Eur. J. Biochem. 194,43-49 (1990)

0FEBS 1990

KRDS, a new peptide derived from human lactotransferrin, inhibits platelet aggregation and release reaction Elisabeth MAZOYER’, Sylviane LEVY-TOLEDANO’, Francine RENDU’, Laurence HERMANT’, He LU’, Anne-Marie FIAT Pierre JOLLES and Jacques CAEN

’,

’

Institut des Vaisseaux et du Sang, Paris, France Unit6 150 de I’Institut National de la Sant6 et de la Recherche Medicale, et Unit6 Associke 334 du Centre National de la Recherche Scientifique, HBpital Lariboisi&re,Paris, France Laboratoire des Proteines, Unit6 Associke 1188 du Centre National de la Recherche Scientifique, Universit6 Paris VII, Paris, France (Received April 23/July 17, 1990) - EJB 90 0463

KRDS (Lys-Arg-Asp-Ser), a tetrapeptide from human lactotransferrin, was tested in vitro on human platelet function, and its effects were compared to those of RGDS, a tetrapeptide from human fibrinogen. Both peptides had a high probability of initiating a p-turn and were highly hydrophilic. KRDS inhibited ADP-induced platelet 360 pM) to a lesser aggregation [median inhibitory concentration (1C5,J 350 pM] and fibrinogen binding extent than RGDS (IC50 75 pM and 20 pM, respectively). Different from RGDS, thrombin-induced serotonin release was inhibited by KRDS (750 pM) on normal platelets (55 loo/,) and type I Glanzmann’s thrombasthenia platelets (43% f 1). However, KRDS had no effect on cytoplasmic Ca2 mobilization, inositol phospholipid metabolism or protein phosphorylation (myosin light chain P20 and P43). In contrast to RGDS, KRDS does not inhibit the binding of monoclonal antibody PAC-1 to activated platelets. KRDS and RGDS inhibited 4P-phorbol12-myristate-13-acetate (PMA)-induced aggregation and fibrinogen binding, while proteins were normally phosphorylated. Thus, the tetrapeptide KRDS is (a) an inhibitor of serotonin release by a mechanism independent of protein phosphorylation and (b) an inhibitor of fibrinogen binding and, hence, aggregation by a mechanism that may not necessarily involve its direct binding to the glycoprotein IIb-IIIa-complex. +

It is well established [l, 21 that fibrinogen interaction with number of similarities have been previously reported between platelets is essential for platelet aggregation and that fibrino- these two clotting processes [6, 71. Moreover, structural homgen binds to a specific receptor on the platelet surface: the ology has been found between cow K-casein and human figlycoprotein IIb-IIIa complex (GPIIb-IIIa). Unstimulated brinogen y-chain [8]. Recently, Jolles et al. [9]showed that both platelets do not bind fibrinogen. In type I Glanzmann’s the natural and the corresponding synthetic undecapeptide thrombasthenia, characterized by the absence of platelet mem- MAIPPKKNQDK of cow K-casein (residues 106- 116) were brane GPIIb-IIIa, fibrinogen binding and ADP- or thrombin- more inhibitory of ADP-induced platelet aggregation and induced platelet aggregation are not observed. Yet the mech- fibrinogen binding than the C-terminal dodecapeptide, anisms of fibrinogen receptor exposure on the platelet surface HHLGGAKQAGDV, of the human fibrinogen y-chain (resiand of fibrinogen interaction with GPIIb-IITa are not com- dues 400-411). Based on these observations, we looked for pletely understood. Two binding sites have been identified on a peptide from a milk protein that is structurally and functhe fibrinogen molecule: a decapeptide in the carboxy-ter- tionally closely related to the sequence of the human fibrinominal region of the y-chain (LGGAKQAGDV; residues gen a-chain tetrapeptide, RGDS. Such a peptide was found 402 - 41 1) and a tetrapeptide in the carboxy-terminal region in human lactotransferrin and its sequence Lys-Arg-Asp-Ser of the a-chain (RGDS; residues 572-575). Both peptides (KRDS) corresponds to residues 39 -42. Previously published inhibit aggregation and fibrinogen binding to ADP-activated reports indicated that (a) RGDS inhibits thrombus formation platelets [3]. Recently it has been shown that RGDS becomes in dog coronary artery [lo], (b) RGDS-containing peptides more chemically cross-linked to residues 109- 171 of GPIIIa prolong bleeding time in severed hamster mesenteric arteries [4] while the y-chain cross-linking site (positions 400 -41 1) [l11and (c) monoclonal antibodies inhibiting fibrinogen binding to the GPIIb-IIIa complex prevent the formation of must reside within residues 294-314 of GPIIb [5]. The clotting of blood and the clotting of milk are two platelet microthrombi in experimental animals [12]. Recently, physiologically important coagulation processes. A large in vivo experiments with laser-induced thrombosis in the rat and the guinea-pig in the presence of KRDS and RGDS have Correspondence to E. Mazoyer, Institut des Vaisseaux et du Sang, been performed, and have shown the antithrombotic effect of 8 rue Guy Patin, F-75010 Paris, France the peptides and their synergistic effects on the thrombosis Abbreviations. PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphos- inhibition [13]. phate; PtdIns4P, phosphatidylinositol 4-phosphate; PtdIns, In this paper we described the in vitro effect of the phosphatidylinositol; Ins( 1,4,5)P3, inositol 1,4,5-trisphosphate; BSA, tetrapeptide KRDS on platelet function: ADP-induced bovine serum albumin; Indo-I-AM, indo-I acetoxymethyl ester; PMA, 4P-phorbol 12-myristate 13-acetate; median inhibitory platelet aggregation and fibrinogen binding, and the thrombin-induced signal-transduction mechanism (hydrolysis of inconcentration; GP, glycoprotein.

44 ml), and in the absence (control) or presence of different concentrations of peptides. No fibrinogen was added for 140-nM-PMA-induced platelet aggregation. Purified human fibrinogen was labelled with l 25I using the modified chloramine T method [20]. Fibrinogen binding was performed according to Marguerie et al. [21], as modified by Lee et al. [22]. Briefly, a platelet suspension (4 x lo8 platelets/ MATERIALS AND METHODS ml) was incubated with lZ5I-fibrinogen (200 pg/ml), in the Muteriuls absence (control) or presence of the peptide. After the addition The type 1 Glanzmann's thrombasthenia patient studied of ADP (10-20 pM final concentration) or PMA (140 nM), in this paper has been previously described [14]. ADP, human the solution was allowed to stand for 30 min at room temperathrombin and bovine serum albumin were purchased from ture. Parallel experiments without inducer were performed as Sigma. Human fibrinogen was obtained from Kabi laboratory control for spontaneous binding. After incubation, triplicate (Stockholm, Sweden). ['4C]Hydroxytryptamine and [32P] samples of 0.1 ml were layered on to 0.5 ml oil (dioctyl phosphoric acid from Oris (CEA, Saclay, France). Metriz- phthalate/dibutyl phthalate, 2: 2.5), centrifuged at 11000 g for amide was purchased from Nyegaard (Oslo, Norway) and 4 min. The supernatant was aspirated and each pellet counted phycoerythrin-streptavidin from Caltag laboratory (San in a Beckman 7000 y counter. The bound 12sI-fibrinogen was Francisco, USA). The synthetic peptide RGDS was purchased expressed as percent inhibition of total '251-fibrinogen bound. from Tebu laboratory (Le Perray en Yvelines, France).

ositol phospholipids, phosphatidic acid synthesis, protein phosphorylation, Ca2 mobilization and serotonin release). Its effects were compared to those of the structurally related RGDS tetrapeptide present in the human fibrinogen a-chain. +

Synthetic pept ide prepaua t ion The synthetic peptides used throughout this study were prepared using a 430A peptide synthetizer (Applied Biosystems). After final deprotection and purification by reversephase HPLC, the free peptide appeared to be homogeneous on thin-layer chromatography and analytical HPLC. The amino acid analyses were performed after total hydrolysis (6 M HCl with 1:2000 2-mercaptoethanol; 18 h at 110°Cunder vacuum) with a Biotronik Autoanalyzer (model LC 6000). The sequence of all peptides was established using a 470 A gas-phase Sequencer (Applied Biosystems). The phenylthiohydantoin amino acid derivatives were automatically identified with an Applied Biosystems 120 A phenylthiohydantoin analyzer used on-line with the sequencer. The distribution of hydrophobic and hydrophilic groups in human lactotransferrin and fibrinogen fragments was calculated by a computer according to the protein sequence plotting program (PRPLOT Dayhoff 1151) and using the parameters of Kyte and Doolittle [ 161. The secondary structure was predicted according to Garnier et al. [17]. Blood sampling

Human blood was collected in 1 : 10 vol. 130 mM citric acid, 124 mM trisodium citrate and 110 mM glucose, and platelet-rich plasma was obtained by centrifugation at 120 g for 15 min at room temperature. Pla tele t uggregu t ion andf ibrinogen binding Platelets in platelet-rich plasma were isolated and washed according to the technique of Patscheke [I 81, as modified by Lee [19], and resuspended in a Tyrode's buffer [I37 mM NaCl, 2 mM KC1, I:! mM NaHC03, 0.3 mM NaH2P04, 2 mM CaC12, 1 mM MgC12, 5.5 mM glucose, 5 mM Hepes, 0.35% bovine serum albumin (BSA), pH 7.41. Platelet aggregation was performed in a Chrono-Log corporation aggregometer. Results were given in terms of change in light transmission 2 min after the addition of the inducer and expressed as percent inhibition of maximal intensity of control. 10 - 20-pM-ADP-induced platelet aggregation was performed in the presence of purified human fibrinogen (200 pg/

Platelet release reuction, inositol phospholipid metabolism and protein phosphorylation Platelet-rich plasma was incubated for 90 min at 37°C either with [14C]serotonin (18.5 kBq/ml platelet-rich plasma) for measurement of platelet serotonin release, or with [32P]phosphoricacid (3700 kBq/ml platelet-rich plasma) for measurement ofinositol phospholipid metabolism and protein phosphorylation. Platelets were isolated and washed by centrifugation on a metrizamide gradient as previously described [23] and finally resuspended in a buffer containing 10 mM Hepes, 140 mM NaCl, 3 mM KC1, 0.5 mM MgC12, 5 mM NaHCO,, 10 mM glucose, pH 7.4. The platelet suspension (4.5 x lo8 platelets/ml) was incubated in an aggregometer while stirring at 37"C, in the absence (control) or presence of the tetrapeptide at different concentrations, before the addition of the inducer (thrombin or PMA). Serotonin release reaction. The [14C]serotonin plateletrelease reaction was performed on the same sample as the aggregation sample. At different times after the addition of the inducer (10, 30, 60 and 120 s) the reaction was stopped by dilution in 1 : 5 vol. ice-cold 0.1 M EDTA and immediate centrifugation for 30 s at 1500 g in an Eppendorf centrifuge. The supernatant was analyzed for serotonin content by measuring radioactivity. The results were expressed as relative release of total ['4C]serotonin incorporated into platelets. Inositol phospholipid vnetabolism and protein phosphorylation. The 32P-labelledsamples were transferred, at the desired time of stimulation, in 3.75 vol. ice-cold chloroform/ methanolll2 M HCl/O.l M EDTA (20:40: 1 :2, by vol.). The samples were partitioned by addition of 1 :25 vol. each of chloroform and distilled water. A clear biphasic system was obtained after centrifugation 10 rnin at 4°C. The upper phase (aqueous) was discarded and the proteins at the interface were solubilized by a 1-h incubation at 6 0 T in 150 p1 Laemmli buffer (0.06 M Tris, 2% SDS, 20% glycerol, 0.01% bromophenol blue). After reduction with 5% 2-mercaptoethanol for 30 min at 60°C, 32P-labelled proteins were analyzed by a discontinuous SDS/polyacrylamide gel using 13% acrylamide in the resolving gel and 5% acrylamide in the stacking gel. After staining with Coomassie brillant blue, gels were dried and exposed to Kodak XAR X-ray film. Autoradiographs were scanned with a LKB ultroscan. The lower phase was evaporated to dryness under N 2 and dissolved in chloroform. Inositol phospholipids were separ-

45 ated into individual phospholipids by means of one-dimensional thin-layer chromatography on silica plates according to Jolles et al. 1241. After being localized by autoradiography, the separated 32P-labelled phospholipids, phosphatidylinositol 4,5-bisphosphate [PtsIns(4,5)P2], phosphatidylinositol 4-phosphate (PtsIns4P), PtdIns and phosphatidic acid, were quantified by scraping the identified spots and measuring radioactivity in a scintillation counter.

1.60

1.60

1.40

1.40

1.20

1.20

1.00

1.00

0.80

0.80

0.60

0.60

0.40 1.60

I

0.40 3 9 4 0 4 1 42

3940 4 1 42 K R D S

K

R

O

S

1.40

Platelet cytoplasmic calcium mobilization

Platelets in platelet-rich plasma were isolated by centrifugation for 15 min at room temperature and resuspended in a modified Tyrode's buffer (NaC1 130mM, KC1 5mM, NaH2P04 1 mM, NaHC03 24 mM, glucose 10 mM, Hepes 10 mM, saccharose 12.5 mM, BSA 0.5%, prostaglandin El 5 pM, pH 7.4). Platelets in suspension (2 x 108/ml)were incubated with 2 pM indo-1-acetoxymethyl ester (indo-1-AM) for 45 min at room temperature. Excess probe was removed by two short washes in the modified Tyrode's buffer without prostaglandin El and BSA. The fluorescence was monitored by using a Spectrofluo JY3D (Jobin Yvon, Division d'instruments SA) in a thermostated 1-cm cuvette at 37°C. Excitation of Indo-1-AM occurred at 331 nm, with emission detected at 410 nm. Fluorescence was continuously monitored before and after thrombin stimulation (0.2 U/ml) in different experimental conditions, either in the presence of added external Ca2+(1 mM) or in the presence of a Ca2' chelator EGTA (0.5 mM) or in the presence of NaCl (0.15 M).

1.20 1 .oo

0.80 3.00

0.60

0.40

I 5 6 .0 39 4 0 41 42 K R O S

i;ipl

3 9 4 0 41 42 K R D S

::;;m

1.20 1 .oo

0.80

0.80

0.60

0.60

0.40

0.40

572 573571575

572 573574575

l ' R G g D S O m R C O S

1.60

0.90

1.30 1.00

-0.50

Monoclonal antibody PAC-I binding to stimulated platelets

0.70

-1.90

Biotin-conjugated PAC-1 was prepared as already published [25]. Human washed platelets were prepared as previously described. After 10 min stimulation with thrombin (0.5 Ujml) at room temperature without stirring, biotinylated PAC-1 (7.7 pg/ml) was incubated for 15 rnin at room temperature with unstimulated and stimulated platelets (1.5 x 108/ml), in the presence or the absence of KRDS (750 pM) or RGDS (500 pM). Platelets were then incubated for 10 rnin with a second fluorescent reagent phycoerythrin-streptavidin and finally fixed (glutaraldehyde 1%). The platelets were analysed for PAC-I binding on a FACstar flow cytometer.

0.40

-3.30

0.10

-4.70

572 575574575

572 573574575

R C O S

R C O S

Fig. 1. Predicted secondary structure and hydropathy of fragments of human lactotransferrin and of human fibrinogen cc-chain. The sequence KRDS corresponds to residues 39 -42 of human lactotransferrin (A - D), and the sequence RGDS corresponds to the residues 572 575 of human fibrinogen (E-H). The potential for forming cc-helix (A, E), /I-turn (B, F), /I-sheet (C, G) was predicted according to Garnier et al. [17], and the hydropathy (D, H) was calculated according to Kyte and Doolittle [I61

RESULTS Amino acid composition and sequence determination of the peptides

Control

The different synthetic peptides were checked for their amino acid composition and sequences, KRDS, RGDS, KRDR, KRDY and KRDSY, and were found to be in excellent agreement with expectations (not shown). Their purities were determined as higher than 98%.

KRDS 750pM Fg ADP

Relationship between secondary structure and hydropathy of the two peptides

-

RGDS 500pM

1 min

The probability concerning the secondary structure and the hydropathy of KRDS and RGDS is indicated in Fig. 1. The peptides KRDS and RGDS both presented a high probability of initiating a /?-turn form inside human lactotransferrin and human fibrinogen (as shown in the Fig. 1 B and F by values higher than the average); while the probability of forming an a-helix (Fig. 1A and E) or /3-sheet (Fig. 1C and

Fig. 2. Inhibition of ADP-inducedplatelet aggregation in the presence of K R D S and RGDS. Human platelets were washed according to the technique described in Materials and Methods. Platelets were aggregated by the addition of fibrinogen (Fg; 200 pg/ml) and ADP (10-20 pM) in the absence (control) or presence of KRDS (750 pM) or RGDS (500 pM). The curves representative of several experiments, show the higher peptide concentrations used during the different tests

Table 1. Structurallfunctionul relationship qf' the inhihition of ADPinducedplatelet aggregation and 'ZSITfihrinogenbinding by KRDS and KRDS analogues Values represent the peptide concentration causing 50% inhibition (ICs0)of ADP-induced platelet aggregation and '251-fibrinogen binding. n.d., not determined Peptide 0

ICSO

Aggregation

1

I I11111[

I

I1111111

I

'ZSI-fibrinogen binding

I I I I I

PM

KRDS KRDSY KRDR KRDY

1o

-~

1o

-~

Peptide

1 o--=

lo-=

(M)

Fig. 3. Concentration-d~pendentinhibition qf ADP-inducedplutelet aggregation and '25Z:fibrinogenbinding by KRDSand RGDS. (A) Aggregation. Various concentrations of peptide were added to human washed platelets prior to the addition of ADP (10-20 pM) and fibrinogen (200 Fgiml). Results are expressed as mean & SEM of inhibition relative to the control maximum. (B) 'Z51-fibrinogen binding. Human washed platelets were incubated for 30 min with '2SI-fibrinogen (200 pg/ml) and various concentrations of peptide after the addition of ADP (10-20 pM). Results, expressed as mean f SEM of inhibition relative to the control '251-fibrinogen bound, are the means of three different experiments for KRDS and two different experiments for RGDS

G) was low. Moreover, KRDS and RGDS peptides were both highly hydrophilic as shown in Fig. 1 D and H.

350 460 > 2000 1200

360 560 n.d. 1000

Table 2. Concentration-dependent inhibition qf KRDS and RGDS on 0.2- U,'ml-thrombin-induced platelet responses Platelets labelled with ['4C]serotonin were washed and stimulated by 0.2 U/ml thrombin. Platelet aggregation and ['4C]serotonin release were recorded. The results, expressed as mean f SD of inhibition, represent the mean of three different experiments (normal platelets) or the mean of a duplicate experiment (type I Glanzmann's thrombasthenia platelets) Peptide

Concentration

Normal platelets

aggregation release

Type I Glanzmann's thrombastenia platelets release

PM

YOinhibition

KRDS

75 350 750

14 25k4 46 f 7

2 13&5 55i10

43+l

RGDS

12 25 50 100 500

15 34 62 83i4 86 & 3

0

0

K R D S and RGDS effect on ADP-indud platelet aggregation and jibuin ogen h inding Both KRDS and RGDS inhibited washed human platelet aggregation induced by ADP (10-20 pM) (Fig. 2). These peptides inhibit the aggregation in a dose-dependent manner. However, the concentrations required to obtain 50% inhibition (IC5,,) of the aggregation intensity were approximately 350 pM and 75 pM, respectively, for KRDS and RGDS (Fig. 3A). 1251-labelled fibrinogen binding to ADP stimulated platelets was inhibited by KRDS and RGDS in a concentration-dependent manner (Fig. 3B). The IC5,, was 360 pM for KRDS and 20 pM for RGDS. The KRDS specificity, in terms of ability to inhibit ADPinduced platelet aggregation and fibrinogen binding, was further analysed with peptides containing modifications of the amino acid sequence (KRDS analogues). Less inhibition of platelet aggregation and fibrinogen binding was found in the presence of KRDY (IC5,, 1.2 mM and 1 mM, respectively) or KRDR (higher than 2 mM for the aggregation); however the KRDSY peptides has an inhibitory activity similar to KRDS (ICs0 460 pM and 560 pM, respectively; Table 1).

K R D S and R G D S effect on thrombin-induced platelet aggregation and serotonin release

0.2-U/ml-thrombin-induced platelet aggregation was inhibited by KRDS in a dose-dependent manner (Table 2). Using a KRDS concentration of 350 pM, this inhibition was less important (25%) compared to the inhibition of ADPinduced platelet aggregation and ADP-induced fibrinogen binding (50%). Inhibition of platelet serotonin release (55%) was associated with inhibition of thrombin-induced platelet aggregation (46%) by 750 pM of KRDS (Table 2). A higher inhibition of serotonin release (70%) was also found with a lower concentration of thrombin (0.05 U/ml; data not shown). By contrast, RGDS, a stronger inhibitor of thrombin-induced platelet aggregation (83% inhibition with 100 pM). was unable to inhibit serotonin release, even at a concentration as high as 500 pM. Using platelets from a type I Glanzmann's thrombasthenia patient, serotonin-release inhibition in the presence of KRDS (750 pM) was in the same range as that with normal platelets (43%; Table 2). This KRDS effect was also observed using a

47 Table 3. Changes in platelet inositolphospholipids induced by 0.2 Wjml thrombin in the absence (control) or presence of KRDS (750 p M ) or RGDS (500 p M ) Platelets labelled with [3ZP]phosphoric acid were stimulated with thrombin. Inositol phospholipids were separated into individual phospholipids by means of thin-layer chromatography on silica plates, were quantified by scraping and measuring radioactivity of the identified spots. Results were expressed as the mean f SEM (n = 3) of resting level [control (0 s), represents loo%]. PtdA, phosphatidic acid Inositol phospholipid measured

Sample

Table 4.Effect of KRDS (750 p M ) and RGDS (500 p M ) on cytoplasmic Ca2+mobilization Platelets were loaded with indo-I-AM. Fluorescence was continuously recorded before (resting [Ca2+])and after thrombin (0.2Ujml) stimulation in different experimental conditions, in the presence of external Ca2+ (1 mM), EGTA (0.5 mM) or NaCl(O.15 M) which were added prior to stimulation. Results show [Ca2+]measured as mean SEM (n = 3)

+

Resting [Ca2+]

Time after addition of thrombin

[Ca"] measured with thrombin (0.2U/ml and Ca2 (1 mM)

EGTA (0.5 mM)

NaCl (0.15M)

491 Jr24 518559 828k25

228+29 204&50 215+50

215k28 226+47 192515

+

5s

10 s

30 s

120 s nM

Ptd(4,5)InsP2

Control KRDS RGDS

PtdA

Control KRDS RGDS

81 k 06 705 11 69f04

63 f 04 72k04 67f05 235 & 39 293 & 42 171 +I4 230k07 252f45 279f01

+

106 09 95503 91 k04 372 46 328k81 455f36

+

118 f06 84f07 97f06 438 & 92 397k37 634f78

Control KRDS RGDS

42+3 50+5 42f2

(500 pM), although 90% inhibition of platelet aggregation and fibrinogen binding were observed, no modification of P43 and P20 phosphorylation could be detected (data not shown). lower concentration of thrombin (50% inhibition with 0.05 U/ ml of thrombin; data not shown). RGDS (500 pM) had again no effect on the serotonin release.

Effect of K R D S and RGDS on cytoplasmic Ca2+ mobilization

The resting cytoplasmic CaZ concentration averaged 42 & 3 nM and was not modified in the presence of either peptides (Table 4). The cytoplasmic Ca2 concentration was studied in the presence of external Ca2+ (1 mM) and in its absence, using EGTA (0.5 mM). The Ca2+ mobilized from internal stores was estimated by the measure in presence of external EGTA (0.5 mM); in these conditions, neither KRDS nor RGDS modified Ca2+ mobilization. The Ca2+ concentration measured in the presence of external Ca2+ was not modified by KRDS, while it was increased in presence of RGDS (Table 4). Furthermore, the kinetics of cytoplasmic Ca2 increase after thrombin stimulation were not delayed in presence of KRDS and RGDS (data not shown). +

Inositol phospholipid metabolism in the presence of KRDS and RGDS

+

Following thrombin addition (0.2 Ujml), the variations of the radioactivity associated with PtdIns(4,5)P2 and phosphatidic acid were shown in Table 3. The transient decrease in PtdIns(4,5)P2 radioactivity (33%) observed 10 s after thrombin addition was modified neither by the presence of KRDS (750 pM) nor RGDS (500 pM). The decreases in PtdIns4P and PtdIns were also unaffected by the peptides. For longer times of activation, the following increase in radioactivity in all three inositol phospholipids was slightly lower in the presence of peptides, compared to control, although the differences were not significant. Moreover [32P]phosphatidic acid synthesis measured after thrombin addition was not Effect of KRDS and RGDS on biotin - PAC-I binding to stimulated platelets modified by the presence of KRDS or RGDS. The monoclonal antibody PAC-1 epitope is located on platelet membrane GPIIb-IIIa near the fibrinogen receptor. Protein phosphorylation in the presence of K R D S and RGDS Its binding is inhibited by both fibrinogen and RGD-containIn the presence of thrombin (0.2 Ujml), two proteins ap- ing peptides [27,28]. The binding of biotin -PAC-1 was studpeared phosphorylated: the myosin light chain P20 and P43. ied on unstimulated platelets and platelets stimulated by These proteins were normally phosphorylated in presence of thrombin (0.5 Ujml). Unstimulated platelets did not bind KRDS (750 pM) and RGDS (500 pM). Over a 2-min period, PAC-1. Using a concentration of 750 pM KRDS, no modifiKRDS and RGDS did not interfere with the hnetics of the cation of PAC-1 binding to stimulated platelets was observed protein phosphorylation (data not shown). compared to control stimulated platelets. However RGDS, at a concentration of 500 pM, induced a 96 & 2% inhibition KRDS and RGDS effect on PMA-inducedplatelet aggregation, (Fig. 4). fibrinogen binding and protein phosphorylations +

It has been demonstrated [26] that PMA induces platelet aggregation and fibrinogen binding. PMA-induced activation is associated mainly with phosphorylation of the P43 and to a much lower extent with the phosphorylation of the P20. A concentration of 750pM KRDS added to washed human platelets induced a 54% inhibition of platelet aggregation and 45% inhibition of fibrinogen binding without affecting P43 and P20 phosphorylations. In the presence of RGDS

DISCUSSION Similarities between blood and milk coagulation processes have previously been noted [6, 71. Structural and functional homology between the dodecapeptide of fibrinogen y-chain (residues 400 -41 1) and the undecapeptide of cow K-casein (residues 106- 116) have been already reported [8, 91. The Nterminal undecapeptide of cow Ic-casein was shown to be three

48 300 I

I

L

W

n

E 3 S

0

Fluorescence intensity Fig. 4. &&r of K R D S (750 p M ) mu' RGDS (500 pA4) on biotinPAC-I binding io 10.5 U j m l ~ -thvombin-stimulated platelets. Unstimdated (.........) and thrombin-stimulated platelets in the absence (- .-.-) and the presence of KRDS (-) or RGDS (-) were incubated with biotin - PAC-1. Phycoerythrin-streptavidin was then added and the platclcts wcrc analysed for PAC-I binding by flow cytometry. This graph is representative of four different experiments - -

times more inhibitory of ADP-induced fibrinogen binding than the C-terminal dodecapeptide of human fibrinogen y-chain. Moreover the decapeptide of fibrinogen y-chain LGGAKQAGDV and the tetrapeptide of fibrinogen a-chain, RGDS, both inhibitors of ADP-induced fibrinogen binding, have been shown to interact with two spatially distinct sites on the GPIIb-IIla complex [29]. The RGDS peptide is more efficiently cross-linked to membrane GPIIIa on stimulated platelets while thc dodecapeptide of the y-chain is cross-linked to membrane GPIlb [4, 51. Therefore, our interest was to look for a tetrapeptide that will be structurally and functionally related to the human fibrinogen x-chain tetrapeptide, RGDS. This peptide was found in human lactotransferrin, and corresponds to the sequence Lys-Arg-Asp-Ser (KRDS; residues 39 -42) close to the N-terminus of the 703 amino acids [30]. This fragment is situated in a /&turn inside the molecule and is highly hydrophilic and polar. The RGDS sequence corresponds to residues 572 - 575 close to the C-terminus of the 610-aminoacid x-chain of fibrinogen; this portion ofthe molecule extends as a high appendage at the end of the fibrinogen molecule and is therefore available to bind to exposed fibrinogen receptor on stimulated platelets [31]. The secondary structure of the RGDS recognition site in proteins involved in cell-surface adhesion is important [32]. Thus, these two tetrapeptides, KRDS and RGDS, have similarities in their sequences, with very close secondary structures as well as similar hydropathy profiles. As previously shown [31,33 - 351, our results confirm that the tetrapeptide RGDS inhibits ADP-induced human platelet aggregation and fibrinogen binding to ADP-activated platelets in a concentration-dependent manner. Gartner et al. [31] showed that the RGDS peptide decreases the affinity of the available fibrinogen receptors without changing their number. The tetrapeptide derived from lactotransferrin, KRDS was also found to inhibit platelet aggregation and fibrinogen binding to ADP-activated platelets. However higher concentrations of KRDS were necessary, compared to RGDS, to obtain the same extent of inhibition of aggregation and fibrinogen binding. With ADP-stimulated human megakaryocytes 1361, KRDS was shown to be a more efficient

inhibitor than RGDS of the binding of P2, a monoclonal antibody which precipitates GPIIb-IIIa and GPIa [37]. Amino acid substitutions within this tetrapeptide sequence, serine by either arginine (KRDR) or tyrosine (KRDY), reduced the observed inhibitory activity, while after the addition of a C-terminal tyrosine (KRDSY), the inhibition remained more similar to that of the KRDS. Among the peptides tested, KRDS was the most specific in term of the highest inhibitory activity, indicating the importance, for tetrapeptide activity, of the integrity of these four amino acids in the sequence. It has been proposed that the activation of the protein kinase C induces conformational modifications of the GPIIbIIIa complex which leads to exposure of the fibrinogen receptor [26, 381. Our results show that, in the presence of KRDS and RGDS, protein kinase C was still activated by PMA, since P43, the main substrate for protein kinase C, was normally phosphorylated, while platelet aggregation and fibrinogen binding were both inhibited. The protein kinase C would fulfill the role of an intraplatelet signal transducer regulating the availability of the receptor for adhesive proteins. Here, the peptides KRDS and RGDS would inhibit the binding of fibrinogen by preventing the conformational modifications of the GPIIb-IIIa molecule necessary for the exposure of the fibrinogen-binding epitope. KRDS inhibited, in a dose-dependent manner, the thrombin-induced platelet release reaction. This is in contrast with RGDS and trigramin peptides shown to inhibit thrombininduced platelet aggregation without preventing the granule release reaction [31, 33, 391. The tetrapeptide KRDS could thus interfere with earlier steps such as the signaling system. Stimulation of platelets by thrombin activates a phospholipase C which cleaves PtdIns(4,5)P2 and thus gives rise to Ins(1,4,5)P3 and diacylglycerol. Ins(1 ,4,5)P3 mobilizes Ca2+ from the dense tubular system which allows myosin-lightchain-kinase activation, and hence the phosphorylation of the myosin light chain (P20); diacylglycerol activates protein kinase C which phosphorylates the 43-kDa protein (P43). It is conceivable that KRDS interferes with one of these plateletactivation steps. The present results do not support this hypothesis since (a) CaZ mobilization remains normal, (b) phospholipase C activation induced by thrombin was not modified, as assessed either by the observed PtdIns(4,5)P2 hydrolysis, as well as by phosphatidic acid synthesis, and (c) phosphorylation of P43 and P20 were normal. However a different hypothesis could be made concerning this peptidegranule-release inhibition. One hypothesis is that KRDS could affect an as-yet-undefined step occurring between protein phosphorylation and granule release. Ferrell and Martin [40] have recently shown that thrombin induces three waves of tyrosine phosphorylation in platelets. RGDS, which does not modify granule release, inhibits the third wave; platelets from Glanzman's thrombasthenia release normally and also do not express this third wave. It could be speculated that KRDS, which inhibits granule release, interferes with one of the first two waves which could be responsible of the granule release. Another hypothesis is that the tetrapeptide KRDS could also modify granule release by an effect independent of protein phosphorylation; such an inhibitory effect has already been shown [41]; however in this report the inhibitory molecule was not a peptide and was demonstrated to induce a membrane structural modification which could lead to modified fusion. KRDS also inhibited thrombin-induced serotonin release from thrombasthenic platelets (lacking platelet membrane GPIIb-IIIa). Thus KRDS would act on platelet membrane at +

49 a different site from RGDS, since RGDS binds to the platelet membrane fibrinogen receptor, GPIIb-IIIa [4,42]. This difference in the platelet membrane binding site was reinforced by the fact that RGDS inhibited the binding of the monoclonal antibody PAC-1 to stimulated platelets, while KRDS did not. Altogether, these results showed that KRDS is an inhibitor of serotonin release without interfering with steps known so far of the platelet signal-transduction mechanism: phospholipase C activation and protein phosphorylations. It is also a mild inhibitor of platelet aggregation and fibrinogen binding by a mechanism that may not necessarily require its direct binding to the GPIIb-IIIa complex. It would be interesting to investigate the mechanism of this GPIIb-IIIa-independent inhibition. It may be that KRDS interferes with one of the platelet aggregation proteins, such as fibrinogen or thrombospondin, or with the fibrinogen-thrombospondin interaction with the platelet, leading to modified platelet aggregation and release. Recently [13], in vivo experiments on laser-induced arteriolar thrombosis in the animal, have shown that KRDS inhibits thrombosis with an of 0.5 mg/kg, while a higher RGDS concentration (1 mg/kg) was necessary to induce the same relative inhibition. More interestingly, the two peptides have synergistic effects on thrombosis: using concentrations that induced less than 10% inhibition, both peptides injected at the same time induced 70% and 38% inhibition in the rat and the guinea pig, respectively. These results on thrombosis inhibition, associated with the in vitro effects, reinforce the hypothesis that the two peptides may act on different sites. For these reasons, we believe that KRDS is a new peptide, different from RGDS, with potential antithrombotic action. We are most grateful to S. Shattil for providing the PAC-2 monoclonal antibody and to E. Savariau for the art work. This work has been supported by a CIDIL-CNIEL grant (no. 1151/86)

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