Integrin-independent Tyrosine Phosphorylation of p125fak in Human Platelets Stimulated by Collagen*

Marcus Achison, Catherine M. Elton, Philip G. Hargreaves, C. Graham Knight, Michael J. Barnes, and Richard W. FarndaleDagger

From the Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, United Kingdom

Received for publication, August 8, 2000, and in revised form, October 24, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Collagen fibers or a glycoprotein VI-specific collagen-related peptide (CRP-XL) stimulated tyrosine phosphorylation of the focal adhesion kinase, p125fak (FAK), in human platelets. An integrin alpha 2beta 1-specific triple-helical peptide ligand, containing the sequence GFOGER (single-letter nomenclature, O = Hyp) was without effect. Antibodies to the alpha 2 and beta 1 integrin subunits did not inhibit platelet FAK tyrosine phosphorylation caused by either collagen fibers or CRP-XL. Tyrosine phosphorylation of FAK caused by CRP-XL or thrombin, but not that caused by collagen fibers, was partially inhibited by GR144053F, an antagonist of integrin alpha IIbbeta 3. The intracellular Ca2+ chelator, BAPTA, and the protein kinase C inhibitor, Ro31-8220, were each highly effective inhibitors of the FAK tyrosine phosphorylation caused by collagen or CRP-XL. These data suggest that, in human platelets, 1) occupation or clustering of the integrin alpha 2beta 1 is neither sufficient nor necessary for activation of FAK, 2) the fibrinogen receptor alpha IIbbeta 3 is not required for activation of FAK by collagen fibers, and 3) both intracellular Ca2+ and protein kinase C activity are essential intermediaries of FAK activation.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Hemostasis, prevention of blood loss from damaged blood vessels, is dependent upon the activation of platelets by subendothelial collagens (types I and III) of the vessel wall. Platelet activation involves shape change, adhesion, aggregation, and secretion of granule contents. These events lead to the formation of a clot at the site of injury (1).

Platelets possess several receptors for collagen, recently reviewed (2-4), including the integrin alpha 2beta 1 (glycoproteins Ia-IIa), CD361 (glycoprotein IV), and glycoprotein VI (GpVI). Platelet activation by collagen is a two-stage process involving sites in collagen, which support platelet adhesion, and others, which support both platelet adhesion and activation (5, 6). At present, alpha 2beta 1 is considered primarily an adhesive co-receptor (7), whereas other collagen receptors, notably glycoprotein VI, activate platelets (8). This study was designed to clarify which collagen receptors transmit signals to the platelet interior, information which is crucial for the development of anti-platelet therapy based on collagen receptor antagonism (9).

Recent evidence suggests that CD36, alpha 2beta 1, and GpVI each contribute to both signaling and adhesion to collagen (10). GpVI, recently cloned (11), acts with the Fc receptor gamma -chain (12, 13) as a crucial signaling receptor complex, and platelets deficient in GpVI fail to aggregate in response to collagen, although the tyrosine kinase c-Src, but not p72syk, is activated (14). The role of alpha 2beta 1 in platelet signaling is unclear: alpha 2beta 1-reactive snake venoms fuel the debate on the integrin's role in platelet signaling (15, 16), and overexpression of alpha 2beta 1 has recently been advanced as a risk factor in myocardial infarction and stroke (17, 18).

We have synthesized a collagen-related peptide (CRP) recognized by GpVI, which shares both the triple-helical structure and activatory characteristics of collagen (19). CRP comprises a repeating GPO motif, a sequence representing about 10% of the primary structure of collagen. CRP, when cross-linked (CRP-XL), is a potent platelet agonist depending only upon GpVI for its activity. Several lines of evidence show that CRP-XL is not recognized by alpha 2beta 1 (19-24).

Recently, we have developed a triple-helical peptide containing the sequence GFOGER, which is a high affinity binding motif for the alpha 2 I domain (25). This peptide, GPC-[GPP]5-GFOGER-[GPP]5-GPC, designated GFOGER-GPP, supports platelet adhesion, as well as collagen, but does not activate platelets in suspension even when cross-linked (GFOGER-GPP-XL), nor does it stimulate obvious tyrosine phosphorylation (23). Its binding to platelets is fully abrogated by the alpha 2-specific monoclonal antibody 6F1. The sequence GFOGER has been co-crystallized with the alpha 2 I domain, demonstrating its binding to the metal ion-dependent adhesion site (26).

CRP-XL and collagen elicit very similar signals from platelets, activating protein kinase C (PKC) (27), mobilizing arachidonic acid from platelet membranes (19), and Ca2+ from intracellular stores (27); CRP-XL activates p38 mitogen-activated protein kinase (28) and p72syk and leads to tyrosine phosphorylation of many platelet proteins, including phospholipase Cgamma 2 (29) and the Fc receptor gamma -chain (12). CRP-XL, like collagen, activates platelet procoagulant expression (24). These studies emphasize the importance of GpVI as a collagen receptor in platelets.

Platelet activation leads to the up-regulation of tyrosine kinases, including FAK (30), p72syk (31), and members of the c-Src family (32). FAK, a 125-kDa cytosolic non-receptor tyrosine kinase, is associated with focal adhesion plaques of adherent cells such as fibroblasts and platelets (33). FAK is of particular interest, because it is considered a key intermediary of signaling through integrins (34-36).

Phosphorylation of FAK occurs at five tyrosine residues and correlates with an increase in FAK tyrosine kinase activity. Autophosphorylation of tyrosine 397 allows it to bind the c-Src family member Fyn (37, 38), whereas phosphorylation of tyrosine 407 and the C-terminal tyrosine 861 may support the interaction of FAK with other signaling molecules (39). Tyrosines 576 and 577 are phosphorylated by c-Src (40) and contribute to the regulation of the catalytic activity of FAK. Another possible regulatory mechanism for FAK is proteolytic cleavage by the Ca2+-dependent protease calpain, which leads to a reduction in its autophosphorylation (41).

Evidence from fibroblasts suggests that occupation of beta 1 integrins is a sufficient stimulus to activate FAK (35, 42, 43). Collagen binding to alpha 2beta 1 in T cells protects them from apoptosis in a FAK-dependent manner (36). Adhesion of platelets to monomeric collagen occurs through alpha 2beta 1. A causal relationship has been proposed between alpha 2beta 1 and FAK activation in platelets adherent to monomeric collagens (44-46). This does not exclude the operation of the two-step, two-site model (6), because other (lower affinity) collagen receptors may come into play only after platelet adhesion via alpha 2beta 1. Secondary binding of sequences within collagen, such as that of the GPO motif to platelet GpVI, is increasingly viewed as an obligatory activatory event (10, 22, 23).

The platelet fibrinogen receptor, integrin alpha IIbbeta 3, is required for FAK activation in platelets stimulated with thrombin (46), although some stimuli, such as cross-linking of Fcgamma RIIA, may activate FAK or other focal adhesion-associated proteins without involving alpha IIbbeta 3 (47, 48). FAK phosphorylation in platelets has been dissociated from both alpha IIbbeta 3 occupancy and focal adhesion formation centering on alpha IIbbeta 3 in the absence of fibrinogen binding (49, 50).

We considered that FAK tyrosine phosphorylation in platelets might be an early event after occupancy of alpha 2beta 1 by collagen fibers, and that we could determine the relative importance of alpha 2beta 1 and GpVI in this process by comparing the capacity of collagen, CRP-XL, and GFOGER-GPP-XL to elicit FAK tyrosine phosphorylation. Synthetic triple-helical peptides have not hitherto been examined in this context. We have applied these ligands to human platelets, immunoprecipitated FAK with specific anti-FAK antibodies, and determined the tyrosine phosphorylation state of the enzyme as an index of its activity.

Furthermore, we have examined the role of intracellular Ca2+ and PKC in platelet FAK activation using the Ca2+ ionophore, ionomycin, to increase the internal platelet Ca2+ concentration and the Ca2+ chelator, BAPTA, to buffer platelet cytosolic Ca2+; PKC was either directly activated using the phorbol ester, TPA, or specifically inhibited using Ro31-8220.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Collagen, as native type I fibers isolated from bovine tendon, was donated by Ethicon Inc., Somerville, NJ. CRP and GFOGER-GPP (Gly-Pro-Cys-[Gly-Pro-Pro]5-Gly-Phe-Hyp-Gly-Glu-Arg-[Gly-Pro-Pro]5-Gly-Pro-Cys) were synthesized and cross-linked using 3-(2-pyridyldithio)propionic acid N-hydroxysuccinamide ester (P-3415, Sigma, UK) as described previously (19, 25). Anti-phosphotyrosine (clone 4G10) was from Upstate Biotechnology Inc., Lake Placid, NY. Anti-FAK (catalog no. F15020/L4) was from Affiniti Research Products, Nottingham, UK, and C-20 (catalog no. sc-558) was from Santa Cruz Biotechnologies, Inc., Santa Cruz, CA. Anti-alpha 2 mAbs, 6F1 and P1E6, were a kind gift from Dr B. S. Coller, School of Medicine, State University of New York, Stony Brook, NY, and obtained from Calbiochem-Novabiochem (UK) Ltd., Nottingham, UK, respectively. The anti-beta 1 mAbs, 2A4 and mAb13, were from Genosys Biotechnologies, Cambridge, UK, and from Becton Dickinson, Oxford, UK, respectively. Horseradish peroxidase-linked anti-mouse whole antibody from sheep (NA931), Rainbow molecular weight markers (RPN756), [32P]orthophosphate, and Hybond C nitrocellulose (RPN303C) were from Amersham Pharmacia Biotech, UK. Thrombin (T-4265), apyrase (A-6535), aspirin (A-5376), 12-O-tetradecanoylphorbol-13-acetate (TPA, P-8139), bovine albumin fraction V (A-4503), Ponceau S (P-3504), leupeptin (L-2884), phenylmethylsulfonyl fluoride (P-7626), benzamidine (B-6506), and luminol (A-4685) were all from Sigma, UK. Chemicals for electrophoresis were Electran grade from BDH Laboratory Supplies, Poole, UK. 4-Iodophenol (I1, 020-1) was from Aldrich Chemical Co., Gillingham, UK. H2O2 (H/1800/07) was from Fisons Scientific Equipment, Loughborough, UK, and RX medical x-ray film was from Fuji Photo Film Co. Ltd., Japan. X-ray developer (LX24) and fixer (FX40) were from Kodak Scientific, Cambridge, UK. BAPTA-AM (1,2-[bis-aminophenoxy]ethane-N,N,N',N'-tetraacetic acid, tetraacetoxy-methyl ester; 196419), Pansorbin (507858), and Ro31-8220 (557520) were from Calbiochem-Novabiochem, Nottingham, UK, and Fura2-AM was from Molecular Probes, Eugene, OR. The fibrinogen receptor antagonist and RGD peptidomimetic, GR144053F, was a generous gift from GlaxoWellcome, Stevenage, UK. All other chemicals were of standard reagent grade.

Platelet Preparation-- Platelet concentrates, less than 24 h-old, pooled from four donors, were obtained from the National Blood Service, Long Road, Cambridge, UK, centrifuged at 250 × g for 15 min to remove red blood cells, leaving platelet-rich plasma, from which the platelets were centrifuged at 700 × g for 15 min. The platelet pellet was resuspended in loading buffer (LB; 145 mM NaCl, 5 mM KCl, 10 mM glucose, 1 mM MgSO4, 0.5 mM EGTA, 10 mM HEPES, pH 7.36). The platelets were pelleted at 700 × g for 10 min and resuspended in LB at 109/ml for immunoprecipitation and at 5 × 108/ml for other work. Aspirin (100 µM) and apyrase (0.25 units/ml) were used where indicated.

Immunoprecipitation-- Platelet-agonist suspensions (500 µl) were mixed with an equal volume of 2 × radioimmune precipitation buffer (2% Triton X-100, 2% sodium deoxycholate, 0.2% SDS (each w/v), 316 mM NaCl, 2 mM EGTA, 20 mM Tris/HCl, pH 7.2, with 10 mg/ml leupeptin, 10 mM benzamidine, 2 mM phenylmethylsulfonyl fluoride, and 2 mM Na3VO4), and incubated on ice for 30 min before centrifugation (13,000 × g) for 5 min at 4 °C. Pansorbin (60 µl/ml lysate) was added to each sample tube and rotated at 4 °C for 60 min. Before use, stock Pansorbin was centrifuged (13,000 × g) for 1 min at 4 °C, resuspended in the original volume of 1 × radioimmune precipitation buffer, and allowed to stand at room temperature for 15 min. It was then centrifuged again and resuspended in 1× radioimmune precipitation buffer containing bovine serum albumin (1% w/v). Samples were centrifuged (13,000 × g) for 1 min at 4 °C, the supernatant was removed, and to it anti-FAK polyclonal antibody (Santa Cruz) was added at 1 µg per ml of platelet lysate, and the sample was rotated for 20 h at 4 °C. Pansorbin (60 µl) was then added to each sample, and the samples were mixed and rotated at 4 °C for 60 min before being centrifuged (13,000 × g) for 1 min at 4 °C. Pellets were washed three times with 800 µl of ice-cold 1× radioimmune precipitation buffer before being resuspended in 80 µl of 1× SDS sample buffer (10% glycerol, 0.002% bromphenol blue, 2% SDS, each w/v, 70 mM Tris/HCl, pH 7.2, with 1% 2-mercaptoethanol, v/v) and boiled for 5 min. Samples were divided in 2× 40 µl, and proteins were separated by 8% SDS-polyacrylamide gel electrophoresis then blotted to nitrocellulose (2 h at 1 mA/cm2, Hoefer TE77 semi-dry blotter). Uniform protein transfer was verified by Ponceau S staining. One blot was incubated with 4G10 (1:2500) and washed with TBST (20 mM Tris/HCl, 136 mM NaCl, 0.1% (w/v) Tween 20, pH 7.6), and anti-phosphotyrosine was detected using horseradish peroxidase-linked anti-mouse antibody (1:10,000) and enhanced chemiluminescence (1.24 mM luminol, 1.63 mM 4-iodophenol, 2.71 mM H2O2). Phosphorylation was quantitated densitometrically using a Leica Q500 image analyzer (51) and is expressed as a percentage change relative to control values. The other blot was probed with monoclonal anti-FAK (Affiniti, 1:1000), to verify uniform recovery of FAK. Each experiment was performed using a different platelet preparation on three separate occasions.

Platelet Aggregation-- Platelets were prepared as for immunoprecipitation and resuspended to 109/ml in LB. 150 µl of suspension was stirred (1100 rpm) in an aggregometer at 30 °C as described (19), and inhibitors or solvent were added and followed 5 min later by ligand in a volume of 3 µl as indicated.

To verify the inhibitory properties of the anti-beta 1 antibody, 2A4, washed platelets were prepared from whole blood (19), and preincubated in the aggregometer as above for 1 min with 2A4 (20 µg/ml), before addition of just sufficient collagen fibers to cause maximal aggregation.

Protein Kinase C Activity-- Platelets were prepared as for immunoprecipitation and resuspended to 109/ml in LB. They were labeled with 32Pi at 100 µCi/ml for 1 h at 30 °C, centrifuged to remove excess radiolabel, and resuspended to 109/ml. Samples (20 µl) were treated with ligand (5 µl), and the reaction was stopped after 2 min using 25 µl of Laemmli buffer (51). Proteins were separated on a 10% polyacrylamide gel, and the phosphorylation of a 47-kDa protein band (p47, presumed to be pleckstrin, the major protein kinase C substrate in platelets (52)) was detected by autoradiography.

Intracellular Ca2+ Measurement-- Platelet concentrates were centrifuged to remove red cells as above and loaded with 2 µM Fura2-AM at room temperature for 45 min. Platelets were pelleted by centrifugation as above and resuspended to 108/ml in LB. The platelet suspensions were transferred to a Spex Fluoromax DM3000CM fluorimeter, and fluorescence excited at 340 and 380 nm was used to calculate the intracellular calcium concentration as described previously (53). Where indicated, BAPTA-AM (20 µM) was preincubated after Fura2 loading for 20 min.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Immunoprecipitation of FAK-- FAK, immunoprecipitated from platelets activated by collagen at 25 µg/ml, showed a time-dependent increase in tyrosine phosphorylation (Fig. 1a). The increase in tyrosine phosphorylation at 60 s was detectable but small; therefore, 5-min incubation, causing a substantial increase in FAK tyrosine phosphorylation, was chosen for subsequent assays. Fig. 1b shows equal recovery of FAK in each sample.



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Fig. 1.   Collagen fibers and CRP-XL activate FAK in a time-dependent fashion. a and b, platelets treated with collagen (25 µg/ml) for the indicated times were lysed, and FAK was precipitated and immunodetected using 4G10 anti-phosphotyrosine (a) or anti-FAK (b) as described. c and d, platelets were treated with CRP-XL (5 µg/ml) and handled otherwise as for a and b.

Immunoprecipitation of FAK from platelets activated by CRP-XL at 5 µg/ml showed a time-dependent increase in tyrosine phosphorylation of FAK (Fig. 1c). Fig. 1d shows immunoprecipitated FAK from CRP-XL-activated platelets, using the Affiniti monoclonal anti-FAK for immunodetection. Again, equal amounts of FAK were demonstrated in each sample.

Effect of Ligand Concentration-- Fig. 2, a and d, shows a concentration-dependent increase in the tyrosine phosphorylation of FAK immunoprecipitated from platelets activated by different concentrations of collagen and CRP-XL. Equal recovery of FAK was demonstrated in all cases (data not shown). Collagen fibers at 25 µg/ml and CRP-XL at 5 µg/ml caused near-maximal increases in FAK tyrosine phosphorylation. Some experiments (data not shown) were performed in the presence of apyrase, which scavenges ADP secreted by activated platelets, and aspirin, which blocks the conversion of arachidonate to thromboxane A2. The inhibitors had no marked effect, indicating that tyrosine phosphorylation of FAK does not depend upon these processes in platelets stimulated by collagen or CRP-XL.



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Fig. 2.   Activation of FAK by collagen fibers and CRP-XL is dose-dependent and independent of alpha 2beta 1 occupancy. Platelets were treated with ligand, as indicated, for 5 min, then FAK tyrosine phosphorylation was determined in immunoprecipitates as described in the legend to Fig. 1. Platelets were treated in parallel experiments with collagen, up to 100 µg/ml, in the absence (a) or presence (c) of 50 µM Ca2+ in excess of the 500 µM EGTA in the buffer. In b, platelets were preincubated with the anti-alpha 2, P1E6 (2 µg/ml), or anti-beta 1, 2A4 (20 µg/ml) as indicated, for 5 min, then treated with collagen fibers (25 µg/ml) for 5 min. In d, platelets were treated with the indicated levels of CRP-XL and were otherwise handled exactly as in a; in e platelets were treated with CRP-XL (5 µg/ml) after preincubation with P1E6 or 2A4 as for b.

In some experiments the basal level of FAK tyrosine phosphorylation was detectable, whereas others (e.g. Fig. 2e) showed negligible FAK tyrosine phosphorylation. This may reflect variation between donors, or in the activation state of resting platelets between experiments, as well as in the immunodetection procedure. Conclusions throughout this study are therefore based on comparisons made within an experiment, and where possible, within immunoblots rather than between blots.

Role of alpha 2beta 1 in FAK Tyrosine Phosphorylation by Collagen and CRP-XL-- When FAK was immunoprecipitated from platelets preincubated with anti-alpha 2 P1E6 or anti-beta 1 2A4 for 5 min before activation with collagen for 5 min, there was no diminution, confirmed by densitometry, in the level of tyrosine phosphorylation of FAK induced by either ligand (Fig. 2b). Similar data were obtained using the anti-beta 1 mAb13 (data not shown) or the anti-alpha 2, 6F1 (Fig. 5b).

CRP-XL induces platelet activation without involvement of alpha 2beta 1 and caused substantial tyrosine phosphorylation of FAK. This shows that ligation of GpVI, the receptor for CRP-XL, induces phosphorylation of FAK. As anticipated, the anti-alpha 2 and anti-beta 1 antibodies had no effect on the tyrosine phosphorylation of FAK by CRP-XL.

Recent work in this laboratory has shown that the affinity of platelet alpha 2beta 1 is dependent upon the presence of micromolar Ca2+ in the suspending medium (54). For this reason, the experiments shown above for collagen were repeated in the presence of a small excess of Ca2+ over EGTA in the buffer, conditions that support alpha 2beta 1-dependent platelet adhesion to immobilized collagens. FAK tyrosine phosphorylation was not enhanced by the presence of Ca2+ compared with the parallel incubation in the absence of Ca2+ (Fig. 2c).

We have recently shown the peptide sequence GFOGER to be a recognition motif in type I collagen for the alpha 2beta 1 I domain (25). Application of the cross-linked triple-helical peptide, GFOGER-GPP-XL, to platelets at up to 50 µg/ml caused no discernible increase in FAK tyrosine phosphorylation (Fig. 3a). In contrast, in this experiment as in Fig. 2a, collagen fibers caused substantial FAK tyrosine phosphorylation. The addition of micromolar Ca2+ to the medium did not support FAK phosphorylation stimulated by even high levels of the peptide (200 µg/ml).



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Fig. 3.   GFOGER-GPP-XL does not elicit FAK phosphorylation in the presence or absence of Ca2+. Platelets were treated with the indicated levels of the alpha 2beta 1-specific peptide, GFOGER-GPP-XL, for 5 min in the absence (a) or presence (b) as indicated, of an excess of Ca2+ as in Fig. 2c. FAK phosphorylation was determined as for Fig. 1. The lane marked col represents a control using collagen fibers at 25 µg/ml.

Functional Verification of the Anti-alpha 2beta 1 Antibodies-- 6F1 as used in the present study completely blocked platelet adhesion to monomeric collagen (54, 55). Similar experiments showed both P1E6 and mAb13 to be effective inhibitors of platelet adhesion to monomeric collagen. The anti-alpha 2, P1E6, blocked the capacity of reconstituted type I collagen fibers to induce platelet aggregation (7). We verified here that both P1E6 and the anti-beta 1, 2A4, could attenuate the platelet aggregation stimulated by threshold concentrations of native collagen fibers (data not shown). Together, these data confirm the functional activity of the antibodies used here.

Functional Verification of the alpha IIbbeta 3 Antagonist GR144053F-- Fig. 4 shows that CRP-XL (5 µg/ml) or thrombin (1 unit/ml), levels of agonist consistent with the rest of the study, aggregated platelets suspended in medium containing 0.5 mM EGTA, but that collagen fibers (25 µg/ml) caused minimal platelet aggregation. Preincubation with the fibrinogen receptor antagonist GR144053F (1 µM) reduced the extent of aggregation to <15% of control values in platelets stimulated by CRP-XL or thrombin.



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Fig. 4.   Platelet aggregation, which occurs in the presence of EGTA with CRP-XL and thrombin, is sensitive to GR144053F. Platelets were prepared as for immunoprecipitation studies and suspended in LB at 1 × 109 per ml. They were preincubated as indicated with GR144053F (GR; 1 µM) for 5 min, stirring at 1100 rpm in the aggregometer, then ligands were added to elicit aggregation. a, thrombin (Thr) 1 units/ml was added; b, CRP-XL (CRP) was added at 5 µg/ml; c, collagen fibers (Col) were added at 25 µg/ml, at times indicated by arrows.

Effect of GR144053F on FAK Tyrosine Phosphorylation-- Fig. 5a shows that preincubation of platelets with 1 µM GR144053F, a level which causes complete blockade of alpha IIbbeta 3 (54), caused a substantial reduction in FAK tyrosine phosphorylation in platelets subsequently stimulated by CRP-XL (77% reduction over four trials) or thrombin (63% over two trials). This effect was of similar order to the inhibition (~85%) of aggregation by GR144053F for CRP-XL or thrombin. In contrast, there was little observable inhibition (15%; five trials) of the action of collagen by GR144053F, even when used in conjunction with alpha 2-blockade by 6F1 (Fig. 5b). Basal phosphorylation of FAK was also inhibited to some extent (30%; four trials), perhaps indicating a degree of activation of platelets under resting conditions, consistent with the suggestion, above, that the basal platelet preparations might to some extent be activated. This effect of GR144053F was minor compared with the marked inhibition of the action of CRP-XL or thrombin.



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Fig. 5.   FAK phosphorylation, stimulated by CRP-XL and thrombin, but not collagen, is sensitive to GR144053F. Platelets were preincubated with GR144053F (1 µM) for 5 min. Ligands were then added for a further 5 min, and FAK was immunoprecipitated as described. a, platelets were stimulated with CRP-XL (5 µg/ml) or thrombin (1 unit/ml) as indicated (bas represents basal controls). b, platelets were stimulated with collagen fibers (25 µg/ml) as indicated. The presence of GR144053F or of the anti-alpha 2 monoclonal antibody, 6F1 at 2 µg/ml in the preincubation is denoted by + beneath the relevant lanes.

Role of Protein Kinase C in FAK Tyrosine Phosphorylation-- To investigate signaling pathways required for FAK tyrosine phosphorylation, we examined the role of protein kinase C. FAK was immunoprecipitated from platelets stimulated for 5 min with collagen (25 µg/ml), CRP-XL (5 µg/ml), or TPA (400 nM). As before, marked tyrosine phosphorylation of FAK was induced by collagen and CRP-XL, whereas control levels were undetectable (Fig. 6a). TPA caused a very minor increase in FAK tyrosine phosphorylation: densitometry showed that CRP-XL and collagen were each about 20 times more effective than TPA.



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Fig. 6.   A role for PKC activity and [Ca2+]i in the tyrosine phosphorylation of FAK. Tyrosine phosphorylation was determined in immunoprecipitates as described, from (a) platelets stimulated with collagen (25 µg/ml), CRP-XL (5 µg/ml), or TPA (400 nM) for 5 min, as indicated. For b, platelets were preincubated for 10 min with Ro31-8220 (5 µM), denoted by +, then treated with collagen (25 µg/ml) or CRP-XL (5 µg/ml) for 5 min. For c FAK tyrosine phosphorylation was determined in platelets treated with 1 µM ionomycin (Io) with or without Ro31-8220 preincubation as above. For d, platelets were preincubated for 20 min with BAPTA-AM (20 µM), then stimulated with collagen or CRP-XL as indicated. e, a Western blot of whole platelet lysates, treated with CRP or collagen after preincubation with BAPTA as indicated, then probed for phosphotyrosine. The position where FAK is expected to run in this blot is indicated.

Effect of Ro31-8220 or BAPTA on Tyrosine Phosphorylation of FAK Stimulated by Collagen, CRP-XL, or Ionomycin-- Fig. 6b shows complete inhibition of tyrosine phosphorylation of FAK immunoprecipitated from platelets after pretreatment with the PKC inhibitor Ro31-8220 (5 µM) prior to activation by collagen (25 µg/ml) or CRP-XL (5 µg/ml) for 5 min. We have shown 5 µM Ro31-8220 to cause complete inhibition of PKC, measured as p47 phosphorylation (56). The calcium ionophore, ionomycin, also caused substantial tyrosine phosphorylation of FAK (Fig. 6c), suggesting a role for calcium signaling in FAK activation, and again, as for collagen and CRP-XL, this action was substantially attenuated by Ro31-8220.

Preincubation of platelets with the Ca2+-chelating agent, BAPTA-AM, to buffer rises in intracellular Ca2+, markedly attenuated the ability of both CRP-XL and collagen fibers to stimulate tyrosine phosphorylation of FAK (Fig. 6d). For comparison, in Western blots prepared from whole platelet lysates there was an inhibition of overall tyrosine phosphorylation stimulated by collagen, CRP-XL and in the control samples of 12, 21, and 7%, respectively (Fig. 6e), when platelets were preincubated with BAPTA-AM. This inhibition indicated that the effects on FAK are highly specific. In contrast, one band of about 38 kDa increased in intensity significantly after BAPTA pretreatment in both collagen- and CRP-stimulated platelets. Note that the effects of BAPTA on the 120-kDa region are minor for collagen, although much more apparent for CRP, suggesting that other bands insensitive to BAPTA comigrate with FAK.

Effect of Ionomycin on PKC Activity-- Fig. 7a shows that ionomycin, from 500 to 2000 nM, was an effective activator of PKC, determined from the phosphorylation of p47. Higher ionomycin levels caused no further increase in phosphorylation of p47 (data not shown).



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Fig. 7.   Ionomycin, collagen, and CRP-XL stimulate PKC activity. a, platelets were labeled with 32Pi as described then treated with increasing levels of ionomycin, as indicated, and an autoradiograph was prepared as under "Experimental Procedures." The position of p47 is indicated on the left. For b and c, platelets were prepared as above, then preincubated with 20 µM BAPTA-AM for 20 min, as indicated, before stimulating with collagen (25 µg/ml), CRP-XL (5 µg/ml), ionomycin (Io; 1 µM), or TPA (200 nM) as shown. Autoradiographs were prepared as above.

Effect of BAPTA-AM on PKC Activity-- Fig. 7b shows the effect of BAPTA-AM (20 µM) on platelets activated by collagen (25 µg/ml) or CRP-XL (5 µg/ml). With or without BAPTA loading, both effectors caused marked activation of PKC, indicated by p47 phosphorylation. The action of TPA (not shown) or CRP-XL, was largely insensitive to the presence of BAPTA; only the action of collagen was noticeably attenuated, but PKC activity persisted at greater than 50% despite BAPTA loading. This suggests the presence of Ca2+-sensitive and -insensitive PKC isoforms activated by collagen receptors in human platelets.

Effect of Ro31-8220 on [Ca2+]i-- Fig. 8 shows time courses for the rise in [Ca2+]i evoked by collagen (a) or ionomycin (b), with and without pre-incubation of platelets with the PKC inhibitor, Ro31-8220. Inhibition of PKC caused a marked increase in both the peak amplitude and duration of Ca2+ signals observed under these conditions. Parallel measurement of [Ca2+]i using Fura2 showed that calcium signaling was abolished by BAPTA loading (data not shown).



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Fig. 8.   Ro31-8220 enhances Ca2+ signals elicited by collagen and ionomycin. Platelets were loaded with Fura2-AM, as described, then stirred in the cuvette of a fluorimeter, after preincubation with Ro31-8220 for 10 min, where indicated. Collagen (a, 90 µg/ml) or ionomycin (b, 1 µM) were added as indicated by the arrows. Intracellular calcium levels were calculated as described under "Experimental Procedures."



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Our aim in this study was to explore the capacity of the platelet integrin alpha 2beta 1 to activate FAK, comparing the efficacy of the synthetic analogue of collagen, CRP-XL, with that of native type I collagen fibers and with the alpha 2beta 1-specific peptide, GFOGER-GPP-XL. Thus, we intended to determine whether alpha 2beta 1 acts as a signaling receptor for collagen in platelets, working from the premise that FAK phosphorylation is an event likely to be integrin-dependent in platelets as well as in other cells.

The first part of the present work addresses the role of the collagen receptor alpha 2beta 1 in the regulation of FAK. Both collagen and CRP-XL activate FAK, as indicated by its tyrosine phosphorylation state, in a concentration- and time-dependent manner. The failure of antibodies against the alpha 2 and beta 1 integrin subunits, which prevent adhesion to collagen (validated as described under "Results") to block FAK activation demonstrates that alpha 2beta 1 occupancy by collagen fibers does not regulate FAK. This result contrasts with the proposed general role of beta 1 integrins in FAK activation (43). These experiments were performed in the presence of micromolar Ca2+, conditions where the integrin is known to be competent to bind collagen (54). The potency of CRP-XL, which does not bind alpha 2beta 1, in stimulating tyrosine phosphorylation of FAK suggests that another collagen receptor, GpVI, initiates FAK activation in platelets.

Recently, we have identified the sequence GFOGER within collagen type I, which binds to the I domain of the integrin alpha 2 subunit (25). This peptide sequence, in triple-helical conformation, binds platelets in alpha 2beta 1-dependent manner and supports purified alpha 2beta 1 binding. By co-crystallization with the recombinant alpha 2 I domain, we have shown that the E residue of the peptide coordinates the divalent cation in the metal ion-dependent adhesion site of the integrin alpha 2 subunit (26). This indicates that the peptide properly replicates the alpha 2beta 1-binding properties of collagen. However, even at levels up to 200 µg/ml with or without micromolar Ca2+ (Fig. 3b), GFOGER-GPP-XL caused no discernible increase in tyrosine phosphorylation of platelet FAK. This shows that neither alpha 2beta 1 occupancy nor clustering by the cross-linked peptide is sufficient to activate FAK in platelets in suspension.

The fibrinogen receptor alpha IIbbeta 3 has attracted most attention as a means of regulating FAK activity in platelets. Collagen and CRP-XL were added to unstirred suspensions of platelets in the presence of EGTA, conditions where alpha IIbbeta 3 is not competent and aggregation is not anticipated (57); therefore, we did not expect alpha IIbbeta 3 to regulate FAK in these experiments. To verify this, we added collagen fibers to platelets stirred in an aggregometer, causing, as anticipated, no significant aggregation. However, despite the presence of EGTA, both CRP-XL and thrombin under similar conditions caused some aggregation, which was highly sensitive to the alpha IIbbeta 3 antagonist, GR144053F (Fig. 4). This indicates that GR144053F as used here is a good antagonist of alpha IIbbeta 3 occupancy.

Partial inhibition of FAK phosphorylation by GR144053F in platelets treated with either CRP-XL or thrombin showed that alpha IIbbeta 3 activation is important in the regulation of FAK, as has been shown previously for thrombin (44). But GR144053F had little effect on the activation of FAK by collagen, consistent with collagen's failure to cause much aggregation under these conditions. (It should be noted that aggregation is more likely to occur during stirring in the aggregometer than in all other components of the study, where platelets were not stirred for more than a second after the addition of ligand.) The inclusion of both GR144053F and 6F1 (Fig. 5b), to provide simultaneous blockade of alpha 2beta 1 and alpha IIbbeta 3, had little effect on FAK tyrosine phosphorylation stimulated by collagen fibers, which is therefore shown to proceed in platelets without the involvement of either integrin under these conditions. Recently, the use of mutant alpha IIbbeta 3 showed that FAK activation could be dissociated from alpha IIbbeta 3 occupancy (49), as we propose here for the regulation of FAK by collagen fibers. Integrin-independent activation of FAK has also been reported in platelets activated using immobilized human IgG (48), an event that depends instead on Fcgamma RIIA.

The identity of the collagen receptors responsible for FAK activation remains to be resolved. Our experiments demonstrate that GpVI occupancy alone, resulting from treating platelets with CRP-XL, is not sufficient to elicit full tyrosine phosphorylation of FAK that is independent of alpha IIbbeta 3. In this respect, CRP-XL shows some similarity to thrombin. Using specific antibodies to cross-link CD36, a candidate receptor along with GpVI, others have discounted CD36 as a regulator of FAK (58), although this technique provides clustering only of CD36 populations rather than of CD36 with other receptors, as we expect will occur with the native collagen fibers used here. Investigation of whether GpVI acts as a co-receptor in regulating FAK in platelets stimulated with collagen fibers, and the possible role of CD36 in these events, must await the development of receptor-specific antagonists.

We sought to identify intracellular signaling events that are involved in the regulation of FAK activity during platelet activation. PKC has been implicated in FAK activation in both platelets (48, 59) and other cells (60, 61) that were adherent to non-collagenous substrates. We found that TPA stimulated only a slight increase in tyrosine phosphorylation of FAK in platelet suspensions. However, pretreatment of platelets with the PKC inhibitor, Ro31-8220, virtually abolished tyrosine phosphorylation of FAK caused by collagen or CRP-XL. This suggests that, although direct stimulation of PKC itself is insufficient to cause major stimulation of FAK, PKC is an important mediator of the tyrosine phosphorylation of FAK stimulated by either collagen or CRP-XL.

Next, we showed that [Ca2+]i is also important in the control of FAK tyrosine phosphorylation, by using ionomycin to elicit Ca2+ mobilization, and BAPTA-AM loading to buffer [Ca2+]i. Ionomycin stimulated tyrosine phosphorylation of FAK, whereas BAPTA-AM completely abolished tyrosine phosphorylation of FAK in platelets stimulated with collagen or CRP-XL, confirming a role for Ca2+. These results contrast with the work of Haimovich et al. (48) who showed that, in IgG-adherent platelets, exposure to BAPTA-AM caused, if anything, increased FAK phosphorylation. The same group reported no effect of BAPTA-AM on FAK phosphorylation in platelets adherent to fibrinogen (59), but in the same paper, they show that BAPTA-AM abolishes the action of thrombin in stimulating FAK in fibrinogen-adherent platelets. A requirement for increased [Ca2+]i in the regulation of FAK was similarly proposed for epinephrine-stimulated platelet suspensions (59). Possibly, the role of Ca2+ in regulating FAK activity may be ligand-specific.

Ionomycin treatment also activated PKC. To resolve the roles of [Ca2+]i and PKC, platelets were first preincubated with Ro31-8220 to inactivate PKC and then treated with ionomycin. FAK tyrosine phosphorylation was virtually abolished, as in platelets stimulated with collagen or CRP-XL after PKC blockade. It is important to note that Ro31-8220 enhanced the increase in [Ca2+]i stimulated by either collagen or ionomycin, very likely as a consequence of inhibiting PKC-dependent Ca2+ ATPases, which export Ca2+ from the cytosol. Reciprocal experiments showed that, although BAPTA blocks FAK phosphorylation, it had little effect on PKC activity. Hence, neither elevated [Ca2+]i or increased PKC activity is sufficient to support FAK phosphorylation, but each is necessary for FAK activation by collagen, CRP-XL, or ionomycin. Such a role for Ca2+ has been proposed for endothelial cell FAK activation consequent to spreading on type IV collagen (62).

In conclusion, our data suggest that the regulation of platelet FAK by native collagen fibers is independent of integrins, occurring despite blockade of alpha 2beta 1 or alpha IIbbeta 3 or both. The activation of phospholipase Cgamma 2 via GpVI (29, 63) causes Ca2+ and PKC signals essential for the regulation of FAK. Yet these signals together, activated by either CRP-XL or thrombin, are not sufficient to elicit full FAK phosphorylation without alpha IIbbeta 3 occupancy. Coordination of signals from alpha IIbbeta 3 and other receptors have been proposed to regulate FAK (34). Our data suggest that collagen, perhaps because it is recognized by different platelet receptor populations in addition to GpVI and alpha 2beta 1, is able to bypass the requirement for integrins. The role and identity of these co-receptors for collagen remain to be elucidated.


    FOOTNOTES

* This work was supported by British Heart Foundation studentships (to M. A. and C. E.), by a Biotechnology and Biological Sciences Research Council studentship (to P. G. H.), and by the Medical Research Council.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Biochemistry, University of Cambridge, Bldg. O, Downing Site, Tennis Court Rd., Cambridge, CB2 1QW, UK. Tel.: 44-1223-766111; Fax: 44-1223-333-345; E-mail: rwf10@mole.bio.cam.ac.uk.

Published, JBC Papers in Press, November 10, 2000, DOI 10.1074/jbc.M007186200


    ABBREVIATIONS

The abbreviations used are: CD36, collagen receptor glycoprotein IV; GpVI, collagen receptor glycoprotein VI; BAPTA, bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; CRP, collagen-related peptide; FAK, focal adhesion kinase (p125fak); GPO, glycine-proline-hydroxyproline; LB, loading buffer; PKC, protein kinase C; TPA, 12-O-tetradecanoylphorbol-13-acetate; mAb, monoclonal antibody; Ro31-8220, 2-{1-[3-[(amidinothio)propyl]-1H-indol-3-yl}-3-(1-methylindol-3-gl)-maleimide methane sulfonate; GR144053F, 4-[4-[4(aminoiminomethyl)phenyl]-1-piperazinyl]-1-piperidineacetic acid, hydrochloride trihydrate.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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