Bioclff~dca et Biophysica Acta. il37 (1992177-81

77

© 1992IEL~-vierSciencePublishersB.V..'L~Ifightsreserved0167-4889/92/$05.00

BBAM~ 10295

Rapid Report

Photoaffinity labeling of ~.ntegr~no~IIb/33 (glycoprotein IIb-IIIa) on intact platelets ~.th 8-azido-[~,-32p]ATP 'i P~,tcr M a y i n g e r a n d M e i n r a d G a w a z • Mrdizinis~.e

~lCYhdkl. Kuldkurn Groflhadern de; Lud~q~ Ya.~rL,rdl~r.s Unk*ersitSt Miinchen, MFmcl-.en.(German}.)

Keywords: Platelet;GPllb-IIla; Integrinalib/$3; Nucleotidebinding;PhotoaffinLWlabeling;,8-azido-ATP The fibdnogen receptor GFllb-!lla plays a crucial role in plateiet aggregation. Here we show that the adenine nucleotide, ~a:zido-ATP, inh~its ADP-indu~.v.dconformational change of the platelct fibrinogen recqator GPllb-llla (integrin aIib/331. Photoafflnity labeling of intact platelcts with 8-azido-[y-3ZP]ATP exclusively modifies two plasma-membraneglyceproteins which are identical with both sebanits of GPllb-llla. The presence of adenine-nucleotide-binding sites on GPllb-llta implies that the platelet fibrlnogen receptor is directly regu!atcd by cxtraccllular adenine 'nucieotides.

Piatelet aggregation is a crucial step in primary he.,nostasis. Although the impoffance of extraceUular adenine nucleot~des, i.e, A D P and ATP, for platelet aggregation is well established [1,2] there are conflicting data concerning the plasma-membrane receptor involved in ADP-induced platelet response [3-5]. The adenosine analog 5"-fluorosuifonylbenzoyl adenosine has been shown to label a 100-kDa protein on intact platetets [4]. Recently, a nucleotide-binding site on glycoprotein lib has been reported by direct photoaffinity labeling with [35S]ATP-rI-S [5]. However, the physiological importam~ of these proteins, for ADPmediated platelet aggregation has not been dearly demonstrated. The photoreactive ATP analog, 8-azidoATP, is a suitable reagent for probing nucleotide-binding sites in a variety, of different biological systems [6,7]. Recently, h was found that o°-bromo-ADP, a compo,~nd with highly related structure, is a strong antagonist of A D P binding to platelet plasma-membrane [~;]. In -his study we investigated the effects of 8*azido-ATP on ADP-induccd conformational change of platelet fibrinogen (tg) re¢c'pF:or GPllb-llla by use of conformation-dependent monoclonal antibodies and flow c y t o m e ~ , in addition, we performed photoaffimty labeling experiments of isolated intact pla'.elets using 8-azido~'y-32p]ATP to study nuclcotide-b[nding sites or, platelet plasma-membrane.

Correspo.~.~ to: IVL Gawaz, Medizinische Klinik l, Klinikum GmShadem. Lmt~g-MerJmilianstJn~-~rsit~iL,Marchio~iv.istl. 15, 8000-M~ac~a 70, Germa~..

Platelets were isolated from freshly drawn citrateanticoagulated human bl,.md by differential centrifugation followed by gel filtration on Sepharose 213 in divalent ion-free Tyrode's buffer containing 0.1% glucose, 2 m m o l / l MgCi2, 137.5 m m o l / l NaCI, 12 mmol/I NaHCO 3, 2.6 m m o l / i KC! (pH 7.4) without bovine serum albumin. For photoaffinity labeling platelets were suspended in divalent ion-free Tyrude's buffer at 4-108 cells/mL Photoaffinity labeling of intact platelets was performed as described [6]. 10 p.i of a freshly isolated platelet suspension were transferred to the wells of a microtiter plate and .~.upplemented with A D P or ATP as indicated. 8-azido-[7-32p]ATP was added in a final concentration of 100 p.M. After 5 rain incubation at 0°C the samples were irradiated individually with a 90-W Hg-lamp. To protect the platelets against p h o t ~ d a t i o n and UV-damage, the probes were flushed with nitrogen prior to irradiation and a chloroform filter was placed between fight source and sample. After labeling experiments plate!ets were solubilized in Tris buffer containing 3% $DS, 1 p.M I.eupeptin, and 1 mM phenyhnetbylsulfonyl fluoride (pH 7.0). Samples to be run under reducing conditions were treated with 2% mercaptoethanol for 3 min at 90°C. Proteins were fractionated by SDS-PAGE on 10% resolving slab gels [11]. Immunoblots were performed on nitrocellulose paper (BioRad) and decorated accoming to standard methods using 2% Tween as blocking r,:agent and peroxidase-conjdgated anti.mouse rabbit ~gG as secondary antibody. FACS analysis was periOrmed as described [10] on a FACScan (Becton Dickinson). 10000 particles were analyzed for each

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Membrane Immunfluor~scence Membrane Immunfluorescence F'tg. I. Effect of 8-azido-A'rP on the conformational change of Worinogenreceptor GP|lb-llla ~ a-gr~qula relez~e ~n~ysed by flow c)1ometri,: techniques. LIBSI and t h r o m ~ i n (TSP) expressi~ were used ~ parameters of ctmformational change of GPllb-lila (A) and a-granula release, re~czlz~.~./[10].Fresh human platelels were incubated with Iq0 p-M ADP (left upper p~nel). 500 pM 8-azido-ATP (right upper panel), 1O0/~M ADP and ! mM 8-azido-A'rP (tlght lower panel), and I(KJ/zM AIDP and I mM ATP (left lower panel). After addition of saturating com:enL,'ationsof fluorescein (FITC)-conjugated anti-LIBS! mAb. samples were incubated for 20 rain at w,mn temperature and a n l i ~ binding was measured by FACS anaIysh. HistogranLsof antibody binding in the presence of ~lenine nuc]eotid~ (solid line) have been superimposed upon hLstograrnsof b~ndingin their absence (dotted line). s a m p l e using C O N S O R T ~ scientific software (Becton Dickinson). m A b s 9 0 B B I 0 ( a n t i - G P l l l a ) a n d 9 8 D F G (anti-GPllb) used for immnnoblotting were generously provided by Prof. V i r t a n e n , University o f Helsinki, Finland. P m t i - L I B S l t a n t i - G P l l l a ) a n d a n t i - T S P l - I ( a n t i - t h r o m b o s p o n d i n ) w e r e t h e g e n e r o u s gift o f Dr. M a r k G i n s b c r g , C o m m i t t e e o n V a . ~ t l a r Biology, Scripps Clinic, L~ Jolla [10]. All m A b s u s e d f o r flow cytometrlc analysis w e r e c o n j u g a t e d with fluorescein a c c o r d i n g to s t a n d a r d methc.ds to achieve a fluoresc e n c e - t o - p r o t e i n r a t i o o f 2 : 3 . R u o r e s c e i n isothioc y a n a t e a n d the n u c l e o t i d c s A D P , A T P , a n d ~ a z i d o A T P w e r e p u ~ h a s e d front Sigma. 8-azido-['),-32P~A._TP w~s synthesized a c c o r d i n g to published m e t h o d [9] with modifications recently descn'bed [6]. X - r a y films (XO m a t A R ) w e r e f r o m K o d a k . All o t h e r r e a g e n t s we~c o f the highest p u r i t y available.

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Fig. 2. Inhibition of ADP-ind~Jcedconfermational change of GPilbIlia (LII~I expression) by A~'P and 8-azido-ATP. Isolated platelcts were incubated with ranting o~lacentrations of ATP and 8-~zido-ATP in the prescl~c of i00 M.M ADP. MBSI expression was determined using saturating :onccntrations of anti-UBSI antibmiy and a n t i b ~ ~,~Iding ,,,'as measared by FACS anabsis as described [IO], Ordinate represents percc~*~,o~eof maximal LIBSI expression ( ~ = LIBSi x IO0/LIBSI m~).

acts as a specific antagonist on ADP-bin,;iag sites iFig. 1A and B). Comparison of concentration-dependent inhibition of ADP-induced LIBS1 expression by 8azido-ATP and ATP indicates that 8-azido-ATP binds with similar affinity as does ATP to specific receptor sites on the platelet surface with half-maximal inhibition of ADP-induced LIBS1 expression at around 100 #M (Fig. 2). Photoaffinity labeling of intact platelets with 100 p,M 8-azido-[y-32p]ATP exclusively yielded two distinct bands of radioactivity with n:uic~ :.::r mobility of 120 and I00 kDa, respectively, under reducing condi*.ions (Fig. 3A). Incorooration of 8-azido-[y-32p]ATP was not observed without irridation (data not shown) and was completely blocked in the presence of 500/tM ATP or 8-azido-ATP, respectively (Fig. 3A). irridation of platelets with 8-azido-ATP ir the presence of 0.1% Triton X-100 results in additional labeling of the cytosl~eletal protein actin, indicating that only surface proteins are accessible to the photoaffinity label whe,,~ detergent is omitted (Fig. 3A). These experiments sb~v that 8-azido-[y-3zP]ATP modifies specifically r~o distinct plasma-membrane proteins on intact platelets

For GPllb-llla conformational change analysis a monoc!onal altibody, anti-LIBSl, was u~se0 that preferentially recognizes an epitope on GPllla which is exposed after ligand-induced conformationa! change of this glycoprotein has occurred [12]. in addition, a monoclonal antibody (anti-TSPi-l) against human platelet thrombospondin, an adhesive protein that is surfaceexpressed during a-granula release, w~,s used [10,13]. Application of both fluoreseein-labeled monoclonal antibodies in flow cytoraet~' ~llewed comparison of nucleotide-induced effects on the contbrmationai change of the fg receptor and on degranulation of plateiets. When platelets were incubated in the pzesence of 100 ttM ADP, a significant increase of ar~ti-LIBSl and anti-TSPl-1 binding can be observed, indicating that both conformafional change of GPllb-ll;a and a-granule release has occurred (Fig. IA and B). However, stimulation of platelets in concentrations between 100 /tM and 1 mivl 8-azido-ATP did not effect anti-LIBSl or anti-TSPl-i antibody binding (Fig. 13. a r J B). f~oin-cubation of 100 ~tM ADP with ! mM 8-azido-ATP results in almost complete inhibition of the ADP-induced platelet response, implying that 8-azido-ATP

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Fig. 3. Photoaflinity labelingof intact platelets with 8-az;flo- ~,-32PJATP.4- l0s p.'atelets/mlwere incubated with 100/xM8-azldo-[y-32P]ATP and itridiated with UV light.The sampleswere analyzedby SDS-PAGEand fluomgraphyusing10% slabgels. Each'lanewas loaded withequal amounlsof protein(30/,tg).(A) Lane I: Cooma.~ietquestain of total platelet proteins;lanes2-4: autorndiographyof photoaffinitylabelingwith 8-azido-h'-32p]ATP(lane 2), in the presenceof i mM ATP (lane 3) and of 0.1% Triton X-100(lane 4). (B) Lanes I and 2: Ct,oma~icI~;nestain of total platelet proteins separat-~l under reducing and ~onreducingconditions, respectively; lanes 3 and 4: conespondingfltc~rographies (reduced and nonreduced,respectively).

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Fig. 4. 8-azido-ATP-sensilivepIatelet membrane-proteinsare identical villi both subunitsof f'mrinogerreceptor GPIIb-illa. 8-azido-[y"2P]ATP photoaffinity-lal~ledintact platelets were soiubilizndand run ol3 i~,c slab gels under recIu~ngand nonreducingconditions. ~ely. Gel trat~fers were probed with monoclonaIantibodies to GPilb (mAb 98DFG)and to GPIIia (mAb~c~3-_nBI0),subsequenlb" (lanes i and 2)- Lanes 3 and ~ are autoradiogramsof the immur.oblot.

which may be involved in ADP-induced platelet aggregation. To further characterize the 8-azido-ATP-seositive membrane proteins, labeled platelets were solubilized and separated on SDS-PAGE under reducing and nonreducing conditions. As shown in Fig. 3B, a marked difference in the molecular mobiliW between nonreduced and reduced samples can be obs~,~ed. Staining of the gel with periodic Schiffs reagent reve~icd tlmt both radiolabeled proteins are glycuproteins (data not shown). Using monocional antibodies directed against the two subunits of the platelet fg receptor, GPllb and GPHla, we found in our immunoblotting studies that the 8-azido-ATP-sensiti~c ~'yc~proteins are identical with the a- and/]-sabunits of the. fg receptor GPllbllla (integnn ailb/]3) (Fig. 4). The major findings of this work are: (a) 8-azido-ATP is a specific antagonist of ADP-ioduced conforma~ional change of platelet fg receptor GPiib-llla (LIBSI express~.on) and a-granula release (TSP expression). (b) Labeling of intact platelets with g-azido-[y-32p]ATP

results in exclusive modification of two plasma-membrane glycoproteins which could be identified as aand/]-subunits of the fg receptor GPllb-llla (integrin allb/]3). A['~bough the hnpor~ance of A D P in platelet function is very well established [1,2] the molecular mechanistns of ADP-induced platelet aggregation and effects of extracellular nucleotides on platelet function is still poorly understood. Using the adenosine analogue FSBA, a 10t)-kDa plasma-membrane could be identified which seems to be related to ADP-induced fg-receptor activation [4]. However, the physiological relevance and the mechanism of activation of this protein has not yet been fully elucidated. Recent!y, a nuclcotida-binding site on glycoprotein lib has been desen'bed by photoaffinity labeling with [35S]ATP-~S [5]. Our investigation of nucleotide-receptor sites on the platelet plasma-membrane using 8-azido-ATP supports the presence of binding sites for adenine nucleotides on fg receptor GPllb-lIla. Moreover, 8-azidc-ATP inh~its ADP-induced conformatinnal change of GPllbIlia and u-granola release in a similar manner to ATP. Thus, 8-azido-ATP seemed to be a suitable reagent for probing adenine-nuc!--~otide-receptor sites on platelet membrane. Photoaffinity labeling of intact piatelets with 8-azido-[y-32p]ATP results in specific and exclusive labeling of both subunits of the fg receptor GPllb-llla. These data confirm the recently described presence of a nucleotide-binding site on GPIIb but in addition show interaction of nuclcot:4es with t h e / ] 3 subunit GPllla. The presence of nnclcotide-binding sites on both fg-receptor ,~abunits and the fact that 8-azido-ATP antagonizes the ADP-induced conformational change of these glycoproteins tempted us to speculate that the fg receptor is directly regulated by extracellular adenine noclcotides. This regulation involves a conformational change of GPilb-llla which pot only results ~ exposure of fg-binding sites but also in cz-granuIa release reaction. However, it still remains to be clarified whether nuclcotide-binding to GPllbllla is involved in A D P - i n d u ~ d plalelet activation or regulates directly fg-receptor function, and thus fg binding to the platelet membrane. Studies with Glanzmann's piatelets which either lack GPiib-lila or the fg-binding site~ [14] are currently in progress to answer this question. GPllb-llla is the most prominent /]3-integrin of platelets and mediates both platelet-platelet and pla~ei,~t-substrata interactions [15]. As a member of the cytoadhesin subfamily (/]3) it has an identical /]mbunit (GPIiIa) as the more wide.ty distributed vitronec~in receptor VnR [15]. Thus, the presence of a nacleotide-binding site in GPilla may also suggest that extracellular nucleotides may be involved in the function of other fg-binding integrins such as VnR on endothelial or melanoma cells [16].

W e thank Caroline Bogner for h e r fine art-wcTk. This work was supported in part by a fellowship from the Dcutsche Forschungsgemeinschaft to Meinrad G a w a z ( G r a n t G a 123/2). T h e p a p e r is dedicated to o u r scienlific t e a c h e r Prof. Dr. Martin Klingenberg.

Refereaces I Born. G.V.R. 11.o62)Nature 194, 927-929. 2 CusacL NJ. and HouranL S.M. 11982) Br, J. Ph~nnacol, 77, 329-333. 3 Macfadane. D.E.. MiUs, DoCB. and Srivaslra, P.C. (1982) BiochemistB, 21. 544-549. 4 Colman, P,.W~(1990) FASEB J. 4,1425-1435. 5 Greco. N J . Yamamoto. N ~ J ~ B.W. Tandon, N.N. Moos, M. and Jam/eson, G.A. (1991}I. Biol. Chem. 266, 13627-13633.

6 Mayinger,P, Winkler E. and KlingenbergM. 11989) FEBS Lett. 244, 421-426. "/ Czarnecki, J., Geahlen, IL and Bond, H, (1979) Me:hods E ~ tool. 56, 642-653. 8 Jefferson, J.R., Harmon, J.T. and Jamieson, G.A. 11988) Blood 71,110-116. 9 Heaton, G.M., Wagenvoord,ILJ. and N/cholls. D.G. (1978) Eur. J. Biochem.82, 515-521. 10 Ginsberg, M.H., Fielinger, A.L.. Lain, S.C.T., Forsy,,h,J., McMillan, R., Plow, E.F. and Shattil, SJ. 11990) Blood 76, 2017-2023. I1 Lanmm|i, U.IC 11970)Nature 227, 680-685. 12 Frelinger, A.L, Lain, S.C.T., Plow, E.F., Smith, M.A., Loftus, J.C. and Ginsberg, M.H. (1988)J. Biol. Chem. 263,12397-12403. 13 Phillips, D.R., Jennings, L.K. and Prasanna, H.IL 11980)J. Biol. Chem. 255,11629-11632. 14 Powling, MJ. and Hardisty, R.~L ~1985)Blood 66, 731-754. 15 Ginsberg, M.H., Loflus, J. Ryckwaert, J.L, Pierschbacher, M., I~tela, IL, Ranslathi, E. and Plow, E-F. (1987) J. Biol. Oten~ 262, 5437-$,/~i. 16 Cheresh, D.A., P~ela, R,, Pierschbacher, M.D., Klier, F.G, Rues-lahti, E. and Reisfeld, R.A. 11987)J. Cell. Biol. 105, 1163!173.

Photoaffinity labeling of integrin alpha IIb beta 3 (glycoprotein IIb-IIIa) on intact platelets with 8-azido-[gamma-32P]ATP.

The fibrinogen receptor GPIIb-IIIa plays a crucial role in platelet aggregation. Here we show that the adenine nucleotide, 8-azido-ATP, inhibits ADP-i...
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