A Novel Peptide Motif for Platelet Fibrinogen Receptor Recognition*

(Received for publication, September 13, 1996, and in revised form, December 27, 1996)

Jun Katada , Yoshio Hayashi , Yoshimi Sato , Michiko Muramatsu , Yoshimi Takiguchi , Takeo Harada , Toshio Fujiyoshi and Isao Uno

From the Life Science Research Center, Advanced Technology Research Laboratories, Nippon Steel Corporation, 1618 Ida Nakahara-ku, Kawasaki 211, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

To develop a specific antagonist of platelet alpha IIbbeta 3 using small linear peptides, we synthesized a series of hexapeptides that did not have an Arg-Gly-Asp (RGD) sequence and examined their anti-platelet activity and their specificity for alpha IIbbeta 3. We found a novel motif sequence, Pro-X1-X2-X3-Asp-X4, where X1 to X4 were all L-form alpha -amino acids, which specifically inhibited aggregation of human platelets at submicromolar concentrations. The Pro residue at the N terminus was essential to the anti-platelet activity, and the acetylation of the imino group of this residue also resulted in the complete loss of the activity. The results of the binding assay using purified human platelet alpha IIbbeta 3 and placental alpha vbeta 3 and those of the cell adhesion assay suggest that this motif peptide is highly specific for platelet alpha IIbbeta 3 among other integrins. Flow cytometric studies using an fluorescein isothiocyanate-labeled RGD peptide showed that this motif peptide inhibited the binding of an RGD peptide to activated platelets, suggesting that it has the same inhibitory mode as RGD peptides. Conformational analysis of this motif peptide and an RGD-containing peptide suggests that the imino group of the Pro residue may substitute for the role of the guanidino group of the Arg residue of the RGD sequence.


INTRODUCTION

Integrins are heterodimeric cell surface receptor molecules and are thought to be particularly important mediators of cell adhesion, cell migration, and adhesion-dependent intracellular signaling. Until now, more than 20 integrin receptors have been identified, and many of them recognize the Arg-Gly-Asp (RGD)1 sequence as a common recognition motif within their putative ligands upon ligand-receptor interaction (1-4). The ligand binding specificity has been studied by many investigators, and it has been revealed that some integrins show apparent redundancy in integrin-ligand interactions. For example, alpha vbeta 3, a so-called vitronectin receptor, can bind several apparently dissimilar ligands such as vitronectin, fibrinogen, fibronectin, von Willebrand factor, osteopontin, and bone sialoprotein, all of which have the RGD sequence (reviewed in Refs. 5 and 6). In addition, the same RGD site within a ligand can be recognized by several different integrins. These apparent redundancies in integrin-ligand interactions are very interesting aspects of integrin receptors and may be beneficial to the cell adhesion of many types of cells to extracellular matrix proteins.

We are interested in developing small peptides that are specific for each integrin as a tool for investigating ligand-integrin interactions and as an agent for therapeutic uses. Recently, many small peptides containing the RGD sequence have been synthesized, and their specificity toward integrins has been examined (7, 8). Most of the linear RGD peptides have shown very low specificity among many types of integrin receptors including platelet alpha IIbbeta 3 (fibrinogen receptor), alpha vbeta 3 (vitronectin receptor), and alpha 5beta 1 (fibronectin receptor). In the case of alpha IIbbeta 3 and alpha vbeta 3, both integrins have the same beta  subunit (beta 3), although they have distinct alpha  subunits. It has been reported that the primary amino sequences of alpha IIb and alpha v are only about 36% homologous (9). By cross-linking studies, it has been shown that RGD peptides were bound and cross-linked with the beta 3 subunit (10, 11). Moreover, single point mutation at several positions within the beta 3 subunit abrogated the binding of RGD peptides to both receptors (12-15). These data indicate that the interaction between the RGD sequence and the common beta 3 subunit is responsible for the ligand recognition by both integrins. However, it has also been reported that some RGD peptides with a cyclic structure show relatively high specificity (16-18). These results suggest that the three-dimensional structure of the RGD site itself and/or its flanking regions are very important for the ligand binding specificity, because cyclic peptides have very rigid structure and their three-dimensional structures are considerably fixed. It is also suggested that the alpha  subunit, the critical difference between both integrins, is associated with such a ligand binding specificity. This idea is supported by the results of experiments in which a decapeptide from the carboxyl terminus of the fibrinogen gamma -chain was relatively specific for platelet alpha IIbbeta 3 and it cross-linked with alpha IIb subunit of this integrin (19, 20). Several peptides without the RGD sequence, which are highly specific for platelet alpha IIbbeta 3, have also been reported. For example, a tick saliva protein, which does not have the RGD sequence, is a potent alpha IIbbeta 3 antagonist and shows high specificity for this integrin (21). Another example is barbourin, which is a member of the disintegrins found in snake venom (22).

The aim of our study is to develop a potent and highly specific alpha IIbbeta 3 antagonist of small linear peptides (less than six residues) consisting of natural amino acids. We found a novel motif sequence of a hexapeptide. One of the motif peptides, Pro-Ser-Hyp-Gly-Asp-Trp (Hyp representing 4-hydroxy-L-proline) (NSL-9511), inhibited platelet aggregation at a submicromolar concentration, although it did not affect adhesion of human endothelial cells to immobilized vitronectin and fibronectin. The results of the solid phase binding assay suggest that this peptide is a specific antagonist of platelet alpha IIbbeta 3. NSL-9511 also exhibited antithrombotic activity in an in vivo thrombosis model.


EXPERIMENTAL PROCEDURES

In Vitro Platelet Aggregation

Platelet aggregation studies were performed in platelet-rich plasma (PRP) obtained from various animal species, including humans. Blood was drawn into plastic syringes containing 1/10 volume of 3.8% trisodium citrate. PRP was prepared by centrifugation of citrated whole blood at 160 × g for 15 min at room temperature. PRP was removed, and the platelet count was determined. Platelet-poor plasma was obtained by centrifugation of the remaining blood at 2000 × g for 15 min. Saline or peptide solution of various concentrations was added to PRP at 37 °C 1 min prior to the initiation of platelet aggregation. Platelet aggregation was initiated with 5 µg/ml collagen, and the aggregation was measured in an aggregometer (NBS Hematracer-601, Nikoh Bioscience Co., Ltd., Tokyo) as an increase in light transmission. Platelet aggregation is presented as the percentage of inhibition of the rate of platelet aggregation compared with control samples, and the IC50 values were calculated from dose-inhibition curves.

Preparation of Integrin Receptors

Vitronectin receptor (alpha vbeta 3) was purified according to the procedures described elsewhere (17, 23) with some modifications. Briefly, human placentas were cut into small pieces and extracted with buffer containing 100 mM octyl glucoside. The extract was centrifugated, and the supernatant was applied to a Sepharose CL4B column and then applied to a GRGDSPK-Sepharose affinity column. The column was washed with five bed volumes of Tris-buffered saline (TBS) containing 3 mM Ca2+ and 50 mM octyl glucoside at 4 °C and then washed with five bed volumes of the same solution at room temperature. Bound vitronectin receptor was eluted with TBS containing 10 mM EDTA and 50 mM octyl glucoside. Finally, Ca2+ was added to the eluted fraction so that the final concentration of the free Ca2+ was 2 mM.

Platelet fibrinogen receptor (alpha IIbbeta 3) was purified from outdated human platelets using a procedure almost identical to that for alpha vbeta 3. Briefly, the platelets were washed three times with TBS containing 1 mM EDTA and 0.2% glucose and then were lysed with TBS containing 100 mM octyl glucoside, 1 mM MnCl2, 1 mM MgCl2, and 0.1 mM phenylmethylsulfonyl fluoride. After the centrifugation at 30,000 × g, the supernatant fraction was applied to a Sepharose 2B column, and the flow-through fraction was then applied to a GRGDSPK-Sepharose affinity column. Bound fibrinogen receptor was eluted with TBS containing 50 mM octyl glucoside and 5 mM GRGDS peptide. The eluted fraction was dialyzed to remove a GRGDS peptide and concentrated on an Amicon YM 30 filter. Purified human alpha IIbbeta 3, purchased from Enzyme Research Laboratories, Inc., was also used.

Solid Phase Binding Assay

The inhibitory effects of the peptides on integrin-ligand interactions were evaluated by using a competitive enzyme-linked immunosorbent assay (ELISA) (17). Briefly, for vitronectin-alpha vbeta 3 ELISA, 96-well microtiter plates were coated overnight at room temperature with 10 µg/ml human vitronectin (Iwaki Glass Co., Ltd., Tokyo), and the plates were washed with TBS containing 0.05% Tween 20. alpha vbeta 3 solution was added to each well, and then the peptide solution of various concentrations was added to each well. The plates were incubated at room temperature for 2 h and washed with TBS containing 0.05% Tween 20. Bound receptor was detected by using anti-human alpha v rabbit polyclonal antibody (Chemicon Int., Inc.) and affinity-purified goat anti-rabbit IgG conjugated to horseradish peroxidase.

Fibrinogen-alpha IIbbeta 3 ELISA was performed using a similar protocol except that microtiter plates were coated with 10 µg/ml human fibrinogen (Sigma) and anti-human alpha II mouse monoclonal antibody was used.

To verify the specificities of ligand-integrin interactions, anti-alpha v monoclonal antibody (23C6; Serotec Ltd., UK) and anti-alpha IIbbeta 3 monoclonal antibody (Becton Dickinson) were used.

Effects on the Binding of Fluorescein Isothiocyanate (FITC)-labeled RGD Peptide to Activated Platelets

The binding of the FITC-labeled RGD peptide to activated platelets was studied by flow cytometry. The activated washed platelets were prepared according to the methods of Frojmovic et al. (24) with some modifications. Human PRP was treated with 0.5 µM prostaglandin E1 (Sigma) at room temperature for 10 min. The pH of the PRP was then adjusted to 6.5 with 1 M citrate solution, and the PRP was centrifugated at 1200 × g for 10 min. The pellet was suspended in Ca2+-free Tyrode's solution containing 0.35% bovine serum albumin and 0.2 units/ml apyrase (grade III; Sigma), and the suspension was centrifugated at 1200 × g for 10 min again. The pellet was resuspended in Tyrode's solution containing 0.35% bovine serum albumin and 0.38% trisodium citrate (termed PRP (1:10)). 100 µM ADP was added to this platelet suspension and incubated at room temperature for 5 min. The platelets were then treated with 0.1 mM FITC-labeled peptide (FITC-WSRGDW) for 25 min at room temperature in the presence or absence of inhibitory peptides at various concentrations. The relative intensity of fluorescence was analyzed in a flow cytometer (Cyto ACE-300, Jasco, Tokyo, Japan).

Cell Adhesion Assay

The adhesion assay was performed in 24-well tissue culture plates. Each well was precoated with 30 µg/ml fibronectin (purified from human plasma, Iwaki Glass Co., Ltd., Tokyo) or 3 µg/ml vitronectin (purified from human plasma, Iwaki Glass Co., Ltd., Tokyo) at 4 °C for 24 h. On the day of the experiment, each well was blocked with 2% bovine serum albumin for 1 h at room temperature.

ECV 304 cells (obtained from Health Science Research Resource Bank, Osaka Japan), a cell line cloned from human umbilical vein endothelial cells (25), were cultured in medium 199 supplemented with 10% fetal calf serum and 50 IU/ml penicillin and 50 µg/ml streptomycin at 37 °C. They were harvested using 0.05% trypsin and 0.5 mM EDTA and washed twice with divalent cation-free phosphate-buffered saline. Finally, they were resuspended in serum-free Eagle's minimum essential medium (5 × 105 cells/ml). The cells were preincubated with appropriate inhibitors for 30 min at 37 °C, and then 200 µl of this cell suspension then was added to each well of the 24-well plates precoated with fibronectin or vitronectin, as mentioned above, and were incubated at 37 °C in a CO2 incubator. After 30 min, each well was washed with serum-free Eagle's minimum essential medium to remove unattached cells, and 100 µl of 65 µg/ml WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt) and 100 µl of 7 µg/ml 1-methoxy-5-methylphenazinium methylsulfate were added to each well. After 1 h, the number of adhered cells was quantified by measuring the absorbance at 450 nm (26). Anti-human alpha vbeta 3 monoclonal antibody, 23C6 (Serotec, Oxford, UK), was used to examine the contribution of alpha vbeta 3 integrin (27). EILDVPST peptide (Funakoshi, Tokyo, Japan), originating from the CS-1 site of fibronectin (28), was used to examine the contribution of the CS-1 site to the cell adhesion to fibronectin.

Guinea Pig Arteriovenous Shunt Model

Male Hartley guinea pigs weighing 230-320 g were anesthetized with pentobarbital (35 mg/kg, intraperitoneal), and an intratracheal cannula was inserted through the incision. To maintain a moderate anesthesia during the experiment, additional pentobarbital administration (intraperitoneal) was done if necessary. The polyethylene tubes filled with saline containing 100 units/ml heparin were inserted into the left jugular vein and right carotid artery, and these two tubes were connected with a silicon tube filled with the same solution. The whole length of this shunt was 6 cm. Each agent was infused via a cannula in a right jugular vein at the rate of 2 ml/h for 70 min. 10 min after the onset of the infusion, the silicon tube was exchanged with a polyethylene tube in which a silk thread was inserted. At every 20 min, the silk thread was exchanged, and the wet weight of the thread was measured. After the termination of the infusion, the thrombus formation was examined for another 60 min. The net thrombus weight was calculated from the thread weights before and after the experiments, and the total thrombus weight during 60 min was the sum of three net thrombus weights during each 20 min.

Peptide Synthesis

All peptides were synthesized by solid phase methods using 9-fluorenylmethoxycarbonyl amino acids on an automatic peptide synthesizer (model 431A, Applied Biosystems, Inc.). The final step of the solid phase synthesis involved the acylation of the N termini with different types of carboxylic acids in the presence of N,N-dimethyl formamide and 1-hydroxybenzotriazole in 1-(3',4'-dichlorophenyl)-2-isopropyl-aminoethanol. A trimethylsilylbromide-thioanisol system was used for the final deprotection. The crude peptides were purified by reverse-phase HPLC using a C18 preparative column. All peptides were characterized by fast atom bombardment mass spectrometry or electrospray ionization mass spectrometry, an amino acid analysis, and an analytical high pressure liquid chromatography. The purity of the peptides was >98%. Labeling of a peptide with FITC was performed using FITC isomer I (Sigma). Free FITC was eliminated by dialysis.

Conformational Analysis

The conformational analysis was conducted on Macro Model software, version 3.5d (29). Fifty thousand conformers were generated by the Monte Carlo method (30) and minimized with a modified Amber* Force field (31) in vacuum to obtain 2200 conformers. The distance between the guanidino group of the Arg residue and the carboxy group of the Asp residue (d1) and the distance between the imino group of the Pro residue and the carboxy group of the Asp residue (d2) were calculated. Conformers with shorter d1 or d2 (<10 Å) were eliminated, based on Kessler's active conformation (32). The electrostatic cut-off distance was set to 5 Å, and the force field was modified in order to not evaluate hydrogen bonds.

Statistics

The statistical significance of the difference between two groups was determined by the Student's t test. The statistical significance of the difference between more than two groups was assessed by a one-way analysis of variance followed by Dunnet's test. Differences were considered to be statistically different when p was <0.05. Dose-inhibition curves were obtained by fitting the data to a logistic curve by the least squares method, and the IC50 values were calculated from the fitted curves.


RESULTS

In Vitro Platelet Aggregation Assay

In the previous study (33), we found that an RGD-containing hexapeptide, PSRGDW, was a very potent inhibitor of human platelet aggregation (IC50 = 0.79 µM). The Pro residue at the N-terminal position of this peptide was essential for its high potency, because the substitution of other amino acids for proline or removal of this Pro residue resulted in a decrease in the anti-platelet activity (e.g. SRGDW was 50 times less potent than PSRGDW). These results suggest that the N-terminal Pro residue is responsible for another interaction between this peptide and the alpha IIbbeta 3 receptor besides the guanidino group and the carboxyl group of the RGD site. However, this peptide was not specific for platelet alpha IIbbeta 3 integrin, although it was very potent. To develop a potent antagonist of platelet alpha IIbbeta 3 with high specificity, we synthesized a series of peptides without the Arg residue. The anti-platelet aggregation activity of these peptides is shown in Table I. A replacement of the Arg residue at the third position with norvaline, which means the removal of the guanidino group of the Arg residue, resulted in a slight increase in the anti-platelet activity, suggesting that the Arg residue of the RGD site is not necessary for the anti-platelet activity of this peptide. L-Proline, L-hydroxyproline, and L-dehydroproline at the third position also reproduced the anti-platelet activity of high potency. We also synthesized N-acetyl PSPGDW, in which the imino group of the N-terminal Pro residue was blocked, to investigate the role of this group and found that the acetylation of the imino group resulted in the complete loss of the activity, suggesting that this free imino group is essential to the anti-platelet activity of this peptide. Among the peptides we synthesized here, PS-Hyp-GDW (Hyp representing 4-hydroxy-L-proline) (NSL-9511) was the most potent inhibitor of the platelet aggregation, and this peptide was used for further experiments.

Table I.

Inhibition of in vitro human platelet aggregation

Collagen (5 µg/ml)-induced platelet aggregation using human platelet-rich plasma is shown.
Peptide Platelet aggregation (IC50)

µM
PSRGDW 0.87
SRGDW 40
PS-Nva-GDWa 0.59
PS-Pro-GDW 0.77
PS-Hyp-GDWb (NSL-9511) 0.39
CH3CO-PSPGDWc >1000
GRGDS 470

a Nva, L-norvaline.
b Hyp, 4-hydroxy-L-proline.
c CH3CO-P, N-acetyl-L-proline.

The anti-platelet activity of NSL-9511 differs apparently among species (Table II). Although NSL-9511 was a potent inhibitor of platelet aggregation in guinea pig PRP, it was much less active in mouse and rat PRP.

Table II.

Inhibition of platelet aggregation by NSL-9511 in PRP from different species

Collagen (5 µg/ml)-induced platelet aggregation using PRP from different species is shown.
Species Platelet aggregation (IC50)

µM
Human 0.39
Guinea pig 0.87
Mouse 40.0
Rat >1000

Inhibition of Fibrinogen Binding to Purified Platelet Fibrinogen Receptor

In our preliminary studies using flow cytometry, we have found that NSL-9511 inhibited the binding of fibrinogen to ADP-activated platelets, suggesting that the anti-platelet activity of NSL-9511 results from the blockage of fibrinogen binding to its receptor on the platelet surface. To clarify the mechanism underlying the anti-platelet activity further, we performed a competitive ELISA in which the binding of purified human platelet fibrinogen receptor, alpha IIbbeta 3, to immobilized human fibrinogen was detected by specific antibody. alpha vbeta 3 ELISA was also performed using alpha vbeta 3 purified from human placenta to examine the binding specificity of NSL-9511. As Fig. 1a shows, 100 µM NSL-9511 almost completely inhibited the binding of alpha IIbbeta 3 to fibrinogen, whereas it did not inhibit the binding of alpha vbeta 3 to vitronectin. An RGD-containing peptide, GRGDS, at the same concentration did inhibit both the binding of fibrinogen to alpha IIbbeta 3 and that of vitronectin to alpha vbeta 3 completely. These results suggest that NSL-9511 is highly specific for platelet alpha IIbbeta 3. Anti-alpha v monoclonal antibody and anti-alpha IIbbeta 3, both of which have been reported as binding-blocking antibody, completely inhibited the binding of vitronectin to alpha vbeta 3 and fibrinogen to alpha IIbbeta 3, respectively. As Fig. 1b shows, NSL-9511 inhibited the binding of fibrinogen to alpha IIbbeta 3 in a dose-dependent manner with an IC50 value of 70 nM. On the other hand, this compound hardly inhibited the binding of vitronectin to alpha vbeta 3 and the percentage of inhibition was only 23% at the highest dose (0.6 mM).


Fig. 1. Effects of NSL-9511 on the ligand binding to integrin receptors. Human fibrinogen or vitronectin was immobilized on microtiter plates, and the binding of solubilized alpha IIbbeta 3 purified from human platelets and alpha vbeta 3 purified from human placenta, in the presence of peptides or monoclonal antibodies, was detected with anti-alpha IIb and anti-alpha v antibodies and a horseradish peroxidase-conjugated anti-IgG antibody. a, effects of monoclonal antibodies and peptides on the binding of alpha vbeta 3 to vitronectin (solid bars) and the binding of alpha IIbbeta 3 to fibrinogen (stippled bars). 50 µg/ml anti-alpha vbeta 3 antibody, 50 µg/ml anti-alpha IIbbeta 3 antibody, 100 µM NSL-9511, and 100 µM GRGDS were used. b, inhibition of the binding of alpha vbeta 3 to vitronectin (open circles) and the binding of alpha IIbbeta 3 to fibrinogen (solid circles) by NSL-9511 at various concentrations. Each value represents the mean ± S.E. of triplicates of one representative experiment.
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Effects on Binding of RGD Peptide to Activated Platelets

Although NSL-9511 is a potent antagonist of alpha IIbbeta 3, it does not possess the RGD sequence. To determine whether NSL-9511 and the RGD peptide are mutually exclusive upon binding to alpha IIbbeta 3, we synthesized FITC-labeled RGD peptide and examined the effects of NSL-9511 on the binding of this RGD peptide to activated platelets. As Fig. 2 shows, activation of washed platelets with ADP increased the mean fluorescence when the platelets were incubated with 100 µM FITC-WSRGDW. This increment in mean fluorescence was inhibited by the addition of excess unlabeled RGD peptide (1 mM WSRGDW) (data not shown), suggesting that it resulted from the specific binding of FITC-labeled WSRGDW to platelet integrins in an activation-dependent manner. An addition of 10 µM NSL-9511 completely inhibited the increase in the mean fluorescence. These results suggest that NSL-9511 and the RGD peptide share the same binding site on alpha IIbbeta 3 or that their binding sites are very close.


Fig. 2. Fluorescence histograms demonstrating the inhibition by NSL-9511 of the binding of a FITC-labeled RGD peptide to activated platelets. Human platelets were activated by 100 µM ADP and incubated with 100 µM FITC-WSRGDW in the presence of 10 µM NSL-9511 at room temperature. Four thousand platelets were analyzed in a flow cytometer to generate each histogram.
[View Larger Version of this Image (22K GIF file)]


Effects on Cell Adhesion to Vitronectin and Fibronectin

The binding specificity of NSL-9511 was examined in a cell adhesion assay, in which ECV304 cells, originating from human umbilical vein endothelial cells, adhered to immobilized vitronectin and fibronectin via integrin receptors. As shown in Fig. 3a, the adhesion of ECV 304 cells to immobilized human fibronectin was inhibited by the GRGDS peptide, and almost complete inhibition was observed at 1000 µM. The EILDVPST peptide, which is the sequence within the CS-1 site of fibronectin and is considered to be another sequence responsible for the adhesion of various cell types to fibronectin (28), did not show such an inhibitory activity up to 100 µM under this assay condition. These results suggest that the adhesion of ECV 304 cells to fibronectin will be mainly mediated by the alpha 5beta 1 (fibronectin receptor)-RGD interactions. On the other hand, the adhesion of ECV 304 cells to immobilized vitronectin was inhibited by GRGDS by about 50% even at 1000 µM (Fig. 3b). An excess amount of 23C6, the monoclonal antibody to alpha vbeta 3 that has been reported to inhibit cell adhesion via alpha vbeta 3 integrin (27), also inhibited the ECV304 adhesion for vitronectin by about 50%. In this adhesion assay, NSL-9511 did not inhibit the adhesion of ECV 304 cells to immobilized fibronectin and vitronectin up to 1000 µM (Fig. 3, a and b), suggesting that NSL-9511 does not have inhibitory actions on integrins such as alpha vbeta 3 and alpha 5beta 1 and that it is highly specific to platelet alpha IIbbeta 3.


Fig. 3. Effects of NSL-9511 on ECV 304 cell adhesion to immobilized fibronectin and vitronectin. ECV 304 cells, harvested with 0.05% trypsin and 0.5 mM EDTA and washed twice with phosphate-buffered saline, were preincubated with each peptide or antibody for 30 min and then allowed to adhere to fibronectin-coated (a) and vitronectin-coated (b) plates. After 30 min, the number of attached cells was quantified by the WST-1 methods and expressed as a percentage of that in the absence of inhibitors. Each bar represents the mean ± S.E. of three experiments.
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Guinea Pig Arteriovenous Shunt Model

To examine the antithrombotic activity of NSL-9511 in vivo, a guinea pig arteriovenous shunt model was used. In this model, a shunt circuit is constructed between a carotid artery and a jugular vein with a silicon tube in which a silk thread is inserted, and the circulation of blood in this shunt results in a platelet-rich thrombus formation around the silk thread. As shown in Fig. 4a, the total thrombus weight over 60 min was 14.8 ± 1.2 mg in the control group where saline was infused via a jugular vein. Infusion of heparin (100 units/kg/h), an anticoagulant, inhibited the thrombus formation by about 50%. NSL-9511, administered by infusion, inhibited the thrombus formation in a dose-dependent manner, and at a dose of 10 mg/kg/hr, no thrombus was formed at all during the infusion. After the termination of the infusion, the thrombus formation was examined for another 60 min (Fig. 4b). Although the antithrombotic effect of heparin continued for at least 60 min after the termination of its infusion, the effect of NSL-9511 disappeared quickly, and the thrombus weight formed during this 60 min was not significantly different from that of the control group even after the administration of 10 mg/kg/h (13.9 ± 1.7 mg in the control group versus 13.1 ± 0.65 mg in the 10 mg/kg/h group). These results suggest that NSL-9511, infused via the jugular vein, inhibited the platelet-rich thrombus formation in vivo, but the duration of its antithrombotic action is very short. RGDS did not inhibit thrombus formation at a dose of 100 mg/kg/h in this model.


Fig. 4. An antithrombotic activity of NSL-9511 in a guinea pig arteriovenous shunt model. NSL-9511 (1.0, 3.0 and 10.0 mg/kg/h), RGDS (100 mg/kg/h), or heparin (100 units/kg/ml) was infused via a cannula in a right jugular vein, and the weight of the thrombi formed during the infusion for 60 min (a) and during a postinfusion period for 60 min (b) was measured. Mean ± S.E., n = 5; *, p < 0.01 (significantly different from the control).
[View Larger Version of this Image (32K GIF file)]



DISCUSSION

We have been developing specific antagonists of integrin receptors such as alpha IIbbeta 3, alpha vbeta 3, or alpha 5beta 1 using small linear peptides as the basic structure, because they are, in general, easy to synthesize by standard solid phase methods, and we can obtain much information about the structure-activity relationship by changing the constituent amino acids. In this study, we have found a series of potent and highly specific antagonists of platelet alpha IIbbeta 3 integrin that are linear hexapeptides without the RGD sequence. Although several peptide alpha IIbbeta 3 antagonists with high specificity for this integrin have already been reported, such as fibrinogen gamma -chain C-terminal decapeptide (19, 20), a tick saliva protein (21), barbourin (22), and synthetic cyclic peptides (16-18), this is the first report of small linear ones using L-form natural amino acids. Scarborough et al. (22) have reported that barbourin, a member of the disintegrin family of snake venom proteins, inhibits the binding of fibrinogen to alpha IIbbeta 3 but does not block vitronectin binding to alpha vbeta 3. Barbourin has a KGD sequence rather than RGD; therefore, the Lys residue is thought to be critical for the binding specificity. These results suggest that for potent antagonism toward integrins that recognize a RGD sequence such as alpha IIbbeta 3, alpha vbeta 3, or alpha 5beta 1, two interacting sites, an acidic moiety and a basic moiety, are necessary, and the substitution for a guanidino group of the Arg residue by the other basic groups can produce the binding specificity. Our results described here also demonstrate that the guanidino group of the Arg residue within the RGD sequence is not unique to the antagonistic activity toward alpha IIbbeta 3, that the imino group of proline may displace the role of the guanidino group, and that the introduction of the Pro residue to the N terminus of the peptide results in more potent activity and binding specificity.

Now we are studying the close structure-activity relationships around this peptide. Because the N-terminal Pro residue and the Asp residue at the fifth position are essential to the activity, the basic structure of the peptides can be summarized to have the following motif, Pro-X1-X2-X3-Asp-X4. The results of the preliminary experiments suggest that 1) a small amino acid such as Ser, Ala, or Gly is preferable at the X1 position, 2) X2 may be any amino acid, 3) X3 must be a small amino acid with a residue such as Gly, or a cyclic amino acid such as Pro, and 4) X4 prefers an amino acid with an aromatic side chain. We are going to discuss the structure-activity relationship of this peptide in detail elsewhere.

One of the motif peptides, NSL-9511, inhibited the binding of fibrinogen to purified human platelet alpha IIbbeta 3 in a dose-dependent manner with an IC50 of 70 nM (Fig. 1). Because NSL-9511 also inhibited in vitro platelet aggregation at submicromolar concentrations (IC50 = 390 nM), the anti-platelet activity of NSL-9511 can be explained by the blockage of the fibrinogen binding to alpha IIbbeta 3. Further, in this binding assay, the concentration of fibrinogen in the incubation medium did not greatly affect the IC50 value of NSL-9511 (data not shown). These results suggest that NSL-9511 inhibits fibrinogen binding, not by interacting with fibrinogen molecules but by binding to alpha IIbbeta 3 integrin as an RGD peptide does. Therefore, we concluded that NSL-9511 is an alpha IIbbeta 3 antagonist.

Because NSL-9511 does not have an RGD sequence and is a much more potent antagonist of alpha IIbbeta 3, we then examined whether the binding mode of NSL-9511 and an RGD peptide is the same. The results of the experiment showing that NSL-9511 dose-dependently inhibited the binding of an FITC-labeled RGD peptide to activated platelets (Fig. 2) suggest that NSL-9511 and the RGD peptide share the same binding site on alpha IIbbeta 3 or that the binding site of NSL-9511 lies so close to the RGD-binding site that NSL-9511 and an RGD peptide are mutually exclusive upon binding to alpha IIbbeta 3.

These findings, that NSL-9511 is an alpha IIbbeta 3 antagonist and possibly has the same binding mode as an RGD peptide and that the N-acetylation of the imino group of the Pro residue results in the complete loss of the anti-platelet activity, lead us to the idea that the imino group of the Pro residue at the N terminus replaces in a complementary manner the function of the guanidino group of the Arg residue within an RGD sequence as a basic moiety essential to the activity. This idea is supported by the conformational analysis of a PSRGDW peptide, which has both the imino group of the Pro residue and the guanidino group of the Arg residue. The spatial distance between the carboxyl group of the Asp residue and the guanidino group of the Arg residue (d1) of stable conformers and the spatial distance between the carboxy group and the imino group of the Pro residue (d2) of stable conformers were calculated (Fig. 5). The largely overlapped area of d1 and d2 distribution suggests that the relative arrangement between the Pro residue and the Asp residue is similar to that of the Arg residue, indicating that the imino group of the Pro residue can occupy the same conformational space as the guanidino group of the Arg residue.


Fig. 5. The conformational analysis of PSRGDW. The conformational analysis was conducted on Macro Model, version 3.5. Fifty thousand conformers were generated by the Monte Carlo method and minimized with a modified Amber* Force field in vacuum to obtain 2200 conformers. The distance between the guanidino group of the Arg residue and the carboxy group of the Asp residue (d1) and the distance between the imino group of the Pro residue and the carboxy group of the Asp residue (d2) were calculated. Conformers with shorter d1 or d2 (<10 Å) were eliminated, based on Kessler's active conformation. The electrostatic cut-off distance was set to 5 Å, and the force field was modified in order to not evaluate hydrogen bonds. The largely overlapped area of d1 and d2 distribution suggests that the relative arrangement between the Pro residue and the Asp residue is similar to that of the Arg residue.
[View Larger Version of this Image (43K GIF file)]


To examine the binding specificities of the motif peptide, we performed the binding assay using purified human placental alpha vbeta 3 and vitronectin and the cell adhesion assay, in which ECV 304 adhered to immobilized fibronectin or vitronectin. In the alpha vbeta 3-vitronectin binding assay, both an anti-alpha vbeta 3 monoclonal antibody and 100 µM GRGDS completely inhibited the binding, suggesting that the binding was mediated by alpha vbeta 3 and the RGD sequence of vitronectin. The inhibition of the binding of vitronectin to alpha vbeta 3 by NSL-9511 was very weak at its highest concentration (Fig. 1b), suggesting that NSL-9511 is highly specific for alpha IIbbeta 3. The results of the cell adhesion assay (Fig. 3) also show that NSL-9511 did not inhibit cell adhesion via other integrins such as alpha vbeta 3 or alpha 5beta 1. ECV304 is a cell line established from human umbilical vein endothelial cells, and we have confirmed the expression of alpha vbeta 3 and alpha 5beta 1 on ECV304 using monoclonal antibodies by flow cytometry (data not shown). Adhesion of ECV304 to vitronectin is thought to be largely mediated by alpha vbeta 3 because the monoclonal antibody to the alpha vbeta 3 complex inhibited the adhesion. However, we could not show the dependence of alpha 5beta 1 on the adhesion of ECV304 to fibronectin, because an anti-alpha 5beta 1 antibody that has been reported to block the binding of fibronectin to alpha 5beta 1 is not available. These results suggest that NSL-9511 is an antagonist highly specific for platelet alpha IIbbeta 3 but not for other integrins such as alpha vbeta 3 or alpha 5beta 1. There are several possible explanations for this high specificity of NSL-9511 for alpha IIbbeta 3. First, it is possible that integrins other than alpha IIbbeta 3 prefer a guanidino group as a basic interaction site upon binding with the ligand, while both a guanidino group and secondary amines such as an imino group of the Pro residue are preferable as a basic moiety upon ligand binding to alpha IIbbeta 3. It is also possible that the active spatial structure for alpha vbeta 3 or alpha 5beta 1 integrin is quite different from that for alpha IIbbeta 3 and that the NSL-9511 cannot conform to this structure, while the RGD peptide can.

NSL-9511 also exhibited the anti-platelet activity in vivo, when infused intravenously (Fig. 4). Infusion of NSL-9511 at a rate of 10 mg/kg/h almost completely inhibited the platelet-rich thrombus formation around the thread, and this antithrombotic activity quickly wore off after the termination of the infusion. Peptides, including NSL-9511 are rapidly degraded in guinea pig plasma at 37 °C by the action of aminopeptidases (data not shown); therefore, this quick disappearance of anti-thrombotic activity would be due to the decrease in plasma concentration of NSL-9511 caused by the enzymatic degradation. The same is true for an RGDS peptide, which did not show any antithrombotic activity in this shunt model. Substantial increases in bleeding time have been an undesirable characteristic exhibited by many alpha IIbbeta 3 antagonists (34) including an anti-alpha IIbbeta 3 monoclonal antibody, 7E3 (35, 36), and this is one of the limitation factors in the use of an alpha IIbbeta 3 antagonist as an antithrombotic agent. Therefore, NSL-9511, which exhibits potent antithrombotic activity in vivo during the infusion, although this activity wears off quickly and the prolongation of bleeding time is not as serious, has a potential for clinical uses, especially as an antithrombotic drug at an acute phase.

Another interest of ours is the possibility that these motif peptides can be produced endogenously. Although many of the human proteins include the sequences homologous to this motif sequence, they do not lie at the N terminus, and the imino group of the Pro residues is not free. To exert anti-platelet activity, these proteins need to be processed so as to expose the Pro residue at a new N terminus. Aminopeptidase P, which has been reported to exist in human cells such as platelets (37), and some virus proteases such as HIV-1 protease (38) are candidates for catalyzing such a reaction.


FOOTNOTES

*   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.
   To whom correspondence should be addressed: Life Science Research Center, Advanced Technology Research Laboratories, Nippon Steel Corp., 1618 Ida Nakahara-ku Kawasaki 211, Japan. Tel.: 81-44-777-4111; Fax: 81-44-752-6352.
1   The abbreviations used are: RGD, Arg-Gly-Asp; FITC, fluorescein isothiocyanate; PRP, platelet-rich plasma; TBS, Tris-buffered saline; ELISA, enzyme-linked immunosorbent assay.

Acknowledgments

We are grateful to Dr. Seiichi Ohkuma (Emeritus Professor of Tokyo Collage of Pharmacy) and Dr. Setsuko Yamazaki (Japanese Red Cross Tokyo Western Blood Center) for the generous gift of outdated platelets and to Dr. Masaaki Ohkuma (Institute of Life Science, Kurume University) for the generous gift of platelet alpha IIbbeta 3-rich membrane fraction. We also thank Emiko Yasuda and Mako Yano for technical assistance in peptide syntheses.


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