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J Biol Chem, Vol. 274, Issue 34, 24321-24327, August 20, 1999


Activation-dependent Adhesion of Human Platelets to Cyr61 and Fisp12/Mouse Connective Tissue Growth Factor Is Mediated through Integrin alpha IIbbeta 3*

Arom JedsadayanmataDagger , Chih-Chiun Chen§, Maria L. Kireeva§, Lester F. Lau§, and Stephen C.-T. LamDagger parallel

From the Departments of Dagger  Pharmacology and § Molecular Genetics, University of Illinois, Chicago, Illinois 60612

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cyr61 and connective tissue growth factor (CTGF), members of a newly identified family of extracellular matrix-associated signaling molecules, are found to mediate cell adhesion, promote cell migration and enhance growth factor-induced cell proliferation in vitro, and induce angiogenesis in vivo. We previously showed that vascular endothelial cell adhesion and migration to Cyr61 and Fisp12 (mouse CTGF) are mediated through integrin alpha vbeta 3. Both Cyr61 and Fisp12/mCTGF are present in normal blood vessel walls, and it has been demonstrated that CTGF is overexpressed in advanced atherosclerotic lesions. In the present study, we examined whether Cyr61 and Fisp12/mCTGF could serve as substrates for platelet adhesion. Agonist (ADP, thrombin, or U46619)-stimulated but not resting platelets adhered to both Cyr61 and Fisp12/mCTGF, and this process was completely inhibited by prostaglandin I2, which prevents platelet activation. The specificity of Cyr61- and Fisp12/mCTGF-mediated platelet adhesion was demonstrated by specific inhibition of this process with polyclonal anti-Cyr61 and anti-Fisp12/mCTGF antibodies, respectively. The adhesion of ADP-activated platelets to both proteins was divalent cation-dependent and was blocked by RGDS, HHLGGAKQAGDV, or echistatin, but not by RGES. Furthermore, this process was specifically inhibited by the monoclonal antibody AP-2 (anti-alpha IIbbeta 3), but not by LM609 (anti-alpha vbeta 3), indicating that the interaction is mediated through integrin alpha IIbbeta 3. In a solid phase binding assay, activated alpha IIbbeta 3, purified by RGD affinity chromatography, bound to immobilized Cyr61 and Fisp12/mCTGF in a dose-dependent and RGD-inhibitable manner. In contrast, unactivated alpha IIbbeta 3 failed to bind to either protein. Collectively, these findings identify Cyr61 and Fisp12/mCTGF as two novel activation-dependent adhesive ligands for the integrin alpha IIbbeta 3 on human platelets, and implicate a functional role for these proteins in hemostasis and thrombosis.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Platelet adhesion to the subendothelial matrix and platelet aggregation are key mechanisms by which platelets participate in hemostasis and thrombosis. Thus, upon vascular injury, platelets adhere to the exposed subendothelial matrix, leading to platelet aggregation caused by the binding of plasma fibrinogen or von Willebrand factor (vWf)1 to the activated platelets. Integrin alpha IIbbeta 3 is the most prominent platelet adhesion receptor, which interacts with several adhesive ligands including fibrinogen, vWf, fibronectin, and vitronectin (1-3). On resting platelets, alpha IIbbeta 3 is present in an inactive conformation incapable of binding soluble adhesive proteins. The formation of platelet agonists such as thrombin at sites of vessel injury induces platelet inside-out signaling, which leads to the binding of soluble fibrinogen and vWf to alpha IIbbeta 3, resulting in platelet aggregation.

In normal blood vessels, the major matrix components in the subendothelium mediating platelet adhesion are thought to be vWf, fibronectin, collagen, and laminin. Initial platelet adherence to damaged vessel walls is thought to involve both non-integrin (e.g. the GPIb-IX-V complex) and integrin adhesion receptors (e.g. alpha 2beta 1, alpha 5beta 1, and alpha 6beta 1) in addition to alpha IIbbeta 3 (2, 4, 5). During the development and progression of atherosclerosis, activation of cellular components in the atherosclerotic plaques may generate as yet undefined substrates that mediate platelet adhesion to ruptured plaque lesions. In this regard, it has recently been shown that activated platelets adhere to osteopontin in atherosclerotic plaques through integrin alpha vbeta 3 (6). Another extracellular matrix-associated protein, connective tissue growth factor (CTGF), was found to be overexpressed in advanced atherosclerotic lesions as compared with normal blood vessels (7).

CTGF belongs to an emerging family of conserved and modular proteins with diverse biological functions (8, 9). Six members of this protein family, including CTGF and Cyr61, have been described to date. Both Cyr61 and Fisp12, the mouse ortholog of CTGF, were identified as products of immediate-early genes transcriptionally induced in fibroblasts in response to serum growth factors (10-13). Upon synthesis, both proteins are secreted and become associated with the cell surface and the extracellular matrix (14, 15). Both Cyr61 and Fisp12/mCTGF have been shown to mediate adhesion and to promote migration in vascular endothelial cells (16-18). Although neither protein alone induces mitogenesis in vascular endothelial cells, both are able to augment growth factor-induced DNA synthesis (15, 16). Furthermore, Fisp12/mCTGF can promote vascular endothelial cell survival under conditions that induces apoptosis (17). All of these activities are pro-angiogenic; indeed, both Cyr61 and Fisp12/mCTGF were found to induce angiogenesis in vivo in corneal micropocket assays (17, 18). While the mechanism through which Cyr61 and Fisp12/mCTGF induce angiogenesis in vivo is not known, biochemical and functional evidence indicate that the integrin alpha vbeta 3 serves as a receptor on endothelial cells for Cyr61 and Fisp12/mCTGF mediating cell adhesion and migration (17-19).

Both Cyr61 and CTGF proteins are present in normal and diseased blood vessel walls (7, 20). Based on the similarity of ligand recognition specificity between integrins alpha vbeta 3 and alpha IIbbeta 3, we postulate that Cyr61 and Fisp12/mCTGF may serve as adhesive substrates for the platelet integrin alpha IIbbeta 3. In the present study, we show that both Cyr61 and Fisp12/mCTGF support the adhesion of platelets in an activation-dependent manner. Furthermore, active but not inactive alpha IIbbeta 3 binds directly to purified Cyr61 or Fisp12/mCTGF in a solid-phase binding assay. Thus, these studies identify two novel activation-dependent adhesive ligands for human platelets, and implicate a functional role for these proteins in hemostasis and thrombosis.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Antibodies, Peptides, and Reagents-- The anti-beta 3 monoclonal antibody mAb15 (21) was provided by Dr. Mark H. Ginsberg of the Scripps Research Institute, La Jolla, CA, and was radioiodinated with carrier-free Na125I (Amersham Pharmacia Biotech) using the IODO-BEADS iodination reagent (Pierce) to a specific activity of approximately 2 µCi/µg. The monoclonal antibodies AP-2 (22) and LM609 (23) were generous gifts of Dr. T. J. Kunicki and Dr. D. A. Cheresh, respectively, of the Scripps Research Institute, La Jolla, CA. Polyclonal anti-Cyr61 and anti-Fisp12/mCTGF antibodies were raised in rabbits as described previously (15), and purified by chromatography on protein A-Sepharose.

Peptide sequences are represented by the single-letter amino acid codes (24). The fibrinogen gamma  chain dodecapeptide H12 with the sequence HHLGGAKQAGDV was purchased from Research Genetics Inc. RGDS and RGES peptides were purchased from Peninsula Laboratories. Echistatin was purchased from Sigma, and fibrinogen was obtained from KabiVitrum, Inc.

Protein Purifications-- Recombinant Cyr61 and Fisp12/mCTGF, synthesized in a baculovirus expression system using Sf9 insect cells, were purified from serum-free conditioned media by chromatography on Sepharose S as described (15, 16). SDS-PAGE analysis of purified Cyr61 and Fisp12/mCTGF revealed the presence of a single Coomassie Blue-stained band of 40 and 38 kDa, respectively. On immunoblots, the purified proteins reacted specifically with their cognate antibodies (15).

Activated alpha IIbbeta 3 was purified by RGD affinity chromatography as described (25). Briefly, outdated human platelets were isolated by differential centrifugation and solubilized in lysis buffer (10 mM HEPES, pH 7.4, 0.15 M NaCl, containing 1 mM CaCl2, 1 mM MgCl2, 100 µM leupeptin, 1 mM phenylmethylsulfonyl fluoride, 10 mM N-ethylmaleimide, and 50 mM octyl glucoside). The octyl glucoside extract was incubated with 1 ml of GRGDSPK-coupled Sepharose 4B overnight at 4 °C. After washing with 15 ml of column buffer (same as lysis buffer except it contained 25 mM octyl glucoside), bound alpha IIbbeta 3 was eluted with 1.7 mM H12 (2 ml) in column buffer. The H12 eluate was applied to a Sephacryl S-300 High Resolution column (1.5 × 95 cm), and alpha IIbbeta 3 was eluted with 10 mM HEPES, pH 7.4, 0.15 M NaCl, 1 mM CaCl2, 1 mM MgCl2 and 25 mM octyl glucoside.

Unactivated alpha IIbbeta 3 was isolated by the method of Fitzgerald et al. (26) with slight modifications. The flow-through fraction of the GRGDSPK-Sepharose column was applied onto a concanavalin A-Sepharose 4B column (1 × 20 cm). Unbound proteins were washed with 50 ml of column buffer, and bound alpha IIbbeta 3 was then eluted with 100 mM mannose dissolved in column buffer. Fractions containing alpha IIbbeta 3 were further purified on a Sephacryl S-300 High Resolution column as described above.

Protein concentrations were determined using the BCA protein assay (Pierce) with bovine serum albumin (BSA) as the standard. In some experiments, to ensure that equal concentrations of activated and unactivated alpha IIbbeta 3 were used, the purified receptor preparations were subjected to SDS-PAGE and densitometric scanning of the silver-stained protein bands was performed.

Platelet Isolation and Adhesion Assay-- Venous blood was drawn from healthy donors and collected into acid-citrate-dextrose. Washed platelets were prepared by differential centrifugation as described (27) and finally resuspended in HEPES-Tyrode's buffer (5 mM HEPES, pH 7.35, 1 mM MgCl2, 1 mM CaCl2, 135 mM NaCl, 2.7 mM KCl, 11.9 mM NaHCO3, 1 mg/ml dextrose, and 3.5 mg/ml BSA). The platelet count was adjusted to 3 × 108 platelets/ml.

Microtiter wells (Immulon 2 Removawell strips, Dynex Technologies, Inc.) were coated with Cyr61, Fisp12/mCTGF, or fibrinogen (25 µg/ml, 50 µl/well) overnight at 22 °C, and then blocked with 3% BSA at 37 °C for 2 h. Washed platelets were added to the wells (100 µl/well) in the presence and absence of platelet agonists and incubated at 37 °C for 30 min. The wells were washed with HEPES-Tyrode's buffer and adherent platelets were detected with 125I-mAb15, an anti-beta 3 monoclonal antibody. Binding of the labeled antibody (50 nM, 50 µl/well) proceeded for 1 h at 22 °C. After extensive washing with HEPES-Tyrode's buffer, bound radioactivity was determined by gamma -counting. In inhibition studies, washed platelets were preincubated with blocking peptides or antibodies at 37 °C for 15 min prior to addition to microtiter wells. In experiments to examine the effect of divalent cation chelation, EDTA (5 mM) was added to suspensions of washed platelets and preincubated at 37 °C for 15 min.

As indicated, adherent platelets were also detected by the acid phosphatase assay (28). Briefly, following the adhesion and washing procedures as described above, the substrate solution (0.1 mM sodium acetate, pH 5.0, 20 mM p-nitrophenyl phosphate, and 0.1% Triton X-100; 150 µl/well) was added and incubated for 2 h at 37 °C. The reaction was stopped by the addition of 20 µl 2 N NaOH, and absorbance at 405 nm was measured.

Solid-phase Binding Assay of Integrin alpha IIbbeta 3 to Cyr61 and Fisp12/mCTGF-- Microtiter wells were coated with purified proteins as described above. Integrin alpha IIbbeta 3 was added to the wells in the presence and absence of inhibitors, and binding proceeded at 37 °C for 3 h. Unbound receptor was removed and the wells were washed twice with HEPES-Tyrode's buffer. Bound receptor was detected with 125I-mAb15 (25) as described above.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Activation-dependent Adhesion of Human Platelets to Cyr61 and Fisp12/mCTGF-- Recently, we reported that vascular endothelial cells adhere to Fisp12/mCTGF and Cyr61 through interaction with integrin alpha vbeta 3 (17, 19). In this study, we investigated whether these proteins could also support the adhesion of blood platelets. Microtiter wells were coated with purified recombinant Fisp12/mCTGF or Cyr61, and the adhesion of isolated platelets to these proteins was detected with 125I-mAb15, an anti-beta 3 monoclonal antibody. It is noteworthy that this antibody binds to both activated and unactivated alpha IIbbeta 3 indifferently. As controls, fibrinogen- and BSA-coated wells were also used. Initially, we compared the adhesion of unactivated versus activated platelets to immobilized Fisp12/mCTGF and Cyr61. To ensure that the platelets were not activated during the washing procedures, PGI2 (100 nM), which inhibits activation by raising platelet cAMP levels, was added to the platelet suspensions. Fig. 1 shows that unactivated platelets failed to adhere to either protein. However, activation of platelets with 0.1 unit/ml thrombin (panel A), 500 nM U46619 (panel B), or 10 µM ADP (panel C) caused a dramatic increase in platelet adhesion to both Fisp12/mCTGF- and Cyr61-coated wells. To confirm that the adhesion process is activation-dependent, PGI2 (100 nM) was added with the agonists to prevent platelet activation. Under these conditions, platelet adhesion to both Fisp12/mCTGF and Cyr61 was significantly inhibited.


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  		Fig. 1.   Adhesion of activated platelets to Fisp12/mCTGF and Cyr61. Washed platelets were stimulated with 0.1 unit/ml thrombin (panel A), 500 nM U46619 (panel B), or 10 µM ADP (panels C and D) and added to microtiter wells coated with 25 µg/ml fibrinogen, Fisp12/mCTGF, Cyr61, or BSA. To prevent platelet activation, 100 nM PGI2 was added to the platelet suspensions in the presence and absence of agonists as indicated. After incubation at 37 °C for 30 min, non-adherent platelets were removed by washing. Adherent platelets were detected by the binding of 125I-mAb15 (panels A-C) or by the acid phosphatase assay (panel D). Data shown are means of triplicate determinations, and error bars represent standard deviations. Representative of five experiments. Open columns, PGI2; solid columns, agonist; striped columns, agonist + PGI2.

For comparison, platelet adhesion to fibrinogen-coated wells was assessed. While unactivated platelets were capable of adhering to immobilized fibrinogen at a low level as previously reported (29-31), platelet adhesion to Cyr61 and Fisp12/mCTGF appeared to be absolutely dependent on cellular activation (Fig. 1). Following platelet activation with strong agonists such as thrombin and U46619, platelet adhesion to Cyr61 and Fisp12/mCTGF was comparable to fibrinogen. However, the weaker agonist ADP caused a lesser response. Since ADP does not induce secretion of alpha -granule proteins from washed human platelets and does not induce platelet aggregation in the absence of exogenous fibrinogen (32), we therefore used ADP to induce platelet adhesion in later experiments.

To further substantiate the activation-dependent adhesion of platelets to these proteins, we performed an independent assay to quantitate the relative numbers of adherent platelets. This assay measured the acid phosphatase activity of adherent platelets. In Fig. 1 (C and D), both 125I-mAb15 binding and acid phosphatase assays were used to assess the adhesion of ADP-stimulated platelets to fibrinogen, Fisp12/mCTGF, and Cyr61, and similar results were obtained. Since the amounts of bound 125I-mAb15 were directly proportional to the numbers of integrin alpha IIbbeta 3 on adherent platelets, we used this method for quantitative studies hereafter.

Fig. 2 (A and B) shows that the adhesion of ADP-activated platelets to Fisp12/mCTGF and Cyr61 was dose-dependent and saturable. Again, in the presence of PGI2, unactivated platelets adhered poorly to both proteins even at high coating concentrations. The specificity of the adhesion process was characterized in inhibition studies using anti-peptide polyclonal antibodies raised against the central variable regions of Fisp12/mCTGF and Cyr61. On immunoblots, anti-Fisp12/mCTGF and anti-Cyr61 reacted specifically with Fisp12/mCTGF and Cyr61, respectively, and no cross-reactivity was observed (15). As shown in Fig. 3, anti-Fisp12/mCTGF inhibited platelet adhesion to Fisp12/mCTGF but not to Cyr61, and likewise, anti-Cyr61 inhibited Cyr61-mediated platelet adhesion but not that mediated by Fisp12/mCTGF. In specificity controls, no inhibition was observed with normal rabbit IgG. Additionally, neither anti-Fisp12/mCTGF nor anti-Cyr61 inhibited platelet adhesion to fibrinogen-coated wells. Thus, these findings indicated that the abilities of Fisp12/mCTGF and Cyr61 to mediate platelet adhesion are intrinsic properties of these proteins.


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  		Fig. 2.   Dose-dependent adhesion of ADP-activated platelets to Fisp12/mCTGF and Cyr61. Washed platelets, incubated with 10 µM ADP or 100 nM PGI2, were added to wells coated with the indicated concentrations of Fisp12/mCTGF (panel A) or Cyr61 (panel B). After incubation at 37 °C for 30 min, adherent platelets were detected with 125I-mAb15. Data shown are means of triplicate determinations, and error bars represent standard deviations


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  		Fig. 3.   Inhibition of platelet adhesion to Fisp12/mCTGF and Cyr61 by anti-Fisp12/mCTGF or anti-Cyr61 antibodies. Microtiter wells coated with 25 µg/ml fibrinogen, Fisp12/mCTGF, or Cyr61 were preincubated with 1 mg/ml rabbit IgG, anti-Fisp12/mCTGF, or anti-Cyr61 at room temperature for 1 h. After three washes with Tyrode's buffer, ADP-activated platelets were added. Platelet adhesion proceeded for 30 min at 37 °C, and adherent cells were detected with 125I-mAb15. Percentage of inhibition was calculated relative to the adhesion of platelets to control wells incubated without antibodies. Data shown are means of triplicate determinations, and error bars represent standard deviations.

Identification of alpha IIbbeta 3 as the Receptor Mediating Platelet Adhesion to Fisp12/mCTGF and Cyr61-- Upon platelet activation, the ligand binding affinities of integrin alpha IIbbeta 3 and alpha vbeta 3 are up-regulated (1, 6). To determine whether these integrin receptors mediate platelet adhesion to Fisp12/mCTGF and Cyr61, we tested the inhibitory effect of peptide antagonists and the divalent cation chelator EDTA. Fig. 4A shows that preincubation of platelets with EDTA at 37 °C completely abolished platelet adhesion to both proteins indicating that the adhesion process is divalent cation-dependent, consistent with the involvement of an integrin receptor. The major platelet integrin, alpha IIbbeta 3, is sensitive to inhibition by RGD-containing peptides and a dodecapeptide (H12) derived from the fibrinogen gamma  chain (33-35). As shown in Fig. 4A, the adhesion of ADP-activated platelets to Cyr61 and Fisp12/mCTGF was specifically inhibited by RGDS but not by RGES. Likewise, the RGD-containing snake venom peptide echistatin (36) also completely blocked platelet adhesion to both proteins. It has been shown that the dodecapeptide H12 preferentially interacts with integrin alpha IIbbeta 3 as compared with integrin alpha vbeta 3 (37, 38). Thus, the observation that H12 inhibited platelet adhesion to Cyr61 and Fisp12/mCTGF (Fig. 4A) suggest that this process is mediated by alpha IIbbeta 3 rather than alpha vbeta 3. Indeed, while the complex-specific monoclonal antibody AP-2 (anti-alpha IIbbeta 3) completely blocked the adhesion of ADP-activated platelets to Fisp12/mCTGF and Cyr61, no inhibition was observed with LM609 (anti-alpha vbeta 3) or with normal mouse IgG (Fig. 4B). In control samples, the adhesion of ADP-activated platelets to fibrinogen was also completely inhibited by EDTA, RGDS, echistatin, H12, or AP-2, but not by RGES or LM609 (data not shown). Taken together, these results indicate that platelet adhesion to these proteins is mediated through interaction with activated integrin alpha IIbbeta 3.


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  		Fig. 4.   Inhibition of platelet adhesion to Fisp12/mCTGF and Cyr61. Washed platelets were preincubated with the indicated inhibitors at 37 °C for 15 min and activated with 10 µM ADP. The platelet suspensions were added to Fisp12/mCTGF- or Cyr61-coated wells and incubated at 37 °C for 30 min. Adherent platelets were detected with 125I-mAb15. A, platelets were preincubated with vehicle buffer (no add), 5 mM EDTA, 1 mM RGDS, 1 mM RGES, 1 µM echistatin, or 1 mM H12. B, platelets were preincubated with vehicle buffer (no add) or with 50 nM mouse IgG, LM609, or AP-2. Data shown are means of triplicate determinations, and error bars represent standard deviations. Figure is representative of two experiments.

Direct Binding of Activated Integrin alpha IIbbeta 3 to Fisp12/mCTGF and Cyr61-- To address whether integrin alpha IIbbeta 3 binds directly to Fisp12/mCTGF and Cyr61, we performed a solid-phase binding assay to detect the receptor-ligand interaction. In these experiments, activated and unactivated alpha IIbbeta 3 were purified from platelet lysates as described under "Materials and Methods," and the binding of purified alpha IIbbeta 3 to Cyr61 or Fisp12/mCTGF immobilized onto microtiter wells was detected with 125I-mAb15.

Both activated and unactivated alpha IIbbeta 3 were indistinguishable on SDS-PAGE analysis as detected by silver staining (Fig. 5A). However, as reported previously (39), activated alpha IIbbeta 3, but not the unactivated receptor, was capable of binding to immobilized fibrinogen. Likewise, we observed higher binding of activated versus unactivated alpha IIbbeta 3 to Fisp12/mCTGF and Cyr61 (Fig. 5B). In contrast, similar background bindings of activated and unactivated alpha IIbbeta 3 to control wells coated with BSA were observed. Thus, these data are consistent with the observation that activated but not unactivated platelets adhered to Cyr61 and Fisp12/mCTGF.


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  		Fig. 5.   The binding of activated and unactivated alpha IIbbeta 3 to immobilized Fisp12/mCTGF and Cyr61. A, activated alpha IIbbeta 3 (lane 1) and unactivated alpha IIbbeta 3 (lane 2) were purified from octyl glucoside extracts of outdated human platelets as described under "Materials and Methods." Proteins were separated by SDS-PAGE on 7.5% polyacrylamide gels under non-reducing conditions, and detected by silver staining. B, 20 nM activated or unactivated alpha IIbbeta 3 was added to microtiter wells coated with fibrinogen, Fisp12/mCTGF, Cyr61, or BSA. Binding proceeded for 3 h at 37 °C. After washing, bound alpha IIbbeta 3 was detected with 125I-mAb15 (50 nM). Data shown are means of triplicate determinations, and error bars represent standard deviations. Figure is representative of two experiments.

To further characterize the interaction of alpha IIbbeta 3 with Fisp12/mCTGF and Cyr61, we performed binding isotherms with varying concentrations of RGD affinity-purified alpha IIbbeta 3. Fig. 6 shows that the dose-dependent binding of activated alpha IIbbeta 3 to Fisp12/mCTGF and Cyr61 was saturable, with half-saturation occurring at 15 nM and 25 nM alpha IIbbeta 3, respectively. Again, no significant binding of alpha IIbbeta 3 to control BSA-coated wells was observed. To demonstrate the specificity of the interaction, inhibition studies were performed. As expected, the binding of activated alpha IIbbeta 3 to Fisp12/mCTGF and Cyr61 was specifically blocked by RGDS but not by RGES (Fig. 7). Furthermore, echistatin and the H12 peptide also effectively inhibited alpha IIbbeta 3 binding to these proteins. These findings are consistent with results obtained in the platelet adhesion assay. Collectively, these functional and biochemical data demonstrate that activated integrin alpha IIbbeta 3 is the receptor mediating activation-dependent platelet adhesion to Cyr61 and Fisp12/mCTGF.


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  		Fig. 6.   Binding isotherms of activated alpha IIbbeta 3 to Fisp12/mCTGF and Cyr61. Microtiter wells were coated with 10 µg/ml Fisp12/mCTGF (panel A) or Cyr61 (panel B). Varying concentrations of activated alpha IIbbeta 3 were added and incubated for 3 h at 37 °C. Bound receptor was detected with 125I-mAb15. Data shown are means of triplicate determinations, and error bars represent standard deviations.


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  		Fig. 7.   Inhibition of alpha IIbbeta 3 binding to Fisp12/mCTGF and Cyr61. Activated alpha IIbbeta 3 (30 nM), preincubated with vehicle buffer (no add), 0.1 mM RGDS, 0.1 mM RGES, 0.1 mM H12, or 0.5 µM echistatin for 15 min at 37 °C, was added to microtiter wells coated with 10 µg/ml fibrinogen, Fisp12/mCTGF, Cyr61, or BSA. Binding proceeded for 3 h at 37 °C, and bound receptor was detected with 125I-mAb15. Data shown are means of triplicate determinations, and error bars represent standard deviations.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The major findings in this study are: 1) human platelets adhere to two novel angiogenic inducers, Cyr61 and Fisp12/mCTGF, in an activation-dependent manner; and 2) platelet adhesion to Cyr61 and Fisp12/mCTGF is mediated through the integrin alpha IIbbeta 3, which serves as a cell surface receptor for these proteins. These results establish Cyr61 and Fisp12/mCTGF as two additional adhesive ligands for integrin alpha IIbbeta 3 on activated platelets. Since both proteins are matrix-associated molecules synthesized by endothelial cells and smooth muscle cells of vessel walls (7, 20), these findings suggest a physiological role for Cyr61 and Fisp12/mCTGF in hemostasis and thrombosis.

Cyr61 and Fisp12/mCTGF are members of a family of multifunctional extracellular signaling molecules (8, 9). Originally identified as products of growth factor-inducible immediate-early genes, these proteins were thought to mediate the biological responses of growth factors. Recent studies have demonstrated the roles of these proteins in cell adhesion, migration, proliferation, survival, and differentiation (16-18, 40). Furthermore, both proteins have been implicated in complex biological processes such as angiogenesis, wound healing, embryogenesis, and tumor growth (17, 18, 40-42). Members of this protein family share four conserved structural domains, which include: 1) an insulin-like growth factor-binding protein homology domain, 2) a von Willebrand factor type C domain, 3) a thrombospondin type 1 repeat homology domain, and 4) a carboxyl-terminal domain with homology to some types of collagens and mucins. Heparin-binding sequence motifs can be found in domains 3 and 4, consistent with the observation that both Cyr61 and Fisp12/mCTGF bind heparin (14, 15). The human ortholog of Fisp12, CTGF, was first identified as a mitogenic factor in the conditioned medium of human umbilical vein endothelial cells (13). However, the mechanism and the receptor mediating its mitogenic activity have not yet been elucidated.

We have previously shown that, on vascular endothelial cells, both Cyr61 and Fisp12/mCTGF interact with integrin alpha vbeta 3, which mediates cell adhesion and migration (17, 19). While integrin alpha vbeta 3 is found in a number of cell types, it is present at a very low copy number on platelets (38, 43). In contrast, a closely related integrin, alpha IIbbeta 3, is the predominant adhesion receptor on blood platelets mediating platelet adhesion and aggregation. On resting platelets, integrin alpha IIbbeta 3 is present in a low affinity state incapable of binding soluble adhesive ligands. Activation of platelets by physiological agonists such as thrombin, ADP, and thromboxane A2 up-regulates the ligand binding affinity of alpha IIbbeta 3 through inside-out signaling processes. In the present study, we found that unactivated platelets failed to adhere to Cyr61 and Fisp12/mCTGF, whereas platelets activated by a variety of agonists adhere strongly to both proteins. Furthermore, we conclude that Cyr61 and Fisp12/mCTGF interact with the platelet integrin alpha IIbbeta 3 based on the observations that RGD-containing peptides and the dodecapeptide H12, as well as the anti-alpha IIbbeta 3 monoclonal antibody AP-2, blocked platelet adhesion to these proteins. Although integrin alpha vbeta 3 on endothelial cells serves as the receptor for Cyr61 and Fisp12/mCTGF, the inability of the anti-alpha vbeta 3 monoclonal antibody LM609 to inhibit platelet adhesion to these proteins may reflect the relatively low abundance of this receptor as compared with integrin alpha IIbbeta 3 on the platelet surface.

Solid-phase binding assays with purified integrin alpha IIbbeta 3 to immobilized Cyr61 and Fisp12/mCTGF confirmed that these proteins are direct ligands of this integrin. Furthermore, consistent with the platelet adhesion data, we observed that activated purified alpha IIbbeta 3, but not the unactivated receptor, binds directly to these proteins. Thus, both Cyr61 and Fisp12/mCTGF are ligands specific for the activated conformer of integrin alpha IIbbeta 3. Interestingly, neither proteins contain the RGD motif or the fibrinogen gamma  chain dodecapeptide sequence recognized by integrin alpha IIbbeta 3. Nevertheless, both peptides were able to inhibit the interaction of alpha IIbbeta 3 with Cyr61 and Fisp12/mCTGF, possibly due to conformation changes induced by peptide binding to alpha IIbbeta 3 (21, 44). These proteins, therefore, represent the first examples of activation-dependent ligands for integrin alpha IIbbeta 3 that do not contain either the RGD or the fibrinogen gamma  chain dodecapeptide sequence motifs.

The expression of both cyr61 and fisp12/mCTGF is developmentally regulated in a tissue-specific and temporally restricted manner during embryogenesis (15, 20, 42). In particular, cyr61 is expressed in developing blood vessel walls. Both Cyr61 as well as human and mouse CTGF proteins can be colocalized with smooth muscle cells of arterial walls (7, 20). In the adult, expression of both cyr61 and fisp12/mCTGF is induced in the granulation tissue of healing cutaneous wounds, consistent with the abilities of these proteins to promote chemotaxis and proliferation of fibroblasts and to induce angiogenesis at the site of wound repair (8, 20, 41). These expression patterns and activities implicate a role for Cyr61 and Fisp12/mCTGF in the development and maintenance of blood vessels.

Platelets play an essential role in hemostasis, the arrest of blood flow from injured vessels. The presence of Cyr61 and CTGF in arterial vessel walls suggests that platelet adhesion to these proteins may contribute to the stability of the hemostatic plug. The initial adhesion of unactivated platelets to injured blood vessels is thought to be due to the interaction of the GPIb-IX-V complex with vWf on the exposed subendothelium (4, 5). Such interaction has been shown to activate the platelet integrin alpha IIbbeta 3, thus allowing alpha IIbbeta 3 to bind to other adhesive ligands including soluble fibrinogen (31, 45). Our present finding that activated alpha IIbbeta 3 also binds immobilized Cyr61 and Fisp12/mCTGF suggest that these proteins may contribute to the tight adhesion of platelets to the subendothelial matrix following the initial GPIb-IX-V interaction with vWf. Furthermore, the generation of thrombin and other platelet agonists would activate circulating platelets, thus allowing their interaction with Cyr61 and CTGF in the injured vessel walls.

CTGF has been shown to be overexpressed in advanced atherosclerotic lesions (7). Specifically, Northern blot analysis shows that the level of CTGF mRNA was expressed 50-100-fold higher in atherosclerotic blood vessels as compared with normal arteries. In advanced atherosclerotic lesions, CTGF protein was highly expressed in vascular smooth muscle cells as well as in endothelial cells at the luminal sites of the vessels and in the vasa vasorum of the plaque lesions. CTGF may have multiple roles in the pathogenesis of atherosclerosis. First, it may act in concert with other growth factors and cytokines to promote cell migration and proliferation. Second, since it is an angiogenic factor, it would likely induce neovascularization of the fibrous plaques. Third, it may also be involved in the formation of occlusive thrombi since retraction or removal of endothelial cells of atherosclerotic plaques would expose CTGF in the underlying subendothelial matrix to which activated platelets could adhere. In this regard, our findings that activated alpha IIbbeta 3 mediates platelet adhesion to CTGF may have important implications in the pathogenesis of acute arterial occlusion resulting from ruptures or fissures of atherosclerotic plaques. Thus, the potential role of CTGF and Cyr61, and perhaps other members of this protein family, in hemostasis and thrombosis as manifested by platelet adhesive functions merits further investigation.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants HL41793 (to S. C.-T. L.) and CA46565 and CA80080 (to L. F. L.).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.

Present address: Laboratory of Molecular Mechanisms of Transcription, NCI, National Institutes of Health, Frederick, MD 21702.

parallel To whom correspondence should be addressed: Dept. of Pharmacology (M/C 868), University of Illinois, 835 S. Wolcott Ave., Chicago, IL 60612. Tel.: 312-413-5928; Fax: 312-996-1225; E-mail: sclam@uic.edu.

    ABBREVIATIONS

The abbreviations used are: vWf, von Willebrand factor; CTGF, connective tissue growth factor; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis; mAb, monoclonal antibody; PGI2, prostaglandin I2.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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