THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc
Vol. 268, No. 19, Issue of July 5, pp. 14322-14328, 1993 Printed in U.S.A.
A Peptide Ligand of the Human Thrombin Receptor Antagonizes a-Thrombin and Partially Activates Platelets* (Received for publication,January 12, 1993)
Ulla B. RasmussenSQ,Christian Gachetlf, YasminSchlesingerS, Daniel HanaulI, Philippe Ohlmannlf, Ellen Van Obberghen-Schilling11 ,Jacques Pouyssegurll, Jean-Pierre Cazenavelf, and Andrea PaviraniS From the $Department ofMolecular and Cellular Biology, Transgene, S. A., I I rue de Molsheim, F-67082 Strasbourg, the llCentre Regional de Transfusion Sanguine, Institut National de la Sante et de la Recherche Medicale U-311, Laboratoire d’Histocompatibilite, F-67085 Strasbourg,and the 11 Centre de Biochimie, Centre National de la Recherche Scientifique-Institut National de la Sante et de la Recherche Medicale, Parc Valrose, F-06108 Nice,France
The peptide YFLLRNP antagonizes the aggregation of humanplatelets when induced by low concentrations of a-thrombin or the thrombin receptor agonist peptide (SFLLRNP), demonstrating that it interacts specifically with the thrombin receptor. Platelets exposed to YFLLRNP show immediate shape change (pseudopod formation) and potentiation of the ADP and plateletactivating factor response, but no Ca2+mobilization, P47 (pleckstrin) phosphorylation, secretion, or aggregation. Thus, YFLLRNP induces a state of partial activation of the platelets. Furthermore, with platelets prestimulated with adrenalin (10 PM), YFLLRNP induces aggregation, but no secretion, and only in the presence of added fibrinogen. We also found that prostacyclin inhibits the YFLLRNP-inducedshape change; but EDTA, aspirin, and apyrase (ADP scavenger) do not. Thus, the thrombin receptor in platelets may communicate, independently of Ca2+mobilization and P47 phosphorylation (protein kinase C activation),with intracellular signaling mechanisms that 1) modulate the cytoskeleton structure, 2) potentiate other platelet responses, and 3) stimulate coupling between the thrombin receptor and fibrinogen binding (the glycoprotein IIb-IIIa complex). YFLLRNP may be useful for differentiatingbetweenseveral possible activation states of the platelet thrombin receptor.
is ultimately followed by irreversible aggregation. A unique activation mechanismof a recently cloned thrombin receptor has made itpossible to studysome of the biological consequences specific for its activation (4-6). The thrombinreceptor, a seven transmembranedomain G proteincoupled receptor, is fully activated by a peptide (SFLLRNP) that consists of the tethered ligand that is exposed after activation by a-thrombin cleavage (4, 7). Receptor activation by this agonist peptide(which must be 5 residues or longer to retain full agonist activity) has revealed that many of the athrombin short-term effects can be attributed to the cloned receptor. In human platelets, this includes activation of phospholipase C and phosphatidylinositol 3-kinase(8);inhibition of adenylate cyclase (9); tyrosine phosphorylation of several proteins (10); and full activation, secretion, and aggregation (4, 11, 12). The agonist peptide also has effects that parallel thea-thrombin-mediatedshort-term effects onfibroblasts (11, 13),erythroleukemia cells (14),endothelial cells (15), glomerular mesangial cells (16), and gastric smooth muscle cells (17). Furthermore, specific activation of the thrombin receptor by the agonist peptide induces growth of vascular smooth muscle cells (18) and causes shape changesin neural cells (19). Structure-activity relationships between various synthetic peptides and the thrombin receptor have beenstudiedin fibroblasts (20), platelets (10,21,22), and cells expressing the recombinantthrombinreceptor (21). We describe here a peptide, YFLLRNP, that antagonizes low concentrations of a-Thrombin, the central enzyme of the coagulation cascade, a-thrombin and the agonist peptide SFLLRNP in platelet is a serine proteinase that also possesses hormonal andgrowth aggregation. Interestingly,YFLLRNPalso induces partial factor activities. It exerts a diverse range of biological func- activation of human platelets, visible by shape change and tions that are essential to hemostasis and wound healing (1). pseudopod formation; but it is unable to fully activate the Characteristic intracellular responses to a-thrombin activa- platelets. We further analyzed some of the intracellular retion in platelets as well as in other cell types include imme- sponses that YFLLRNPinduces and itseffect on platelets in diate activation of phospholipases C and A, via G proteins combination with otherreagents. (2). Phospholipase C activation causes formation of 1,Z-diacylglycerol and inositol 1,4,5-trisphosphate. 1,2-DiacylglycMATERIALSANDMETHODS eroi activates protein kinase C, which in platelets results in Preparation and Aggregation of Human Platelets-Six volumes of phosphorylation of a 47-kDaprotein(P47orpleckstrin); blood were collected from healthy donors (no medication for at least inositol 1,4,5-trisphosphate mobilizes intracellular Ca2+, 10 days before donation) into 1 volume of acid/citrate/dextrose which activates Ca2+-dependentenzymes (3). Platelet activa- anticoagulant, and twice-washed plateletsuspensions were prepared tion by a-thrombin causes secretionof granule contents and as described (23). The final suspension medium was Tyrode’s buffer (137 mM NaCl, 2 mM KCI, 12 mM NaHC03,0.3 mM NaH2P0,1 mM * The costs of publication of this article were defrayed in part by MgC12, 2 mM CaCl,, 5.5 mM glucose,pH 7.35) containing 0.35% the payment of page charges. This article must therefore be hereby human albumin (Centre R6gionalde Transfusion Sanguine, Strasmarked “aduertisement” in accordance with 18 U.S.C. Section 1734 bourg, France)and 2 pg of apyrase/ml, a concentration that converted 0.25 PM ATP to AMP within 2 min at 37 “C. Apyrase (Centre solely t o indicate this fact. This work is dedicatedto the memory of the late Jean-Pierre Rigional de Transfusion Sanguine) was extracted from potato as described (24) and was added to avoid ADP receptor desensitization. Lecocq, without whom this work would not have been possible. Towhom correspondence should be addressed. Tel.: 33-88-27- Platelets were kept at 37 ‘C throughout the experiments. Platelet count was adjusted in the final suspensionto 3 X 105/pl. Aggregation 91-00; Fax: 33-88-22-58-07.
14322
A Thrombin Antagonist Peptide was measured a t 37 "C by a turbidimetric method in a dual-channel Payton aggregometer in a final volume of 0.5 ml with the indicated additions and with continuous stirring at 1100 rpm. In some experiments, platelets were treated with 1 mM aspirin for 15 min and subsequently centrifuged and resuspended in Tyrode's buffer. The efficacy of the aspirin treatment was checked by measuring arachidonic acid-induced platelet aggregation, which is fully abolished after treatment. The following reagents were used in the aggregation assays: purified human a-thrombin (Centre RBgional de Transfusion Sanguine); human fibrinogen (AB Kabi, Stockholm, Sweden); PAF' (Bachem, Bubendorf, Switzerland); PGI,, adrenalin, and ADP (Sigma); and synthetic peptides >95% pure (Neosystem, Strasbourg, France) solubilized in double-distilled water. Preparation of Human Platelets for Transmission Electron Microscopy-Samples of the platelet suspension (1ml) were stirred at 37 "C in the aggregometer and activated by addition of a-thrombin or peptides at the indicated concentrations. At the indicated times, samples were fixed by adding an equal volume of fixative solution previously warmed to 37 "C and composed of 1.5% glutaraldehyde in 0.1 M sodium cacodylate buffer containing 1%sucrose, pH 7.3. After 5 min, the mixtures were centrifuged for 15 s at 8000 X g in an Eppendorf centrifuge. Supernatants were discarded, and the platelet pellets were resuspended and further fixed for 1 h a t 37 'C with the same fixative solution. After an additional centrifugation, the platelets were incubated for 1 h at room temperature with 1%tannic acid in 0.05 M sodium cacodylate buffer, pH 7.0. The fixed platelet suspensions were then post-fixed for 1 h at 4 "C with 1% osmium tetroxide in 0.1 M sodium cacodylate buffer, pH 7.3. They were then washed once in 0.1 M sodium cacodylate buffer, pH 7.3, for 10 min at room temperature and thereafter stained for 1 h at 4 "C with a 1% aqueous solution of uranyl acetate, followed by dehydration in successively increasing concentrations of ethanol (50, 70, 80, 95, and 100%). Finally, theplatelets were incubated overnight in Epona/ absolute alcohol (1:1,v/v) and embedded in
[email protected] sections stained with lead citrate were examined under a Siemens Elmiscop 102 electron microscope (60 kV). Intracellular CaZ+Mobilization-The cytoplasmic free Ca" concentration ([Ca"],) was measured using the fluorescent Ca2+indicator fura-2. The platelets were loaded with fura-2 by incubation at 37 "C with 1 p~ fura-2/acetoxymethyl ester (Molecular Probes, Inc., Eugene, OR) for 45 min during the first step of the washing procedure. The first two washes were performed with Tyrode/Hepes buffer (5 m M Hepes) without Ca" or albumin. The platelets were finally suspended in Tyrode/Hepes buffer containing 0.1% bovine albumin and 2 pg of apyrase/ml, and the platelet count was adjusted to 3 X 105/pl. Two-ml aliquots of the platelet suspension were transferred into a quartz cuvette with a magnetic stirrer, which was placed in a thermostatically controlled chamber at 37 "C in a PTI Delta-Scan-1 fluorescence spectrophotometer (Photon Technology International, Inc., Princeton, NJ). Reagents were added directly into the cuvette at the indicated times and concentrations. During the experiments, the excitation wavelengths were altered between 340 and 380 nm (for calculation of [Ca"],) or between 340 and 360 nm (for visualization of Mn2+quenching). The fluorescence was measured by recording the emitted light at 510 nm. Bandwidths were 4 nm on both excitation and emission monochromators. Maximum (1mM Ca") and minimum (10 mM EGTA) fluorescence levels were determined at the end of each experiment by sequentially adding Triton X-100 (0.1% final concentration) and EGTA to thecuvette. The ratio of the fluorescence readings was calculated as R = 340/380 nm and processed according tothe equation [Ca'+], = KD((R - Rmin)/(Rmax- R ) ) ( S m / S b 2to) calculate the intraplatelet free Ca2+ concentration (25). The KD for fura-2 was assumed to be 224 nM. RmaXand Rminare the maximum and minimum fluorescence ratios measured a t the end of the experiment, respectively; Sm and S b 2 are the fluorescence values at 380 nm in the absence and presence of saturating [Ca2+], respectively. Pleckstrin Phosphorylation-Freshly prepared platelets from 150 mlof bloodwere washed in phosphate-free Tyrode/Hepes buffer containing 0.35% human albumin and 1p~ PGI, and resuspended in the same buffer containing 2 mCi of carrier-free 3'POi!+. The platelet suspension was incubated for 1 h at 37 "C. The platelets were then washed in the same buffer and finally resuspended in Tyrode/Hepes buffer, 0.1% human albumin, and 2 pg apyrase/ml at a concentration of 5 X 10' platelets/pl. One-ml samples of the platelet suspension were stirred (1100 rpm) at 37 "C in the aggregometer cuvette and
' The abbreviations used are: PAF, platelet-activating factor; PGI,, prostacyclin; SCCS, surface-connected canalicular system; GP, glycoprotein.
Partially Activates Platelets
14323
activated as indicated. The reaction was stopped by transferring 0.9 ml of platelet suspension into 0.1 ml(3 N) of HClO, and rapid mixing. Precipitated protein was centrifuged at 8000 x g for 45 s; washed in Tyrode's buffer; and dissolved in 150 p1 of 2% SDS, 40 mM Tris, 25% glycerol, 2 mM N-ethylmaleimide, and 5% mercaptoethanol. After heating (100 "C, 15 min), samples (each containing 50 pg of protein) were then electrophoresed with size markers on discontinuous SDSpolyacrylamide (7-13%) gel, which was stained, dried, and exposed to Kodak X-Omat films. Bands of interest were excised and incubated in 30% H,O, at 40 "C overnight before liquid scintillation counting. RESULTS
Inhibition of Platelet Aggregation-We have previously tested a series of peptides for their ability to activate the thrombin receptorby comparing stimulationof phospholipase C, inhibition of adenylate cyclase, and stimulation of DNA synthesis in fibroblasts (20). Peptides that displayed no agonist activity or with decreased activity compared to thewildtypepeptide(SFLLRNP) were YFLLRNP,propFLLRNP (theNH,-terminalserine exchanged with propionic acid), FLLRNP, SALLRNP, SLLRNP, and SFLRNP. These peptides were all tested(200 p ~for) potential antagonisticeffects on a-thrombin-induced (0.25 nM) platelet aggregation. Only the heptapeptide YFLLRNP antagonized this effect. Fig. 1 shows that YFLLRNP at 100 p~ or higher concentrations attenuates platelet aggregation when induced by low concentrations of a-thrombin (0.1-0.5nM) (Fig. 1, A and B ) or SFLLRNP (5-10 p ~ (Fig. ) IC). Inhibition of the SFLLRNPa-thrombin-induced and aggregation indicates that YFLLRNPinteracts specifically with the cloned platelet thrombin receptor. Furthermore, YFLLRNP induced platelet shape change as monitoredby an increase in turbidity of the platelet suspension when 250 p~ peptide was added (Fig. 1A). The peptide was then testedfor a potential agonisteffect on platelets at concentrations up to 400 pM. However, no platelet aggregation was observed with stirring in the presence of 0.8 mg of fibrinogen/ml during continuousrecording for 30 min a t 37 "C, conditions under which the antagonisteffect of YFLLRNP remains unchanged (data not shown). UltrastructuralAspects of Unstimulated and StimulatedHuman Platelets-Unstimulated human platelets (Fig. 2A ) have a typical discoid appearance supported by a circumferential bundle of microtubules located just under the platelet surface. Granules,electron-dense bodies,occasional mitochondria, and glycogen particlesaredispersedthroughoutthecytoplasm. Channels of the surface-connected canalicular system (SCCS) are continuous with the cell surface and appear as unconnected vacuoles or canaliculi in the cytoplasm. The lumina of theSCCSarenotstainedintheunstimulated platelets. Platelets treated for 10 s with a 200 p~ concentration of an unrelated peptide show no change in morphology (Fig. 2, compare B with A). However, addition for 10 s of 200 p~ YFLLRNP has a specific effect on the platelets, which can be visualized by shape change and formation of pseudoa centralization of the pods (Fig. 2C). Insomeplatelets, granules occurs, but no change in either granule contents or morphology of the SCCS is seen. When the fixation of the YFLLRNP-treatedplatelets is performed2 minafterthe addition of the peptide, the changes in platelet morphology appear less pronounced (Fig. 2 0 ) . Addition for 10 s of 5 p~ SFLLRNP (Fig. 2E) or 0.2nM a-thrombin (Fig. 2F) induces pseudopod formation; intensely stained granules (by tannic acid/osmium); and communicationbetween granules and the SCCS, which becomes dilated and filled with a fibrillar electron-dense material that sometimes appears t o be extruded from the plateletsurface. Intracellular Responses to YFLLRNP-Since Figs. 1 and 2 show that YFLLRNP partially activates platelets but that it is also an antagonist to a-thrombin and SFLLRNP,we ana-
A Thrombin Antagonist
14324
Pepticie Partially Activates Platelets
Ca2+release nor Mn'+ entry (no quenching of signal), but it inhibits bothwhen platelets are subsequently stimulated with 0.25 nM a-thrombin (Fig. 3B). A similar result was obtained when platelets were stimulated with5 p~ SFLLRNP instead of a-thrombin (data not shown). These results show that 200 pM YFLLRNP does not induce intraplatelet Ca2+ mobilization;rather,itinhibitsthea-thrombin-andSFLLRNPinduced intraplatelet Ca2+release and Ca2+ entry at concentrations at which it inhibits aggregation. t P47 (Pleckstrin) Phosphorylation-The47-kDa protein 200 0,50 pleckstrin is a substrate for protein kinase C, for which three isoformshavebeen characterized in platelets to date (29). 20 0.13/ Protein kinase C isoforms are activated by several different platelet agonists, including a-thrombin, PAF, collagen, and 200 arachidonic acid, but not adrenalin (30). Fig. 4 shows that PM 10 200 pM YFLLRNP does not induce phosphorylation of pleckYFLLRNP 40 0,13 a-thrombin strinduring a 2-minincubation period,whereas 5 WM SFLLRNP and 20.2 nM a-thrombin do. All samples were analyzed at 20 s (shown in Fig. 4), 1 min, and 2 min after stimulation; but only minor differences in the intensities of 50 0.13 the bands were noted. In addition, ADP, adrenalin, and the t ) YFLLRNP (200 p ~ ) combination of adrenalin (10 p ~ and 200 do not cause pleckstrin phosphorylation, irrespective of the order in which they are added to the platelets (Fig. 4). 100 0.13 Potentiation of Platelets by YFLLRNP-Adrenalin does not cause Ca2+mobilization (or pleckstrin phosphorylation) (Fig. 4) in platelets; but it potentiates the effect on Ca2+ release, phosphorylation, fibrinogen binding, and aggregation of many 2do 0,13 t other agonists(30). Fig. 5 showsthat when 200 p~ YFLLRNP PM nM 200 YFLLRNP a-thrombin PM is added to the platelets in the presence of 0.8 mg/ml fibrinYFLLRNP ogen, shape change (but noaggregation) occurs, and a subsequent addition of 10 p~ adrenalin stilldoes not induce aggre1 min. I gation (Fig. 5A, trace b). When 10 p~ adrenalin is added first 10 and then followed by 200 p~ YFLLRNP, immediate andfull rM SFLLRNP aggregation takes place, but only when fibrinogen is present FIG. 1. Inhibition of platelet aggregation by YFLLRNP. (Fig. 5A, traces a and c). This suggests that activation of the The platelets were preincubated with YFLLRNP at the indicated a,-adrenergicreceptor potentiatesthethrombin receptor, concentrations before aggregation was initiated by adding a-thrombin consistent with earlier findings (30). However, it seems that ( A and B ) or SFLLRNP (C) at theindicated concentrations. A , dose the YFLLRNP-induced shape change renders the thrombin response of YFLLRNP inhibition with a fixed concentration of athrombin (0.13 nM); B, dose response of wthrombin-induced aggre- receptor inaccessible tosubsequentadrenalinpotentiation gation with a fixed concentration of YFLLRNP (200 p ~ ) C; , dose (Fig. 5A, trace b). response of SFLLRNP-induced aggregation with a fixed concentraADP is a platelet agonist thatinduces rapid transient Ca2+ tion of YFLLRNP (200 PM). The results are representative of three mobilization and activation of G proteins (31), but nopleckseparate experiments that showed slight variations in the dose re- strin phosphorylation (Fig. 4). In the presenceof fibrinogen, sponses, depending on the platelet donor. Note the immediate change it causes reversible platelet aggregation (32). Fig. 5B shows in turbidity of the platelet suspension when 250 PM YFLLRNP was the reversible aggregation when 5 p~ ADP is added to the added. platelets (trace b); but when 200 p~ YFLLRNP is added to lyzed the intracellular responses that this peptide may induce the platelets prior to ADP, anirreversible aggregation occurs (trace a). Thus, YFLLRNP potentiates the ADP signaling or inhibit. Intraplatelet Cu2+Mobilization-Intraplatelet Ca2+mobili- pathway. Moreover, this effect is not specific for the ADP zation is believed to be a necessary step to aggregation (26). response since the platelet PAF response is also potentiated Fig. 3A shows that in the presence of 1 mM Ca2+, 200 pM by YFLLRNP, although to a lesser degree at the concentraYFLLRNP induces no change in the intraplatelet Ca2+ con- tions used (Fig. 5B, traces c and d ) . Secretion of the platelet granule content was assayed by centration 50 s after its addition. Furthermore, Ca2+mobilization is inhibitedby -50% when 200 p~ YFLLRNP is added measuring secreted tritiated 5-hydroxytryptamine. Whereas to the plateletsbefore 0.25 nM a-thrombin (Fig. 3A) or 5 pM 0.2 nM a-thrombin and 5 p~ SFLLRNP induced -80% seSFLLRNP (data notshown). 200 p~ YFLLRNP, the concen- cretion, neither200 p~ YFLLRNP alone nor the combination tration at which it inhibitedaggregation (Fig.l),also inhibited of 0.8 mg of fibrinogen/ml, 10 p~ adrenalin, and 200 p M YFLLRNP induced secretion of 5-hydroxytryptamine,althe a-thrombin- and SFLLRNP-induced intraplatelet Ca2+ release when external Ca2+was absent (in the presence of 1 though aggregation occurred (data not shown). Inhibition of YFLLRNP Effect-Both the shape change and mM EGTA) (data not shown). Mn2+ quenches the fluorescence of Ca2+/fura-2, and it has been shown to enter platelets the YFLLRNP-inducedaggregation of platelets preincubated in the same manner as Ca2+ when stimulated by agonists (27, with fibrinogen and adrenalin are unaffected by the ADP 28). Fig. 3B shows the fluorescence a t two excitation wave- scavenger apyrase (10 pg/ml, a concentration that blocked lengths, 340 nm (Ca2+-dependent) and 360 nm(Ca2+-inde- the aggregation induced by 5 p~ ADP). This indicates that caused by ADPsecretion when pendent), when platelets are stimulated with YFLLRNP andthe aggregation isnot As YFLLRNP is added (data not shown). The YFLLRNP-ina-thrombin in the presenceof 100 p~ Mn2+, but no Ca2+. duced shape change is independent of Ca2+ in the medium expected, 200 p~ YFLLRNPinducesneitherintraplatelet
v "'
Ib
f
-
Thrombin A Antagonist Peptide Partially Activates Platelets
14325
0
r
F
FIG. 2. Platelet ultrastructure before and after stimulation. Platelets were prepared for electron microscopy as described under "Materials and Methods." They were stimulated with incubation buffer (control) ( A ) , 200 PM unrelated peptide (8 amino acids) ( B ) , 200 p M YFLLRNP (C and D),5 PM SFLLRNP ( E ) ,and 0.2 nM a-thrombin (F).D was fixed 2 rnin after addition of the peptide, whereas all other samples shown were fixed a t 10 s after stimulation. Arrows indicate the SCCS; open arrowheads indicate storage granules; and closed arrowheads indicate pseudopods. Note the discoid appearance of the platelets in A and I3 and thepresence of pseudopods in C and D, but no degranulation or dilation of the SCCS. In E and F,there are pseudopods and degranulation and dilation of the SCCS in which lumina are and X 35,000 ( E and F). filled with a fibrillar material. Magnifications are X 21,000 (A-C), X 28,000 (D),
Thrombin A Antagonist Peptide Partially Activates
14326
Platelets
a-lhrmbln O
l
, 50150
, 100
,
,
I
l
,
,
250
200 Smds
a-lhrombin
0
50
100
150
Seconds
FIG. 3. Effect of YFLLRNP on platelet Ca2+ mobilization. A, effects of a-thrombin (0.25 nM) (thick trace) and of YFLLRNP ) subsequent a-thrombin stimulation (0.25 nM) (thin (200 p ~ with trace) on intraplatelet calcium concentration in the presence of 1mM CaCI2 in the medium. The intraplatelet Ca2+ concentration was calculated as described under “Materials and Methods.” B, effects of ) a-thrombin (0.25 nM) (thick traces) and of YFLLRNP (200 p ~with subsequent a-thrombin stimulation (0.25 nM) (thin traces) on fura-2 fluorescence in the presence of 100 pM MnCI2 and no CaCI2 in the medium. Lower traces are at an excitation wavelength of 340 nm (Ca”-dependent), and upper traces are a t 360 nm (Ca2+-independent) (Sharp peaks appearing at thetimes of addition are due to insertion of the pipette tip into thecuvette.) The results are representative of three experiments with platelets obtained from different donors.
FIG. 4. P47 (pleckstrin) phosphorylationin platelets. Shown is an autoradiograph of :’2P-labeledpolypeptides obtained after stimulation of platelets for 20 s with 200 p~ YFLLRNP incubation buffer (Control); 5 p M SFLLRNP; 0.2 nM a-thrombin; 2.0nM a-thrombin; 10 pM adrenalin (Adr.); 200 p M YFLLRNP ( Y F ) followed by 10 p~ adrenalin; 10 p~ adrenalin followed by 200 p~ YFLLRNP; and 5 p~ ADP, respectively. All samples were analyzed after stimulation for 20 s, 1 min (data not shown), and 2 min (data not shown), but only minor differences in induction of pleckstrin phosphorylation were observed. The counts obtained from the excised pleckstrin bands and values obtained from scanning of the autoradiographs were consistent with the results shown. The same results were obtained with platelets prepared separately from two different donors.
of the -CH,OH group. This modification changes the peptide froma powerful plateletagonist into a weak a-thrombin antagonist unable to induce platelet aggregation even at 400 pM (5 pM is sufficient for SFLLRNP) in the presence of fibrinogen and after prolonged incubation periods (30 min). YFLLRNP probably binds to the receptor with low affinity, resulting in only weak antagonism. Although YFLLRNP is an a-thrombin and SFLLRNP antagonist (Fig. l), it also induces a transient shapechange in platelets, which revert to a stable morphology, but no secretion or aggregation (Fig. 2). since it occurs in the presence of 5 mM EDTA; however, no In addition, it potentiates the ADP- and PAF-induced platelet aggregation is induced when the platelets are preincubated responses. When the platelets are preincubated with fibrinowith fibrinogen and adrenalin, probably because of the re- gen and adrenalin, it induces aggregation (Fig. 5 ) , but without quirement of Ca2+for fibrinogen binding (Fig. 5C, truces u measurable secretion of granule content. These effects of and b). YFLLRNP areclearly demonstrated. In addition, it does not PGI2 is known to raise the intraplateletCAMP level and to activate protein kinase C (no pleckstrin phosphorylation) or inhibit platelet aggregation. PG12 totally inhibits the induce Ca2+ mobilization, butrather inhibitsa-thrombinYFLLRNP-induced platelet shape change (Fig. 5C, truce c) induced intracellular Ca2+release and entry (Figs. 3 and 4). and also the YFLLRNP-induced aggregation of platelets The side groups in the agonist peptide that are necessary preincubated with fibrinogen and adrenalin (truce d ) . Thus, for activation of the thrombin receptor are theNH2-terminal the platelet response to 200 p~ YFLLRNP is caused by amino group (but not the side chain); the aromatic group on factors that arecontrolled by CAMP-mediated effects. the second-position phenylalanine, which is the most imporAspirin inhibits the cyclooxygenaseand endoperoxide path- tant for the activating capacity of the agonist peptide; and ways (formation of thromboxane A2 from arachidonic acid). the fourth- and/or fifth-position leucine and arginine side A control showed that aggregation was completely inhibited chains (10, 20-22). A specific conformation of these side when 100 p~ arachidonic acid was added to the aspirin- groups and the NH2-terminal amino group are likely to fit in treated platelets (data not shown). Aspirin-treated platelets a pocket formed by the receptor. The most plausible interpreshow shape change when stimulated with 200 p~ YFLLRNP tation of the effects of YFLLRNP on the thrombin receptor (Fig. 5C, truce e); but the aggregation is inhibited when the is therefore that thearomatic side chain of the tyrosine “fits” platelets are preincubated with fibrinogen and adrenalin in a place where it is able to prevent a complete conforma(truce f ) , although 400 p~ YFLLRNP overcomes the aspirin tional change of the receptor, which is necessary for aggreinhibition (data not shown). gation, while a partial change is possible. This “partial conformational change” of the thrombin receptor leads to platelet DISCUSSION shape change and potentiation of other platelet responses, YFLLRNP is a peptide that differs from the platelet throm- but not to activation of G protein-coupled Ca2+mobilization bin receptor agonist peptide SFLLRNP in that it has an and protein kinase C activation (via phospholipase C) (2, 3). aromatic side group on the NH2-terminal amino acid instead The effects of a partial activationof the thrombin receptor
A Thrombin Antagonist Peptide Partially Activates Platelets
a. H f g EDTA
pY F
Buf b.
i t
\
“yF Hfg P G l s ~ u f
t l
d.
C.
Buf Adr yF Hfg
.%t ’\ mi,
Y F
Adr
Hz ASP
e.
\
-
1 min.
FIG. 5. Potentiation and inhibition of the platelet YFLLRNP response. The indicated reagents were added to platelet suspensions in the aggregometer cuvette at the following final concentrations: 0.8 mg of human fibrinogen (Hfg)/ml, 200 PM YFLLRNP ( Y F ) ,10 PM adrenalin (Adr), 5 PM ADP, 20 nM PAF, 5 mM EDTA, 0.1 PM PGI,, and incubation buffer (BJ).Aspirin (Asp) treatment of the platelets was performed as described under “Materials and Methods.” The same resultswere obtained from two or more different donors.
14327
exposure of the fibrinogen-binding sites either (since no aggregation occurs), but it does when the platelets are prestimulated with adrenalin (Fig. 5 A ) . In other words, it is likely that the aggregation that is caused by YFLLRNP (but only when the platelets are pretreated with adrenalin)is initiated by fibrinogen binding to the GPIIb-IIIa complex as soon as binding site exposureis induced by YFLLRNP binding to the thrombin receptor (Fig. 5 A ) . Fibrinogen binding to its receptor on the platelet surface is a key step in the aggregation process. Platelets from patients lacking the fibrinogen receptor (Glanzmann’s thrombasthenia) are unable to aggregate in response to any agonist, Thefibrinogen receptor in platelets, GPIIb-IIIa (also called arr&),is a Caz+-dependent heterodimer complex and a member of the integrinfamily of adhesion receptors (33). The mechanism leading to the activation of this receptor that enables it to bindfibrinogen after platelet stimulation by an agonist is notfully understood. NeitheradrenalinnorYFLLRNP (or thecombination) activatesproteinkinase C (Fig. 4), which is essentialto platelet secretion (26, 34). The platelet aggregation that is induced by YFLLRNP after preincubation with fibrinogen and adrenalin alsooccurs without secretion. Asimilar platelet response (aggregation without secretion) is induced by fibrinogen and ADP (Fig. 5B, truce b ) (32), and thiswas shown to involve activation of an as yet unidentified G protein (31). How the combination of adrenalin and YFLLRNP causes platelet aggregation and evidently activation of the GPIIbIIIa complex in the presenseof fibrinogen still requires investigation. It is likely that it involves activation of a G protein different from the G protein thatcouples to phospholipase C. Thus, YFLLRNP is useful for discriminating between these two signaling pathways that are activated by the thrombin receptor. In summary, we have shown that the peptide YFLLRNP 1) is an antagonist to a-thrombin and SFLLRNP (thrombin receptor agonist peptide)in human platelets,2) induces plateletshapechangewithoutCa2+ mobilization orpleckstrin phosphorylation, 3) is able to potentiate other agonist-induced platelet responses, and 4) induces adrenalin-treated platelets to aggregate without secretion and only in the presence of fibrinogen. Thus, YFLLRNP may be a useful tool for differentiating betweenseveralpossible activation states of the humanplateletthrombin receptor, andit may provide a structural template for developing more efficient antagonists targeted to the thrombinreceptor. Acknowledgments-The cooperation of D. Colin (Institute of Mi-
may allow us to differentiate some of the steps in the normallycrobiology, Strasbourg, France) with regard to the fluorescence specrapid andcomplex platelet activation and aggregation process. trophotometry is highly appreciated. We also thank K.-U. Lentes and R.Bischoff for valuable discussions, B. Heller for preparing the First,plateletshapechangeorcytoskeletalrearrangement figures, and M. Courtney for critically reading the manuscript. can occur without Ca2+ mobilization and protein kinase C activation. Besidesbeingcoupled to the classicG proteinREFERENCES mediatedsignalingpathways,thethrombinreceptor may 1. Fenton, J. W., I1 (1988) Semin. Thromb. Hemostasis 14,234-240 thereforemodulateother cellular components involved in 2. Pouyssbgur, J., and Seuwen, K. (1992) Annu. Reu. Physiol. 64,195-210 3. Lapetina, E. G . (1990) FEBS Lett. 268,400-404 cytoskeleton rearrangement and potentiationof other recep4. Vu, T.-K. H., Hung, D. T., Wheaton, V. I., and Coughlin, S. R. (1991) Cell tors. Modification of cellular architecture asa consequence of 64, 1057-1068 U. B., Vouret-Craviari, V., Jallat, S., Schlesinger, Y., Pages, thrombin receptor activation has also been observed in neural 5. Rasmussen, G., Pavirani, A., Lecocq, J.-P. Pouyssigur J., and Van Obberghencells. Cell rounding and neurite retractionoccur independent Schilling, E. (1991) FEBS Lett. 288, 123-12k 6. Zhong, C., Hayzer, D. J., Corson, M. A,, and Runge, M. S. (1992) J. Biol. of Ca2+ mobilization and protein kinase C activation when Chem. 267, 16975-16979 neural cells are exposed to the thrombin receptor agonist 7. Vu, T.-K. H., Wheaton, V. I., Hung, D. T., Charo, I., and Coughlin, S. R. (1991) Nature 363,674-677 peptide, amorphological responsethat was showntobe 8. Huang, R., Sorisky, A,, Church, W. R., Simons, E. R., and Rittenhouse, S. blocked by inhibitors to tyrosine phosphatases and to broad E. (1991) J. Biol. Chem. 266, 18435-18438 9. Seiler, S. M., Michel, I. M., and Fenton, J. W.,I1 (1992) Biochem. Biophys. specificity kinases (19). Second, potentiation of platelet reRes. Commun. 182, 1296-1302 sponses by adrenalin may involve stimulation of the coupling 10. Vassallo, R. R., Jr., Kieber-Emmons, T., Cichowski, K., and Brass, L. F. (1992) J. B i d . Chem. 267,6081-6085 between the thrombin receptor and the fibrinogen receptor 11. Vouret-Craviari, V., Van Obberghen-Schilling, E., Rasmussen, U. B., Pa(the GPIIb-IIIacomplex) (33). Adrenalin alone, in our hands, virani, A,, Lecocq, J.-p., and Pouyssegur, J. (1992) Mol. Bid. Cell 3,95102 does not expose fibrinogen-binding sites on the GPIIb-IIIa 12. Hung, D. T.,Vu, T.-K. H., Wheaton, V. I., Ishii, K., and Coughlin, S. R. complex (30). YFLLRNP alone probably does not lead to (1992) J. Clin. Inuest. 89, 1350-1353
14328
A Thrombin Antagonist Peptide Partially Activates Platelets
13. Vouret-Craviari, E., Van Obherghen-Schilling, E., Scimeca, J. C., Van Obberghen, E., and Pouyssegur, J. (1993) Biochem. J. 2 8 9 , 209-214 14. Brass, L. F. (1992) J. B i d . Chem. 267,6044-6050 15. Ngaiza, J. R., and Jaffe, E. A. (1991) Blochem. Biophys. Res. Commun. 17Q 1CC.C-lCCl A , V I LYYY-lYYl
16. Troyer, D., Padilla, R., Smith, T., Kreisberg, J., and Glass, W., I1 (1992) J. B~ol.Chem. 267. 20126-20131 17. Hollenberg, M. D.', Yang, S. G., Laniyonu, A. A,, Moore, G. J., and Saifeddine M. (1992) Mol. Pharmacol. 42, 186-191 18. Herbert, L. M., Lamarche, I., and Dol, F. (1992) FEBS Lett. 301,155-158 19. Suidan. H. S.. Stone. S. R.. Hemmines. - . B. A.. andMonard. D. (1992) Neuron 8,363-375' 20. Van Ohher&y-Schi!ling, E: Rasmussen, U. B., Vouret-Craviari, V., Lentes, U., Pavlranl, A. andPouyssegur, J. (1993) Eiochem. J., in press 21. Scarborough, R. M., Naughton, M. A,, Teng, W., Hung, D. T., Rose, J., Vu, T.-K. H., Wheaton, V. I., Turck, C. W., and Coughlin, S. R. (1992) J. Biol. Chem. 267,13146-13149 22. Chao, B. H.,Kalkunte, S., Maraganore, J. M., and Stone, S. R.(1992) Biochemistry 31,6175-6178
23. Cazenave, J.-P., Hemmendinger,S., Beretz, A,, Sutter-Bay, A,, and Launay, J. (1983) Ann. Biol. Clin. 4 1 , 167-179 24. Molnard, J., and Lorand, L. (1961)Arch. Biochem. Biophys. 93,353-363 25. Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) J. Biol. Chem. 2 6 0 , 3440-3450 26. Siess, W. (1989) Physiol. Reu. 6 9 , 58-178 27. Neylon, C. B., and Irvine, R. F. (1991) J. Biol. Chem. 266,4251-4256 28. Merritt, J. E., and Hallam, T. J. (1988) J. B i d . Chem. 263, 6161-6164 29. Grabarek, J., Raychowdhury, M., Ravid, K., Kent, K. C., Newman, P. J., and Ware, J. A. (1992) J. Biol. Chem. 2 6 7 , 10011-10017 30. Lanza, F., Beretz, A., Stierle, A., Hanau, D., Kuhina, M., and Cazenave,J.P. (1988) Am. J. Physiol. 255, H1276-H1288 31. Gachet, C., Cazenave, J.-P., Ohlmann, P., Hilf,G., Wieland, T., and Jakohs, K. H. (1992) Eur. J. Biochem. 2 0 7 , 259-263 32. Gachet, C., and Cazenave, J.-P. (1991) Nouu. Reu. Fr. Hematol. 3 3 , 347358 33. Phillips, D. R., Charo, I. F., and Scarborough, R. M. (1991) Cell 6 5 , 359362 34. Nishizuka, Y. (1986) Science 2 3 3 , 305-312
