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Keywords:

  • CD148;
  • collagen;
  • kinase;
  • phosphatase;
  • platelets;
  • Src

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References
  10. Supporting Information

Summary. Background: We have previously shown that the receptor-like protein tyrosine phosphatase (PTP) CD148 is essential for initiating glycoprotein VI (GPVI) signaling in platelets. We proposed that CD148 does so by dephosphorylating the C-terminal inhibitory tyrosine of Src family kinases (SFKs). However, this mechanism is complicated by CD148-deficient mouse platelets having a concomitant reduction in GPVI expression. Objectives: To investigate the effect of CD148 on GPVI signaling independent of the decrease in GPVI expression and to further establish the molecular basis of the activatory effect of CD148 and downregulation of GPVI. Methods: CD148-deficient mouse platelets were investigated for functional and biochemical defects. The DT40/NFAT-lucifierase reporter assay was used to analyze the effect of CD148 on GPVI signaling. CD148–SFK interactions and dephosphorylation were quantified using biochemical assays. Results: CD148-deficient mouse platelets exhibited reduced collagen-mediated aggregation, secretion and spreading in association with reduced expression of GPVI and FcR γ-chain and reduced tyrosine phosphorylation. The phosphorylation status of SFKs suggested a global reduction in SFK activity in resting CD148-deficient platelets. Studies in a cell model confirmed that CD148 inhibits GPVI signaling independent of a change in receptor expression and through a mechanism dependent on tyrosine dephosphorylation. Recombinant CD148 dephosphorylated the inhibitory tyrosines of Fyn, Lyn and Src in vitro, although paradoxically it also dephosphorylated the activation loop of SFKs. Conclusions: CD148 plays a critical role in regulating GPVI/FcR γ-chain expression and maintains a pool of active SFKs in platelets by directly dephosphorylating the C-terminal inhibitory tyrosines of SFKs that is essential for platelet activation.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References
  10. Supporting Information

Src family kinases (SFKs) play a critical role in initiating signaling from various tyrosine kinase-linked platelet receptors. They have also been shown to play a minor signaling role downstream of some G protein-coupled receptors, including the thrombin receptor PAR-1 and the thromboxane A2 (TxA2) receptor TP [1]. SFKs are highly active, promiscuous enzymes that are tightly regulated by tyrosine phosphorylation [2]. Phosphorylation of the tyrosine residue in the C-terminal tail by Csk and the related kinase Ctk/Chk maintains SFKs in an inactive conformation through an intra-molecular interaction with the SH2 domain that masks the active site [2]. Dephosphorylation of the C-terminal tail relieves this interaction and renders the kinase active. Maximal activation is attained after trans-autophosporylation of the activation loop tyrosine that stabilizes the active conformation [3]. Although a great deal is known about which signaling pathways SFKs are involved in regulating, little is known about how protein tyrosine phosphatases (PTPs) regulate SFK activity in platelets.

PTPs have long been hypothesized to be important regulators of signal transduction in platelets [4]. To date, four classic non-transmembrane PTPs, PTP-1B, Shp1, Shp2 and MEG2 and one classic receptor-like PTP (RPTP), CD148 (also referred to as DEP-1, PTPRJ and HPTPη) have been identified in platelets. PTP-1B is the most well studied PTP in platelets and has been shown to be an essential positive regulator of αIIbβ3-associated Src [5–8]. The biologic and molecular functions of other non-transmembrane PTPs in platelets remain largely undefined.

CD148 consists of a large, glycosylated extracellular region containing eight fibronectin type III repeats, a single transmembrane domain and a single cytoplasmic PTP domain. CD148 is expressed in many cell types, including epithelial and endothelial cells, fibroblasts and most hematopoietic cells. Recent work has shown that CD148 and the structurally distinct RPTP CD45 have overlapping functions in B cell and macrophage immunoreceptor signaling [9]. CD45 is highly and exclusively expressed in all hematopoietic cells, except erythrocytes and platelets. CD148- and CD45-deficient B cells and macrophages have impaired B cell and Fcγ receptor proximal signaling, respectively [9]. These defects are exacerbated in CD45:CD148 double-deficient B cells and macrophages [9]. The molecular mechanism can at least partially be explained by hyper-phosphorylation of the C-terminal inhibitory tyrosine of SFKs, rendering the SFKs catalytically less active than in wild-type cells. Based on previous findings from our group, we proposed that CD148 initiates glycoprotein VI (GPVI) and αIIbβ3 signaling in platelets by a similar mechanism [10].

The main objective of this study was to investigate how CD148 regulates SFK activity in platelets. We focused on collagen signaling, because SFKs are essential for initiating signaling from collagen receptors, GPVI and the integrin α2β1. GPVI mediates platelet activation via the immunoreceptor tyrosine-based activation (ITAM)-containing FcR γ-chain, and the integrin α2β1 primarily mediates adhesion and spreading on collagen, but also exhibits weak outside-in signaling [11]. Both GPVI and α2β1 signal through the sequential activation of SFKs, Syk tyrosine kinase and phospholipase Cγ2. In the present study, we demonstrate that CD148 maintains a pool of active SFKs in resting platelets that are essential for an optimal platelet response to collagen.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References
  10. Supporting Information

Materials

Sources of all chemicals and reagents used in this study can be found in Supplemental Methods.

Mice

CD148 transmembrane-knockout (CD148 TM-KO) mice on a C57Bl/6 background were generated as previously described [9]. All procedures were undertaken, with United Kingdom Home Office approval in accordance with the Animals (Scientific Procedures) Act of 1986.

Preparation of washed human platelets

Washed human and mouse platelets were prepared, as previously described [12].

Platelet aggregation and ATP secretion

Platelet aggregation and ATP secretion were measured using a Chrono-Log lumi-aggregometer (Havertown, PA, USA), as previously described [10].

Platelet static adhesion and spreading assay

This assay was performed, as previously described with minor modifications (Supplemental Methods) [12,13].

DT40/NFAT luciferase assay

This assay was performed, as previously described (Supplemental Methods) [14].

Platelet biochemistry

Washed human and mouse platelet whole cell lysates (WCLs) were prepared and western blotted, as previously described [12].

In vitro substrate-trapping assay

This assay was performed, as previously described (Supplemental Methods) [15].

In vitro dephosphorylation of SFKs

This protocol can be found in Supplemental Methods.

Kinetics of dephosphorylation of SFK-derived phospho-peptides

The kinetics of dephosphorylation of synthetic phospho-peptides corresponding to the activation loop and C-terminal inhibitory tyrosine residues of SFKs (Supplemental Table S1) by recombinant CD148 were measured in real-time using the EnzChek Phosphatase Assay kit (Invitrogen, Paisley, UK), as previously described [16].

Statistical analysis

All statistical analysis was performed with GraphPad Prism version 4. Data shown in Fig. 1 were analyzed using Student’s t-test. Data in Fig. 3 were analyzed by two-way anova followed by Tukey’s multiple comparison test. Data were considered statistically significant if P < 0.05.

image

Figure 1.  Impaired collagen-mediated functional responses of CD148-deficient platelets. (A) Platelet aggregation and ATP secretion were measured using a lumi-aggregometer. Washed platelets (2 × 108 mL−1) from wild-type (WT) and CD148 transmembrane-knockout (KO) mice were stimulated with 10 μg mL−1 collagen in the absence (Ai) and presence (Aii) of 10 μM indomethacin (cyclooxygenase inhibitor) and 2 U mL−1 apyrase (ADP scavenger). Representative images are shown from six mice per genotype per condition. (Bi) Washed platelets (2 × 107 mL−1) from WT and KO mice were pre-treated with either 0.1% DMSO, a combination of 2 U mL−1 apyrase and 10 μM indomethacin or 0.1 U mL−1 thrombin for 10 min before being placed on a collagen-coated coverslips (10 μg mL−1 collagen) for 45 min at 37 °C. Images were captured by differential interference contrast microscopy. Representative images are shown from three mice per genotype per condition. Scale bar equals 5 μm. (Bii) The mean surface area of platelets was quantified (±standard deviation) for 50–150 platelets from three randomly selected views for each mouse. (Biii) The mean number of platelets per field of view (±standard deviation) from three randomly selected views for each mouse. Data sets were compared using Student’s t-test (***P ≤ 0.001).

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image

Figure 3.  Catalytically active CD148 enhances glycoprotein VI (GPVI) signaling in DT40 cells. DT40 chicken B cells were transiently transfected with various combinations of the following expression plasmids: wild-type CD148 [CD148(WT)], catalytically inactive CD148 [CD148(C/S)], GPVI and FcR γ-chain (GPVI/FcRγ), and the NFAT luciferase reporter. Transfected cells were stimulated with 1 μg mL−1 collagen for 6 h at 37 °C and subsequently lysed. Luminescence is directly proportional to the amount of GPVI signaling. NFAT luciferase activity was normalized to activity in GPVI/FcR γ-chain transfected cells stimulated with 1 μg mL−1 collagen in each experiment. Data are presented as mean ± standard deviation (five independent experiments). Data were found to be significant by two-way anova, both in terms of treatment and expression of different constructs (P ≤ 0.0001). Statistical significance between various conditions was subsequently determined by Tukey’s multiple comparison test (*≤ 0.05).

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Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References
  10. Supporting Information

Reduced collagen reactivity of CD148-deficient mouse platelets

As the collagen response of platelets is heavily dependent on released ADP and TxA2, we started by investigating aggregation and ATP secretion of platelets from CD148-deficient mice in the presence and absence of the ADP scavenger apyrase and cyclooxygenase inhibitor indomethacin. Reduced collagen-mediated aggregation and ATP secretion exhibited by CD148-deficient platelets was further reduced in the presence of apyrase and indomethacin, demonstrating that released ADP and TxA2 partially mask these defects (Fig. 1Ai, ii).

We next analyzed the ability of CD148-deficient platelets to adhere and spread on a collagen-coated surface, in order to determine whether integrin α2β1 function was abrogated in the absence of CD148. Optimal platelet adhesion and spreading on collagen is dependent on GPVI-mediated inside-out signaling; integrin α2β1-mediated adhesion, spreading and weak outside-in signaling; and released ADP and TxA2 that enhance the collagen activation signals. CD148-deficient platelets exhibited reduced spreading on collagen under basal conditions that was further reduced in the presence of apyrase and indomethacin (Fig. 1Bi, ii). Thrombin pretreatment enhanced adhesion and spreading of CD148-deficient platelets to collagen, however, they still did not spread to the same extent as control platelets under the same conditions (Fig. 1Bi, ii). CD148-deficient platelets adhered normally to collagen under all conditions tested (Fig. 1Biii).

CD148-deficient platelets have concomitant reduction in GPVI/FcR γ-chain expression

GPVI is constitutively associated with the ITAM-containing FcR γ-chain on the platelet surface. GPVI expression is also directly dependent on FcR γ-chain expression. Previous findings from our group demonstrated that CD148-deficient platelets have approximately 58% reduction in GPVI surface expression that may contribute to the collagen phenotype, particularly at low concentrations of collagen [10]. In the present study, we checked whether CD148-deficient platelets had a concomitant reduction in FcR γ-chain expression. Whole cell lysates prepared of resting control and CD148-deficient platelets were western blotted with anti-GPVI and anti-FcR γ-chain antibodies. GPVI and FcR γ-chain expression were reduced in CD148-deficient platelet lysates compared with control platelet lysates (Fig. 2).

image

Figure 2.  Reduced glycoprotein VI (GPVI) and FcR γ-chain expression in CD148-deficient platelets. Whole cell lysates prepared of washed platelets (5 × 108 mL−1) from wild-type (WT) and CD148 transmembrane knockout (KO) mice were western blotted with anti-CD148, -GPVI, -FcR γ-chain and -actin antibodies. Results are representative of ten mice per genotype.

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Catalytically active CD148 enhances GPVI signaling

As CD148-deficient platelets have a concomitant reduction in GPVI/FcR γ-chain expression, the DT40/NFAT-luciferase reporter assay was used to determine whether CD148 catalytic activity enhances GPVI signaling. This assay has been demonstrated to provide a sensitive readout of sustained ITAM signaling [14]. DT40 chicken B cells were transiently transfected with the GPVI/FcR γ-chain complex on its own or in combination with either wild type or catalytically inactive CD148 and the NFAT luciferase reporter. GPVI/FcR γ-chain expression (C1239S) was the same under all conditions tested (data not shown). Cells were grown overnight and stimulated the following day with 1 μg mL−1 collagen for 6 h. Luciferase activity of the lysates was measured and used as a direct readout of GPVI signaling. Co-expression of wild-type CD148 with the GPVI/FcR γ-chain complex enhanced collagen-induced NFAT reporter activity by approximately 2.5-fold compared with the GPVI/FcR γ-chain complex alone (Fig. 3). In contrast, catalytically inactive CD148 had no effect on GPVI signaling in the same assay (Fig. 3). These findings demonstrate that CD148 catalytic activity enhances GPVI signaling.

Impaired collagen signaling in CD148-deficient platelets

We next tested whether collagen signaling was impaired in CD148-deficient platelets. We focused on early time points (0–30 s) as previous findings demonstrated a block in GPVI proximal signaling in CD148-deficient platelets [10]. A high concentration of collagen (100 μg mL−1) was used to minimize the effect of reduced GPVI/FcR γ-chain expression in CD148-deficient platelets. Stimulations were performed in the presence of apyrase, indomethacin and lotrafiban to inhibit the effects of secondary mediators and integrin αIIbβ3 outside-in signaling. Whole cell lysates were western blotted with an anti-phosphotyrosine antibody. A dramatic reduction in the number and intensity of the majority of bands was observed in CD148-deficient platelets compared with wild-type platelets, including phosphorylation of the FcR γ-chain, although this is complicated by the reduction in FcR γ-chain expression (Figs 2 and 4Ai). However, hyper-phosphorylated bands were consistently detected at 60–53 kDa (where SFKs migrate) and 24–22 kDa (doublet) in resting and collagen-stimulated CD148-deficient platelets that may represent CD148 substrates (Fig. 4Ai). Longer stimulations (90–300 s) demonstrated that collagen signaling was severely impaired, but not abolished in CD148-deficient platelets (Fig. 4B).

image

Figure 4.  Impaired proximal collagen-induced signaling in CD148-deficient platelets. Washed platelets (5 × 108 mL−1) from wild-type (WT) and CD148 transmembrane knockout (KO) mice were stimulated with 100 μg mL−1 collagen for various lengths of time (s) in the presence of 2 U mL−1 apyrase, 10 μM indomethacin and 10 μM lotrafiban. (Ai and B) Platelets were lysed and whole cell lysates (WCLs) western blotted with an anti-phosphotyrosine (p-Tyr) antibody. Positions of Src family kinases (SFKs) and potential substrates of CD148 are indicated at 60–53 and 24–22 kDa (arrows and ?). The position of the FcR γ-chain is also indicated at 14 kDa. Membranes were stripped and re-blotted with an anti-actin antibody. WCLs were also immunoblotted with: (Aii) SFK activation loop p-Tyr, (Aiii) Lyn p-Tyr-507 (inhibitory site), (Aiv) Fyn p-Tyr-530 (inhibitory site), (Av) Src p-Tyr-529 (inhibitory site) and (C) Syk p-Tyr-352 antibodies.

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As SFKs are essential for initiating ITAM and integrin signaling and their activity is tightly regulated by phosphorylation, we next checked the phosphorylation status of SFKs in mutant platelets using phospho-specific antibodies. SFKs were found to be hypo-phosphorylated on their activation loop tyrosines in resting and collagen-stimulated CD148-deficient platelets compared with control platelets (Fig. 4Aii). Fyn, Lyn and Src were concomitantly hyper-phosphorylated at their C-terminal inhibitory tyrosines p-Tyr-507, p-Tyr-530 and p-Tyr-529, respectively, suggesting that these sites may be substrates of CD148 (Fig. 4Aiii, v). Interestingly, Lyn was more highly phosphorylated at its inhibitory site compared with either Fyn or Src in CD148-deficient platelets.

A critical early signaling event in GPVI signaling is the recruitment and activation of the tyrosine kinase Syk to the phosphorylated FcR γ-chain. This interaction is mediated by the tandem SH2 domains of Syk, which bind to the FcR γ-chain ITAM. Syk is also essential for initiating integrin α2β1 signaling. Using a phospho-specific antibody, we found that Syk was hypo-phosphorylated at Tyr-352 in collagen-stimulated CD148-deficient platelets (Fig. 4C). Phosphorylation of Syk Tyr-352 is a SFK-mediated event that enhances phosphorylation and activation of PLCγ [17,18].

Collectively, these findings suggest that SFK activity is reduced in resting and collagen-stimulated CD148-deficient platelets, resulting in the attenuation of all downstream signaling events.

Platelet-derived SFKs interact with recombinant CD148 in vitro

An in vitro substrate-trapping/pull down assay was used to identify potential substrates and interacting proteins of CD148 in platelets. MBP-tagged fusion proteins of the cytoplasmic tail of wild-type [MBP:CD148(WT)], catalytically inactive [MBP:CD148(C/S)] and substrate-trapping forms of CD148 [MBP:CD148(D/A)] were coupled to amylose-resin and incubated with pervanadate-stimulated human platelet lysates. Interacting proteins were eluted from the fusion proteins and western blotted with an anti-phosphotyrosine antibody. Prominent bands migrating at 90, 56, 43 and 40 kDa were specifically pulled down with MBP:CD148(D/A) (Fig. 5Ai). Several minor bands were also identified at 160, 130 and 100 kDa (Fig. 5Ai).

image

Figure 5.  Src family kinases (SFKs) interact with recombinant CD148 in vitro. MBP-tagged fusion proteins of the cytoplasmic tail of wild-type, catalytically inactive (C1239S) and substrate-trapping (D1205A) forms of CD148 [MBP:CD148(WT), MBP:CD148(C/S) and MBP:CD148(D/A), respectively] were coupled to amylose-resin and used to pulldown interacting proteins from pervanadate (100 μM) stimulated human platelet lystates. (A) Proteins eluted from the resins were western blotted with: (i) anti-phosphotyrosine (p-Tyr), (ii) anti-Lyn (Lyn), (iii) anti-Fyn (Fyn), (iv) anti-Src (Src) and (v) anti-MBP (MBP) antibodies. (Ai) Arrows indicate phospho-proteins specifically pulled down with the substrate-trapping mutant. Positions of MBP:CD148 fusion proteins and Src family kinases (SFKs) are also indicated. (B) MBP:CD148(D/A) was pre-treated with (+) or without (−) 2 mM vanadate prior to being used in the pulldown assay. Proteins eluted off of the MBP:CD148(D/A)-coated amylose-resin were western blotted with: (i) anti-p-Tyr, (i) anti-Lyn, (iii) anti-Fyn and (iv) anti-Src antibodies. Results are representative of 3–4 experiments.

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Based on the biochemical data from CD148-deficient platelets described earlier, we hypothesized that SFKs are direct substrates of CD148. In support of this hypothesis, Fyn, Lyn and Src were all pulled down with the CD148 substrate-trapping mutant (Fig. 5Aii, iv). Further, these interactions were blocked with the phosphate analogue vanadate, which binds to the active site of recombinant CD148 (Fig. 5B).

Recombinant CD148 dephosphorylates platelet-derived SFKs in vitro

We tested whether recombinant CD148 could dephosphorylate SFKs in vitro. Pervandate-treated platelet lysates were incubated with the catalytically active recombinant CD148 PTP domain then western blotted with phospho-specific antibodies to the C-terminal inhibitory or activation loop tyrosines of SFKs. The catalytically inactive CD148 cytoplasmic tail was used as a negative control. Under these conditions, the CD148 PTP domain preferentially dephosphorylated the activation loop tyrosines of all SFKs and the C-terminal inhibitory tyrosine of Lyn (Tyr-507) (Fig. 6i, ii). The CD148 PTP domain was less efficient at dephosphorylating the C-terminal inhibitory tyrosine of Src (Tyr-529) and Fyn (Tyr-530) (Fig. 6iii, iv). Catalytically inactive CD148 did not dephosphorylate any of these sites. These results suggest that CD148 differentially dephosphorylates the activation loop and inhibitory tyrosines of SFKs.

image

Figure 6.  CD148 dephosphorylates the activation loop and C-terminal inhibitory tyrosines of Src family kinases (SFKs) in vitro. Lysates prepared of pervandate (100 μM) treated platelets were incubated with either recombinant MBP, catalytically active wild-type CD148 PTP domain [CD148(WT)] or catalytically inactive (C1239S) MBP:CD148 [MBP:CD148(C/S)] fusion proteins. Lysates were subsequently western blotted with phospho-specific antibodies against: (i) phosphorylated activation loop tyrosine of all SFKs (SFKs activation loop p-Tyr), (ii) phosphorylated Lyn C-terminal inhibitory tyrosine (Lyn p-Tyr-507), (iii) phosphorylated Src C-terminal inhibitory tyrosine (Src p-Tyr-529) and (iv) phosphorylated Fyn C-terminal inhibitory tyrosine (Fyn p-Tyr-530). Corrected band intensities indicated below each panel were quantified using Adobe Photoshop cs version 8.0 (arbitrary units after subtraction of background staining). Representative data from two experiments.

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Kinetics of dephosphorylation of SFK-derived phospho-peptides

The kinetics of dephosphorylation of a panel of synthetic SFK-derived phospho-peptides (Supplemental Table S1) by the recombinant CD148 PTP domain was measured in real-time using the EnzCheck spetrophotometric assay. The recombinant CD148 PTP domain preferentially dephosphorylated phospho-peptides corresponding to the activation loop of Lyn and Src compared with the inhibitory sites of Lyn, Src and Fyn (Fig. 7Ai). Dephosphorylation of the Lyn inhibitory site was marginally better than dephosphorylation of the Src and Fyn inhibitory sites, which correlated with what was observed in the in vitro dephosphorylation assay (Fig. 6). The PTP-1B catalytic domain was also tested in this assay as PTP-1B is highly expressed in platelets and is an essential positive regulator of αIIbβ3-associated Src in platelets (Fig. 6Aii) [7]. Interestingly, the PTP-1B catalytic domain had the opposite specificity to that of CD148, preferentially dephosphorylating SFK C-terminal inhibitory peptides rather than activation loop peptides (Fig. 7Aii). These findings suggest that CD148 and PTP-1B differentially regulate SFK activity.

image

Figure 7.  CD148 differentially dephosphorylates Src family kinase activation loop and inhibitory tyrosines in vitro. (A) The kinetics of dephosphorylation of synthetic Src family kinase (SFK)-derived phospho-peptides corresponding to the activation loop (activation peptide, AP) and inhibitory site (IS) of SFKs by recombinant CD148 and PTP-1B PTP domains were measured. Reactions contained either (Ai) recombinant CD148 PTP domain or (Aii) PTP-1B PTP domain. Inorganic phosphate released from phospho-peptides was measured as an increase in absorbance at 360 nm (Abs 360 nm) over 5 min. (B) Initial rates of dephosphorylation of phospho-peptides corresponding to the activation loop and inhibitory site of Lyn by recombinant CD148 PTP domain were measured. Calculated Vmax (Abs 360 nm s−1) and Km (μM) for Lyn phospho-peptides are shown. Representative data are shown from three separate experiments.

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The calculated Vmax [absorbance (Abs) 360 nm s−1] of dephosphorylation of Lyn phospho-peptides by the CD148 PTP domain was comparable for the activation loop and inhibitory sites (5.6 ± 0.5 Abs 360 nm s−1 vs. 5.3 ± 0.7 Abs 360 nm s−1, respectively) (Fig. 7B). However, the Km for the Lyn activation loop phospho-peptide was half of that for the Lyn inhibitory site phospho-peptide (301 ± 56 μM vs. 621 ± 144 μM, respectively), demonstrating a 2-fold higher affinity for the activation loop phospho-peptide compared with the inhibitory site phospho-peptide (Fig. 7B).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References
  10. Supporting Information

The present study builds on previous findings demonstrating that CD148 is a critical positive regulator of collagen-mediated platelet activation [10]. The phosphorylation status of SFKs in resting and collagen-activated CD148-deficient platelets suggests that they were predominantly inactive compared with control platelets. In vitro data demonstrates that CD148 directly dephosphorylates the inhibitory and activation sites of SFKs, however, the net effect of CD148 in platelets is to maintain a pool of active SFKs that allows platelets to respond optimally to collagen. Although reduced SFK activity is central to the mechanism underlying how CD148 regulates platelet reactivity to collagen, it can not fully explain the defects observed in CD148-deficient platelets. Other mechanisms including reduced GPVI/FcR γ-chain expression and other substrates probably contribute to the overall reduction in collagen reactivity of CD148-deficient platelets.

All collagen-mediated functional responses of CD148-deficient platelets were reduced compared with control platelets. Aggregation, secretion and spreading defects were partially masked by released ADP and TxA2, which signal through the G protein-coupled P2Y1 and P2Y12 ADP receptors and TP TxA2 receptor. Previous findings demonstrated that ADP-mediated aggregation and secretion are normal in CD148-deficient platelets, whereas U46619 (TxA2 analogue)-mediated responses are marginally reduced [10]. Despite exhibiting reduced spreading on collagen, CD148-deficient platelets adhered normally to collagen under all conditions tested suggesting that the molecular basis of the spreading defect was primarily a block in collagen signaling, rather than defective integrin α2β1 expression and function.

Findings from the present study demonstrate that CD148 positively regulates GPVI/FcR γ-chain expression as well as GPVI proximal signaling. Reduced expression of GPVI in CD148-deficient platelets may be indirectly due to reduced expression of the FcR γ-chain expression and is not likely due to increased trafficking, shedding or internalization. Reduced GPVI/FcR γ-chain expression may partially contribute to the reduced reactivating of mutant platelets to low concentrations of collagen [10]. However, findings from the DT40/NFAT luciferase assay demonstrate that CD148 also directly regulates GPVI proximal signaling. Reduced SFK activity combined with reduced GPVI/FcR γ-chain expression in mutant platelets resulted in a general reduction in whole cell phosphorylation in mutant platelets, with the exception of hyper-phosphorylated bands migrating at 60–53 kDa and 24–22 kDa that may represent CD148 substrates. Phosphorylation of several key components of the GPVI and α2β1 signaling pathways were reduced in collagen-stimulated mutant platelets, including Syk and PLCγ2 (Fig. 4 and data not shown). Phosphorylation of the FcR γ-chain also appeared to be reduced in collagen-stimulated mutant platelets, however, this is complicated by the reduced levels of GPVI and FcR γ-chain. Hyper-phosphorylated bands migrating at 60–53 kDa are at least partially comprised of SFKs, whereas the identity of the 24–22 kDa doublet is not known.

CD148 directly dephosphorylates the C-terminal inhibitory and activation loop sites of SFKs in vitro, suggesting that it may both activate and attenuate SFK activity depending on which site it dephosphorylates first. It has been proposed that net activation of SFKs by CD45 and other RPTPs requires a degree of separation between the RPTP and the SFK to promote dephosphorylation of the C-terminal tail while simultaneously allowing autophosphorylation of the activation loop tyrosine [19–21]. Conversely, elevated expression of a RPTP or close proximity to a SFK will have a net inhibitory effect on SFK activity by dephosphorylating the activation loop tyrosine [19]. The fact that CD148 is membrane localized and has a low copy number relative to SFKs in platelets may explain why it has a net positive regulatory role on SFKs rather than an inhibitory role.

The pool of active SFKs present in resting platelets allows platelets to respond rapidly to contact with collagen. Recent findings by Schmaier et al. [22] demonstrate that GPVI-associated Lyn is constitutively active, through a mechanism that involves disruption of the intra-molecular interaction between the SH3-domain and the proline-rich linker region of SFKs by the proline-rich domain of GPVI. Interestingly, GPVI-associated Lyn is not phosphorylated on its inhibitory site (Tyr-507), suggesting that a PTP is essential for maintaining GPVI-associated Lyn in an active conformation [22]. Platelets do not spontaneously activate despite containing this pool of active SFKs because signaling proteins and complexes must be brought into, the correct membrane compartments, spatial orientations and proportions through ligand-mediated receptor clustering. It was recently demonstrated that the CD3ε ITAM of the T cell receptor (TCR) is embedded in the lipid bi-layer of the plasma membrane, away from active SFKs in unstimulated T cells [23]. Ligand-mediated engagement and clustering of the TCR exposes the ITAM to active SFKs, which phosphorylate it and initiate downstream signaling [23]. A similar mechanism may explain why the GPVI/FcR γ-chain does not spontaneously signal in resting platelets. Endothelial-released nitric oxide and prostacyclin, and the immune receptor tyrosine-based inhibitory motif (ITIM)-containing receptors PECAM-1 and G6b-B contribute to maintaining platelets in a resting state.

The partial block in collagen signaling in CD148-deficient platelets is likely due to redundancy between CD148 and other PTPs. Possible candidates include the non-transmembrane PTPs, PTP-1B, Shp1 and Shp2. PTP-1B is highly expressed in platelets and is essential for activating αIIbβ3-associated Src [7]. However, PTP-1B-deficient platelets bound soluble fibrinogen normally in response to the GPVI agonist convulxin, suggesting that GPVI-mediated inside-out integrin αIIbβ3 signaling is normal [7]. Differences in the functional roles of CD148 and PTP-1B in platelets may be due to differences in compartmentalization and the level of expression. Evidence supporting the role of Shp1 in activating SFKs in platelets include it dephosphorylating the C-terminal inhibitory tyrosine of Src in vitro and attenuating GPVI signaling in Shp1-deficient mouse platelets [24,25].

Findings from this study demonstrate that CD148 enhances platelet responsiveness to collagen by maintaining a pool of active SFKs in platelets. Although regulation of SFK phosphorylation is central to the mechanism of how CD148 regulates the platelet response to collagen, we suspect other mechanisms including reduced GPVI/FcR γ-chain expression and hyper-phosphorylation of other substrates may contribute to the overall reduction in collagen reactivity of CD148-deficient platelets.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References
  10. Supporting Information

We would like to thank S. P. Watson and M. G. Tomlinson for valuable discussions, and J. Ullah from the Birmingham Biomedical Sciences Unit for exceptional technical assistance. S. Ellison performed this work as part of a British Heart Foundation (BHF) PhD Studentship (FS/05/085/19460). Y. A. Senis is a BHF Intermediate Research Fellow (FS/08/034/25085). Supported was also provided by BHF Project grant PG/07/034/22775.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References
  10. Supporting Information

Table S1. Synthetic SFK-dervied phospho-peptides.

Data S1. Methods.

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FilenameFormatSizeDescription
JTH_3865_sm_Supplemental-Methods.doc51KSupporting info item
JTH_3865_sm_Table1.tif801KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.