Thrombspondin Acts via Integrin-associated Protein to Activate the Platelet Integrin alpha IIbbeta 3*

(Received for publication, April 1, 1997)

Jun Chung , Ai-Guo Gao and William A. Frazier Dagger

From the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Integrin-associated protein (IAP or CD47) is a receptor for the cell/platelet-binding domain (CBD) of thrombospondin-1 (TS1), the most abundant protein of platelet alpha  granules. Although it associates with alpha IIbbeta 3, IAP has no known function in platelets. TS1, the CBD, and an IAP agonist peptide (4N1K) from the CBD of TS1 activate the platelet integrin alpha IIbbeta 3, resulting in platelet spreading on immobilized fibrinogen, stimulation of platelet aggregation, and enhanced tyrosine phosphorylation of focal adhesion kinase. Furthermore, 4N1K peptide selectively stimulates the phosphorylation of LYN and SYK and their association with FAK. The phosphorylation of SYK is blocked by pertussis toxin, implicating a Gi-like heterotrimeric G protein. IAP solublized from membranes of unstimulated platelets binds specifically to an affinity column of 4N1K peptide. Both alpha IIb and beta 3 integrin subunits and c-Src bind along with IAP. This complex of proteins is also detected with immunoprecipitation. Activation of platelets with the agonist peptide 4N1K results in the association of FAK with the IAP-alpha IIbbeta 3 complex. Thus an important function of TS1 in platelets is that of a secreted costimulator of alpha IIbbeta 3 whose unique properties result in its localization to the platelet surface and the fibrin clot.


INTRODUCTION

Integrin-associated protein (IAP)1 is important in host defense where it is required for integrin-dependent functions of polymorphonuclear leukocytes (1). IAP also appears to be important in modulating integrin function in other cells (2) and in signal transduction upon ligand binding by certain integrins with which it associates (3, 4). We have recently discovered that IAP is a receptor for the COOH-terminal cell binding domain (CBD) of the thrombospondins (TSs) including TS1 (5), the most abundant protein of platelet alpha  granules (6). A peptide from the CBD, kRFYVVMWKk (4N1K) has been identified as an IAP agonist (2, 5). TS1 is thought to have a role in augmenting platelet aggregation (7, 8). A mAb (C6.7) against the IAP-binding domain of TS1 can block secretion-dependent platelet aggregation (8), but the mechanism of this effect has remained obscure (9). IAP is present on platelets (10), but it was initially reported to have no functional role in platelet activation or aggregation (11). However, Dorahy et al. (12) have recently reported that the 4N1K agonist peptide, which we had identified in the CBD of TS1, can activate washed platelets causing their aggregation. In nucleated cells, IAP associates with alpha vbeta 3 and modulates its function (3, 4). For example, the CBD of TS1 and the 4N1K peptide stimulate the chemotaxis of endothelial cells on RGD-containing substrata, and this effect is blocked specifically by mAbs against IAP and alpha vbeta 3 (5). TS1, its CBD, and 4N1K peptide all stimulate the rapid spreading of C32 melanoma and NIH3T3 cells on sparse vitronectin substrata, which support only weak, slow spreading of these cells in the absence of TS1 (2). This stimulation of alpha vbeta 3-dependent spreading is specifically inhibited by pertussis toxin, indicating the participation of a heterotrimeric Gi-like protein in a pathway linking IAP to a common cellular pathway resulting in protein kinase C activation, which leads to cell spreading (2) and motility (5). This sort of stimulation of an integrin-dependent function via G protein-dependent pathways is reminiscent of the costimulation of alpha IIbbeta 3 function in platelets by agents that act via heptahelical or seven transmembrane spanning receptors such as ADP, epinephrine, and thrombin (13, 14). Here we have examined the hypothesis that IAP ligation by TS1 has a role in modulating alpha IIbbeta 3 function. We find that IAP stimulation by its agonist 4N1K activates alpha IIbbeta 3 as judged by enhanced binding of the conformationally sensitive mAb PAC-1 (13-15) resulting in spreading of platelets on fibrinogen-coated surfaces, aggregation of stirred platelets (12), and assembly of a signaling complex containing IAP, the integrin, c-Src, FAK, and SYK. All of these actions of IAP are blocked by pertussis toxin, indicating the essential participation of a Gi-like heterotrimeric G protein (2). In unstirred, washed platelets where the integrin is not engaged, 4N1K stimulates the tyrosine phosphorylation of SYK, an early event in platelet activation by many agonists (17). This activation of SYK is blocked by pertussis toxin but not by inhibitors of downstream signaling events. These results provide a novel explanation for the role of TS1 in platelet function and establish IAP as signaling coreceptor for platelet stimulation.


EXPERIMENTAL PROCEDURES

Apyrase, bovine serum albumin, cytochalasin D, human fibrinogen, indomethacin, and prostaglandin E1 were obtained from Sigma. Epinephrine, D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone, calphostin C, herbimycin A, and wortmannin were from Calbiochem. Pertussis toxin was from Life Technologies, Inc. BAPTA was from Molecular Probes (Eugene, OR). Monoclonal antibody PAC1 was a gift from Dr. Sanford J. Shattil. Anti-human IAP mAbs, B6H12, and 2D3 were kindly provided by Dr. Eric J Brown. mAb against c-Src was a generous gift of Dr. D. Lublin. mAbs against FAK (clone 77 for Western blotting) and phosphotyrosine (PY20) were products of Transduction Laboratory (Lexington, KY). mAbs against FAK (2A7 for immunoprecipitation), and phosphotyrosine (4G10) and anti-SYK polyclonal IgG were from Upstate Biotechnology Inc. Anti-LYN polyclonal IgG was from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). Rabbit anti-human integrin beta 3 polyclonal antibody and mouse anti-integrin alpha IIb mAb were purchased from Novous Molecular, Inc. (San Diego, CA). All peptides used were synthesized and purified by the Protein and Nucleic Acid Chemistry Laboratory of Washington University as described previously (2). TS1 and its cell binding domain were prepared as described (2).

Blood was obtained from healthy human donors in 0.15 volume of acid-citrate/dextrose solution supplemented with 1 µM prostaglandin E1 and 1 unit/ml apyrase (14). Platelet-rich plasma was separated from whole blood at 1000 rpm in a Beckman GP table top centrifuge for 30 min and further centrifuged at 2000 rpm for 30 min to pellet the platelets. Pelleted platelets were washed once and resuspended in incubation buffer (137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 5.6 mM glucose, 1 mg/ml bovine serum albumin, 3.3 mM NaH2PO4, and 20 mM Hepes, pH 7.4) at concentration of 2 × 108 platelets/ml. Apyrase was then added to a final concentration of 10 units/ml to remove ADP. All other inhibitors were preincubated for 15 min except for pertussis toxin (preincubated for 2 h). Platelet adhesion to fibrinogen was studied on Lab-Tek 8 chamber slides (VWR Scientific), which were precoated with human fibrinogen (100 µg/ml, Sigma) overnight at 4 °C and blocked with 10 mg/ml bovine serum albumin. After 60 min at room temperature, adherent platelets were fixed with paraformaldehyde, permeabilized with 0.1% Triton X-100, and stained with rhodamine-phalloidin to determine the degree of platelet spreading on fibrinogen.

To detect phosphorylation events at various time points, adherent platelets were lysed in RIPA buffer and cell lysates were subjected to immunoprecipitation and immunoblot analysis as described (2). The extent of activation of alpha IIbbeta 3 was determined with PAC-1 mAb binding using a FACStar flow cytometer as described (14). Aggregation of freshly collected, washed platelets were performed in a Chrono-Log aggregometer (14).

For affinity chromatography of IAP protein complexes, Ni-NTA-agarose beads (0.25 ml, Qiagen) were charged with His-tagged 4N1K (His6-KRFYVVMWKK) or His-tagged 4NGG (His6-KRFYGGMWKK) peptide (1 µmol) in Hepes-buffered saline (HBS), and washed extensively in HBS. Washed platelets were lysed with 1% Triton X-100 in HBS containing protease and phosphatase inhibitors (2). Lysates were centrifuged at 13,000 rpm in a refrigerated microcentrifuge for 30 min. The soluble fraction was mixed with the peptide-charged agarose beads for 3 h at 4 °C. The beads were rinsed into a Quik Snap column (0.6 × 6 cm, MidWest Scientific, St. Louis, MO) and washed extensively with 1% Triton X-100 in HBS (20 ml), followed by 20 mM imidazole (20 ml) and 100 mM imidazole (20 ml) in the same buffer. Proteins bound via the His tag were eluted with buffer containing 1 M imidazole. 100-µl fractions were collected, 5 × SDS sample buffer containing 2-mercaptoethanol was added, and fractions were immediately boiled and subjected to SDS gel electrophoresis and immunoblot analysis with the indicated antibodies.


RESULTS

We have employed a system used by Shattil and co-workers (14, 15) to assess alpha IIbbeta 3 activation in which washed platelets are allowed to adhere to a fibrinogen-coated surface in the presence of apyrase, an ADP scavenger. In the absence of a costimulator, the platelets adhere but cannot spread (Fig. 1A). Epinephrine (Fig. 1C), thrombin receptor peptide (Fig. 1D), and ADP itself bind to heptahelical receptors that are coupled via G proteins to the generation of lipid intermediates including arachidonate, prostaglandins, leukotrienes, and diacylglycerols (13). These ultimately activate protein kinase C, which leads in an as yet unknown way to activation of the integrin resulting in the spreading of the platelets on fibrinogen (Fig. 1, C and D) (13, 14). With this assay, we find that TS1, the recombinant CBD, and active IAP binding peptides such as 4N1K (Fig. 1B) specifically stimulate the spreading of platelets adherent to fibrinogen. At its maximum, the stimulation by 4N1K peptide is equal to or greater than that achieved with either epinephrine or thrombin receptor peptide (Fig. 2A). The IAP dependence of the stimulation by 4N1K is demonstrated by the inhibition of the effect by the function blocking anti-IAP mAb B6H12 but not by mAb 2D3, which binds IAP but fails to block function (16). The same results were obtained with F(ab)'2 fragments of the mAbs (not shown). That the stimulation by 4N1K might be due to generation of active thrombin was ruled out by the finding that sufficient D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone to inhibit activation by 1 unit/ml active thrombin had no effect on the action of 4N1K (not shown).


Fig. 1. The TS1 peptide 4N1K stimulates platelet spreading on immobilized fibrinogen. Washed, freshly drawn human platelets treated as in Ref. 14 were incubated in Hepes-buffered saline containing 10 units/ml apyrase to scavenge secreted ADP along with the additions indicated below and allowed to adhere to fibrinogen-coated slides as described under "Experimental Procedures." After 1 h at room temperature, adherent platelets were fixed with paraformaldehyde, permeabilized with Triton X-100, and stained with rhodamine-phalloidin to visualize actin filaments. All fluorescence micrographs were exposed and developed identically to facilitate comparisons. Final magnification is 2,000×. A, apyrase alone. B, 4N1K peptide, 50 µM. C, epinephrine, 10 µM. D, thrombin receptor peptide, 5 µM. E, 4N1K + indomethacin, 10 µM. F, 4N1K + NDGA, 20 µM. G, 4N1K + both indomethacin and NDGA. H, 4N1K + cytochalasin D, 10 µM.
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Fig. 2.

A, specificity of the stimulation of spreading by 4N1K. Platelet spreading on fibrinogen was performed as described in the legend to Fig. 1. After fixing and staining with rhodamine-phalloidin, five randomly selected fields were counted for total platelets (more than 100) and spread platelets. 4N1K was present as indicated at 50 µM and the antibodies B6H12, 2D3, and mouse IgG at 100 µg/ml. Epi, 10 µM epinephrine; TRP, 5 µM thrombin receptor peptide, SFLLRN. Apyrase was present in all samples at 10 U/ml. B, effect of inhibitors on platelet spreading stimulated by 4N1K. Stimulation with 4N1K in all cases as in A with apyrase as a scavenger (-, apyrase alone). Treatments were pertussis toxin for 2 h at room temperature (PT, 100 ng/ml, the B oligomer caused no inhibition under these conditions, not shown); herbimycin A (HA, 27 µM); indomethacin (Indo, 10 µM); NDGA (20 µM); cytochalasin D (cyto D, 10 µM); wortmannin (10 nM); calphostin C (Calp. C, 100 nM); BAPTA (10 µM). Details of incubations as in Ref. 2. C, effect of platelet costimulators on alpha IIbbeta 3 activation determined by PAC-1 binding. alpha IIbbeta 3 activation was measured by mAb PAC-1. Bound PAC-1 was determined by FACScan analysis using a fluorescent secondary antibody as in Ref. 14. The mean fluorescence intensity (arbitrary units) of the brightest 85% of the counted platelets is graphed here. (-), 10 units/ml apyrase (in all samples) present alone; Epi, 10 µM epinephrine; TRP, 5 µM thrombin receptor peptide. 4N1K (KRFYVVMWKK), 4N7G (KRFYVVMGKK), and 4NGG (KRFYGGMWKK) were all at 50 µM.


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Fig. 2B summarizes the effects on 4N1K-stimulated platelet spreading of a number of inhibitors previously shown to inhibit alpha IIbbeta 3-dependent spreading of platelets and/or alpha IIbbeta 3 activation (13, 14). As we had found for the TS and 4N1K stimulation of C32 cells on low density VN surfaces (2), pertussis toxin, but not the carbohydrate binding B oligomer of the toxin, blocks more than 65% of the 4N1K-stimulated platelet spreading on fibrinogen with only a 90-min preincubation. As noted by others (17), the tyrosine kinase inhibitor herbimycin A effectively blocks spreading, reflecting the several steps at which tyrosine kinase activation has been implicated (13, 18). Interestingly, indomethacin (a cyclooxygenase inhibitor) and nordihydroguaretic acid (NGDA, a lipoxygenase inhibitor) each have a partial effect alone (Figs. 1, E and F, and 2B), but together totally obliterate spreading (Figs. 1G and 2B). This indicates that both pathways are activated by 4N1K and that each contributes to the effect. This sort of bifurcation can presumably add redundancy to the response. As noted by others (14), cytochalasin D effectively blocks spreading (Figs. 1G and 2B) by preventing actin filament polymerization. Inhibition of PI 3-kinase by wortmannin partially blocks the response to 4N1K, whereas the protein kinase C inhibitor calphostin c and the calcium chelator BAPTA are quite effective as noted by Shattil et al. (14) for epinephrine costimulation. Thus the properties of the CBD peptide stimulated spreading on fibrinogen mirror those of classical costimulators of alpha IIbbeta 3 function such as ADP, epinephrine and thrombin (13). Comparable data were obtained for the recombinant CBD of TS1 (not shown).

As an independent index of alpha IIbbeta 3 activation, we tested the ability of 4N1K to promote the binding of mAb PAC1 to its activation-dependent epitope in the ligand binding site of alpha IIbbeta 3 (14). As seen in Fig. 2C, 4N1K, at maximum, was able to stimulate a level of PAC1 binding greater than that of either epinephrine or thrombin receptor peptide. The specificity of the effect is demonstrated by the partial activity of peptide 4N7G (KRFYVVMGKK) and the complete lack of activity of peptide 4NGG (KRFYGGMWKK). In other IAP-dependent assays such as chemotaxis and cell spreading, 4N7G is partially active, whereas 4NGG is completely inactive (2). Further, the development of PAC1 binding in the presence of 4N1K was blocked by either calphostin C or indomethacin (not shown), indicating that it resulted from stimulation of intracellular signaling pathways and was not mediated by an extracellular effect of 4N1K. The classical costimulators of alpha IIbbeta 3 also initiate platelet aggregation, and we have previously shown that mAb C6.7 directed against the TS1 CBD inhibits secretion-dependent platelet aggregation (8). Therefore, we tested 4N1K for its ability to cause aggregation of washed platelets in an aggregometer assay. Fig. 3 (A and B) show that the maximal response of the platelets to 4N1K was equal to that for thrombin receptor peptide and greater than that for epinephrine, whereas the inactive control peptide 4NGG was without effect. Walz et al. (9) and Tuszynski et al. (19) have previously reported that whole TS1 can augment the aggregation of gel-filtered platelets. The inhibition of platelet aggregation by antibodies against the NH2-terminal heparin binding domain of TS1 (20) suggests that binding of TS1 to negatively charged glycans helps to tether the protein to the platelet surface.


Fig. 3. Effect of 4N1K on platelet aggregation. Freshly collected, washed human platelets (8) were equilibrated in the aggregometer cuvette at 37 °C for 5 min and then stimulated with thrombin receptor peptide (TRP, 5 µM), epinephrine (Epi, 10 µM), the control peptide 4NGG (50 µM) (A), or 4N1K (50 and 100 µM) and 4NGG as a control (B). The concentrations of thrombin receptor peptide and epinephrine used were determined to give maximal stimulation for each reagent in preliminary experiments.
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We next examined an intracellular event related to the activation of platelet spreading and aggregation: phosphorylation of proteins on tyrosine residues (15, 21). Preliminary experiments with platelets spreading on fibrinogen-coated surfaces revealed that 4N1K could greatly enhance the rate and extent of tyrosine phosphorylation of several proteins previously identified as early targets of tyrosine kinases in activated platelets (15, 18, 21). Fig. 4A shows the time course of tyrosine phosphorylation of FAK in platelets attached to fibrinogen in the presence of apyrase with (left) and without (right, mirror image) 4N1K. At the earliest times of 1 to 2 min of 4N1K treatment, FAK contains significantly elevated levels of phosphotyrosine that increase throughout the time course. In these experiments we noted the early appearance of a tyrosine phosphorylated protein of molecular mass of approximately 70 kDa whose phosphorylation and association with FAK was much more prominent in 4N1K-treated platelets than in control or epinephrine-stimulated platelets. Fig. 4B shows that this protein is the 72-kDa tandem SH2 domain kinase SYK, whose phosphorylation on tyrosine has been identified as a very early event in platelet activation (13, 17). 4N1K treatment results in a significant stimulation of SYK association with FAK compared with epinephrine (lane 4). As seen in Fig. 4B, the association of SYK with FAK is selectively augmented by 4N1K compared with epinephrine. The association of LYN with FAK (Fig. 4C) is also selectively stimulated with a time course similar to that of SYK. In B cells, LYN has been found to phosphorylate sites in ITAM-containing cytoplasmic domains of receptors thus creating docking sites for SYK (22). It is interesting that FAK phosphorylation increases until 60 min, whereas SYK and LYN association with FAK is highest at 30 min and decreases by 60 min of spreading.


Fig. 4. 4N1K stimulates the tyrosine phosphorylation and association of FAK, SYK, and LYN. A-C, platelets spreading on fibrinogen as in Fig. 1 in the presence and the absence of 4N1K (50 µM) were dissolved in RIPA buffer at the indicated times and the lysate immunoprecipitated with mAb PY-20 (antiphosphotyrosine) (A) or mAb 2A7 (anti-FAK) (B and C). After SDS-polyacrylamide gel electrophoresis the blot in A was stained with anti-FAK mAb (clone 77). The band at 125 kDa is FAK. Staining of B was with anti-SYK polyclonal IgG. Staining of C was with anti-LYN polyclonal IgG. The methods were detailed in Ref. 5. D, suspended, unstirred platelets (in the presence of apyrase) were treated with 4NGG or 4N1K (50 µM) for the indicated times and then lysed, electrophoresed, and Western blotted as above with anti-SYK mAb. E, suspended, unstirred platelets were preincubated with apyrase and the indicated inhibitors as in Ref. 14. 4N1K (lanes 3-11) or 4NGG (lane 2) were added at 50 µM 5 min before lysis with RIPA buffer. Inhibitors were: none (lanes 1, 2, and 7); 10 µM BAPTA (lane 3); 100 nM calphostin C (lane 4); 10 nM wortmannin (lane 5); 10 µM cytochalasin D (lane 6); 4N1K alone (lane 7); 20 µM NDGA (lane 8); 10 µM indomethacin (lane 9); 1 µM prostaglandin E1 (lane 10); 100 ng/ml pertussis toxin (lane 11) was preincubated with platelets for 2 h prior to the addition of 4N1K. Platelets were lysed with RIPA buffer at indicated times, and lysates were immunoprecipitated with mAb PY-20. Subsequent immunoblotting was with anti-SYK.
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To determine the events connecting 4N1K-IAP binding with SYK phosphorylation, suspended, unstirred washed platelets were stimulated with 4N1K. These conditions prevent platelet aggregation and hence fibrinogen binding by alpha IIbbeta 3, which leads to outside in signaling and the initiation of a cascade of downstream events including a massive wave of tyrosine phosphorylation and cytoskeletal reorganization (13). In these unstirred platelets the 4N1K-stimulated tyrosine phosphorylation of SYK is maximal at 3-5 min and declines thereafter (Fig. 4D). At later times, when slow platelet aggregation begins to occur despite the lack of stirring, SYK phosphorylation again increases as previously reported (Ref. 17 and data not shown). Again, the control peptide 4NGG is inactive. These results indicate that platelet aggregation and signaling downstream of alpha IIbbeta 3 ligation are not required for SYK activation. Using this same protocol we tested the effects of a number of inhibitors on the ability of 4N1K to stimulate tyrosine phosphorylation of SYK (Fig. 4E). Interestingly, only pertussis toxin strongly inhibited SYK phosphorylation at 5 min in response to 4N1K. Cytochalasin D and prostaglandin E1, which elevates cAMP, gave partial and variable inhibition, whereas other inhibitors did not reduce SYK phosphorylation (three experiments gave comparable results). Significantly, indomethacin and NDGA had no effect, nor did calphostin c or BAPTA, protein kinase C inhibitors. Because BAPTA is a Ca+2 chelator, it seems unlikely that the effect of 4N1K on SYK phosphorylation is mediated by Ca+2 flux, a known action of IAP in other cell types (23, 24). These data indicate that SYK phosphorylation occurs quite proximally to IAP activation and requires a heterotrimeric Gi-like protein but not other downstream effectors of platelet activation. Similar results in terms of SYK phosphorylation have been obtained upon stimulation of unstirred platelets via activation of thrombin, collagen, and ADP receptors (25-27). The robust stimulation of SYK phosphorylation perhaps indicates a special role for TS1 in activating SYK kinase. The mechanism of this effect is under investigation.

To gain insight on the proteins that might initiate IAP signaling, we have used an affinity column strategy to ask which proteins, if any, are in a preformed complex with IAP in resting platelets. To do this, unstimulated platelets were dissolved in a buffer containing Triton X-100 (1% v/v), and the soluble extract was applied to Ni-NTA agarose beads, which had been charged with 4N1K peptide containing a hexahistidine tag at its amino terminus. The control was His-tagged 4NGG peptide charged beads. After washing with 0.2 M imidazole, the IAP and other specifically bound proteins were eluted with 1 M imidazole. Because only hexahistidine-tagged proteins or peptides remain bound to Ni-NTA matrix after washing with 0.2 M imidazole, it is likely that proteins that elute with M imidazole are bound via interactions with IAP that binds 4N1K. As seen in Fig. 5A, no IAP is eluted from the 4NGG column (fractions 1 through 7 are shown), whereas IAP is readily detectable in fractions eluted from the 4N1k column. Both integrin subunits, alpha IIb and beta 3 coeluted with IAP, confirming that under these rather stringent conditions in the presence of Triton X-100, IAP is complexed with the integrin heterodimer. Western blots of these fractions were probed with antibodies against tyrosine kinases involved in platelet signaling. Interestingly, only a mAb specific for c-Src versus other Src-type kinases gave a strong signal (Fig. 5A) while FYN, LYN, and YES were not detected in the eluate (not shown). FAK was marginally detectable (Fig. 5A) and SYK was not detected in the column eluate even on prolonged exposures (not shown). These results suggest that the integrin and IAP are in a complex with c-Src, and perhaps FAK but not SYK in resting platelets. However, the data in Fig. 4A indicate that FAK becomes phosphorylated in response to 4N1K stimulation. To determine if FAK could become associated with the integrin-IAP complex after 4N1K stimulation, suspended, unstirred platelets were stimulated with 4N1K as above, and at various times samples were lysed in Triton X-100 buffer and immunoprecipitated with an anti-beta 3 mAb, run on SDS-polyacrylamide gel electrophoresis, and blotted. In agreement with the results in Fig. 5A, lysates from platelets treated with the control peptide 4NGG (at 5 min) displayed relatively little association of FAK with the integrin-IAP complex (Fig. 5B). However, as early as 1 min after the addition of 4N1K, a significant increase in the amount of FAK associated with the complex was detected (Fig. 5B). IAP and c-Src were also detected in the beta 3 immunoprecipitate when platelets were treated with either 4NGG or 4N1K (not shown) in further agreement with the results in Fig. 5A. The same increase in FAK association with the integrin-IAP complex was also detected in anti-IAP immunoprecipitates (not shown).


Fig. 5. IAP associates with alpha IIbbeta 3, c-Src, and FAK. A, 1% Triton X-100 soluble material from unstimulated, suspended platelets was applied to Ni-NTA agarose columns charged with either His-tagged 4NGG (left) or His-tagged 4N1K (right) peptides. After washing the columns with 0.2 M imidazole to remove nonspecifically bound proteins, specifically bound proteins were eluted with 1 M imidazole, and 100-µl fractions were collected (fraction numbers are indicated above the figure). After SDS-polyacrylamide gel electrophoresis, immunoblots were probed with the indicated antibodies. B, suspended platelets were treated for the indicated times with either 4NGG or 4N1K and lysed with 1% Triton X-100, and the lystates immunoprecipitated with anti-beta 3 antibody. Immunoblots of the SDS gels were probed with the indicated antibodies.
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DISCUSSION

These data provide a new paradigm for the action of TS1 at the site of its greatest physiological concentration, the platelet. Upon platelet activation, TS1 is secreted and binds to the platelet surface via a number of receptors (9, 28-31). These include sulfatides, which bind to the NH2-terminal heparin binding domain (32), platelet gpIV, or CD36, which binds to a region within the type 1 repeats in the central stalk or "core" of the TS1 subunit, and alpha vbeta 3 and alpha IIbbeta 3, both of which have been shown to bind TS1 in an RGD-dependent manner (29, 33). The binding of TS1 to resting platelets does not require Ca2+ ions, whereas binding to the receptors exposed on activated platelets is blocked by EDTA (9, 12, 20, 30). We have reported that binding of cells to the CBD and 4N1K peptide and affinity labeling of IAP by 4N1K are not sensitive to EDTA (2, 5), suggesting, as noted by Dorahy et al. (12), that IAP is the primary TS1 receptor on resting platelets. Our present data indicate that the CBD of TS1 can bind to platelet IAP and that the peptide 4N1K is a specific agonist for IAP signaling. Because the RGD sequence of TS1 is just amino-terminal to the CBD, the binding of TS1 to IAP may facilitate the exposure of the RGD site and its binding to either alpha vbeta 3 or alpha IIbbeta 3 associated with IAP. This could have consequences for modulation of fibrinogen binding by alpha IIbbeta 3 and may allow TS1 to participate directly in platelet-platelet cross-linking. This is likely to be the case in washed platelets where TS1 is much more abundant than fibrinogen after alpha -granule discharge. Whether or not the same TS1 molecule engages IAP and associated alpha IIbbeta 3, the surface bound TS1 would appear to serve the function of a tethered costimulator of alpha IIbbeta 3 activation and hence of ligand binding and platelet cross-linking. The previous model for TS1 action in platelets emphasized the role of TS1 as a cross-linker of fibrinogen bridges between platelets (7, 34). Our data indicate that TS1 can strengthen ligand binding to alpha IIbbeta 3 and hence platelet cross-linking, by a direct effect on the classical intraplatelet costimulatory signaling cascade resulting in an enhancement of the affinity/avidity of alpha IIbbeta 3 (13).

The experiments reported here are in fact the first report of a detergent-stable complex containing the integrin alpha IIbbeta 3 and IAP obtained from platelets. An early report showed coimmunoprecipitation of IAP with an anti-beta 3 mAb (3), but because platelets contain alpha vbeta 3, this does not prove association with alpha IIbbeta 3. Dorahy et al. (12) used "disrupted platelet membranes" obtained by sonicating platelets that were then passed over a 4N1K affinity column. IAP and other proteins including beta 3 subunits were eluted with RFYVVM peptide. Because no detergent was employed, these data cannot argue for complex formation. We have shown by both affinity chromatography and immunoprecipitation of detergent solubilized platelet membranes that alpha IIbbeta 3 and IAP do indeed exist in a complex. Because there are approximately equal numbers of alpha IIbbeta 3 and IAP molecules in the platelet membrane (3), each IAP may be associated with an alpha IIbbeta 3 molecule. However, because IAP appears to activate alpha IIbbeta 3 via a signaling pathway, direct physical association of each alpha IIbbeta 3 with an IAP may not be required. Further we find that c-Src is also resident in this complex with alpha IIbbeta 3 and IAP. At least five members of the Src-type kinase family are expressed in megakaryocytes and platelets (36), but only c-Src could be detected in the alpha IIbbeta 3-IAP complex. Even though c-Src is present in platelets greater abundance than any other family member, a clear functional role for c-Src in platelets has not been identified. Thus we suggest that at least one role of c-Src in platelets is to initiate the IAP-dependent tyrosine phosphorylation of SYK. 4N1K stimulates the tyrosine phosphorylation of SYK in unstirred platelets (Fig. 4D), and this is inhibited by pertussis toxin and to a lesser extent by cytochalasin D but not by other inhibitors of platelet activation (Fig. 4E). This is precisely the inhibitor profile found for thrombin stimulation of SYK phosphorylation by Clark et al. (26), who reported that c-Src and SYK are phosphorylated in a first wave of tyrosine phosphorylation that does not require integrin engagement or platelet aggregation. There is ample precedent for the stimulation of Src-like tyrosine kinases by receptors that act through heterotrimeric G proteins including Gi (37-40); however, the precise mechanism(s) linking G protein subunits to activation of Src-type kinases is unknown (41). For example, it has recently been reported that Gi derived Gbeta gamma activates c-Src-dependent tyrosine phosphoryation of the epidermal growth factor receptor (41). These authors propose that PI 3-kinase may mediate the activation of c-Src, and in fact, Gbeta gamma activation of PI 3-kinase in platelets can occur (41, 42). However, the IAP-dependent stimulation of SYK phosphorylation that we observe upon treatment of platelets with 4N1K is not sensitive to inhibition by wortmannin (Fig. 4E), a property of all three PI 3-kinase isoforms (43). The mechanism of c-Src activation by IAP is currently under investigation.

As seen in Fig. 5 (A and B), relatively small amounts of FAK are associated with the integrin-IAP complex extracted from resting platelets, but FAK association with this complex is rapidly increased upon agonist activation of IAP. Furthermore, SYK associates with FAK with equal rapidity (Fig. 4B),2 and this association is blocked by pertussis toxin (not shown). Taken together our data suggest that ligation of IAP with the agonist peptide 4N1K activates a heterotrimeric pertussis toxin-sensitive Gi protein. Through an as yet unknown mechanism, which does not appear to involve PI 3-kinase activation, the G protein may activate c-Src, which in turn could phosphorylate SYK and FAK. The SYK homolog ZAP-70 functions in T cell activation where it is initially activated by binding to ITAM motifs that are phosphorylated on tandem tyrosines by the Src-type kinase, FYN (44). The resultant clustering of ZAP-70 molecules presumably allows intermolecular tyrosine phosporylation to occur. The phosphorylated and activated ZAP-70 can then phosphorylate a number of downstream targets leading to T cell proliferation and other responses. A similar ITAM-dependent mechanism has been proposed for SYK activation in B cells (45). SYK activation in platelets has remained unexplained. Recent reports (46, 47) indicate that the cytoplasmic domain of the integrin beta 3 subunit can be tyrosine phosphorylated, but it is not clear that the two tyrosines in that sequence can function as an ITAM motif. Although the spacing between the tyrosines is precisely that of a cannonical ITAM, the residues in the Y+3 positions are not well conserved (44). It has been reported that cross-linking of platelet Fc receptors, Fcgamma RII, leads to phosphorylation of SYK, even in the absence of alpha IIbbeta 3 (48). Tyrosine residues in the cytoplasmic domains of Fcgamma RII chains occur in sequence contexts (YXXL) reminiscent of ITAMs but the spacing of the tyrosines is somewhat different (44). The possibility remains that a tyrosine kinase binds via a single SH2 domain to one of these phosphorylated sites in the integrin or the Fc receptor tail, which could then phosphorylate an ITAM-like motif in another protein, perhaps FAK itself.

In summary, we have identified TS1 as the natural ligand for IAP and 4N1K as a peptide agonist (2, 5). It is now possible to isolate the IAP costimulatory pathway and study its function. The role for TS1 suggested by our experiments is that of an autocrine or juxtacrine costimulator of alpha IIbbeta 3 function whose ability to bind to the platelet surface (9, 28, 30) and fibrin clot (35) differentiates TS1 from small molecule and other protein costimulators. The robust effect of 4N1K on LYN and SYK phosphorylation and FAK association (Fig. 4) suggests that this may be a unique consequence of TS1-IAP mediated signaling. Finally the modulatory effect of TS1/IAP on alpha IIbbeta 3 function presents a novel and potentially efficacious point at which to "tune down" the function of alpha IIbbeta 3 without totally obliterating it, thus perhaps controlling acute thrombotic events without precipitating dangerous bleeding episodes.


FOOTNOTES

*   This work was supported by a grant from Monsanto-Searle. A preliminary report of this data was presented at a meeting: The Thrombospondin Gene Family, June 19, 1996, University of Washington, Seattle, WA.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 and Molecular Biophysics, Box 8231, Washington University Medical School, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-3348; Fax: 314-362-7183; E-mail: frazier{at}biochem.wustl.edu.
1   The abbreviations used are: IAP, integrin-associated protein; CBD, cell binding domain; TS, thrombospondin; mAb, monoclonal antibody; HBS, Hepes-buffered saline; NGDA, nordihydroguaretic acid; FAK, focal adhesion kinase; PI, phosphatidylinositol; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid; Ni-NTA, nickel nitrilotriacetic acid; ITAM, immunoreceptor tryosine-based activation motif.
2   J. Chung, A.-G. Gao, and W. A. Frazier, unpublished results.

ACKNOWLEDGEMENTS

We thank Drs. Fred Lindberg and Eric Brown for many insightful discussions and anti-IAP mAbs; Dr. Sam Santoro for help with aggregometry; Dr. Sandy Shattil for mAb PAC1 and helpful advice; Tom Broekelmann for help with fluorescence microscopy; and the Protein and Nucleic Acid Chemistry Laboratory of Washington University for peptide synthesis, purification, and characterization. Julie Dimitry prepared recombinant CBD as in Ref. 2, and Mary Beth Finn obtained platelets and prepared platelet thrombospondin as described previously (8). We thank Anna Goffinet for preparation of the manuscript.


REFERENCES

  1. Lindberg, F. P., Bullard, D. C., Caver, T. E., Gresham, H. D., Beaudet, A. L., and Brown, E. J. (1996) Science 274, 795-798 [Abstract/Free Full Text]
  2. Gao, A.-G., Lindberg, F. P., Dimitry, J. M., Brown, E. J., and Frazier, W. A. (1996) J. Cell Biol. 135, 533-544 [Abstract]
  3. Brown, E., Hooper, L., Ho, T., and Gresham, H. (1990) J. Cell Biol. 111, 2785-2794 [Abstract]
  4. Lindberg, F. P., Gresham, H. D., Schwarz, E., and Brown, E. J. (1993) J. Cell Biol. 123, 485-496 [Abstract]
  5. Gao, A. G., Lindberg, F. P., Finn, M. B., Blystone, S. D., Brown, E. J., and Frazier, W. A. (1996) J. Biol. Chem. 271, 21-24 [Abstract/Free Full Text]
  6. Baenziger, N. L., Brodie, G. N., and Majerus, P. W. (1971) Proc. Natl. Acad. Sci. U. S. A. 68, 240-243 [Abstract]
  7. Leung, L. L. (1984) J. Clin. Invest. 74, 1764-1772 [Medline] [Order article via Infotrieve]
  8. Dixit, V. M., Haverstick, D. M., O'Rourke, K. M., Hennessy, S. W., Grant, G. A., Santoro, S. A., and Frazier, W. A. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3472-3476 [Abstract]
  9. Walz, D. A., Zafar, R. S., and Zeng, Z. (1993) in Thrombospondin (Lahav, J., ed), pp. 149-164, CRC Press, Boca Raton, FL
  10. Rosales, C., Gresham, H. D., and Brown, E. J. (1992) J. Immunol. 149, 2759-2764 [Abstract/Free Full Text]
  11. Fujimoto, T., Fujimura, K., Noda, M., Takafuta, T., Shimomura, T., and Kuramoto, A. (1995) Blood 86, 2174-2182 [Abstract/Free Full Text]
  12. Dorahy, D. J., Thorne, R. F., Fecondo, J. V., and Burns, G. F. (1997) J. Biol. Chem. 272, 1323-1330 [Abstract/Free Full Text]
  13. Shattil, S. J., Ginsberg, M. H., and Brugge, J. S. (1994) Curr. Opin. Cell Biol. 6, 695-704 [Medline] [Order article via Infotrieve]
  14. Shattil, S. J., Haimovich, B., Cunningham, M., Lipfert, L., Parsons, J. T., Ginsberg, M. H., and Brugge, J. S. (1994) J. Biol. Chem. 269, 14738-14745 [Abstract/Free Full Text]
  15. Haimovich, B., Lipfert, L., Brugge, J. S., and Shattil, S. J. (1993) J. Biol. Chem. 268, 15868-15877 [Abstract/Free Full Text]
  16. Cooper, D., Lindberg, F. P., Gamble, G. R., Brown, E. J., and Vadas, M. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 3978-3982 [Abstract/Free Full Text]
  17. Schwartz, M. A., Schaller, M. D., and Ginsberg, M. H. (1995) Annu. Rev. Cell Biol. Dev. Biol. 11, 549-599 [CrossRef][Medline] [Order article via Infotrieve]
  18. Huang, M.-M., Lipfert, L., Cunningham, M., Brugge, J. S., Ginsberg, M. H., and Shattil, S. J. (1993) J. Cell Biol. 122, 473-483 [Abstract]
  19. Tuszynski, G. P., Rothman, V. L., Murphy, A., Siegler, K., and Knudsen, K. A. (1988) Blood 72, 109-115 [Abstract]
  20. Gartner, T. K., Walz, D. A., Aiken, M., Starr-Spires, L., and Ogilvie, M. L. (1984) Biochem. Biophys. Res. Commun. 15, 290-295
  21. Lipfert, L., Haimovich, B., Schaller, M. D., Cobb, B. S., Parsons, J. T., and Brugge, J. S. (1992) J. Cell Biol. 119, 905-912 [Abstract]
  22. Nagai, K., Takata, M., Yamamura, H., and Kurosaki, T. (1995) J. Biol. Chem. 270, 6824-6829 [Abstract/Free Full Text]
  23. Schwartz, M. A., Brown, E. J., and Fazeli, B. (1993) J. Biol. Chem. 268, 19931-19934 [Abstract/Free Full Text]
  24. Tsao, P. W., and Mousa, S. A. (1995) J. Biol. Chem. 270, 23747-23753 [Abstract/Free Full Text]
  25. Taniguchi, T., Kitagawa, H., Yasue, S., Yanagi, S., Sakai, K., Asahi, M., Ohta, S., Takeuchi, F., Nakamura, S., and Yamamura, H. (1993) J. Biol. Chem. 268, 2277-2279 [Abstract/Free Full Text]
  26. Clark, E. A., Shattil, S. J., Ginsberg, M. H., Bolen, J., and Brugge, J. S. (1994) J. Biol. Chem. 269, 28859-28864 [Abstract/Free Full Text]
  27. Keely, P. J., and Parise, L. V. (1996) J. Biol. Chem. 271, 26668-26676 [Abstract/Free Full Text]
  28. McGregor, J. L., and Boukerche, H. (1993) in Thrombospondin (Lahav, J., ed), pp. 111-127, CRC Press, Boca Raton, FL
  29. Lawler, J., and Hynes, R. O. (1989) Blood 74, 2022-2027 [Abstract]
  30. Aiken, M. L., Ginsberg, M. H., Byers-Ward, V., and Plow, E. F. (1990) Blood 76, 2501-2509 [Abstract]
  31. Asch, A. S., Barnwell, J., Silverstein, R. L., and Nachman, R. L. (1987) J. Clin. Invest. 79, 1054-1061 [Medline] [Order article via Infotrieve]
  32. Roberts, D. D. (1993) in Thrombospondin (Lahav, J., ed), pp. 73-90, CRC Press, Boca Raton, FL
  33. Lawler, J., Weinstein, R., and Hynes, R. O. (1988) J. Cell Biol. 107, 2351-2361 [Abstract]
  34. Gartner, T. K., Gerrard, J. M., White, J. G., and Williams, D. C. (1981) Nature 289, 688-690 [Medline] [Order article via Infotrieve]
  35. Bale, M. D., Westrick, L. G., and Mosher, D. F. (1985) J. Biol. Chem. 260, 7502-7508 [Abstract/Free Full Text]
  36. Kefalas, P., Brown, T. R. P., and Brickell, P. M. (1995) Int. J. Biochem. Cell Biol. 27, 551-563 [CrossRef][Medline] [Order article via Infotrieve]
  37. Chen, Y., Pouyssegur, J., Courtneidge, S. A., and Van Obberghen-Schilling, E. (1994) J. Biol. Chem. 269, 27372-27377 [Abstract/Free Full Text]
  38. Luttrell, L. M., Hawes, B. E., van Biesen, T., Luttrell, D. K., Lansing, T. J., and Lefkowitz, R. J. (1996) J. Biol. Chem. 271, 19443-19450 [Abstract/Free Full Text]
  39. Ishida, M., Marrero, M. B., Schieffer, B., Ishida, T., Bernstein, K. E., and Berk, B. C. (1995) Circ. Res. 77, 1053-1059 [Abstract/Free Full Text]
  40. Ptasznik, A., Traynor-Kaplan, A., and Bokoch, G. M. (1995) J Biol. Chem. 270, 19969-19973 [Abstract/Free Full Text]
  41. Luttrell, L. M., Della Rocca, G. J., van Biesen, T., Luttrell, D. K., and Lefkowitz, R. J. (1997) J. Biol. Chem. 272, 4637-4644 [Abstract/Free Full Text]
  42. Thomason, P. A., James, S. R., Casey, P. J., and Downes, C. P. (1994) J. Biol. Chem. 269, 16525-16528 [Abstract/Free Full Text]
  43. Wymann, M. P., Bulgarelli-Leva, G., Zvelebil, M. J., Pirola, L., Vanhaesebroeck, B., Waterfield, M. D., and Panayotou, G. (1996) Mol. Cell. Biol. 16, 1722-1733 [Abstract]
  44. Cambier, J. C. (1995) J. Immunol. 155, 3281-3285 [Medline] [Order article via Infotrieve]
  45. Kurosaki, T., Takata, M., Yamanashi, Y., Inazu, T., Taniguchi, T., Yamamoto, T., and Yamamura, H. (1994) J. Exp. Med. 179, 1725-1729 [Abstract]
  46. Blystone, S. D., Lindberg, F. P., Williams, M. P., McHugh, K. P., and Brown, E. J. (1996) J. Biol. Chem. 271, 31458-31462 [Abstract/Free Full Text]
  47. Law, D. A., Nannizzi-Alaimo, L., and Phillips, D. R. (1996) J. Biol. Chem. 271, 10811-10815 [Abstract/Free Full Text]
  48. Haimovich, B., Regan, C., DiFazio, L., Ginalis, E., Ji, P., Purohit, U., Rowley, R. B., Bolen, J., and Greco, R. (1996) J. Biol. Chem. 271, 16332-16337 [Abstract/Free Full Text]

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