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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.
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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.