The Tetrapeptide Analogue of the Cell Attachment Site of Fibronectin Inhibits Platelet Aggregation and Fibrinogen Binding to Activated Platelets*

binding. These data suggest that a region near the carboxyl-terminus of the a-chain of fibrinogen interacts with the fibrinogen receptor on activated platelets. The data also support the concept that the sequence Arg-Gly- Asp-Ser on the aggregation of platelets in plasma stimulated by 2 p~ ADP. In the middle tracing, exogenous fibrinogen was added raising the final fibrinogen concentration to 17.6 p ~ . At this fibrinogen concentration and in the absence of Arg-Gly-Asp-Ser, platelet aggregation proceeded normally. B, inhibitory effect of Arg-Gly-Asp-Ser on the aggregation of ADP-stimulated gel-filtered platelets. Platelets were gel-filtered as described under “Experimental Procedures” and 0.5 mM CaCh and purified human fibrinogen (final concentration, 200 pg/ml or 0.6 pM) were added to the suspensions. Platelet aggregation was stimulated by 10 p~ ADP in the presence or absence of various concentrations of Arg-Gly-Asp-Ser. Platelet aggregation in the absence of the tetra- peptide was designated 100% aggregation.

Plasma and cell surface fibronectins are glycoproteins that mediate the attachment of cells to extracellular matrices (1). Recently Pierschbacher and Ruoslahti (2) demonstrated that a peptide with the sequence Arg-Gly-Asp-Ser has the cell attachment function of plasma fibronectin. This finding has been supported by observations of Yamada and Kennedy (3).
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.  The sequence Arg-Gly-Asp-Ser is also found in at least six other proteins including the a-chain of fibrinogen (4). Fibrinogen is required for platelet aggregation and binds to receptors on the platelet surface exposed by platelet stimulation (5-7). There is evidence that the carboxyl-terminus of the fibrinogen y-chain interacts with the platelet fibrinogen receptor (8)(9)(10). However, other data suggest that portions of the fibrinogen a-chain are also involved in this interaction (11,12). This evidence led Pierschbacher and Ruoslahti to query whether Arg-Gly-Asp-Ser might also be recognized by the platelet fibrinogen receptor. We examined this question by measuring the effect of the tetrapeptide on platelet aggregation stimulated by a variety of agonists and on the binding of fibrinogen to ADP-stimulated platelets.

EXPERIMENTAL PROCEDURES
Peptide Preparation-The peptides Arg-Gly-Asp-Ser and Arg-Gly-Tyr-Ser-Leu-Gly were prepared by Peninsula Laboratories, Belmont, CA. The preparations were homogeneous when analyzed by high performance liquid and thin layer chromatography. Amino acid analysis of an acid hydrolysate of the peptides was also consistent with their predicted composition. Prior to use, the peptides were dissolved at a concentration of 5 mg/ml in a buffer consisting of 150 mM NaC1, 50 mM sodium phosphate, pH 7.4.
Platelet Preparation-Platelet-rich plasma was obtained by differential centrifugation at 25 "C of fresh, whole human blood anticoagulated with 0.1 volume of 0.13 M sodium citrate. For studies using gel-filtered platelets, 4-ml aliquots of the platelet-rich plasma were applied to 40-ml columns of Sepharose 2B (Pharmacia) equilibrated with an elution buffer containing 137 mM NaC1, 2.7 mM KC1, 1 mM MgC12, 5.6 mM glucose, 0.35 mg/ml bovine serum albumin (Fraction V, Sigma), 3.3 mM NaH2P04, and 4 mM Hepes,' pH 7.4. Fractions containing the highest platelet concentrations were pooled and the platelet count was determined with a Coulter Model ZB particle counter.
Studies of Platelet Function-Platelet aggregation studies were performed in a Chrono-log Aggregometer at 37 "C (13). For aggregation studies using platelet-rich plasma, aliquots of the peptide solutions were added directly to 0.5 ml of platelet-rich plasma 3 min prior to platelet stimulation. For aggregation studies using gel-filtered platelets, 200 ,ug/ml fibrinogen (Kabi) and 0.5 mM CaClz were added to the platelet suspensions. Both fibrinogen and CaC1, were omitted from the reaction mixtures when thrombin was the platelet agonist. Thrombin-stimulated platelet secretion was studied using platelets preloaded with ['*C]serotonin. For these studies, platelet-rich plasma was incubated with 0.2 &i/ml ['4C]serotonin (New England Nuclear) for 30 min at 25 "C and then gel-filtered. Aliquots (300 pl) of the labeled platelet suspensions were incubated for 3 min at 37 "C in the presence or absence of the peptides before a-thrombin (Parke-Davis) was added. After an additional 3 min, the incubations were stopped by sedimenting the platelets through a mixture of silicone oils (Hi-Phenyl silicone/Methyl silicone, 4:1, William F. Nye, Inc., Fairhaven, MA). The quantity of [14C]serotonin secreted by the stimulated platelets was calculated after counting a portion of the supernatant buffer for 14C. Platelet shape change was evaluated by examining the platelet aggregation tracings and by observing the platelets with a phase contrast microscope. Measurement of Fibrinogen Binding to ADP-stimulated Platelets-Fibrinogen binding to ADP-stimulated platelets was measured as described previously (6). Briefly, purified human fibrinogen was radiolabeled with lZ5I by the iodine monochloride technique (14). Suspensions of gel-filtered platelets (108/ml) were mixed with various concentrations of the 1251-fibrinogen and 0.5 mM CaCl,. To initiate fibrinogen binding, the platelets were stimulated with 10 p~ ADP and were incubated at 37 "C for 3 min without stirring. To terminate The abbreviation used is: Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid. the binding reaction, the platelets were sedimented through silicone oil in an Eppendorf centrifuge (Brinkman Instruments). The tips of the centrifuge tubes containing the pelleted platelets were cut off and counted for lZ5I. Fibrinogen nonspecifically bound to the pelleted platelets was measured by performing the fibrinogen binding assays in the presence of a 15-fold excess of unlabeled fibrinogen. Nonspecific binding represented 10% or less of the total '%I-fibrinogen associated with the stimulated platelets. The effect of the peptides on fibrinogen binding was measured by adding the dissolved peptides to the platelet suspensions 3 min prior to the addition of ADP. Analysis of the binding data was aided by the use of a Texas Instruments TI programmable 58 calculator.

RESULTS
The effect of the tetrapeptide Arg-Gly-Asp-Ser on platelet aggregation was examined in both platelet-rich plasma and suspensions of gel-filtered platelets. In platelet-rich plasma, 100-300 p~ Arg-Gly-Asp-Ser inhibited platelet aggregation stimulated by ADP, collagen, and y-thrombin. As seen in A , inhibitory effect of 120 p~ Arg-Gly-Asp-Ser on the aggregation of platelets in plasma stimulated by 2 p~ ADP. In the middle tracing, exogenous fibrinogen was added raising the final fibrinogen concentration to 17.6 p~. At this fibrinogen concentration and in the absence of Arg-Gly-Asp-Ser, platelet aggregation proceeded normally. B, inhibitory effect of Arg-Gly-Asp-Ser on the aggregation of ADP-stimulated gel-filtered platelets. Platelets were gel-filtered as described under "Experimental Procedures" and 0.5 mM CaCh and purified human fibrinogen (final concentration, 200 pg/ml or 0.6 p M ) were added to the suspensions. Platelet aggregation was stimulated by 10 p~ ADP in the presence or absence of various concentrations of Arg-Gly-Asp-Ser. Platelet aggregation in the absence of the tetrapeptide was designated 100% aggregation. tion and Fibrinogen Binding did not demonstrate this effect. Because the addition of approximately a 3-fold excess of exogenous human fibrinogen to the platelet-rich plasma, but not the addition of an equivalent concentration of the protein casein, nearly reversed the inhibitory effect of Arg-Gly-Asp-Ser, it seemed likely that the tetrapeptide was interfering with the ability of fibrinogen to bind to the platelet fibrinogen receptor. However, the plasma concentration of fibrinogen (~9 p M ) is nearly 90-fold greater than the dissociation constant of the fibrinogen receptor (0.1 p M ) (6). TO study the effect of Arg-Gly-Asp-Ser at fibrinogen concentrations nearer the K d of the fibrinogen receptor, the aggregation studies were repeated using gel-filtered platelets suspended in a buffer containing 0.6 p~ fibrinogen. Under these conditions, Arg-Gly-Asp-Ser was a more potent inhibitor of aggregation (Fig. 1B). Platelet aggregation was progressively inhibited as the concentration of the tetrapeptide was increased. Fifty per cent inhibition occurred at Arg-Gly-Asp-Ser concentrations of 60-80 p~ and aggregation was largely prevented a t 100-200 p~.
Because the effect of Arg-Gly-Asp-Ser on platelet aggregation might have been due to interference with platelet activation, we examined the effect of the tetrapeptide on three other manifestations of platelet activation: shape change, secretion, and prostaglandin synthesis. The inhibition of aggregation was not associated with an inhibition of platelet shape change (Fig. IB), a finding confirmed by observing the activated platelets with a microscope. Platelet secretion was measured as thrombin-stimulated [14C]serotonin secretion because it does not depend on prior platelet aggregation (15). As seen in Table I, 230 p~ Arg-Gly-Asp-Ser had no effect on thrombin-stimulated platelet [14]serotinin secretion. Finally, the inhibitory effect of Arg-Gly-Asp-Ser did not depend on platelet prostaglandin synthesis because the tetrapeptide still inhibited primary aggregation in the presence of cyclooxygenase inhibitor indomethacin (data not shown). Thus, Arg-Gly-Asp-Ser appears to be a specific inhibitor of the aggregation phase of platelet function.
Fibrinogen binding to receptors on the platelet surface is a prerequisite for platelet aggregation (16). Therefore, we examined the ability of Arg-Gly-Asp-Ser to inhibit fibrinogen binding to activated platelets. The binding of lZ5I-labeled human fibrinogen to ADP-stimulated, gel-filtered human platelets was measured as a function of fibrinogen concentration both in the absence and in the presence of the tetrapeptide. As seen by the binding isotherms in Fig. 2 A , Arg-Gly-Asp-Ser inhibited the specific binding of 1251-fibrinogen to ADP-stimulated platelets. When these data were analyzed as

FIG. 2. Inhibition of lZ6I-fibrinogen binding to ADP-stimulated platelets by Arg-Gly-Asp-Ser (RGDS).
Various concentrations of lZ5I-labeled human fibrinogen were incubated with suspensions of gel-filtered human platelets (10s platelets/ml) at 37 "C in the presence or absence of Arg-Gly-Asp-Ser. The platelets were then stimulated with 10 p~ ADP. After 3 min, specific fibrinogen binding was measured as described under "Experimental Procedures." A, binding isotherms: specific lBI-fibrinogen binding measured in the absence of Arg-Gly-Asp-Ser double reciprocal plots (Fig. 2 B ) the plots intersected on the vertical axis, consistent with a competitive type of inhibition. This suggests that the presence of Arg-Gly-Asp-Ser decreased the affinity of the available fibrinogen receptors without changing their number. Fibrinogen binding measurements were also performed in the presence of the peptide Arg-Gly-Tyr-Ser-Leu-Gly. This peptide at concentrations as high as 472 p~ did not inhibit fibrinogen binding to the stimulated platelets (data not shown). T o study the inhibitory effect of Arg-Gly-Asp-Ser on fibrinogen binding in greater detail, the fibrinogen binding measurements were repeated as a function of the tetrapeptide concentration. The specific binding of 1251fibrinogen to ADP-stimulated platelets decreased as the concentration of Arg-Gly-Asp-Ser increased (Fig. 3A). However, the tetrapeptide was unable to completely prevent fibrinogen binding such that a plateau of residual binding was always present even at the highest tetrapeptide concentrations. These data were also analyzed on Dixon plots (17) (Fig. 3 B ) . The Dixon plots intersected above the horizontal axis, behavior consistent with a competitive type of inhibition and indicated a Ki for Arg-Gly-Asp-Ser of approximately 25 p~. However, because high concentrations of Arg-Gly-Asp-Ser were unable to completely abolish fibrinogen binding, the data indicate that the tetrapeptide is a partial competitive inhibitor. This implies that fibrinogen receptors containing Arg-Gly-Asp-Ser can still interact with fibrinogen but that the presence of the tetrapeptide lowers their affinity for fibrinogen (18). This interpretation is consistent with the notion that several regions on the fibrinogen molecule are involved in the binding of fibrinogen to its platelet receptor.

DISCUSSION
The tetrapeptide Arg-Gly-Asp-Ser inhibits cellular adhesion mediated by fibronectin and also appears to be a specific inhibitor of platelet aggregation. Although it is possible that Arg-Gly-Asp-Ser exerts its effect on platelets by preventing platelet activation, this is unlikely because other platelet functions that require agonist stimulation, such as platelet shape change and platelet secretion, proceed normally in the presence of the tetrapeptide. Also, the inhibitory effect of the tetrapeptide is not due to inhibition of platelet prostaglandin synthesis because Arg-Gly-Asp-Ser was still active in the presence of the cyclooxygenase inhibitor indomethacin. However, the possibility remains that the effect of Arg-Gly-Asp-Ser does not depend upon its unique amino acid sequence, but rather upon the relatively high concentrations of tetrapeptide required to demonstrate the effect. Again, this is unlikely because equivalent concentrations of the peptide Arg-Gly-Tyr-Ser-Leu-Gly did not affect platelet function.
Arg-Gly-Asp-Ser appears to exert its effect on platelet aggregation by inhibiting fibrinogen binding to activated platelets. Fibrinogen binds to receptors on the platelet surface that are exposed by platelet activation. This receptor-bound fibrinogen promotes platelet aggregation, perhaps by crosslinking adjacent platelets. The inhibitory effect of Arg-Gly-Asp-Ser on fibrinogen binding is complex. Analysis of our fibrinogen binding data on double-reciprocal and Dixon plots indicated that Arg-Gly-Asp-Ser is a competitive inhibitor. Thus, Arg-Gly-Asp-Ser decreases the affinity of the available fibrinogen receptors without altering their number. However, higher Arg-Gly-Asp-Ser concentrations did not completely inhibit fibrinogen binding, indicating that the tetrapeptide is a partial inhibitor (18) and that fibrinogen receptors containing the tetrapeptide can still interact with fibrinogen to some extent but with a decreased affinity. Other data demonstrating that portions of the fibrinogen y-chain also interact with the fibrinogen receptor are consistent with this interpretation. Kloczewiak and co-workers (9,lO) have shown that synthetic pentadecapeptide and dodecapeptide analogues of the carboxyl-terminus of the fibrinogen y-chain inhibit platelet aggregation and fibrinogen binding with an IC5,, of 28 p~, a value similar to the Ki of Arg-Gly-Asp-Ser. The fact that high concentrations of either the a or the y peptide analogues are required to inhibit fibrinogen binding and platelet aggregation also suggests that other factors, such as the three-dimensional structure of the intact fibrinogen molecule, are important in fibrinogen binding to its platelet receptor. Our conclusion that a segment near the carboxyl-terminus of the fibrinogen a-chain is involved in fibrinogen binding to the fibrinogen receptor is supported by other evidence in the literature. For example, Kloczewiak and co-workers demonstrated that polymerized fibrinogen a-chains support platelet aggregation, although not as well as polymerized y-chains (11). Earlier studies by Niewiarowski et at. (12) had also shown that plasmin-generated fragments of fibrinogen lacking intact carboxyl-termini of the a-chains were up to 8-10 times less potent in supporting platelet aggregation than intact fibrinogen.
The peptide sequence we have studied is present as residues 572 through 575 near the carboxyl-terminus of the 610 residue fibrinogen a-chain (19). This portion of the a-chain extends as a highly polar appendage from each end of the fibrinogen molecule (19) and, therefore, is available to bind to exposed fibrinogen receptors on adjacent platelets. The tetrapeptide sequence is also present in fibronectin, most likely in a hydrophilic loop on the surface of the moIecule (2). Of considerable interest, Ginsberg and his colleagues (20-22) have reported that the tetrapeptide Arg-Gly-Asp-Ser inhibits fibronectin binding to activated platelets. Thus, it appears that the tetrapeptide sequence we have studied is involved in the interaction of both fibrinogen and fibronection with the plate-let surface. Moreover, because the amino acid sequence Arg-Gly-Asp-Ser is involved in adhesive reactions mediated by two different macromolecules and can be found on at least five other proteins such as the X phage receptor of Escherichia coli and the Sindbis virus coat protein, our data lend support to the concept that this amino acid sequence has been conserved to support cellular adhesion in a variety of biological processes (2, 23).