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Keywords:

  • platelet;
  • PI3K;
  • RAFTK/Pyk2;
  • SHP-2;
  • thrombin receptor

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Platelet activation by different agonists initiates a signalling cascade involving the phosphorylation of several protein kinases, which control key regulatory events. Previously, we demonstrated that the related adhesion focal tyrosine kinase (RAFTK, Pyk2) was involved in an early phase of platelet activation, independent of integrin and glycoprotein IIb–IIIa activation. In this study, we demonstrate that RAFTK is co-immunoprecipitated with phosphoinositide 3-kinase (PI3K) upon platelet activation, and that thrombin, ADP and collagen induced the phosphorylation of both PI3K and RAFTK. A low dose of thrombin (0·015 U/ml) induced RAFTK phosphorylation and platelet aggregation in a PI3K activity-dependent manner, whereas a high dose of thrombin (0·1 U/ml) induced these events in a PI3K activity-independent manner. ADP and collagen also induced RAFTK phosphorylation and platelet aggregation in a PI3K activity-dependent manner, similar to that of the low-dose thrombin. Furthermore, protein tyrosine phosphatase activity was associated with RAFTK in response to platelet activation, and was found to be that of protein tyrosine phosphatase-2 (SHP-2). The association of SHP-2 with RAFTK was PI3K-dependent and was increased upon RAFTK phosphorylation. Taken together, our results strongly suggest that the involvement of RAFTK in platelet activation is mediated via the PI3K pathway.

Platelets are activated at sites of vascular injury by exposure to extracellular matrix, locally generated thrombin and ADP released from damaged cells, and other activated platelets. The functional responses of platelets to agonist-induced activation lead to platelet adhesion and aggregation at the site of injury.

Thrombin binds to and cleaves the NH2 terminus of two G protein-coupled receptors, protease-activated receptor-1 (PAR1) and protease-activated receptor-4 (PAR4) (Molino et al, 1997; Covic et al, 2000). Recently, the significance of a dual thrombin-receptor system in platelets and differences in signalling triggered by PAR1, the high-affinity receptor, and PAR4, the low-affinity receptor, have been reported (Covic et al, 2000). Activation of PAR1 and PAR4 evokes two distinct waves of intracellular Ca2+ in platelets. PAR1 triggers a very early calcium spike response, followed by a prolonged Ca2+ signal arising from PAR4. Also, it is PAR4 and not PAR1 that is responsible for the strong autocrine Ca2+ response from secreted ADP. However, the signalling events localized further downstream of these two different G protein-coupled receptors are largely unknown.

Activation of platelets can be induced by weak agonists, such as ADP and collagen, through different signalling pathways. The full aggregation response of platelets to ADP involves two G protein-coupled receptors: P2Y1, which triggers calcium signalling, shape change and initial aggregation; and P2Y12 (also known as P2YT or P2TAC), which is connected to the inhibition of adenylate cyclase, and potentiates and completes the initial response (Daniel et al, 1998). The third ADP receptor on platelets, P2X1, is a Ca2+ channel (Sun et al, 1999). Collagen activates platelets through the integrins, α2β1 and αIIbβ3, and the cell surface glycoproteins, GPIb and GPVI, none of which has a link to G proteins (Kehrel, 1995; Kehrel et al, 1998).

Although a number of studies have defined, to some extent, the role of particular receptors, G proteins and downstream molecules in platelet aggregation, there are still many gaps in the knowledge of signalling pathways linked to specific platelet agonists. However, a central role in the signal transduction of platelet activation seems to be assigned to phosphoinositide 3-kinase (PI3K) (Rittenhouse, 1996), as lipid products of PI3K are required for the cytoskeletal rearrangements necessary for platelet aggregation (Hartwig et al, 1996). Thrombin is a potent physiological agonist that stimulates many different signalling pathways in platelets, including Ca2+ mobilization, protein kinase C (PKC) activation and protein–tyrosine phosphorylation. However, the tyrosine kinase that mediates PI3K activation in response to thrombin has not yet been identified.

Previously, we reported the integrin- and glycoprotein IIb–IIIa-independent tyrosine phosphorylation of related adhesion focal tyrosine kinase [RAFTK, also known as Pyk2, cell adhesion kinase-β (CAK-β) and calcium-dependent tyrosine kinase (CADTK)] during an early phase of platelet activation (Raja et al, 1997). To further study the signalling mechanism of RAFTK during platelet activation, we examined RAFTK-associated molecules and their role in RAFTK phosphorylation and platelet activation. We found that PI3K was co-immunoprecipitated with RAFTK. The induction of RAFTK phosphorylation was dependent on PI3K activity upon stimulation with a low dose of thrombin, collagen and ADP, but was independent of PI3K activity upon stimulation with a high dose of thrombin. We also found that RAFTK is associated with the protein tyrosine phosphatase 2 (SHP-2). The association between RAFTK and SHP-2 was also PI3K-dependent. Taken together, these data suggest that RAFTK involvement in platelet activation is mediated by PI3K.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Reagents and antibodies The monoclonal antibody (mAb) to p85 α was obtained from Upstate Biotechnology, (Lake Placid, NY, USA). The mAB to PY99 phosphotyrosine and rabbit anti-SHP-2 (SH-PTP2) antibody were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). LY294002, RGDS and pervanadate were from Sigma Chemical Co. (St. Louis, MO, USA); thrombin was purchased from Enzyme Research Laboratories (South Bend, IN, USA). Akt and phospho-specific Akt antibodies were from New England BioLabs (Boston, MA, USA). RAFTK antiserum was raised against a glutathione S-transferase-fusion protein containing the C-terminus (681–1009 amino acid residues) of human RAFTK cDNA (Avraham et al, 1995).

Platelet preparation Human platelets were isolated from blood obtained from healthy volunteer donors using gradient centrifugation on Cellsep Platelets according to the manufacturer's instructions (Larex, St. Paul, MN, USA) with 3·8% sodium citrate as an anticoagulant. The protocol ensures a platelet population with an expected purity of > 99%. For all experiments, platelets were suspended in Hepes–Tyrode buffer (120 mmol/l NaCl, 9 mmol/l NaHCO3, 3 mmol/l KCl, 0·8 mmol/l NaH2PO4, 1·7 mmol/l MgCl2, 5·5 mmol/l dextrose and 1 mmol/l Hepes) at a concentration of 2–3 × 108/ml.

Platelet activation and aggregation assays Prior to treatment, 1 ml aliquots of platelets were preincubated in a Lumi-aggregometer (Chronolog, Havertown, PA, USA) for 10 min at 37°C, and then stirred at 37°C in the presence of 0·1 U/ml or 0·015 U/ml of thrombin, 10 μmol/l ADP with 5 μg/ml fibrinogen or 5 μg/ml collagen. In some experiments, prior to stimulation with agonists, platelets were pretreated with the PI3K inhibitor LY294002 (10 μmol/l) for 30 min at 37°C. For the aggregation assays, data were expressed as the percentage of light transmission, with the Hepes–Tyrode buffer equal to 100%. Platelet aggregation was recorded for at least 7 min. Duplicates of samples analysed in an aggregometer were used for the Western blot analysis. Platelets, activated for 2 min with various agonists with and without LY294002, were lysed in 0·5 ml of ice-cold lysis buffer (75 mmol/l Hepes, 0·3 mol/l sodium fluoride, 30 mmol/l sodium pyrophosphate, 6 mmol/l sodium orthovanadate, 6 mmol/l EDTA, 6 mmol/l sodium molybdate, 3% NP-40, 0·45 U/ml aprotinin, 12 μg/ml leupeptin, 3 mmol/l phenylmethylsulphonyl fluoride) and then incubated for 30 min on ice. Lysates were then clarified by centrifugation at 10 000 g at 4°C for 5 min.

Immunoprecipitation of platelet lysates Lysates were immunoprecipitated with 10 μl of polyclonal antiserum to RAFTK, 2 μg of p85 or with normal rabbit serum (overnight at 4°C), followed by 1 h incubation with 30 μl of protein A/G agarose at 4°C. Immunoprecipitates were washed five times with lysis buffer, eluted with 2× reducing Laemmli sample buffer by boiling for 5 min, and analysed using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) (Laemmli, 1970).

Western blot analysis Total lysates of platelets (20 μg of protein per lane) were separated using 7·5% SDS–PAGE and transferred to polyvinylidenedifluoride (PVDF) membrane (BioRad, Melville, NY, USA) by electroblotting. Immunoblots were then probed with the appropriate antibodies. Bands were visualized using horseradish peroxidase (HRP)-conjugated anti-rabbit, anti-goat or anti-mouse Ab and the Renaissance Chemiluminescence reagent plus system (NEN, Boston, MA, USA), according to the manufacturer's instructions.

In vitro RAFTK kinase assay The immunoprecipitated complexes, obtained by immunoprecipitating platelet lysates with anti-RAFTK antibodies, were washed three times with lysis buffer and twice in kinase buffer [20 mmol/l Hepes (pH 7·4), 50 mmol/l NaCl, 5 mmol/l MgCl2, 5 mmol/l MnCl2, 1 mmol/l Na3VO4 and 20 μmol/l ATP]. The kinase assay was initiated by incubating the immune complex in kinase buffer containing 25 μg of poly (Glu/Tyr, 4:1; 20–50 kDa; Sigma) and 185 kBq [γ-32P]-ATP at room temperature for 30 min. Reactions were terminated and analysed as described (Avraham et al, 1995; Ganju et al, 1997).

In vitro phosphatase assay Immunoprecipitates of RAFTK and control antibodies were assayed for in vitro PTPase activity using the p-nitrophenyl phosphate assay in 50 mmol/l buffer (sodium acetate pH 4·0–5·5, magnesium sulphate (MES) pH 5·5–6·5, Tris pH 7·0–8·5), 0·5 mg/ml of bovine serum albumin (BSA), 0·5 mmol/l dithiothreitol (DTT) and 5 mmol/l p-nitrophenylphosphatase (pNPP). The reaction was stopped with 1/10th volume of 13% K2HPO4. The absorbance was measured at 405 nm, and the concentration of p-nitrophenylate ion produced was calculated using a molar absorptivity of 1·78 × 104 mol/l/cm. PTPase activity was expressed as increased absorbance at 405 nm. The phosphatase activity was analysed in the absence (–) or presence (+) of pervanadate (PerV, 0·1 mmol/l).

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

RAFTK tyrosine phosphorylation and association with PI3K during platelet activation

Platelets were stimulated with thrombin, followed by analysis of RAFTK tyrosine phosphorylation. Figure 1 shows the tyrosine phosphorylation (A) and activation (B) of RAFTK upon thrombin stimulation. To investigate the signalling mechanism of RAFTK, we examined the tyrosine-phosphorylated proteins that co-immunoprecipitated with RAFTK upon platelet activation. Figure 2A shows three major tyrosine-phosphorylated proteins co-immunoprecipitated with RAFTK, which were identified as the PI3K β subunit p110, PI4K and the PI3K α subunit p85. We confirmed the association of PI3K with RAFTK by a reciprocal immunoprecipitation with p85 specific antibody. These results showed that both RAFTK and p85-PI3K were co-immunoprecipitated and phosphorylated upon platelet activation.

image

Figure 1.  The tyrosine phosphorylation of RAFTK in thrombin-activated platelets. (A) Platelets were unstimulated or stimulated with thrombin (0·1 U/ml, 2 min). Platelet lysates (5 × 108/ml) were immunoprecipitated with anti-RAFTK antibody or normal rabbit serum (NRS) as a control. The immunoprecipitates were resolved using SDS–PAGE, followed by protein transfer to PVDF membrane (Immobilon). Tyrosine phosphorylation was detected with anti-phosphotyrosine antibody (PY99), followed by protein identification with anti-RAFTK antibody (R4250). WB, Western blot analysis; IP, immunoprecipitation. (B) Thrombin stimulates the kinase activity of RAFTK. Platelets were unstimulated (UN) or stirred in the presence of thrombin (0·1 U/ml, 2 min). Total lysates were prepared, immunoprecipitated with anti-RAFTK antibody, and then subjected to an in vitro kinase assay, as described in Materials and methods.

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image

Figure 2. Co-immunoprecipitation of PI3K with RAFTK in platelets. Thrombin-stimulated platelets (0·1 U/ml, 2 min) were lysed with radioimmunoprecipitation assay (RIPA) buffer, followed by immunoprecipitation with antibodies against (A) RAFTK and (B) p85 or normal rabbit serum (NRS) as a control. Immunoprecipitates were resolved using 7·5% SDS–PAGE, and analysed by blotting with anti-phosphotyrosine antibody (PY99), followed by identification of the co-immunoprecipitated proteins with the indicated antibodies. WB, Western blot analysis; IP, Immunoprecipitation.

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Effects of PI3K activity on RAFTK phosphorylation upon thrombin, collagen and ADP stimulation of platelets

To elucidate the effect of PI3K activity on RAFTK during platelet activation, we used the PI3K-specific inhibitor LY294002. Platelets were stimulated with thrombin, collagen or ADP in the presence or absence of LY294002, and then RAFTK phosphorylation was analysed. Because it has been reported that there are two thrombin receptors with different affinities, we used two different concentrations of thrombin, 0·1 U/ml for the low-affinity receptor PAR4 and 0·015 U/ml for the high-affinity receptor PAR1 as shown in Fig 3. RAFTK phosphorylation was induced by both high and low doses of thrombin, collagen or ADP (Fig 3). Blocking of PI3K activity with LY294002 inhibited the RAFTK phosphorylation induced by the low-dose thrombin (0·015 U/ml), collagen or ADP stimulation of platelets, indicating that PI3K is located upstream of RAFTK phosphorylation (Fig 3). However, activation of platelets with a high concentration of thrombin (0·1 U/ml) led to RAFTK phosphorylation that was independent of PI3K activity. This suggests the existence of PI3K-independent signalling cascade(s), leading to RAFTK phosphorylation upon activation of the low-affinity thrombin receptor, PAR4. PI3K was phosphorylated upon stimulation with the two different doses of thrombin, collagen or ADP in a PI3K activity-dependent manner. The PI3K downstream-signalling molecule, Akt, showed a parallel response to these stimuli. These results indicate that RAFTK phosphorylation is induced upon stimulation with low-dose thrombin, collagen or ADP via PI3K, whereas high-dose thrombin stimulation induces RAFTK phosphorylation in a PI3K-independent manner.

image

Figure 3.  Effect of the PI3K inhibitor LY294002 on RAFTK phosphorylation in platelets. Platelets were stimulated for 2 min with thrombin (0·1 U/ml or 0·015 U/ml), ADP (10 μmol/l) or collagen (5 μg/ml) in the presence or absence of LY294002 (10 μmol/l). Platelet lysates (5 × 108/ml) were immunoprecipitated with anti-RAFTK or p85 antibody. Tyrosine phosphorylation was analysed with anti-phosphotyrosine antibody (PY99), followed by protein identification with the indicated antibodies. Control represents resting platelets. WB, Western blot analysis; IP, immunoprecipitation.

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PI3K is required for platelet aggregation in response to thrombin, collagen and ADP

To assess the role of PI3K activity in platelet aggregation, we examined the effect of the PI3K inhibitor LY294002 on platelet aggregation in response to a high (0·1 U/ml) or low (0·015 U/ml) dose of thrombin, 5 μg/ml collagen or 10 μmol/l ADP in the presence of 5 μg/ml fibrinogen. In samples treated with a low concentration of thrombin, collagen and ADP, we observed complete inhibition of platelet aggregation by the PI3K inhibitor (Fig 4), whereas a high dose of thrombin induced the aggregation of platelets independent of PI3K activity (Fig 4A). The results showing the PI3K inhibitor effects on platelet aggregates were in correlation with the tyrosine phosphorylation of RAFTK (Fig 3). These data suggest that platelet aggregation is induced by low-dose thrombin, collagen and ADP via PI3K, whereas platelet aggregation upon high-dose thrombin stimulation is independent of PI3K.

image

Figure 4.  Involvement of PI3K in platelet aggregation in response to various agonists. Platelets were analysed in a Lumi-aggregometer in the presence of thrombin (0·1 U/ml or 0·015 U/ml), ADP (10 μmol/l) or collagen (5 μg/ml). In parallel experiments prior to stimulation with agonists, platelets were pretreated with the PI3K inhibitor LY294002 (10 μmol/l). Data are expressed as the percentage of light transmission, with Hepes–Tyrode buffer equal to 100% transmission.

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Association of protein tyrosine phosphatase activity with RAFTK during platelet activation

To further analyse the signalling mechanism of RAFTK in platelet activation, we examined the existence of a protein tyrosine phosphatase, a negative modulator of tyrosine phosphorylation. In vitro protein tyrosine phosphatase (PTPase) assays revealed that PTPase activity was associated with immunoprecipitates of RAFTK in thrombin-stimulated platelets (Fig 5). The phosphatase inhibitor pervanadate (0·1 mmol/l), which was used as a control for the phosphatase-specific activity, inhibited the phosphatase activity induced by thrombin. This result suggests the association of a negative regulatory PTPase with RAFTK in platelet activation.

image

Figure 5.  Association of protein tyrosine phosphatase activity with RAFTK during platelet activation. Unstimulated or thrombin-stimulated (0·1 U/ml, 2 min) platelets (5 × 108/ml) were lysed with RIPA buffer, followed by immunoprecipitation with anti-RAFTK antibody or normal rabbit serum (NRS) as a control. Immunoprecipitates were analysed for in vitro PTPase activity using the p-nitrophenyl phosphatase assay. PTPase activity was expressed as increased absorbance at 405 nm. The phosphatase activity was analysed in the absence (–) or presence (+) of pervanadate (PerV; final concentration of 0·1 mmol/l). Sepharose beads and NRS immunoprecipitates were used as negative controls.

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Association of SHP-2 with RAFTK during platelet activation

To identify the phosphatase associated with RAFTK in platelets, we analysed RAFTK immunoprecipitates from platelets with or without thrombin stimulation using SDS–PAGE. A co-immunoprecipitated protein with a molecular mass of 72 kDa was detected and identified as SHP-2, using a specific antibody (Fig 6A). The association of SHP-2 with RAFTK was increased upon thrombin stimulation of platelets. To assess the effect of PI3K on the RAFTK/SHP−2 association, we examined this association in the presence or absence of the PI3K inhibitor, LY294002, upon thrombin stimulation. As shown in Fig 6B, LY294002 inhibited the thrombin-induced association of RAFTK and SHP-2. These data suggest a PI3K-dependent increase in the association of SHP-2 with RAFTK upon RAFTK phosphorylation.

image

Figure 6.  Association of SHP-2 with RAFTK is PI3K-dependent. (A) Platelets were isolated, unstimulated or stimulated with thrombin (0·1 U/ml) for 2 min, and then lysed in RIPA buffer. (B) Platelets were stimulated with thrombin (0·1 U/ml, 2 min) in the absence or presence of LY294002 and then lysed in RIPA buffer. Lysates were immunoprecipitated with anti-RAFTK antibody. NRS was used as the immunoprecipitation controls. Immunoprecipitates were resolved using SDS–PAGE and probed with anti-phosphotyrosine (PY-20), anti-RAFTK (R4250) or anti-SHP-2 antibody. The protein bands were visualized using enhanced chemiluminescence (ECL) reagents. UN, unstimulated platelets without stirring; 0, unstimulated platelets with stirring; 2, thrombin-stimulated (2 min) platelets with stirring; WB, Western blot analysis; IP, immunoprecipitation.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

In this report, we characterized RAFTK signalling and its association with PI3K and SHP-2 in platelet activation. Previously, we have shown that the tyrosine phosphorylation of RAFTK, induced upon thrombin stimulation of platelets, was an integrin- and glycoprotein IIb–IIIa-independent event (Raja et al, 1997). Here, we show that RAFTK was constitutively associated with PI3K and that its tyrosine phosphorylation was induced upon platelet activation. Activation of p85/p110 PI3K in platelets is an early integrin-independent signalling event. In addition, tyrosine phosphorylation and activation of RAFTK are early phosphorylation events in platelets (Raja et al, 1997). The association of RAFTK with PI3K could be detected in unstimulated platelets and was increased upon thrombin stimulation (Fig 2). RAFTK was associated with PI3K in unstimulated platelets and, after activation of platelets, there was an increase in the tyrosine phosphorylation and activity of RAFTK (Figs 1, 2 and 3), which may contribute to the increased activation of PI3K.

RAFTK has been reported to form complexes with several signalling molecules in various cell types, and has also been suggested to act as a platform for protein–protein interactions (Ganju et al, 1997; Ohmori et al, 1999; Zrihan-Licht et al, 2000). Interestingly, we found significant differences in the effects of the PI3K inhibitor on RAFTK phosphorylation and platelet aggregation depending on the dose of thrombin. Stimulation of platelets with a low concentration of thrombin resulted in RAFTK phosphorylation and platelet aggregation that were dependent on PI3K activation. However, high-dose thrombin induced RAFTK phosphorylation that was independent of PI3K activity (Fig 3). These results suggest that RAFTK phosphorylation correlates with platelet aggregation, and that PI3K plays a key role in this phosphorylation and platelet aggregation upon stimulation with low-dose, but not high-dose, thrombin. The difference in the dependency of RAFTK phosphorylation on PI3K at various thrombin concentrations could be a reflection of differential signalling through the two thrombin receptors, PAR1 and PAR4. It has been demonstrated that a low concentration of thrombin leads to the cleavage of PAR1, which causes a rapid Ca2+ response in the early phase of the platelet activation process (Rand et al, 1996). The higher concentration of thrombin generated by factor Xa during blood clotting (Rand et al, 1996) activates PAR4 and produces sustained high intracellular Ca2+ levels. Therefore, these data suggest that the high-affinity thrombin receptor, PAR1, leads to RAFTK phosphorylation in a PI3K-dependent mode, whereas the low-affinity receptor, PAR4, activates RAFTK in a PI3K-independent manner. This RAFTK activation may be mediated by the PAR4-induced autocrine increase in intracellular Ca2+ concentration.

We observed a lesser amount of p85 immunoprecipitated from the thrombin-treated platelet lysates compared with the control samples (Fig 2A). Our hypothesis that PI4K could also be forming a complex with PI3K was based on the assumption that co-localization of PI3K and PI4K would increase substrate availability for the kinases. PI4K performs the central role of signalling, as its products contribute to multiple pathways (PI3K and phosphatidylinositol-4-P 5-kinases). Upon immunoprecipitation of p85 from the lysates of resting and thrombin-aggregated platelets, we detected PI4K among the co-precipitated proteins (Fig 2). The strong induction of PI4K activity in aggregated platelets is probably linked to calpain activation.

Platelet aggregation in response to a high or low concentration of thrombin in the presence of the PI3K inhibitor paralleled RAFTK phosphorylation under similar conditions. These data suggest the existence of distinct signalling pathways upon different doses of thrombin. This is consistent with a recent report showing that PI3K-dependent events play a central role in platelet aggregation only when low concentrations of thrombin are used, although the mechanism underlying the phenomenon is not known (Lauener et al, 1999). Lipid products of PI3K are also reported to be involved in ADP-induced spreading of adherent platelets (Heraud et al, 1998).

Protein tyrosine phosphatases play critical roles in the regulation of tyrosine phosphorylation signalling cascades. SHP/SH-PTPs are cytoplasmic tyrosine phosphatases containing src homology 2 (SH2) domains. SHP-2/SH-PTP2 is expressed broadly in various tissues and is suggested to be involved in the regulation of cell spreading and migration. We found the existence of PTPase activity in the immunoprecipitates of RAFTK, and observed this activity to be associated with SHP-2. The association of SHP-2 with RAFTK was inducible and dependent on PI3K activity. To our knowledge, this report is the first to demonstrate the involvement of SHP-2 during platelet activation, and its association with RAFTK. Our results are supported by a recent study on Convulxin-induced platelet activation, which demonstrated the essential roles of PI3K and PTPase(s) in platelet aggregation (Lagrue et al, 1999).

In summary, our results indicate the presence of distinct signalling pathways in platelets upon stimulation with collagen, ADP or different doses of thrombin. RAFTK phosphorylation was induced by these agonists via PI3K with the exception of the high dose of thrombin. In addition, thrombin induced the activation and association of RAFTK with SHP-2, which were dependent on PI3K activity. Our results suggest that the involvement of RAFTK signalling in platelet activation is mediated via PI3K. RAFTK and PI3K activation are early events independent of subsequent platelet aggregation. These results provide insight into the mechanism leading to the activation of p85/p110 PI3K after activation of G protein-coupled receptors in platelets, upon thrombin stimulation.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

We thank Drs Jadwiga Grabarek, Ewa Matczak and Jakub Golab for their much appreciated help in this study. We are also grateful to Daniel Kelley and Sarah Evans for preparing the figures and Janet Delahanty for editing the manuscript.

This work was supported in part by National Institutes of Health Grants HL55445, DAMD 17-98-1-8032, DAMD 17-99-1-9078, and CA76226. This work was performed during the term of an established investigatorship (HA) from the American Heart Association.

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  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
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