Article |
Address correspondence to Osamu Inoue, Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, OX1 3QT, UK. Tel.: 44-1865-271592. Fax: 44-1865-271853. E-mail: osamu.inoue{at}pharm.ox.ac.uk
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Abstract |
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Key Words: integrin 2ß1; blood platelets; cell spreading; PLC
2; FAK
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Introduction |
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Collagen interacts with platelets through direct and indirect mechanisms. At the medium and high shear rates found in arterioles and damaged vascular beds, von Willebrand factor (vWf)* bridges newly exposed collagen fibers to the GPIbIXV complex on the platelet surface. This interaction is facilitated by a fast on-rate of association between vWf and GPIb and is essential for the initial tethering of platelets by collagen at medium to high rates of shear (Savage et al., 1996, 1998). The interaction is opposed by a rapid off-rate of dissociation such that platelets translocate (or roll) for several minutes on vWf in the absence of other proteins before forming stable adhesion through activated
IIbß3 (Savage et al., 1996). The situation in vivo however is very different, as a number of extracellular matrix proteins work in tandem with vWf to promote stable adhesion, notably collagen.
Platelets interact with collagen through two major surface receptors, the integrin 2ß1 and GPVI, as well as with a number of minor receptors of uncertain significance (for review see Watson and Gibbins, 1998). For many years, the interaction of collagen with
2ß1 was thought to be essential for adhesion, whereas the role of GPVI was to mediate activation. The understanding of these events has changed considerably, however, after a number of recent observations using function blocking antibodies and genetically modified mice. In particular, the observation that platelets show dramatically impaired adhesion to collagen under static and shear conditions in the absence of functional GPVI demonstrates a critical role for the glycoprotein in platelet adhesion (Nieswandt et al., 2001a). This can be explained by the loss of inside-out signals from GPVI, which promote
2ß1 activation and enable it to bind to collagen (Jung and Moroi, 1998, 2000). More recently, GPVI has been shown to play a critical role in the interaction of platelets with vWf in vitro (Goto et al., 2002) and with a denuded endothelial carotid artery in vivo (Massberg et al., 2003), demonstrating that it is required at the very initial stage of thrombus formation.
These observations highlight a central role for GPVIFc receptor (FcR) -chain in the interaction of platelets with collagen but do not explain why mice deficient in the glycoprotein show only a minor increase in bleeding times (Nieswandt et al., 2001b). It therefore seems prudent to suggest that other matrix proteins play a role in promoting thrombus formation and can thereby compensate for the absence of GPVI. In this context, it is pertinent to consider the role of
2ß1 in greater detail, because not only is it a major receptor for collagen but it can also be activated by G proteincoupled receptor agonists independent of GPVI.
The role of 2ß1 in plateletcollagen interactions is critically dependent on experimental conditions. A universal finding of
2ß1 blockade is a delay in response to collagen, although in many cases the final extent of activation is not altered. However, under certain experimental conditions, blockade of the integrin can lead to an abolition of adhesion and activation. This is illustrated by the contrasting reports of Chen et al. (2002) and Holtkötter et al. (2002) on the adhesion of
2-deficient murine platelets to collagen under flow. Chen et al. (2002) reported a dramatic inhibition of adhesion to collagen, using washed platelets in a low Ca2+-containing buffer, conditions that favor the interaction with the integrin. In contrast, Holtkötter et al. (2002) reported a negligible effect of
2 ablation on adhesion, using plasma and a physiological concentration of Ca2+. A similar observation has also been reported in ß1-deficient murine platelets in the presence of plasma (Nieswandt et al., 2001a). In a recent follow-up to this study, however, the same group described an increased tendency of the ß1-deficient thrombi to fragment at later times in the experiment compared with those formed by wild-type platelets (Kuijpers et al., 2003). Careful examination of these thrombi revealed that they were more loosely packed than those found in control cells (Kuijpers et al., 2003). This observation demonstrates an unexpected role for
2ß1 in the later stages of hemostasis that is critical for thrombus stability, even though it has no role in the initial events that underlie adhesion.
Suzuki-Inoue et al. (2001) have recently reported spreading of human platelets on Fab fragments of an 2ß1-activating antibody, TS2/16. In light of this, we wondered whether the increased embolization of the ß1-deficient thrombi was caused by a loss of integrin-mediated intracellular signals that mediate remodeling of the cytoskeleton and thereby contribute to thrombus stability. In the present study, we show that a collagen peptide that binds exclusively to
2ß1 generates tyrosine kinasebased intracellular signals that underlie platelet spreading. Importantly, a similar set of observations are seen with collagen in murine platelets deficient in GPVIFcR
-chain. Both sets of responses are inhibited by
2ß1 blockade. Strikingly, the intracellular signaling cascade used by
2ß1 shares many of the features of the GPVI signaling cascade, including participation of Src kinases and PLC
2. The observation that engagement of
2ß1 can induce a similar set of signals to GPVI provides a new insight into the role of
2ß1 in platelet activation by collagen and may explain the relatively minor effect of depletion of GPVIFcR
-chain on bleeding times (Nieswandt et al., 2001b) and the increased tendency of ß1-deficient thrombi to embolize (Kuijpers et al., 2003).
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Results |
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Platelets underwent time-dependent adhesion and formation of filopodia and lamellipodia on a collagen surface (Fig. 1, A and B) . The majority of platelets had spread fully after 30 min, although a number showed intermediate changes in morphology most likely because of a more recent contact with the collagen surface. Because the changes in morphology occurred in the presence of inhibitors, it is suggested that adhesion and spreading are directly mediated through collagen receptors. In contrast, very few platelets settled on a BSA-coated surface and those that did, did not undergo a change in morphology (Fig. 1 A).
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GPVIFcR -chaindeficient murine platelets spread on collagen in the presence of ADP
Spreading of mouse platelets on a collagen-coated surface was investigated in the presence of the above cocktail of inhibitors. The platelets underwent time-dependent adhesion and formation of filopodia and lamellipodia on the collagen surface (Fig. 2 A). Murine platelets deficient in the GPVIFcR -chain complex showed a marked reduction in adhesion to collagen of >60% and underwent only limited spreading (Fig. 2 B, 1 [i and ii]). The interaction of collagen with
2ß1 is increased by inside-out activation of the integrin by stimuli such as ADP and PMA (Jung and Moroi, 1998, 2000). ADP (Fig. 2 B, 1 [iii]) or PMA (unpublished data) restored adhesion and spreading of FcR
-chaindeficient platelets, although this was ablated in the presence of an
2ß1-blocking antibody (Fig. 2 B, 1 [iv]) but not by an isotype-matched control (unpublished data). These findings demonstrate that
2ß1 is able to support spreading on collagen in the absence of GPVI and that either the integrin and/or the stimulus used to promote inside-out signaling is able to mediate spreading.
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Human and murine platelets adhere and undergo extensive spreading on GFOGER-coated surfaces in the presence of indomethacin, ADP receptor antagonists, and lotrafiban (Fig. 3
A, 1 and 2, and B, 1). The formation of filopodia and lamellipodia can be clearly seen (Fig. 3 A, 3). Adhesion of human platelets to the GFOGER monolayer was ablated in the presence of the 2ß1-blocking antibody 6F1 (Fig. 3 A, 1 and 2), but not in the presence of an isotype-matched control or the anti-GPIb mAb 6D1 (Fig. 3 A, 4). The latter provides evidence of specificity in the action of mAb 6F1, especially bearing in mind that GPIb is present at an
10 times greater level than the integrin. Adhesion of murine platelets to GFOGER is inhibited by an
2ß1-blocking antibody (Fig. 3 B, 1 and 2) but not by an isotype-matched control (unpublished data). A similar number of platelets also undergo adhesion and spreading from wild-type, GPVIFcR
-chaindeficient, and
IIb-deficient mice, demonstrating that adhesion is not dependent on GPVI or the integrin
IIbß3 (Fig. 3 B). Consistent with this, GFOGER peptide did not induce significant tyrosine phosphorylation of the FcR
-chain in human platelets (Fig. 3 A, 5), as measured by precipitation with a GSTSyk SH2 domain fusion protein (Gibbins et al., 1996). Platelet adhesion to a monolayer of CRP served as a positive control for these studies (Fig. 3 A, 5). These findings are consistent with reports that GFOGER peptide is unable to bind GPVI (Knight et al., 1998, 2000) and demonstrate that engagement of
2ß1 by a fibrillar collagen binding sequence mediates spreading.
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GFOGER stimulated a marked increase in tyrosine phosphorylation at 15 and 30 min when presented as a monolayer but not as a suspension (Fig. 5 A). The latter is in agreement with a previous study (Knight et al., 1999). These observations demonstrate a fundamental difference in the behavior of adherent and suspended platelets upon exposure to GFOGER. Tyrosine phosphorylation was dramatically inhibited in the presence of PP2, although increases in a major band of 60 kD and a more minor band of 125 kD were observed at later times of stimulation (Fig. 5 A).
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We sought to further investigate the PP2-independent tyrosine phosphorylation of FAK by GFOGER. It has been previously shown that G proteincoupled receptor agonists are able to potentiate integrin-dependent tyrosine phosphorylation of FAK in platelets (Shattil et al., 1994). ADP potentiated tyrosine phosphorylation of FAK by a GFOGER monolayer in the presence of PP2, whereas it had no effect on phosphorylation of PLC2 (Fig. 5 C). Tyrosine phosphorylation of FAK by a GFOGER monolayer in the presence of ADP was maintained in the presence of a supramaximal concentration of PP2 (50 µM; unpublished data). On the other hand, tyrosine phosphorylation of FAK by GFOGER was not observed in suspension studies in the presence of ADP (Fig. 5 C).
These results demonstrate that adhesion to GFOGER leads to tyrosine phosphorylation of FAK and PLC2 through a Src kinasedependent pathway. In addition, FAK is partially regulated by a Src kinaseindependent pathway that is potentiated by ADP.
GFOGER peptide stimulates tyrosine phosphorylation of Src, Syk, SLP-76, and plasma membrane calcium ATPase (PMCA)
We sought to further characterize a number of the other tyrosine-phosphorylated proteins induced by the GFOGER monolayer and, in particular, to compare these with the sequence of events mediated by the major platelet integrin IIbß3. Obergfell et al. (2002) has recently shown a key role for Src in
IIbß3-dependent spreading. This is consistent with the observation that PP2 and SU6656 partially inhibit spreading and protein tyrosine phosphorylation induced by GFOGER and suggests that the two integrins may signal in a similar way. Obergfell et al. (2002) used a phosphospecific antibody to Tyr-416 to demonstrate activation of Src during spreading on fibrinogen. GFOGER stimulated a 1.5-fold increase in phosphorylation on Tyr-416 at 5 and 10 min, the earliest time points that could be investigated because of the low number of adherent cells (Fig. 6
A). The smaller response to GFOGER relative to that induced by adhesion to fibrinogen (Obergfell et al., 2002) is likely to reflect the 50-fold lower density of
2ß1 relative to that of
IIbß3. Adhesion to fibrinogen induces tyrosine phosphorylation of Syk and SLP-76 but not the adaptor LAT, even though this is a prominent phosphorylated band in GPVI-activated platelets (Judd et al., 2000, 2002; Obergfell et al., 2001; Wonerow et al., 2002). GFOGER also stimulated marked tyrosine phosphorylation of Syk and SLP-76, whereas tyrosine phosphorylation of LAT was not detected (Fig. 6 B). Recently, it has been reported that the plasma membrane calcium ATPase (PMCA) on platelet membranes, which mediates calcium efflux from the cytoplasm, is phosphorylated on tyrosine by FAK and that this leads to its inhibition (Wan et al., 2003). PMCA is also phosphorylated after adhesion to GFOGER (Fig. 6 B, 4). These results demonstrate that activation of Src is an early event in platelet adhesion to GFOGER and that this is associated with tyrosine phosphorylation of Syk, SLP-76, and PMCA, but not LAT, as well as FAK and PLC
2, as described in the previous section.
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Discussion |
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Suzuki-Inoue et al. (2001) originally described spreading of human platelets on a surface coated with the F(ab')2 fragments of the antiintegrin 2ß1 antibody TS2/16. More recently, Guidetti et al. (2002) have provided evidence that the small proteoglycan decorin induces tyrosine phosphorylation of Syk and PLC
2 in platelets through integrin
2ß1. In the present study, we have demonstrated spreading of human and murine platelets on an
2ß1-specific peptide, GFOGER, that is unable to bind to GPVI or integrin
IIbß3. Spreading on GFOGER is observed in the presence of a cyclooxygenase inhibitor and antagonists at
IIbß3 and ADP receptors, demonstrating a direct role for
2ß1 in mediating platelet activation. We also observed spreading of murine platelets deficient in GPVIFcR
-chain on collagen. These studies were performed in the presence of ADP or PMA to induce activation of
2ß1 and thereby promote adhesion (Jung and Moroi, 1998, 2000).
Spreading on GFOGER is associated with a marked increase in tyrosine phosphorylation of Src, Syk, SLP-76, PLC2, FAK, and PMCA and elevation of intracellular Ca2+. Tyrosine phosphorylation of PLC
2 is abolished in the presence of PP2 while phosphorylation of FAK is substantially inhibited. Spreading is reduced in the presence of Src kinase inhibitors and in PLC
2-deficient platelets and abolished by chelation of intracellular Ca2+. These observations demonstrate that
2ß1 mediates spreading through a Src kinasedependent pathway that lies upstream of PLC
2 and Ca2+. Syk and SLP-76 are likely to be downstream targets of Src by analogy with the signaling events mediated by
IIbß3 (Judd et al., 2000; Obergfell et al., 2001, 2002). The limited degree of spreading observed in the presence of Src kinase inhibition may be mediated through phosphorylation of PMCA by FAK, leading to inhibition of Ca2+ extrusion (Wan et al., 2003) and a slow elevation in the intracellular levels of the cation. A residual increase in Ca2+ mobilization (unpublished data) is seen in the presence of PP2 on a GFOGER-coated surface and is therefore consistent with this possibility. These potential pathways are summarized in Fig. 9
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It is of considerable interest that the mechanism of signaling by 2ß1 is similar to that of
IIbß3. Recently, Woodside et al. (2002) have reported that the NH2-terminal SH2 domain and linker region of Syk bind with high affinity to the tail of the ß3 integrin subunit. In the same study, the authors demonstrated that Syk binds with a slightly lower affinity to the tail of the ß1 integrin subunit. This gives rise to speculation that the signaling pathways between the two integrin ß subunits are conserved, and that the sequence of events that have been established for
IIbß3 may apply to other integrins, including
2ß1. Consistent with this, both integrins are located outside of membrane rafts (Wonerow et al., 2002), and both regulate Syk, SLP-76, FAK, and PLC
2, but not the raft-associated adaptor LAT.
The observation that 2ß1 mediates spreading adds to the growing list of platelet receptors that are able to mediate this response, including GPVI,
IIbß3, and G proteincoupled receptor stimuli. This apparent redundancy brings into question the significance of the present observations. Importantly, however, the role of soluble mediators during thrombus growth is compromised by their rapid removal in the flowing blood and by recruitment of further platelets into the developing thrombus. In this context,
2ß1 may play an important role in the regulation of spreading, especially bearing in mind the high collagen content of the subendothelial matrix.
Additional work is required to establish the physiological and pathological significance of the 2ß1-dependent signals reported in the present study. Although mice deficient in
2ß1 do not exhibit a marked increase in tail bleeding times (Nieswandt et al., 2001a; Holtkötter et al., 2002), the integrin may play an important role in vascular beds that are high in collagen or that have a different type of lesion. In this context, the observation of increased embolization of ß1-deficient thrombi is of particular interest (Kuijpers et al., 2003). The significance of
2ß1-dependent signals may also be masked in the presence of GPVI. Indeed, the converse may also apply in that the relatively small increase in bleeding times observed in GPVI-deficient platelets (Nieswandt et al., 2001b) may be due to
2ß1-mediated intracellular signals. Strikingly, the two receptors regulate a common set of proteins, including Src kinases and PLC
2. Signaling through
2ß1 may also have a significant role in pathological thrombosis, and this could explain the increase in thrombotic risk that is seen in individuals with polymorphisms that lead to an increase in expression of the integrin
2ß1 (Kunicki, 2002). Agents that prevent signaling by
2ß1 therefore represent potential novel anti-thrombotics. PLC
2 also appears to be a particularly good target in that it lies downstream of the integrins
2ß1 and
IIbß3, as well as the major receptor for collagen, GPVI.
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Materials and methods |
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Preparation of human and murine platelets
Venous blood from healthy drug-free volunteers was taken into 10% sodium citrate. Platelet-rich plasma was obtained after centrifugation at 200 g for 20 min with 15% acidcitratedextrose. EGTA (1 mM) was added, and the platelet-rich plasma was centrifuged at 640 g for 15 min. Murine blood was drawn from CO2 terminally anesthetized mice by cardiac puncture and taken into 100 µl of acidcitratedextrose, and washed platelets were prepared as previously described (Snell et al., 2002). Human and murine platelets were resuspended in modified Tyrodes buffer (137 mM NaCl, 11.9 mM NaHCO3, 0.4 mM Na2HPO4, 2.7 mM KCl, 1.1 mM MgCl2, 5.6 mM glucose, pH 7.3) at a cell density of 0.24 x 108/ml. Platelet suspensions were left for 30 min and then incubated with 10 µM lotrafiban, 10 µM indomethacin, 100 µM EGTA, and, where necessary, 1 mM A3P5P and 1 µM AR-C67085 for 10 min before experimentation. The concentration of DMSO in the incubation never exceeded 0.2%, and an equivalent volume of DMSO was present in controls.
Platelet spreading
For the spreading experiments, 50 µg/ml collagen or 50 µg/ml cross-linked GFOGER peptide was incubated on a coverslip overnight at 4°C. After washing twice with PBS, the coverslip was blocked with 1% fatty acidfree purified BSA in PBS for 2 h at room temperature and washed twice with modified Tyrodes. Washed murine platelets (3 x 107/ml) and washed human platelets (2 x 107/ml) were incubated on the coverslips at 30°C. After removal of unbound platelets by washing with modified Tyrodes buffer, adhered platelets were fixed, permeabilized, and stained by TRITC-conjugated phalloidin, as described previously (Suzuki-Inoue et al., 2001). Platelets were viewed on an inverted fluorescent microscope (Carl Zeiss MicroImaging, Inc.), and digital imaging (x400) was performed using Openlab software for Macintosh. The number and diameter of platelets were counted and measured by a ruler on the printed images (0.028 mm2/image). Statistical significance was evaluated by the t test using a software for statistics, T-Kentakun. In each case, P < 0.05 was taken as the minimum value to indicate statistical significance.
For phase contrast imaging, glass plates were incubated with 50 µg/ml of cross-linked GFOGER peptide overnight. The plates were then blocked with 1% BSA as described above. Platelets were viewed on a phase contrast microscope (Carl Zeiss MicroImaging, Inc.), and digital imaging (x600) was performed using LaserSharp 2000 software.
Protein precipitation studies
Six-well flat-bottomed plates were coated with cross-linked GFOGER peptide (50 µg/ml) overnight at 4°C. The plates were washed and blocked, as described above, before incubation with 500 µl washed platelets (4 x 108/ml) at 30°C for 1530 min. Unbound platelets were removed by aspiration, and adhered cells were solubilized with 500 µl Laemmli sample buffer. A sample of the suspension was taken at the same time and used as a control. Protein concentrations were determined by RC DC protein assay kit II according to the manufacturer's instruction, and matched amounts of protein were separated by one-dimensional SDS-PAGE. Protein tyrosine phosphorylation was measured by Western blotting with 4G10. Protein loading was measured by Coomassie brilliant blue staining.
Immunoprecipitation studies were performed as previously described (Suzuki-Inoue et al., 2002). 500 µl of 2x ice-cold lysis buffer (2% vol/vol Nonidet P-40, 20 mM Tris, 300 mM NaCl, 2 mM EDTA, 2 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 2 mM Na3VO4, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 µg/ml pepstatin A, pH 7.3) was added to each well without removing unbound platelets. After removing debris and preclearing, the supernatants were incubated with 6 µl anti-FAK pAb, 2 µl anti-PLC2 pAb, 2 µl anti-Src mAb, 2 µl anti-Syk pAb, 2 µl antiSLP-76 pAb, 4 µl anti-LAT pAb, or 20 µl anti-PMCA pAb and 30 µl protein ASepharose at 4°C overnight. FcR
-chain was precipitated using glutathione-agarose conjugated with GSTSyk SH2 as previously described (Gibbins et al., 1996). After centrifugation, the pellet was washed and then solubilized by addition of 2x Laemmli sample buffer, and proteins were separated by SDS-PAGE on 8% gels. Protein tyrosine phosphorylation was detected by blotting with 4G10 or antiSrc Tyr416 pAb. Gels were reprobed with anti-FAK mAb, anti-PLC
2 pAb, anti-Src mAb, anti-Syk mAb, antiSLP-76 mAb, anti-LAT pAb, or anti-PMCA mAb. Phosphorylation of Src Tyr416 was quantified using Quantity One software for Macintosh.
Intracellular Ca2+ measurement
Platelet suspensions (5 x 108/ml) were loaded with 10 µM Fura2-AM at 30°C for 1 h. Platelets were washed and resuspended to 2 x 107/ml in modified Tyrodes buffer with 1 mM EGTA, 10 µM indomethacin, 10 µM lotrafiban, 1 mM A3P5P, and 1 µM AR-C67085. 2 x 106 platelets were added to a GFOGER-coated coverslip, and fluorescence imaging was performed using Openlab software for Macintosh as described previously (Mountford et al., 1999).
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Footnotes |
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Acknowledgments |
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This work was supported by grants from the British Heart Foundation, Japan Clinical Pathology Foundation for International Exchange, and Mochida Memorial Foundation for Medical and Pharmaceutical Research, Japan. S.P. Watson holds a British Heart Foundation Chair in Cardiovascular Sciences.
Submitted: 7 August 2002
Revised: 17 January 2003
Accepted: 23 January 2003
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