 12.5% SDS-PAGE (B), transferred to nitrocellulose membranes, and immunoblotted with anti-GPVI IgG (A) or anti-FcRγ IgG (B). Molecular mass markers are indicated in kD on the right of panels.

Figure 2

Tyrosine phosphorylation of proteins coprecipitated with the GPVI–FcRγ complex in collagen- and convulxin-stimulated platelets. Gel-filtered platelets were unstimulated (lane 1) or stimulated for 1 min at 37°C with 50 μg/ml of collagen (lane 2) or 50 ng/ml of convulxin (lanes 3 and 4) and lysed in Triton X-100 lysis buffer. Precipitated proteins with convulxin-coupled Sepharose 4B (lane 1–3) or Sepharose 4B (lane 4) were resolved by 10% (A, top, B, and C) or 12.5% SDS-PAGE (A, bottom and D), transferred to nitrocellulose membranes, and immunoblotted with Abs against phosphotyrosine (A), Syk (B), GPVI (C), and FcRγ (D). Molecular mass markers are indicated in kD on the right of panels. In A (bottom) and D open and closed arrows mark unphosphorylated and phosphorylated FcRγ, respectively.

Figure 3

Coprecipitation of Fyn and Lyn, but not Yes, Src, and Fgr, with the GPVI–FcRγ complex from platelet lysates. Gel-filtered platelets were unstimulated (lanes 1 and 2) or stimulated for 1 min at 37°C with 50 μg/ml of collagen (lane 3) or 50 ng/ml of convulxin (lanes 4 and 5) and lysed in Triton X-100 lysis buffer (lanes 2–5) or directly in SDS sample buffer (lane 1). Whole lysates (lane 1) and precipitated proteins with convulxin-coupled Sepharose 4B (lanes 2–4) or Sepharose 4B (lane 5) were resolved by 10% SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with Abs against Fyn (A), Lyn (B), Yes (C), Src (D), and Fgr (E).

Figure 4

Coimmunoprecipitation of Fyn and Lyn with anti-FcRγ IgG and coimmunoprecipitation of GPVI and FcRγ with anti-Fyn and anti-Lyn Abs. Unstimulated gel-filtered platelets were lysed directly in SDS sample buffer or in Triton X-100 lysis buffer. (A–C) Whole lysates (lane 1) and immunoprecipitates with preimmune rabbit IgG (lane 2) and anti-FcRγ IgG (lane 3) were resolved by 10% SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with Abs against Fyn (A), Lyn (B), and Src (C). (D and E) Whole lysates (lane 1) and immuno-precipitates with Abs against Fyn (lane 2), Lyn (lane 3), Yes (lane 4), and Src (lane 5) were resolved by 10% (D) or 12.5% (E) SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with anti-GPVI IgG (D) and anti-FcRγ IgG (E).

Figure 5

Kinase activity of Fyn and Lyn in platelets stimulated by collagen, convulxin, or anti-GPVI F(ab′)₂. Gel-filtered platelets were stimulated for the indicated periods at 37°C with collagen (A, 50 μg/ml), convulxin (B and D, 50 ng/ml), or anti-GPVI F(ab′)₂ (C, 150 μg/ml) and lysed in RIPA buffer. Fyn (A–C) and Lyn (D) were immunoprecipitated from lysates and subjected to in vitro kinase assay with enolase as an exogenous substrate, followed by 10% SDS-PAGE. Radioactivity of each protein was visualized (A–D) and quantified with the Bio-Imaging Analyzer BAS-2000II. Kinase activity toward enolase was calculated (H). Positions of Fyn, Lyn, and enolase are indicated on the right.

Figure 6

The Src family-specific inhibitor PP1 dose-dependently inhibits collagen- or convulxin-induced tyrosine phosphorylation of whole lysates. Gel-filtered platelets were preincubated for 3 min at 37°C with 0.25% DMSO (lanes 1 and 2) or the indicated concentrations of PP1 (lanes 3–7). Platelets were unstimulated (lane 1) or stimulated with 50 μg/ml of collagen for 30 s (A, lanes 2–7) or 50 ng/ml of convulxin for 15 s (B, lanes 2–7), directly lysed in SDS sample buffer, resolved by 10% SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with anti-phosphotyrosine mAb. Molecular mass markers are indicated in kD on the right of panels. Arrows mark the position of Src.

Figure 6

The Src family-specific inhibitor PP1 dose-dependently inhibits collagen- or convulxin-induced tyrosine phosphorylation of whole lysates. Gel-filtered platelets were preincubated for 3 min at 37°C with 0.25% DMSO (lanes 1 and 2) or the indicated concentrations of PP1 (lanes 3–7). Platelets were unstimulated (lane 1) or stimulated with 50 μg/ml of collagen for 30 s (A, lanes 2–7) or 50 ng/ml of convulxin for 15 s (B, lanes 2–7), directly lysed in SDS sample buffer, resolved by 10% SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with anti-phosphotyrosine mAb. Molecular mass markers are indicated in kD on the right of panels. Arrows mark the position of Src.

Figure 7

PP1 dose-dependently inhibits collagen-induced Syk recruitment and tyrosine phosphorylation of FcRγ and PLCγ2. Gel-filtered platelets were preincubated for 3 min at 37°C with 0.25% DMSO (lanes 1 and 2) or the indicated concentrations of PP1 (lanes 3–6). Platelets were unstimulated (lane 1) or stimulated with 50 μg/ml of collagen for 30 s (lanes 2–6) and lysed in Triton X-100 lysis buffer. Precipitated proteins with convulxin-coupled Sepharose 4B (A–E) or immunoprecipitates with anti-PLCγ2 Ab (F and G) were resolved by 10% (A, B, and E–G) or 12.5% (C and D) SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with Abs against phosphotyrosine (A, C, and F), Syk (B), FcRγ (D), GPVI (E), and PLCγ2 (G). In H the band densities from tyrosine-phosphorylated Syk (A), FcRγ (C), and PLCγ2 (F) were quantified and graphed as a percentage of the band density in lane 2 of each panel. The position of each protein is indicated on the right of panels.

Figure 8

PP1 dose-dependently inhibits collagen- or convulxin- induced aggregation and ATP release reaction. Gel-filtered platelets were preincubated for 3 min at 37°C with 0.25% DMSO or various concentrations of PP1 (μM) as indicated. Platelets were stimulated with 2 μg/ml of collagen (A and a), 10 ng/ml of convulxin (B and b), or 0.1 U/ml of thrombin (C and c) under constant stirring conditions. Aggregation curves (A–C) and ATP release (a–c) were monitored. This experiment is representative of four similar experiments from different donors.