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Monoclonal antibody AG-1 initiates platelet activation by a pathway dependent on glycoprotein IIb–IIIa and extracellular calcium
Biochimica et Bfoplwsica Acttt, I 137 ( l STY2)248-25(I © 1992 El~vier Sci¢n¢¢ Publishers U.V. All righL~reserved n167-4889/n2/$n5.sn
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Monoclonal antibody AG-1 initiates platelet activation by a pathway dependent on glycoprotein IIb-IIIa and extracellular calcium M i c h a e l H . K r o l l ~a, M i c h a e l E . M e n d e l s o h n h J o n a t h a n L , M i l l e r ~, K a r e n K . B a l l e n g. J a n e t K . H r b o l i e h a,h a n d A n d r e w I. S c h a f e r a,h = VA Medical C'enrerand Bayloy College of Medidne, Ho.s~r~ T X (LISA), b Brigham and Women 'x Ho%pltal and Harl,ard Medicat School, Boston, MA (USA) and • Department of Pafkology. SUNY Heallh Science Center, Syrac~l~, N Y ~O.fA)
(Received 15 May 1'992)
Key words: Thrombosis; Calcium; CDg; Glycopmlein: Phospholipase C
The biochemical resl~nses of intact human piatelets to the monoelonal antibody (mAb) AG-I wcrc investigated. AG-[ is a muting igG mAb thal recognizes a series of platelet membrane glycoproteins (Gp) from M~ 21090 to 29 0 ~ , one of which is the M, 24000 (p24) receptor for anti-CD9 mAbs. AG-I causes platelet aggregation and secretion. Platctcls binding AG-I demonstrate a dose- and time-dependent breakdown of phosphatidylinosilol 4,5-bisphosphate (PIFz), production of diaeylglycerol, and generation of phosphatidie acid (PA). These events are associated with the activation of protein kinase C (PKC), an increase in intraeellular ca]ciltm, and fibrinogen binding. Platelet Pat generation and PKC activation in response to AG-I are inhibited by mAbs to piatglet Gpllb-Illa or by extraceliular EGTA, but not by a mAb to ptalclct Gplb or by inhibiting plnt¢lgt Na÷./H * exchange with 54N-ethyI-N-isopropyl)amiloride. Platclet cytoplasmic free calcium ([Ca2+]i) is elevated in response to AG-I, and this elevation is inhibited by mAbs to Gpllb-[Ila, an R G D S pcptide or by ehelatiag extracellular b.alcium. These resu,ats suggest that AG-I binding to a unique platelet-surfac¢ gl~/coprotein ~niliates plalelct responses through the activation of PlPz-speeifie phospholipase C, and that this occurs through a signal pathway that is dependent on Gpllb-Illa and extracellular calcium.
Introduction A G - I is a m u t i n g m o n o c l o n a i antibody ( m A b ) that recognizes a series o f low-molecular-weight pl at e l e t m e m b r a n e glycoproteins ( G p ) in the M r 2 1 0 0 0 to 2 9 0 0 0 range, with the most p r o m i n e n t b a n d o c c u r r i n g at M~ 21 000 it}. It is an I g O originally raised in mice i m m u n i z e d with w a s h e d platelets from patients with
Correspondence to: M.H Emil. Hematology Section. Baylor College of Medicine, MS fO~_ 6565 Fanain Street. Houston. TX 77030. U S A Abbre~iatlons: Plp~. phosphatidylinositol 4.5-bisphosphal¢: Gp. glycoprotein. [Ca2* ]t cytoplasmic free calcium; EGTA, [ethylen=bis(oxyethylenenit rilo)] tetraaeetic acid; PLC, phospholipase C; [P3, inns;to] 1.4,5-trisphosphate; DG, ~n-i,2-diawlglyceroi; PKC, protein kinase C; CP, croat;no phosphate; CPK, croat;no phosphokin ~ ¢ ; USA, I~vin¢ seTum albumin; nlAh monoctonal antibody; ACD, acid-citrate-dextrose; PRP. pI~t¢I~t-rich plasma; SDS PAGE. ~dium dodccyl sulfal¢-pulya~='rylamide gel ¢lecnoPhoresis; p47. M t 47000 protein; pZ0~ M, 20(300 pt'otein~ PA, pho~phatidic acid; [pH] i, cytoplasmic pH; EtA. 5-(N.ethyl-N-isopropyI}amiloride; BCECF-AM.
2'.?'-bis (carl~ethyl)5,h-carbo~fluote~cein
acetoo~methyl ester.
platclct-type yon Willebrand disease, a congenital platelet a bnor m a l i t y characterized by the substitution of valine for lysine at amino-acid residue 233 of glycoprotein ( G o ) Ibm, which is associated with a heighte n e d interaction b e t w e e n the platelet ( 3 p l b , / I X complex a n d n o r m a l v a n W i l l e b r a n d factor molecules [2]. A G - I b i n d i n g to intact h u m a n platelets causes aggregation a n d secretion that are inhibited by cyclic A M P , E D T A , a n d antibodies to G p I l b - I l l a [1], T h e s e obs0rvations indicate that A G - 1 b i n d i n g to h u m a n plate[ors activates signal-transducing pathways f u n d a m e n t a l to the d e v e l o p m e n t o f a s u b s e q u e n t functional response. Several o t h e r plateiet activating m A b s , d e s i g n a t e d as C D 9 antibodies, have been f o u n d to b i n d to a single b a n d of M r 2 4 0 0 0 (refer~-ed to as p 2 4 / C D 9 ) o n oned i m e n s i o n a l W e s t e r n blots [3-7]. A G - I c o m p e t e s with p 2 4 / C D g - s p c o i f i c antibody d e s i g n a t e d m A b 7 [6] for b i n d i n g to a platelet protein o f M T 24000 (p2d). b u t m A b 7 a p p e a r s unable to c o m p e t e with A G - I for b i n d i n g to o t h e r M r 2 1 0 0 0 - 2 9 0 0 0 constituents (,leanings, L.K~, Fox, C.F. and Miller, J.L., u n p u b l i s h e d
249 observations). These findings suggest that platelet activation induced by AG-I may involve different signalling pathways initiated by its interaction with a protein or proteins other than those recognized by such p24/CD9 antibodies. Platelet activation is a complex network of interacting intraeellular biochemical reactions resulting from an extraeellular agnnist binding to the plate!et surface [8]. Receptor occupancy causes the dissoc~arion of a heterotrimeric GTP-binding regulatory prote;n that activates phospholipase C (PLC). PLC catalyzes the hydrofysis of phosphatidylinositol 4,5-bisphosphatc (PIP~) tt: the second messengers inositol 1,4,5-trisphosphate /IPO and sn-l,2-diacylglyeerol (I)(3). IP3 causes the release of calcium (Ca 2.) from storage organelles and DG causes the activation of protein kina~ C (PKC). These events then stimulate the functional responses of aggregation and secretion by inducing structural changes in substrates directly, or indirectly through the activation of specific protein kinases [8]. The purpose of this report is to elucidate the molecular signalling events responsible for AG-I-induced platelet activation. Exl~rimca tal procedures
Materials Human alpha thrombin was from LIS Biochemlcals (Cleveland, OH). Creatinine phosphate (CP), creatinine phosphokinase (CPK). Sepharose 2B-30~, Nonidet P40 (NP40). fatty-acid-free bovine serum albumin (BSA). and indnmethacin were from Sigma (St. Louis. Me). Lipid standards were from Sigma and Calhiochem (San Diego, CA). |-~-"P]Orthophosphorie acid, [ u4C]araehidonie acid, and aZ~l(sodium salt) were from New England Nuclear (Boston, MA). Furs-2 aeetooxymcthyl ester and 2',7°-bis (carhoxyethyl)5,6carboxyfiuorescein (BCECF) acetooxymethyl ester were from Molecular Probes (Eugene, OR). Silica Gel G plates were from Analtech (Newark, DE). Whatman silica gel K5 and LKSD plates were from Fisher Scientific (Fairlawn, NJ). HPLC-grade organic solvents were from American Burdiek and Johnson, (Muskegon, MI). MonoclonaI antibodies (mAb) 7E3 and 10E5 (directed against GpIIb-ll|a) were obtained from Dr. Barry Culler (SUNY, Stony Brook, NY) and mAb AS7 (directed against the amino-termlnal M r apprrtx. 45000 region of Gplb) was produced by one of us (JLM). mAb IV-3 (directed against the M~ 40000 ptatelet FeyRll) was purchased from Medarex (West Lebanon, NH). S-(N-ethyI-N-isopropyl)amiloride (EIA) was obtained from EJ. Cragoe of Merck, Sharp and Dohme (West Point, PA)and was dissolved in dimethyl sulfoxide. Human fibrinogen containing no contaminating plasminogen or yon Willebrand factor was obtained from Enzyme Research Labs (South Bend, IN).
Preparation of monocloaal antibody AG-I Balb/c mice were immunized with washed platelets from patients with platelet-tVpe yon Willebrand disease [2]. Spleen cells from the immunized mi¢¢ were then fused with P3 NS/Ag 4.1 myeloma cells [9]. Culture supernatants from the resulting hybridomas were screened for their ability to inhibit the agglutination of these patients" platelcts by asialo yon WiUebrand factor in the presence of 5 mM EDTA [10]. Several hybridoma supcraatants actually augmented this resl.xmse and in subsequent testing were found capable of inducing aggregation of normal p[atelets without the addition of other ~gents. One of theses hybridomas (AG-I) was cloned by limiting dilution and the cells then grown in plistane-primed Batb/e mice. The resulting ascites was precipitated with ammonium sulfate and affinity purified using the MAPS Affl-gel system (Biorad, Richmond, CA). Ouchterlony analysis using antisera purchased from Serotee (Oxen, UK) showed AG-I to be an lgG of the 5"1 subclass, Fab fragment, of AG-I were prepared by pepsin digestion and subsequently purified as previously descrihed [1]. Absence of intact antibody or of contaminating Fc fragments in the purified Fab preparation was established by SDS-PAGE analysis. Fab fragments were radioiodinated and binding studies performed as previously described ill. Platetet preparation Whole blood Item volunteer donors who had not taken aspirin or other drugs affecting platelet function for at least 10 days prior to donation was collected in 15% acid-citrate-dextrose (ACD). This was centrifuged at 180 Xg for 15 rain. The platelet-rich plasma (PRP) was acidified to pH 6.5 with ACD, CP/CPK (1 raM/25 U / m l , res0ectively) added, and then layered over an albumin gradient and centrifuged at 1500 × g as described by Walsh e t a l . [I I]. Except where noted, the interface platelcts were collected in buffer A (6 mM glucesc, 1.30mM NaCI, 9 mM NaHCO~, 10 mM sodium citrate, 10 mM Tris base, 3 mM KCI, 0,81 KH2PO, and 0.9 mM MgCIz, pH 7.35) and re-centrifuged through the albumin gradient. The collected plat¢lets were suspended in a small volume of buffer A (except where nor'~d) and either radiolabeled or loaded with furs-2 or BCECF by incubating the platelets with the .ppropriate reagent for I h at 37°C in a geotly shaking water bath. Platelets were radiolabeled with 0,5 mCi [S2p]orthophesphat¢ or 20 ~Ci [t4C] arachidonic acid; or were loaded with 2 v,M furs-2 aeetoox'/methyl ester or 6 ~ M BCECF acetooxymethyl ester. Following this. the platelet suspension was gel-filtered through Sapharose 2B-300 equilibrated with buffer A, and sosuspended in buffer A containing I mM CaCI2 at a concentration of 2.5.10s platelets/ml (except where
25O noted). All experiments were carried out in stirring platelet aliquots at 37"C (except where noted), Plarefet lipid measurements Piatelct lipids were extracted according to the method of Bligh and Dyer [121 and analyzed by thinlayer chromatography (T1,C). [32P]Orthophosphatelabeled phospholipids were separated on Whatvaan K5 plates dipped in 1% potassium oxaiate/2 ram EDTA using the solvent system chloroform/acetone/methanol/glacial acetic acid/water (40:15:13: 12:7, v / v ) according to the method of Van Dnngen e t a l . [13]. [14C]Arachidonate-labeled p|atelets were assayed for [l"C]diacylglyeerol formation using silica gel G plates with the solvents benzene/diethyl ether/NH40H (100:80:O:0.2, v / v ) as described by Rittenhouse-Simmons [14] and for [l')C]arachidonie acid and its radiolaholed metabolites on Whatman LK,SD plates using the mlvents diethyl ether/hexane/glacial acetic acid (60:40: I, v/v) as described by Salmon and Flower [15]. Lipid hands wen= detected by autoradiography, idcotifl~d by co-ehromatograpby with unlabeled standards, scraped, and counted for radioactivity. Platelet protein phosptzorylation [~PlOrthophosphate-labeled platelets were stivaalated as described and the reactions stopped by the addition of a solution of 511% glycerol, t0% dithiothreitol, 8% sodium dodecyl sulfate (SDS). and trace bromphenol blue, followed by immediate boiling for 5 rain. SDS-polyacrylamide gel dectrophoresis (SDSPAGE) was performed on 7-17% gradient gels as described by Laemndi [16]. The gels were stained with Coovaassie brilliant blue R, destained, fbled and dried. The phospborylated proteins were located by autoradiography and relevant bands quantified by transmittance densitomctqt on a LKB Ultrascan XL laser dcnsitometer. /Ca z +]~ measurements Washed platelets were suspended in buffer B (145 mM NaCI, 5 mM KCt. 1 mM MgClz, 6 mM glucose, 10 mM Hopes, pH 7At without Ca -'+ and incubated with fura-2 (vide supra). Fura-2-loaded piatelets were gelfiltered and changes in [CaZ+]~ monitored in a PTI Deltascan spectrofluorometer, with dual wavelength excitation capability, using 1.5-ml aliquots of 2.0- Ill" platelets/ml buffer B containing I mM CaCIz or 2 m M lethylenubis(o~ethylenenitrilo)] tetraacetie acid (EGTAt added 1 vain before the addition of agnnist. [CaZ+L was calculated from the ratio of fluorescence emission at 510 nm, following the excitation of fura-2qoaded platelcts at 340 nm and 380 nm. using a K,~ for fura-2 of 224 nM, as previously desex'thud ~17].
Fibrinogen binding assay Human fibrinogen was purified chromatographically, including lysine-Sepharose column separation of contaminating plasminogen and immunoaffinity column separation of contaminating yon Willebrand factor. Fihrinogen was radloiodinated as described by Loscaizo et aL [18]. Fibrinogen binding studies w e ~ performed at 22°C as described by Hawiger et al, [19]. T/lromboxalte measurements
Production of plate[el thromboxane A z was quantitared by radiogmvaunoassay of its stable breakdown product thromboxane B z as previously described [171. pHj rtleosurc/ff~Hl$ Washed platelets were suspended in buffer B withoat Ca -'÷ and incubated with BCECF-AM (vide supra). BCECF-Ioadod plate!ors were gel-filtered and changes in ~toplasmic pH (pH i) measured in a PTI Deltascan spectrofluorometer, using 1,5-ml al~quots of 2.0-i0 s plarelets/ml buffer B plus 1 mM CaCI z, according to the methods of Zavoico etal. [20].
To examine pathways of AG-l-induced platelet activation we first measured the dose- and time-response of intraplatelet signal generation following AO-I binding, The ECs. for platelut phosphoinositide turoover, p47 phosphorylation (M, 47000 substrate for PKC), and p20 phosphorylation (M, 200ID substrate for Ca2+/calmedulin-dependent protein kinase; myosin light chain) is 2-3 ttg/mL This is consistent with the EC~, observed for functional platelet responses it[. The lag phase and the magnitude of these responses are concentration-dependent. Fig. I demonstrates the time-course of PIP, turnover, DG production and the generation of phosphatidie acid (PAt in platclets stimulated by 3 / z g / m l AG-L Protein phosphorylation following AG.I (3 ~ g / m l ) binding to plalelets is represented in Fig. 2. Synchronous mcasorcvaeuts of PIP, and protein phospborylation show that p47 and p20-phosphorylation occur after the hreakdow~ of PIP2. The phosphorylation of p47 indicates that PKC is activated and the phosphoryla'.ion of p20 indicates that intracellular Ca z* ([CaZ"]=) rises in response to AG-1. This latter conclusion was corroborated by directly measuring [Ca-'+]i following AG-i binding (vide infre). The biochemical responses demonstrated above are associated with the release of arachidonle acid and its conversion to active metabolites. Thus, platelet thromboxane B_,~the stable breakdown product of throvaboxane A~, increases from a basal level of 168 p g / m l to 2373 p g / m l 30 s following AG-i stimulation (3 ~ g / m l )
251
and persists at levels greater than 2000 p g / m [ for 5 min fol[owing the. addition o f A G - L A G - I causes the exposure of fibrinogen binding sites on platelet surfaces. Fig. 3A shows the tirae-course of fihrinogen binding to platelets stimulated by AG-1 and Fig. 3B shows that fibrinogen binding to A G - I stimulated platelets is saturable+ In an attempt to define the proaimal signal-generating steps resulting from the A G - l / r e c e p t o r coupling event, a series of experiments was performed to examine the role o f ptatelet-surface glycoproteins in A G - I induced PLC activation. Previous data show that AG- 1 iraraunoprecipitates a series of tow-molecular-weight proteins from the surface of platelets that are distinct from the 1~ chain o f G p l b [1]+ We additionally observe minimal inhibition of AG-l-indu~ed platelet protein phosphorylation and PA generation when the platelets are pretreated with AST, a mAb to the a chain of
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Fig. 2. AG-I (3 gg/ml) stimulates the time-dependent pho~phowlalion of pIatclct Mr 47000 protein (p47) and M+ 21)01)0protcln (p2a). [32P]Orthophosphate-loadcd platelcts were Iicated with AG-I and protein phospho~'lation measured at 1he designated time points (as descried in Ealmrimcatal prom.glurgs). Tngs~ data are expn:ss~d relative to I]% pho~phoIMation at time 0 and are repTe~nlative of f,Lx separate experiments.
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•r i m ( r a i n ) Fig. L A G - I (3 p g / m l ) cauy~5 platelet phosphatidyiirlositol~l,5-bis +
phosphate (PIP2) brea]~down and reSynlhesis, and the generation of diacylglye~rol (DG, top half): and AG-I (3 .ug/'ml) causes Ihe production of plat¢let phosphotidi¢ acid (PA, bottom hal0. PIP, and Pat in response to AG-I were measured synchronously in the same prcpar~ltion of -~2P-lahclc~ plait:lois (as. dcscdh~d in Experimenla[ I)rov.cdure~). DO m¢i~rement~ were made in sel~t-ate plat¢let aliquo~s labeled with [x4C]araehidonicacid (as descrff~cd in Experimental pro~duresl. Dala arc expressed relative tO 100% measure. n~ents at time 0 (PI~). or 0% measurement at time 0 (DG, PAl, and are re~esentalive of 3 separate eqlerimenLs.
G p l b [21] (Fig. 4 and Table l). In contrast to this, two different mAhs to G p l l b - l I l a , designated 7E3 [22] and 10E5 [23], markedly inhibit platelet protein pbesphowlation gad P A production resulting from AG-1 binding (Fig. 4 and T a b l e D. Because previous d a t a indicated that A G - I does not bind to G p l l b - i l l a on Western blots [I], and a mA.b to G p l l b - [ f I a does not inhibit AG-1 Fab binding to intact human platelets (data not shown), these new results suggest that G p l l h - l l l a plays an indirect role in mediating AO-l-induced responses by coupling the platelet receptor for AG-1 with the activation of PLC. Table I demonstrates the effect o f antibodies to G p l b and G p i i b - I I I a on AG-l-indnced platelet PA production. This table also shows that E G T A , which chelates ex-ttacellular Ca 2", is inhibitory to A G - l - i n duced PA production, but indomethacin has little effect on AG-l-inducvd PA production, 2 ram E G T A was added 1 rain before A G - ! to avoid dissociation o f platclct O p l l b - I I I a that occurs with longer incubations with calcium chelators [24]. These results are not likely to be due to the effect of egtracellular calcium on A O - I binding to its platelet receptor(s), because E D T A had an effect on the binding o f [IZSl]AG-I Fab to platelels (data not shown). E I A , which inhibits platelet N a ÷ / H ÷ exchange and abolishes platelet cytoplasmic alkalinization in response to throrabin [20], causes no change o f AG-l-inducad P A production (vide Jaffa). T h e platelet M r 4 0 0 0 0 F e y R I I is considered to be the mediator o f platelet responses caused by some mAbs sp~:ifie for the platelet CD9 complex [4,25,26]. We therefore examined the effect of the m A b I V 3 (anti-Pc~,RII [27]) on A G - l - i n d u c e d platelet signalling. IV.3 (20 p g / m l , - 5 min) completely inhibits platelet P A production and protein phuspho~lation 5 rain follow-
252
ing 3 $zg/ml of AG-I. This is cousistent with the inhibitor, effect of IV.3 o n AG-l-induced aggregation (data not shown) a n d secretion [28]. To determine if AG- l binding to the platclct FcyRII is the so,e mechanism by which AG* 1 activates platelcts, Fab fragments of AG-I . . . . inct,hatcd for 5 mln with "2P-labelled platclcts for PA mcagargments. Fab fragments of AG-I g e n e r a t e R significant quantity of PA, although the ECs~, is ~pp . . . . 5-fold higher than that of the intact antibody: 15 /zg/ml AG-I Fab causes PA production at 5 rain that is 157 =i:61% (mean _+S.E.)above control.
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Fig. 4. Monoclonalanzihodicsto plateh~t GFJIIb-lllainhlbilplat¢lct protein phosphotylatDn in rcslam~ tu AG-I. ~ZP-labeltdph~t¢lcts
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"/E3(3 gg/rnl) or IIiF~(5/zg/lr~llw¢[¢ incubated with th~ plalelcLs for 30 rain and 1 rain. respectively,prior l¢1lh¢ additi{m 0f AG-L Ptutelet pd7 and p2,~phnsphowlalion 1 rain fidh~wingthrombin, and 5 rain I'ollowii~gbutter, al'¢ sh~n fur compa~son. These data are
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600
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Fig. 3. Effectof AG-I on [ t~llfibrinogenbinding In human pi~ltg|gl.S at 22~C. (AI Timc-coulrs¢of [11~l]fibfinugenbinding to platclct.~ activaled I~ AG-I. Gel-filtered plat¢lels weT¢ incubated with 3 ~g/ra[ AO-I fe~ the times noted. Fibrilmgen hou ad is q:xp[¢~sedas a
rl-ilclion oi"or tOtalbindingc~grvcd at 3a rain (B/B m). (B) Eft©c!of [I-~|iiffhrtnogcn concentration on AG-I-induccd platclet fibrinngcn binding. Gel-filtered platclets ~ucr¢ incnbal~d for 20 rain wilh 3 ,agOrot AG-I at incma~;ing cuncent~'alions o; Ubrln0gen and platelet-~ouad fibnnugcn~vagmeasuL'ed ag degct'ih~d in F~perimental prnccdur~s. Dala ]'¢presenl spedfi¢ binding and are means of duplical¢ determinations.
in platclct calcium homeostasis. G p l l b - l l l a is the primary surface binding site for Ca ~÷ on unstimulated platelers [29]. It may be important in the transmcmbraanus movement of Ca z+ during platelet activation although the precise role of this cation flux in platelet activation remains unknown [30-36]. To examine AGl-induced platelet calcium responses and to determine how GpllL~llla may affect them. a series of spcetrofluorometu experiments was performed on platelets loaded with the calcium fluorophore tara-2. The dosea n d time-response of AG-l-induced elevations of IC~2*lt parallel that observed for PIP2 turnover. DG and PA generation, and protein phospho~lation (data not shown). Fig. ~ shows that 2 raM E G T A added 1 rain before AG-I (which chelates extracallular Ca 2.
253
AG-1
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5
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Fig, 5. The effect of extracellular Ca2+ and Gpl|b-nla on the platelet intracelIularCa2+ ([Ca2+ ]~} response to AG-I. In Ibis speclrofluorOmeldc tracing, the absci~.~ais time (mini and the ordinate is ICa" ~II. calculated from the ratio of fluorescence emission (at 511)am) following tl~edeal exeitatiorlof fura-2-1oaded platelets tat 341)and 380 aam),as deseril~ed in E~perimeraal procedures. Unstirred platelet aliqttot= in tmfler at 37"C were p~e-lncubated ~ilh eaCI z (I raM. --I mini. EGTA (2 raM, --I mini, 7E3 (3 p.g/ml, -311 mini. or RGDS (200 ~g/ml, - 5 mini and elevations of [Ca-'+ li measured in slitting plate-let aliquots at 37°(2slimulated by AG-I (3/xg/ml) or Ihmrabin ( I U/ml), These tracings are representative of Cr,'eseparale experiments. w i t h o u t affecting the functional integrity of p l a t e l e t G p l l b - I l l a [24]) inhibits t h e elevation o f [CaZ+]+ observed following p l a t e l e t binding to A G - I . This is different from t h e effect o f E G T A o n [CaZ+]i responses to thrombiu, where extracellular C a 2+ has only a small impact on t h e maximal extent of the rise in [Ca2+]i (Fig. 5)- Fig. 5 also shows t h a L in the presence o f extraeellular C a 2+, a m A b to G p l l b - l l l a (7F,3) abolishes the [Ca2+]i response to A G - I , but has no effect on the [Ca 2 +]i respous¢ to thrombin+ T h e t e t r a p c p t i d e R G D S (arg-gly-asp-scr), which binds to G p l l h - l l l a and TABLE I 3"~P-lab~:led nlatel¢ltSweL'e incubated with AO-I for 5 mln and the reactions terminated and assayed for PA production as descn~oed in Exlmriracnlal procedures, 7E3 (anti Gpllb-ltla), 11)E5(anti-Gpllb+.tin) alld AS7 (anti-Gplb) were pneincubated with platelets as follows: 7E3, 3 ~tg/ml, - 3 0 win: 10F-5, 5 ~g/mt. - 1 rain: AST. 5 ttg/ml. - 5 miix 2 mM EGTA was added I rain prior to AGq. 270 gtM indomethacin was added 2 rain prior Io AG-I. To block Na +/H + exchange, 411 FM EtA w-~ added 24) s prior to AG-I. The data s h ~ n am mca~:tS.[L of l~r~nt platelet PA ploductlon above conttgl levels (PA p~ttzced at ;5rain in anstinlulated stirring platelet aliquots at 37'~L Treatment
n
Pet
AG- 1:4/xg/ml +7F--3 ÷ leES +AS7 + EGTA + indomclha~ n + EIA
6 3 3 6 6 1~ 5
247.64-_40 -4.0+ 16 * 68,3+58 * 195.0+51 98.2+35 * 174.(1± 71 200J0+_64
• P > n.05 IWtwoda1)ed/-lest tunpaired means), in comparison wilh enntrol leve*s.
inhibits its interaction with R G D - c o u t a i n i n g ligands such as fibrino.~en [37], also completely inhibits p]atelet C a 2+ responses to AG-1 (Fig. 5). These observations demonsh, ate that A G - I - i n d u c e d signalling may require a functional G p l l b - l l l a complex to effect the transmembranous influx o f calcium. The observation that A G - I initiates platelet phos. pholipase C activation by a mechanism that is dependent on G p l i b - l l l a is similar to those o[ B a n g a e t al., who examined epinephrine-induced platelet activation [38]+ T h e s e authors demonstrated that epinephrine causes an initiation signal for p l a t e l e t activation through the pbospbolipase A2-mediated release of small amounts of arachidonic acid. This is a well-established concept that forms the basis for the dependency o f weak agonists on intact cyelooxygenase metabolism to effect a full platelet response [8]. A s previously described by Sweatt e t al., this signal is dependent on N a + / H * exchange [39,40]: B a n g a et al. reproduced these earlier observations and showed t h a t a n antibody to G p l l b - I l l a inhibited both cytoplasmic alkalinization and phosphatidic acid production in response to epinephrine, suggesting thai G p l I b - i l l a cou!3ies ~2 re" ceptor occupancy to N a + / H ~ exchange and cydooxygenase-dependent platelet activation [38]. Because A G - l - i n d u c e d P A production is not inhibited by indomethaein, it is likely that this ligand stimulates platelets through pathways distinct from those activated by the weak agonist epinephrine. T o directly assess the role of N a + / H + exchange in A G - l - i n d u c e d platelct reslxmses, p H i was measured in BCECF-Ioaded platelets stimulated by AG-1. These experiments showed that A G - i causes no change in platelet p H i from resting
254 levels. Consistent with thes~ findings are observations that EIA, which inhibits platelet Na+/H + exchange. does not inhibit AG-I -induced PA production (Table t). DiscnsMon
AG-I is a m A b that binds to a series of Iow-molccular-weight platelet-surface proteins having M~ 21 000 to 29000, only one of which is the p24/CD9 antigen. Recent eDNA cloning of the p24/CD9 protein demonstrates that this protein has one N-glycosylation site [41,42], suggesting that the degree of glycosylation cannot account for the different species of platelet proteins that are immunoblotted by AG-I. Therefore, it is likely that AG-I binds also to other platclet-snrface proteins, raising the possibility that AG-1 is the ligand for a platelet membrane signal-transducing protein that is different from p24/CD9. The binding of A(J-1 to human platclcts causes platelut activation of a magnitude comparable to that observed with the most potent physiological platelet agonists. Studies of the biochemical mechanisms of AG-lqnduce~d platolet activation presented here provide new information about fundamental signal-response COUldingmechanisms that may be relevant to platelet activation in rive. We have shown that AG-I stimulates phospholipase C, activates protein kinasc C, cause~ an elevation of [Ca2+]~, and initiates the expression of fibrinogen binding sites. AG-l-induced signals for platelet activation are inh~ited by monoclonal antibodies to Gpllb-llla and by cxtracellular ECITA, suggesting Ihat AG-! initiates platelet phuspholipase C activation through a process that is dependent on ex. tracellular calcium and a functional membrane GpIlb-?lla complex. AG-I initiated platelet activation is different from that observed with a 2 receptoc-initiutod platclet phospholipase C activation because it is not dependent on N a ~ / H + ¢~change and not inhibited by indometbaein, indicating that the initiation of platelet phospholipase C activation by AG-I does not follow the Na*/H+ exchange-dependent release and metabolism of arachidonic acid. Indomethacin does, however, inhibit AG1-induced 5-hydro~tr~tamine secretion, suggesting that an amplification response to AG-I depends ou intact arachidonic acid metabolism ]1]. Data showing that AG-I induces the production of large amounts of platelet thromboxaoc A2 are consistent with this hypothesis. Platelet aggregation induced by anti-CD9 mAbs was first reported by Boucheix and colleagues in 1983 [3], who more recently showed that their p24/CD9 mAb causes platelet phosphatidic acid formation and PKCand Ca2+-dupendent protein phosphorylation that is not dependent on extracellular Ca"* but may be de-
pendent on the activation of PLC [43]. Hate nt al. have reported a p24/CD9-specifie mAb that activates platelets and induces expression of the fibrinogen receptor;, this activation is independent of extracellular Ca z÷ [5,44]. The mechanism of fibrinogen binding site expression induced by p24/CD9 antibodies is unknown, but Slupsky el. el. demonstrated that platelet p24 associates with Gpllb-IIIa following iLs binding to ligand [45]. There is no evidence for this occurring in response to A(/-I, and present data indicate that ACI-I does not interact directly with platclet Opllb-ltla and that platel~.t Gpllb-llla remains acccsslble to fibrinogun binding following platelet incubation with AG-1. An alternative explanation for the basis of AG-l-induced fibrinogen binding is, however, suggested by this study: AG-1 initiates fibrinogen binding to platelets by causing intracellular signals that directly induce the Gplib-llla receptor for fibrinogen on activated human platelets [46]. Jannings ct at. and Worthington et al. have reported p24/CD9 mAbs that cause platclct PLC activation [25,26]. In contrast to our results with AG-I, they observe that this is not dependent on Gpllb-Illa, but find that this involves Gplb [25,26]. While these groups have reported that antibodies to Gplb completely inhibit anti-CEO-induccd plat¢lct activation, we observe tha* AS7 (aati-Gplb) does star inhibit AG-l-induced olatelet activation. We also find that AG-l-indueed platelet activation is inhibited by an antibody to the M r 40000 platelct Fc~,RII receptor, and this is consistent with that observed with other p24/CD9 mAbs [25,261. In addition, AG-I appears to initiate platelet signalling independent of the platelet Fe-~Rll receptor, because AG-I Fab fragments stimulate PA production. These obsercations, in conjunction with previous observations that AG-1 Fab does not stimulate platelet aggregation ill, suggest that, as has been described with epinephrine and endoperoxidc analogues [47], PLC-madiated platelet intracellular signals in response to AG-1 Fab do not reach a magnitude sufficient to cause aggregation. The activation pathway initiated by A G d binding to human platelets appcat~ to differ in important ways from platelet activation pathways induced by p24/CD9 antibodies. It is possible that AG-1 binding to other constituents of the M, 21000 to 29000 family of platelet memblane proteins that it immunoprecipJtatus triggers cellular processes that act in concert, or perhaps even antagonistically, to those initiated by its binding to the p24/CD9 protein. AG-l-induced platelet activation is completely depqndent on Opllb-llla. This result supports the by. pothcsis that Gpllb-ttta is important in mediating platelet stimulation, perhaps by regulating the transmembranous influx of calcium [48]. This mechanism of Gplib-Illa function is consistent with two hypotheses
255 proposed by others: (1) G p l [ h - l l l a is structurally a n d / o r functionally related to platelet calcium channels [30-33] and (2) calcium channels are important in platelet activation I34-36]. Evidence in support of these hypotheses cominues to ace,qae, although the mechanism by which G p l l b - l l l a regulates platelet [Ca2÷], is not fully elucidated. Recently published d a t a demonstrate that G p l I b - l l l a regulates voltage-independent C a 2+ currents in platelet plasma membranes and t h a t ligand binding (mAb o r R O D S peptide) decreases channel aetiviW [491. T h e r e are, in addition, recent d a t a thai platclet [Ca2*|i responses to shear stress result from the transmembranous inGux of Za 2~, and t h a t mAb o r peptide inhibition o f G p l l b - I l l a function decreases these responses [50]. Precise eharaeterization of the gtrstetaral relationships between the occupied A G - I receptor, p 2 4 / C D 9 , Gpllb-llla~ and phespholipase C will be possible only when the primary structure o f the functional A G - I rec~plor is determined. A M r 2 1 0 0 0 m e m b r a n e protein immunoprecipitated by A G - I is, in partieular, a good candidate for a signal transducing protein: not only is this the major platelet-surface glyeoprotein protein recognized by A G - I , but, in addition, this protein (or a coprecipitating protein of similar molecular weight) hag been d e m o n s t r a t e d to be a GTP-binding protein {51|. Acknowledgements T h e a u t h o r s t h a n k D r . S t e p h e n Peiper f o r h e l p f u l discussions and D e a n n a G o l d e n for the preparation of this manuscript. Supported by N I H grants H L 0 2 3 1 1 , HL02154, HL328S3, and HL3604S, the V A Merit Review Board, Granls-in-Aid from t h e Texas and Massachusetts Affiliates of the A.H.A., and the Biomedical R e s e a r c h Support G r o u p of Baylor College of Medicine.
Referenoes I Miller, $.L~ Kupinski, J.M. and Hastad, K.O. rig86) Blood ~8. 743-75 L 2 Miller. J.L, Cunningham, I), Lyle. V.A. and Finch, C.A. (1991) Frog. Natl. Aead. Set. USA kS, 47Sl-4765 3 Bouchetx. C., 5aria, C., Mi~hahi, M., Sofia, J., Per/at, J,-Y., Fourier, N., Pdlard. M. and Ro~nfcld, C. 119831 FEBS Len. 161,289-295. 4 Carroll. R.C., Worthington, R.E. and Boueheix, C. (19~) Biochem. J. 266, 52"/-535. 5 Halo, T,, Iked;*, let-,Vas1~kawa,M, Watanabe, A. and Kobayashi, Y. (19881 Blood 72, 224~z?.g. 6 J,JnrdslSs, LIC. Fox. C.F., Kouns. W.C., McKay, CP.. Bailou, L.R. and $ehulhZ.i-I.E. 119991J. Biol. Chem. 265. 3815-3g22. 7 ttigashihara, M, Takahata. Y, Nakahara, g., Ku¢okawa. K. (1990) FEBS LeU. 264, 270-274. 8 Kroll, M.H. and Srhafer, A.I. 119891Blood 74, 1181-1195. 9 at. V.T. and Herzenherg. L..,L (tgSo) in Seleclod Methods in
Cellular Imrnuanlogy (B.B. Mish~:lland S.M. Shiigi. eds.I p. 351. WIHI Fleeman and Co.. San Fransls¢o. In Miller, J.L., Ruggeril Z.M. and Lyl~, V.A. (1987) Bkloa 7n. 18114-1809. I I Wakh. P.N., Mills. D.C.B. and White, J.G. (1977~.BL J. Haemaiol. 36, 281 296. 12 Bligh, E.G. and Dyer. W.J. (lgsg) Can. J- Biachem. Ph~iol. 37. 191-lgT. 13 Van Dtmgen, C.$., gwi,~r:i. H. and Oi~pen, W,H. (I';SD Anal. Biochem. 144. :134 1119. 14 RhtenhotLse-gimmon~. S. (1981)J_ Biol. Chem. 2S6. 4153-415S. 15 Sulmoll, J.A and Rowers, RJ. (I9821 in Methods in Enzymology (W.E.M. Lands and W.L. Smith. eds.I Vol. 86, pp. 477-492, Academic Press. New York. I11 Laemmli, U,K. 119701Nature 227, 680-685. 17 Kloll, M.H., Zavoien, G.B. and Schafer, A.I. 119891J. Cell. Physiol. 139.558-S64. 18 Loscalzo,J., lnhal. A. and Handin, R.I. 1198@J. Clin. Invest. 78. 1112-1119. 19 Ilawlger, r., Parkinson. S. and Timmoag S. (19801Nature 283. 195-197. 20 Zavoico. G.B., Cragoe, E.J., JL and Feinstein, M.B. (lg86) ,I. Biol. Chem. 2111,[3160-13167. 21 Miller) &L., Htlslad, K.O., Kupinski, $.M., L?d¢, V.A., and Kunicki. TJ. (It)~l} Br. J. Haeraalol. 74, 313-31g. 22 Colter, B.S., Peerschk¢, E.I., Scudder. L.E. and Sullivan, C.A. (19831J. Ciin. Invest. 72, 325-338, 23 Caller, B.S. (19851J. Clio. Invest. 76. IOl-10g. 24 Fitzgerald, L.A. and. Phillips, D.IL 119851J. Biol. Chem. 260. 1136fi-11774. 25 Jennings, L.K, Kouns. W.C and Fox, C.F. 11989) Blood 74 (suppl. l) A 633. 26 Worthington, R.E., Carroll, R.C, and I~ucheis, C. (19~1) gr. J. HaeraatoL 74, 218-222. 27 nosenfeld, S.I., Ryan, D.H.~Looney. RJ.. Anderson, C.L, Abraham. G.N., I.eddy. J.P. 11~87lJ. lmmunoL 138, 2869-2873. 28 Horsewood, P.. Hay,~eard.C.P.M.. Wartenkin, T.E. and Kelton. J.G. (1991l Blood 78. 1019-1026 2g Brass, LF. and Shanil, S.J. (1984l J. Clin, Invest 73. 6211-632. 30 Powling, MIJ. and Hardisty, ILM. (Ig85) Blood 66, 731-734. 31 Rybak, M.E., Renzttlli, LA., Brans, M.J. and Cahaly, D,P. (198R) Blood 72, 714-720. 32 Yamaguchi, A., Yamamoto, N., Kitagawa, H.. Tanoue. K. gad Yamazaki, H. (19871FEBS Lett. 225;,228-232. 33 SinlgagIia, F., "ford. M., Ramaschi, G. and Balduini, C. (19891 gioehim. Biophys. Acla Q84~225-230. 34 Hallam, T.J. and Rink, TAr. (19S3) FIzBS L~Ir. 186, 175-179. 35 Zschauer, A.. van Breeman, C,, Bnhlcr, F.R. and Nclsa~n, M.T. t1~181 Natut~ 334, 703-705, 36 Suldan, Z. and Brass, L.F. (1~1) Blood 781 2887-2893. 37 Phillips, D.R., Charo, I.F., parise, L.V. and Fitzgerald, I-A. (1988) Blond 71,831-g43. 3S Bansa, H.S., $imons, B,R, Brass. L.F. and Ratenhouse, S.E. t1986) pix3c.Natl. Acad. Sc[. USA 83, 9197-9201. 39 Swean, J.D., Blair, I.A., Cragoe. E.J. and Limbird. L.I:, tl'~Ss) J. Biol. Chem. 261, 8660-8665, 4{I SweaR, J~D,, Connolly, T,M,~ CraSo¢, E,.L and Lirahird, L.IL ( 1g~161J. Biol. Chera, :261,8667-8673. 41 Lanza, F.~ Wolf~ D, Fox, C.F., Kicffer, N., Seyer, J.M., Fried, V.A., Coaghlin, $.R., Phillips, D.R. and Jeanings. LK. l199D J. Biol. Chem. 266, lQ638-10645. 42 noucheix, C., nenoit, P., Ftachet, P., Billard, M., Worthinl~lOn. R.E., Ga~non, J. and Uzan. G. t 19911J.Biol.Chem. 266,117-122. 43 Rends, F., Boaeheix, C., Lebi'¢1, MI. Bourdeau, N.. Benoit, P,, Maclour, J., Sofia, C. and Le~/-Toledano, S. 11987) Biochem. Biopbys. Res. Commun. 146, 1397-1404.
256 44 Ham. T~ Sumida, M., Yzsukawa, M., Walanabe, A~ Okuda, H. and Kobayashi, Y. ( l ~ ) BMod 75, lg87-109L 45 SJUlaS~, J.R., Scchaf=r, J.G, Tang, S-C., Ma~llls-Smlth. A. and S]law, A,R.E. (I989) J. BioL Chem. 264, 1~-289-t2293. 46 $11attil. $d, and Brass, L.F. (1987) J, Biol. Chcm. 2621 tJo2-11Xl0, 47 Ware, J.A. Smith, M. and Salzman, E.W. (198T) J. Clin. Invest. ~0, 2&7-27L 48 Phillips, D . R . Charo, I.!E aad Scarborough. R.M. (t991) Cell 65. 359-36--..
-
49 Fujimota, T . Fujimum, IL and Kuram(~,_~, A. (1991) J. Biol. Cltem. 266. 1637~-16375_ SO Chow. T.W_, Hellums~ J.D.~ Moak¢, J . L and Kroll, M.H. (1OO2) BlmvJ g0. 113-120. 5L K='olI, M,H., Claur¢, R,E,, Miller, & L 0990} Bit,chem. Biophys, Rcs. Commun. 17L,1252-t257.
24~
Monoclonal antibody AG-1 initiates platelet activation by a pathway dependent on glycoprotein IIb-IIIa and extracellular calcium M i c h a e l H . K r o l l ~a, M i c h a e l E . M e n d e l s o h n h J o n a t h a n L , M i l l e r ~, K a r e n K . B a l l e n g. J a n e t K . H r b o l i e h a,h a n d A n d r e w I. S c h a f e r a,h = VA Medical C'enrerand Bayloy College of Medidne, Ho.s~r~ T X (LISA), b Brigham and Women 'x Ho%pltal and Harl,ard Medicat School, Boston, MA (USA) and • Department of Pafkology. SUNY Heallh Science Center, Syrac~l~, N Y ~O.fA)
(Received 15 May 1'992)
Key words: Thrombosis; Calcium; CDg; Glycopmlein: Phospholipase C
The biochemical resl~nses of intact human piatelets to the monoelonal antibody (mAb) AG-I wcrc investigated. AG-[ is a muting igG mAb thal recognizes a series of platelet membrane glycoproteins (Gp) from M~ 21090 to 29 0 ~ , one of which is the M, 24000 (p24) receptor for anti-CD9 mAbs. AG-I causes platelet aggregation and secretion. Platctcls binding AG-I demonstrate a dose- and time-dependent breakdown of phosphatidylinosilol 4,5-bisphosphate (PIFz), production of diaeylglycerol, and generation of phosphatidie acid (PA). These events are associated with the activation of protein kinase C (PKC), an increase in intraeellular ca]ciltm, and fibrinogen binding. Platelet Pat generation and PKC activation in response to AG-I are inhibited by mAbs to piatglet Gpllb-Illa or by extraceliular EGTA, but not by a mAb to ptalclct Gplb or by inhibiting plnt¢lgt Na÷./H * exchange with 54N-ethyI-N-isopropyl)amiloride. Platclet cytoplasmic free calcium ([Ca2+]i) is elevated in response to AG-I, and this elevation is inhibited by mAbs to Gpllb-[Ila, an R G D S pcptide or by ehelatiag extracellular b.alcium. These resu,ats suggest that AG-I binding to a unique platelet-surfac¢ gl~/coprotein ~niliates plalelct responses through the activation of PlPz-speeifie phospholipase C, and that this occurs through a signal pathway that is dependent on Gpllb-Illa and extracellular calcium.
Introduction A G - I is a m u t i n g m o n o c l o n a i antibody ( m A b ) that recognizes a series o f low-molecular-weight pl at e l e t m e m b r a n e glycoproteins ( G p ) in the M r 2 1 0 0 0 to 2 9 0 0 0 range, with the most p r o m i n e n t b a n d o c c u r r i n g at M~ 21 000 it}. It is an I g O originally raised in mice i m m u n i z e d with w a s h e d platelets from patients with
Correspondence to: M.H Emil. Hematology Section. Baylor College of Medicine, MS fO~_ 6565 Fanain Street. Houston. TX 77030. U S A Abbre~iatlons: Plp~. phosphatidylinositol 4.5-bisphosphal¢: Gp. glycoprotein. [Ca2* ]t cytoplasmic free calcium; EGTA, [ethylen=bis(oxyethylenenit rilo)] tetraaeetic acid; PLC, phospholipase C; [P3, inns;to] 1.4,5-trisphosphate; DG, ~n-i,2-diawlglyceroi; PKC, protein kinase C; CP, croat;no phosphate; CPK, croat;no phosphokin ~ ¢ ; USA, I~vin¢ seTum albumin; nlAh monoctonal antibody; ACD, acid-citrate-dextrose; PRP. pI~t¢I~t-rich plasma; SDS PAGE. ~dium dodccyl sulfal¢-pulya~='rylamide gel ¢lecnoPhoresis; p47. M t 47000 protein; pZ0~ M, 20(300 pt'otein~ PA, pho~phatidic acid; [pH] i, cytoplasmic pH; EtA. 5-(N.ethyl-N-isopropyI}amiloride; BCECF-AM.
2'.?'-bis (carl~ethyl)5,h-carbo~fluote~cein
acetoo~methyl ester.
platclct-type yon Willebrand disease, a congenital platelet a bnor m a l i t y characterized by the substitution of valine for lysine at amino-acid residue 233 of glycoprotein ( G o ) Ibm, which is associated with a heighte n e d interaction b e t w e e n the platelet ( 3 p l b , / I X complex a n d n o r m a l v a n W i l l e b r a n d factor molecules [2]. A G - I b i n d i n g to intact h u m a n platelets causes aggregation a n d secretion that are inhibited by cyclic A M P , E D T A , a n d antibodies to G p I l b - I l l a [1], T h e s e obs0rvations indicate that A G - 1 b i n d i n g to h u m a n plate[ors activates signal-transducing pathways f u n d a m e n t a l to the d e v e l o p m e n t o f a s u b s e q u e n t functional response. Several o t h e r plateiet activating m A b s , d e s i g n a t e d as C D 9 antibodies, have been f o u n d to b i n d to a single b a n d of M r 2 4 0 0 0 (refer~-ed to as p 2 4 / C D 9 ) o n oned i m e n s i o n a l W e s t e r n blots [3-7]. A G - I c o m p e t e s with p 2 4 / C D g - s p c o i f i c antibody d e s i g n a t e d m A b 7 [6] for b i n d i n g to a platelet protein o f M T 24000 (p2d). b u t m A b 7 a p p e a r s unable to c o m p e t e with A G - I for b i n d i n g to o t h e r M r 2 1 0 0 0 - 2 9 0 0 0 constituents (,leanings, L.K~, Fox, C.F. and Miller, J.L., u n p u b l i s h e d
249 observations). These findings suggest that platelet activation induced by AG-I may involve different signalling pathways initiated by its interaction with a protein or proteins other than those recognized by such p24/CD9 antibodies. Platelet activation is a complex network of interacting intraeellular biochemical reactions resulting from an extraeellular agnnist binding to the plate!et surface [8]. Receptor occupancy causes the dissoc~arion of a heterotrimeric GTP-binding regulatory prote;n that activates phospholipase C (PLC). PLC catalyzes the hydrofysis of phosphatidylinositol 4,5-bisphosphatc (PIP~) tt: the second messengers inositol 1,4,5-trisphosphate /IPO and sn-l,2-diacylglyeerol (I)(3). IP3 causes the release of calcium (Ca 2.) from storage organelles and DG causes the activation of protein kina~ C (PKC). These events then stimulate the functional responses of aggregation and secretion by inducing structural changes in substrates directly, or indirectly through the activation of specific protein kinases [8]. The purpose of this report is to elucidate the molecular signalling events responsible for AG-I-induced platelet activation. Exl~rimca tal procedures
Materials Human alpha thrombin was from LIS Biochemlcals (Cleveland, OH). Creatinine phosphate (CP), creatinine phosphokinase (CPK). Sepharose 2B-30~, Nonidet P40 (NP40). fatty-acid-free bovine serum albumin (BSA). and indnmethacin were from Sigma (St. Louis. Me). Lipid standards were from Sigma and Calhiochem (San Diego, CA). |-~-"P]Orthophosphorie acid, [ u4C]araehidonie acid, and aZ~l(sodium salt) were from New England Nuclear (Boston, MA). Furs-2 aeetooxymcthyl ester and 2',7°-bis (carhoxyethyl)5,6carboxyfiuorescein (BCECF) acetooxymethyl ester were from Molecular Probes (Eugene, OR). Silica Gel G plates were from Analtech (Newark, DE). Whatman silica gel K5 and LKSD plates were from Fisher Scientific (Fairlawn, NJ). HPLC-grade organic solvents were from American Burdiek and Johnson, (Muskegon, MI). MonoclonaI antibodies (mAb) 7E3 and 10E5 (directed against GpIIb-ll|a) were obtained from Dr. Barry Culler (SUNY, Stony Brook, NY) and mAb AS7 (directed against the amino-termlnal M r apprrtx. 45000 region of Gplb) was produced by one of us (JLM). mAb IV-3 (directed against the M~ 40000 ptatelet FeyRll) was purchased from Medarex (West Lebanon, NH). S-(N-ethyI-N-isopropyl)amiloride (EIA) was obtained from EJ. Cragoe of Merck, Sharp and Dohme (West Point, PA)and was dissolved in dimethyl sulfoxide. Human fibrinogen containing no contaminating plasminogen or yon Willebrand factor was obtained from Enzyme Research Labs (South Bend, IN).
Preparation of monocloaal antibody AG-I Balb/c mice were immunized with washed platelets from patients with platelet-tVpe yon Willebrand disease [2]. Spleen cells from the immunized mi¢¢ were then fused with P3 NS/Ag 4.1 myeloma cells [9]. Culture supernatants from the resulting hybridomas were screened for their ability to inhibit the agglutination of these patients" platelcts by asialo yon WiUebrand factor in the presence of 5 mM EDTA [10]. Several hybridoma supcraatants actually augmented this resl.xmse and in subsequent testing were found capable of inducing aggregation of normal p[atelets without the addition of other ~gents. One of theses hybridomas (AG-I) was cloned by limiting dilution and the cells then grown in plistane-primed Batb/e mice. The resulting ascites was precipitated with ammonium sulfate and affinity purified using the MAPS Affl-gel system (Biorad, Richmond, CA). Ouchterlony analysis using antisera purchased from Serotee (Oxen, UK) showed AG-I to be an lgG of the 5"1 subclass, Fab fragment, of AG-I were prepared by pepsin digestion and subsequently purified as previously descrihed [1]. Absence of intact antibody or of contaminating Fc fragments in the purified Fab preparation was established by SDS-PAGE analysis. Fab fragments were radioiodinated and binding studies performed as previously described ill. Platetet preparation Whole blood Item volunteer donors who had not taken aspirin or other drugs affecting platelet function for at least 10 days prior to donation was collected in 15% acid-citrate-dextrose (ACD). This was centrifuged at 180 Xg for 15 rain. The platelet-rich plasma (PRP) was acidified to pH 6.5 with ACD, CP/CPK (1 raM/25 U / m l , res0ectively) added, and then layered over an albumin gradient and centrifuged at 1500 × g as described by Walsh e t a l . [I I]. Except where noted, the interface platelcts were collected in buffer A (6 mM glucesc, 1.30mM NaCI, 9 mM NaHCO~, 10 mM sodium citrate, 10 mM Tris base, 3 mM KCI, 0,81 KH2PO, and 0.9 mM MgCIz, pH 7.35) and re-centrifuged through the albumin gradient. The collected plat¢lets were suspended in a small volume of buffer A (except where nor'~d) and either radiolabeled or loaded with furs-2 or BCECF by incubating the platelets with the .ppropriate reagent for I h at 37°C in a geotly shaking water bath. Platelets were radiolabeled with 0,5 mCi [S2p]orthophesphat¢ or 20 ~Ci [t4C] arachidonic acid; or were loaded with 2 v,M furs-2 aeetoox'/methyl ester or 6 ~ M BCECF acetooxymethyl ester. Following this. the platelet suspension was gel-filtered through Sapharose 2B-300 equilibrated with buffer A, and sosuspended in buffer A containing I mM CaCI2 at a concentration of 2.5.10s platelets/ml (except where
25O noted). All experiments were carried out in stirring platelet aliquots at 37"C (except where noted), Plarefet lipid measurements Piatelct lipids were extracted according to the method of Bligh and Dyer [121 and analyzed by thinlayer chromatography (T1,C). [32P]Orthophosphatelabeled phospholipids were separated on Whatvaan K5 plates dipped in 1% potassium oxaiate/2 ram EDTA using the solvent system chloroform/acetone/methanol/glacial acetic acid/water (40:15:13: 12:7, v / v ) according to the method of Van Dnngen e t a l . [13]. [14C]Arachidonate-labeled p|atelets were assayed for [l"C]diacylglyeerol formation using silica gel G plates with the solvents benzene/diethyl ether/NH40H (100:80:O:0.2, v / v ) as described by Rittenhouse-Simmons [14] and for [l')C]arachidonie acid and its radiolaholed metabolites on Whatman LK,SD plates using the mlvents diethyl ether/hexane/glacial acetic acid (60:40: I, v/v) as described by Salmon and Flower [15]. Lipid hands wen= detected by autoradiography, idcotifl~d by co-ehromatograpby with unlabeled standards, scraped, and counted for radioactivity. Platelet protein phosptzorylation [~PlOrthophosphate-labeled platelets were stivaalated as described and the reactions stopped by the addition of a solution of 511% glycerol, t0% dithiothreitol, 8% sodium dodecyl sulfate (SDS). and trace bromphenol blue, followed by immediate boiling for 5 rain. SDS-polyacrylamide gel dectrophoresis (SDSPAGE) was performed on 7-17% gradient gels as described by Laemndi [16]. The gels were stained with Coovaassie brilliant blue R, destained, fbled and dried. The phospborylated proteins were located by autoradiography and relevant bands quantified by transmittance densitomctqt on a LKB Ultrascan XL laser dcnsitometer. /Ca z +]~ measurements Washed platelets were suspended in buffer B (145 mM NaCI, 5 mM KCt. 1 mM MgClz, 6 mM glucose, 10 mM Hopes, pH 7At without Ca -'+ and incubated with fura-2 (vide supra). Fura-2-loaded piatelets were gelfiltered and changes in [CaZ+]~ monitored in a PTI Deltascan spectrofluorometer, with dual wavelength excitation capability, using 1.5-ml aliquots of 2.0- Ill" platelets/ml buffer B containing I mM CaCIz or 2 m M lethylenubis(o~ethylenenitrilo)] tetraacetie acid (EGTAt added 1 vain before the addition of agnnist. [CaZ+L was calculated from the ratio of fluorescence emission at 510 nm, following the excitation of fura-2qoaded platelcts at 340 nm and 380 nm. using a K,~ for fura-2 of 224 nM, as previously desex'thud ~17].
Fibrinogen binding assay Human fibrinogen was purified chromatographically, including lysine-Sepharose column separation of contaminating plasminogen and immunoaffinity column separation of contaminating yon Willebrand factor. Fihrinogen was radloiodinated as described by Loscaizo et aL [18]. Fibrinogen binding studies w e ~ performed at 22°C as described by Hawiger et al, [19]. T/lromboxalte measurements
Production of plate[el thromboxane A z was quantitared by radiogmvaunoassay of its stable breakdown product thromboxane B z as previously described [171. pHj rtleosurc/ff~Hl$ Washed platelets were suspended in buffer B withoat Ca -'÷ and incubated with BCECF-AM (vide supra). BCECF-Ioadod plate!ors were gel-filtered and changes in ~toplasmic pH (pH i) measured in a PTI Deltascan spectrofluorometer, using 1,5-ml al~quots of 2.0-i0 s plarelets/ml buffer B plus 1 mM CaCI z, according to the methods of Zavoico etal. [20].
To examine pathways of AG-l-induced platelet activation we first measured the dose- and time-response of intraplatelet signal generation following AO-I binding, The ECs. for platelut phosphoinositide turoover, p47 phosphorylation (M, 47000 substrate for PKC), and p20 phosphorylation (M, 200ID substrate for Ca2+/calmedulin-dependent protein kinase; myosin light chain) is 2-3 ttg/mL This is consistent with the EC~, observed for functional platelet responses it[. The lag phase and the magnitude of these responses are concentration-dependent. Fig. I demonstrates the time-course of PIP, turnover, DG production and the generation of phosphatidie acid (PAt in platclets stimulated by 3 / z g / m l AG-L Protein phosphorylation following AG.I (3 ~ g / m l ) binding to plalelets is represented in Fig. 2. Synchronous mcasorcvaeuts of PIP, and protein phospborylation show that p47 and p20-phosphorylation occur after the hreakdow~ of PIP2. The phosphorylation of p47 indicates that PKC is activated and the phosphoryla'.ion of p20 indicates that intracellular Ca z* ([CaZ"]=) rises in response to AG-1. This latter conclusion was corroborated by directly measuring [Ca-'+]i following AG-i binding (vide infre). The biochemical responses demonstrated above are associated with the release of arachidonle acid and its conversion to active metabolites. Thus, platelet thromboxane B_,~the stable breakdown product of throvaboxane A~, increases from a basal level of 168 p g / m l to 2373 p g / m l 30 s following AG-i stimulation (3 ~ g / m l )
251
and persists at levels greater than 2000 p g / m [ for 5 min fol[owing the. addition o f A G - L A G - I causes the exposure of fibrinogen binding sites on platelet surfaces. Fig. 3A shows the tirae-course of fihrinogen binding to platelets stimulated by AG-1 and Fig. 3B shows that fibrinogen binding to A G - I stimulated platelets is saturable+ In an attempt to define the proaimal signal-generating steps resulting from the A G - l / r e c e p t o r coupling event, a series of experiments was performed to examine the role o f ptatelet-surface glycoproteins in A G - I induced PLC activation. Previous data show that AG- 1 iraraunoprecipitates a series of tow-molecular-weight proteins from the surface of platelets that are distinct from the 1~ chain o f G p l b [1]+ We additionally observe minimal inhibition of AG-l-indu~ed platelet protein phosphorylation and PA generation when the platelets are pretreated with AST, a mAb to the a chain of
iL/ 11111
+
-o----.,+ . . . . . . . .
+
........,+0
I
i
0
I 2
I
4
I 8
I 8
I
10
Time (raM)
Fig. 2. AG-I (3 gg/ml) stimulates the time-dependent pho~phowlalion of pIatclct Mr 47000 protein (p47) and M+ 21)01)0protcln (p2a). [32P]Orthophosphate-loadcd platelcts were Iicated with AG-I and protein phospho~'lation measured at 1he designated time points (as descried in Ealmrimcatal prom.glurgs). Tngs~ data are expn:ss~d relative to I]% pho~phoIMation at time 0 and are repTe~nlative of f,Lx separate experiments.
,~ 110
->'%/,.t-'~b
6.~ 100
~
\,
E
..°---o+•
+,
10 '++
0
+o,0
l 1
l 2
% 12G
+l~.::lk. ~
l 4
I S
Time (rm.)
1000.
"t OLf
I
I
I
I
I
0
1
2
3
4
5
..
I
10
•r i m ( r a i n ) Fig. L A G - I (3 p g / m l ) cauy~5 platelet phosphatidyiirlositol~l,5-bis +
phosphate (PIP2) brea]~down and reSynlhesis, and the generation of diacylglye~rol (DG, top half): and AG-I (3 .ug/'ml) causes Ihe production of plat¢let phosphotidi¢ acid (PA, bottom hal0. PIP, and Pat in response to AG-I were measured synchronously in the same prcpar~ltion of -~2P-lahclc~ plait:lois (as. dcscdh~d in Experimenla[ I)rov.cdure~). DO m¢i~rement~ were made in sel~t-ate plat¢let aliquo~s labeled with [x4C]araehidonicacid (as descrff~cd in Experimental pro~duresl. Dala arc expressed relative tO 100% measure. n~ents at time 0 (PI~). or 0% measurement at time 0 (DG, PAl, and are re~esentalive of 3 separate eqlerimenLs.
G p l b [21] (Fig. 4 and Table l). In contrast to this, two different mAhs to G p l l b - l I l a , designated 7E3 [22] and 10E5 [23], markedly inhibit platelet protein pbesphowlation gad P A production resulting from AG-1 binding (Fig. 4 and T a b l e D. Because previous d a t a indicated that A G - I does not bind to G p l l b - i l l a on Western blots [I], and a mA.b to G p l l b - [ f I a does not inhibit AG-1 Fab binding to intact human platelets (data not shown), these new results suggest that G p l l h - l l l a plays an indirect role in mediating AO-l-induced responses by coupling the platelet receptor for AG-1 with the activation of PLC. Table I demonstrates the effect o f antibodies to G p l b and G p i i b - I I I a on AG-l-indnced platelet PA production. This table also shows that E G T A , which chelates ex-ttacellular Ca 2", is inhibitory to A G - l - i n duced PA production, but indomethacin has little effect on AG-l-inducvd PA production, 2 ram E G T A was added 1 rain before A G - ! to avoid dissociation o f platclct O p l l b - I I I a that occurs with longer incubations with calcium chelators [24]. These results are not likely to be due to the effect of egtracellular calcium on A O - I binding to its platelet receptor(s), because E D T A had an effect on the binding o f [IZSl]AG-I Fab to platelels (data not shown). E I A , which inhibits platelet N a ÷ / H ÷ exchange and abolishes platelet cytoplasmic alkalinization in response to throrabin [20], causes no change o f AG-l-inducad P A production (vide Jaffa). T h e platelet M r 4 0 0 0 0 F e y R I I is considered to be the mediator o f platelet responses caused by some mAbs sp~:ifie for the platelet CD9 complex [4,25,26]. We therefore examined the effect of the m A b I V 3 (anti-Pc~,RII [27]) on A G - l - i n d u c e d platelet signalling. IV.3 (20 p g / m l , - 5 min) completely inhibits platelet P A production and protein phuspho~lation 5 rain follow-
252
ing 3 $zg/ml of AG-I. This is cousistent with the inhibitor, effect of IV.3 o n AG-l-induced aggregation (data not shown) a n d secretion [28]. To determine if AG- l binding to the platclct FcyRII is the so,e mechanism by which AG* 1 activates platelcts, Fab fragments of AG-I . . . . inct,hatcd for 5 mln with "2P-labelled platclcts for PA mcagargments. Fab fragments of AG-I g e n e r a t e R significant quantity of PA, although the ECs~, is ~pp . . . . 5-fold higher than that of the intact antibody: 15 /zg/ml AG-I Fab causes PA production at 5 rain that is 157 =i:61% (mean _+S.E.)above control.
.~ "~ :~ .It."~ ~
~ ~ "~l ~
'~~ 12
0 ~ ~ - - ~
h. 0.)
7
~"i~
_,,^p47-
A.
t~l~ L.U ~ '~-
is involved in AG-I*indaged signal generation, one must take into account the potential ro!e o f Gpllb-Illa
0.8
8 __ ~
~
0.6 0.4
~
0.2 0,@
[J"U0
5
10
I l 20 2S (mln)
15
Incubation Time
~
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*
.~
30
Fig. 4. Monoclonalanzihodicsto plateh~t GFJIIb-lllainhlbilplat¢lct protein phosphotylatDn in rcslam~ tu AG-I. ~ZP-labeltdph~t¢lcts
B. 4O
~_--
~
wcP¢ incubated with AG-I for S rain and the reactions slopped and ;.Lssayed as descril~d in F~!~zrim¢otalpr~x~durcs. The ;~Jltil~3dics
"/E3(3 gg/rnl) or IIiF~(5/zg/lr~llw¢[¢ incubated with th~ plalelcLs for 30 rain and 1 rain. respectively,prior l¢1lh¢ additi{m 0f AG-L Ptutelet pd7 and p2,~phnsphowlalion 1 rain fidh~wingthrombin, and 5 rain I'ollowii~gbutter, al'¢ sh~n fur compa~son. These data are
ao
repres¢ntativ¢ of 4 sepa~kt¢ L'Xp~Tim~nLs.
I
I
I
4100
600
I I~0
1000
~qUdao~a (p~m~)
Fig. 3. Effectof AG-I on [ t~llfibrinogenbinding In human pi~ltg|gl.S at 22~C. (AI Timc-coulrs¢of [11~l]fibfinugenbinding to platclct.~ activaled I~ AG-I. Gel-filtered plat¢lels weT¢ incubated with 3 ~g/ra[ AO-I fe~ the times noted. Fibrilmgen hou ad is q:xp[¢~sedas a
rl-ilclion oi"or tOtalbindingc~grvcd at 3a rain (B/B m). (B) Eft©c!of [I-~|iiffhrtnogcn concentration on AG-I-induccd platclet fibrinngcn binding. Gel-filtered platclets ~ucr¢ incnbal~d for 20 rain wilh 3 ,agOrot AG-I at incma~;ing cuncent~'alions o; Ubrln0gen and platelet-~ouad fibnnugcn~vagmeasuL'ed ag degct'ih~d in F~perimental prnccdur~s. Dala ]'¢presenl spedfi¢ binding and are means of duplical¢ determinations.
in platclct calcium homeostasis. G p l l b - l l l a is the primary surface binding site for Ca ~÷ on unstimulated platelers [29]. It may be important in the transmcmbraanus movement of Ca z+ during platelet activation although the precise role of this cation flux in platelet activation remains unknown [30-36]. To examine AGl-induced platelet calcium responses and to determine how GpllL~llla may affect them. a series of spcetrofluorometu experiments was performed on platelets loaded with the calcium fluorophore tara-2. The dosea n d time-response of AG-l-induced elevations of IC~2*lt parallel that observed for PIP2 turnover. DG and PA generation, and protein phospho~lation (data not shown). Fig. ~ shows that 2 raM E G T A added 1 rain before AG-I (which chelates extracallular Ca 2.
253
AG-1
AG-I 3gglml
Thrombin 1UIml
3pg[m!
4.ca{It2 FIE3 11]0~
SOt
~ 4 t 1
2
3
i
4
5
0
-tCaCI2 ,-rE
1
Tim(mi e n) 2
3
4
5
1
2
3
4
§
Fig, 5. The effect of extracellular Ca2+ and Gpl|b-nla on the platelet intracelIularCa2+ ([Ca2+ ]~} response to AG-I. In Ibis speclrofluorOmeldc tracing, the absci~.~ais time (mini and the ordinate is ICa" ~II. calculated from the ratio of fluorescence emission (at 511)am) following tl~edeal exeitatiorlof fura-2-1oaded platelets tat 341)and 380 aam),as deseril~ed in E~perimeraal procedures. Unstirred platelet aliqttot= in tmfler at 37"C were p~e-lncubated ~ilh eaCI z (I raM. --I mini. EGTA (2 raM, --I mini, 7E3 (3 p.g/ml, -311 mini. or RGDS (200 ~g/ml, - 5 mini and elevations of [Ca-'+ li measured in slitting plate-let aliquots at 37°(2slimulated by AG-I (3/xg/ml) or Ihmrabin ( I U/ml), These tracings are representative of Cr,'eseparale experiments. w i t h o u t affecting the functional integrity of p l a t e l e t G p l l b - I l l a [24]) inhibits t h e elevation o f [CaZ+]+ observed following p l a t e l e t binding to A G - I . This is different from t h e effect o f E G T A o n [CaZ+]i responses to thrombiu, where extracellular C a 2+ has only a small impact on t h e maximal extent of the rise in [Ca2+]i (Fig. 5)- Fig. 5 also shows t h a L in the presence o f extraeellular C a 2+, a m A b to G p l l b - l l l a (7F,3) abolishes the [Ca2+]i response to A G - I , but has no effect on the [Ca 2 +]i respous¢ to thrombin+ T h e t e t r a p c p t i d e R G D S (arg-gly-asp-scr), which binds to G p l l h - l l l a and TABLE I 3"~P-lab~:led nlatel¢ltSweL'e incubated with AO-I for 5 mln and the reactions terminated and assayed for PA production as descn~oed in Exlmriracnlal procedures, 7E3 (anti Gpllb-ltla), 11)E5(anti-Gpllb+.tin) alld AS7 (anti-Gplb) were pneincubated with platelets as follows: 7E3, 3 ~tg/ml, - 3 0 win: 10F-5, 5 ~g/mt. - 1 rain: AST. 5 ttg/ml. - 5 miix 2 mM EGTA was added I rain prior to AGq. 270 gtM indomethacin was added 2 rain prior Io AG-I. To block Na +/H + exchange, 411 FM EtA w-~ added 24) s prior to AG-I. The data s h ~ n am mca~:tS.[L of l~r~nt platelet PA ploductlon above conttgl levels (PA p~ttzced at ;5rain in anstinlulated stirring platelet aliquots at 37'~L Treatment
n
Pet
AG- 1:4/xg/ml +7F--3 ÷ leES +AS7 + EGTA + indomclha~ n + EIA
6 3 3 6 6 1~ 5
247.64-_40 -4.0+ 16 * 68,3+58 * 195.0+51 98.2+35 * 174.(1± 71 200J0+_64
• P > n.05 IWtwoda1)ed/-lest tunpaired means), in comparison wilh enntrol leve*s.
inhibits its interaction with R G D - c o u t a i n i n g ligands such as fibrino.~en [37], also completely inhibits p]atelet C a 2+ responses to AG-1 (Fig. 5). These observations demonsh, ate that A G - I - i n d u c e d signalling may require a functional G p l l b - l l l a complex to effect the transmembranous influx o f calcium. The observation that A G - I initiates platelet phos. pholipase C activation by a mechanism that is dependent on G p l i b - l l l a is similar to those o[ B a n g a e t al., who examined epinephrine-induced platelet activation [38]+ T h e s e authors demonstrated that epinephrine causes an initiation signal for p l a t e l e t activation through the pbospbolipase A2-mediated release of small amounts of arachidonic acid. This is a well-established concept that forms the basis for the dependency o f weak agonists on intact cyelooxygenase metabolism to effect a full platelet response [8]. A s previously described by Sweatt e t al., this signal is dependent on N a + / H * exchange [39,40]: B a n g a et al. reproduced these earlier observations and showed t h a t a n antibody to G p l l b - I l l a inhibited both cytoplasmic alkalinization and phosphatidic acid production in response to epinephrine, suggesting thai G p l I b - i l l a cou!3ies ~2 re" ceptor occupancy to N a + / H ~ exchange and cydooxygenase-dependent platelet activation [38]. Because A G - l - i n d u c e d P A production is not inhibited by indomethaein, it is likely that this ligand stimulates platelets through pathways distinct from those activated by the weak agonist epinephrine. T o directly assess the role of N a + / H + exchange in A G - l - i n d u c e d platelct reslxmses, p H i was measured in BCECF-Ioaded platelets stimulated by AG-1. These experiments showed that A G - i causes no change in platelet p H i from resting
254 levels. Consistent with thes~ findings are observations that EIA, which inhibits platelet Na+/H + exchange. does not inhibit AG-I -induced PA production (Table t). DiscnsMon
AG-I is a m A b that binds to a series of Iow-molccular-weight platelet-surface proteins having M~ 21 000 to 29000, only one of which is the p24/CD9 antigen. Recent eDNA cloning of the p24/CD9 protein demonstrates that this protein has one N-glycosylation site [41,42], suggesting that the degree of glycosylation cannot account for the different species of platelet proteins that are immunoblotted by AG-I. Therefore, it is likely that AG-I binds also to other platclet-snrface proteins, raising the possibility that AG-1 is the ligand for a platelet membrane signal-transducing protein that is different from p24/CD9. The binding of A(J-1 to human platclcts causes platelut activation of a magnitude comparable to that observed with the most potent physiological platelet agonists. Studies of the biochemical mechanisms of AG-lqnduce~d platolet activation presented here provide new information about fundamental signal-response COUldingmechanisms that may be relevant to platelet activation in rive. We have shown that AG-I stimulates phospholipase C, activates protein kinasc C, cause~ an elevation of [Ca2+]~, and initiates the expression of fibrinogen binding sites. AG-l-induced signals for platelet activation are inh~ited by monoclonal antibodies to Gpllb-llla and by cxtracellular ECITA, suggesting Ihat AG-! initiates platelet phuspholipase C activation through a process that is dependent on ex. tracellular calcium and a functional membrane GpIlb-?lla complex. AG-I initiated platelet activation is different from that observed with a 2 receptoc-initiutod platclet phospholipase C activation because it is not dependent on N a ~ / H + ¢~change and not inhibited by indometbaein, indicating that the initiation of platelet phospholipase C activation by AG-I does not follow the Na*/H+ exchange-dependent release and metabolism of arachidonic acid. Indomethacin does, however, inhibit AG1-induced 5-hydro~tr~tamine secretion, suggesting that an amplification response to AG-I depends ou intact arachidonic acid metabolism ]1]. Data showing that AG-I induces the production of large amounts of platelet thromboxaoc A2 are consistent with this hypothesis. Platelet aggregation induced by anti-CD9 mAbs was first reported by Boucheix and colleagues in 1983 [3], who more recently showed that their p24/CD9 mAb causes platelet phosphatidic acid formation and PKCand Ca2+-dupendent protein phosphorylation that is not dependent on extracellular Ca"* but may be de-
pendent on the activation of PLC [43]. Hate nt al. have reported a p24/CD9-specifie mAb that activates platelets and induces expression of the fibrinogen receptor;, this activation is independent of extracellular Ca z÷ [5,44]. The mechanism of fibrinogen binding site expression induced by p24/CD9 antibodies is unknown, but Slupsky el. el. demonstrated that platelet p24 associates with Gpllb-IIIa following iLs binding to ligand [45]. There is no evidence for this occurring in response to A(/-I, and present data indicate that ACI-I does not interact directly with platclet Opllb-ltla and that platel~.t Gpllb-llla remains acccsslble to fibrinogun binding following platelet incubation with AG-1. An alternative explanation for the basis of AG-l-induced fibrinogen binding is, however, suggested by this study: AG-1 initiates fibrinogen binding to platelets by causing intracellular signals that directly induce the Gplib-llla receptor for fibrinogen on activated human platelets [46]. Jannings ct at. and Worthington et al. have reported p24/CD9 mAbs that cause platclct PLC activation [25,26]. In contrast to our results with AG-I, they observe that this is not dependent on Gpllb-Illa, but find that this involves Gplb [25,26]. While these groups have reported that antibodies to Gplb completely inhibit anti-CEO-induccd plat¢lct activation, we observe tha* AS7 (aati-Gplb) does star inhibit AG-l-induced olatelet activation. We also find that AG-l-indueed platelet activation is inhibited by an antibody to the M r 40000 platelct Fc~,RII receptor, and this is consistent with that observed with other p24/CD9 mAbs [25,261. In addition, AG-I appears to initiate platelet signalling independent of the platelet Fe-~Rll receptor, because AG-I Fab fragments stimulate PA production. These obsercations, in conjunction with previous observations that AG-1 Fab does not stimulate platelet aggregation ill, suggest that, as has been described with epinephrine and endoperoxidc analogues [47], PLC-madiated platelet intracellular signals in response to AG-1 Fab do not reach a magnitude sufficient to cause aggregation. The activation pathway initiated by A G d binding to human platelets appcat~ to differ in important ways from platelet activation pathways induced by p24/CD9 antibodies. It is possible that AG-1 binding to other constituents of the M, 21000 to 29000 family of platelet memblane proteins that it immunoprecipJtatus triggers cellular processes that act in concert, or perhaps even antagonistically, to those initiated by its binding to the p24/CD9 protein. AG-l-induced platelet activation is completely depqndent on Opllb-llla. This result supports the by. pothcsis that Gpllb-ttta is important in mediating platelet stimulation, perhaps by regulating the transmembranous influx of calcium [48]. This mechanism of Gplib-Illa function is consistent with two hypotheses
255 proposed by others: (1) G p l [ h - l l l a is structurally a n d / o r functionally related to platelet calcium channels [30-33] and (2) calcium channels are important in platelet activation I34-36]. Evidence in support of these hypotheses cominues to ace,qae, although the mechanism by which G p l l b - l l l a regulates platelet [Ca2÷], is not fully elucidated. Recently published d a t a demonstrate that G p l I b - l l l a regulates voltage-independent C a 2+ currents in platelet plasma membranes and t h a t ligand binding (mAb o r R O D S peptide) decreases channel aetiviW [491. T h e r e are, in addition, recent d a t a thai platclet [Ca2*|i responses to shear stress result from the transmembranous inGux of Za 2~, and t h a t mAb o r peptide inhibition o f G p l l b - I l l a function decreases these responses [50]. Precise eharaeterization of the gtrstetaral relationships between the occupied A G - I receptor, p 2 4 / C D 9 , Gpllb-llla~ and phespholipase C will be possible only when the primary structure o f the functional A G - I rec~plor is determined. A M r 2 1 0 0 0 m e m b r a n e protein immunoprecipitated by A G - I is, in partieular, a good candidate for a signal transducing protein: not only is this the major platelet-surface glyeoprotein protein recognized by A G - I , but, in addition, this protein (or a coprecipitating protein of similar molecular weight) hag been d e m o n s t r a t e d to be a GTP-binding protein {51|. Acknowledgements T h e a u t h o r s t h a n k D r . S t e p h e n Peiper f o r h e l p f u l discussions and D e a n n a G o l d e n for the preparation of this manuscript. Supported by N I H grants H L 0 2 3 1 1 , HL02154, HL328S3, and HL3604S, the V A Merit Review Board, Granls-in-Aid from t h e Texas and Massachusetts Affiliates of the A.H.A., and the Biomedical R e s e a r c h Support G r o u p of Baylor College of Medicine.
Referenoes I Miller, $.L~ Kupinski, J.M. and Hastad, K.O. rig86) Blood ~8. 743-75 L 2 Miller. J.L, Cunningham, I), Lyle. V.A. and Finch, C.A. (1991) Frog. Natl. Aead. Set. USA kS, 47Sl-4765 3 Bouchetx. C., 5aria, C., Mi~hahi, M., Sofia, J., Per/at, J,-Y., Fourier, N., Pdlard. M. and Ro~nfcld, C. 119831 FEBS Len. 161,289-295. 4 Carroll. R.C., Worthington, R.E. and Boueheix, C. (19~) Biochem. J. 266, 52"/-535. 5 Halo, T,, Iked;*, let-,Vas1~kawa,M, Watanabe, A. and Kobayashi, Y. (19881 Blood 72, 224~z?.g. 6 J,JnrdslSs, LIC. Fox. C.F., Kouns. W.C., McKay, CP.. Bailou, L.R. and $ehulhZ.i-I.E. 119991J. Biol. Chem. 265. 3815-3g22. 7 ttigashihara, M, Takahata. Y, Nakahara, g., Ku¢okawa. K. (1990) FEBS LeU. 264, 270-274. 8 Kroll, M.H. and Srhafer, A.I. 119891Blood 74, 1181-1195. 9 at. V.T. and Herzenherg. L..,L (tgSo) in Seleclod Methods in
Cellular Imrnuanlogy (B.B. Mish~:lland S.M. Shiigi. eds.I p. 351. WIHI Fleeman and Co.. San Fransls¢o. In Miller, J.L., Ruggeril Z.M. and Lyl~, V.A. (1987) Bkloa 7n. 18114-1809. I I Wakh. P.N., Mills. D.C.B. and White, J.G. (1977~.BL J. Haemaiol. 36, 281 296. 12 Bligh, E.G. and Dyer. W.J. (lgsg) Can. J- Biachem. Ph~iol. 37. 191-lgT. 13 Van Dtmgen, C.$., gwi,~r:i. H. and Oi~pen, W,H. (I';SD Anal. Biochem. 144. :134 1119. 14 RhtenhotLse-gimmon~. S. (1981)J_ Biol. Chem. 2S6. 4153-415S. 15 Sulmoll, J.A and Rowers, RJ. (I9821 in Methods in Enzymology (W.E.M. Lands and W.L. Smith. eds.I Vol. 86, pp. 477-492, Academic Press. New York. I11 Laemmli, U,K. 119701Nature 227, 680-685. 17 Kloll, M.H., Zavoien, G.B. and Schafer, A.I. 119891J. Cell. Physiol. 139.558-S64. 18 Loscalzo,J., lnhal. A. and Handin, R.I. 1198@J. Clin. Invest. 78. 1112-1119. 19 Ilawlger, r., Parkinson. S. and Timmoag S. (19801Nature 283. 195-197. 20 Zavoico. G.B., Cragoe, E.J., JL and Feinstein, M.B. (lg86) ,I. Biol. Chem. 2111,[3160-13167. 21 Miller) &L., Htlslad, K.O., Kupinski, $.M., L?d¢, V.A., and Kunicki. TJ. (It)~l} Br. J. Haeraalol. 74, 313-31g. 22 Colter, B.S., Peerschk¢, E.I., Scudder. L.E. and Sullivan, C.A. (19831J. Ciin. Invest. 72, 325-338, 23 Caller, B.S. (19851J. Clio. Invest. 76. IOl-10g. 24 Fitzgerald, L.A. and. Phillips, D.IL 119851J. Biol. Chem. 260. 1136fi-11774. 25 Jennings, L.K, Kouns. W.C and Fox, C.F. 11989) Blood 74 (suppl. l) A 633. 26 Worthington, R.E., Carroll, R.C, and I~ucheis, C. (19~1) gr. J. HaeraatoL 74, 218-222. 27 nosenfeld, S.I., Ryan, D.H.~Looney. RJ.. Anderson, C.L, Abraham. G.N., I.eddy. J.P. 11~87lJ. lmmunoL 138, 2869-2873. 28 Horsewood, P.. Hay,~eard.C.P.M.. Wartenkin, T.E. and Kelton. J.G. (1991l Blood 78. 1019-1026 2g Brass, LF. and Shanil, S.J. (1984l J. Clin, Invest 73. 6211-632. 30 Powling, MIJ. and Hardisty, ILM. (Ig85) Blood 66, 731-734. 31 Rybak, M.E., Renzttlli, LA., Brans, M.J. and Cahaly, D,P. (198R) Blood 72, 714-720. 32 Yamaguchi, A., Yamamoto, N., Kitagawa, H.. Tanoue. K. gad Yamazaki, H. (19871FEBS Lett. 225;,228-232. 33 SinlgagIia, F., "ford. M., Ramaschi, G. and Balduini, C. (19891 gioehim. Biophys. Acla Q84~225-230. 34 Hallam, T.J. and Rink, TAr. (19S3) FIzBS L~Ir. 186, 175-179. 35 Zschauer, A.. van Breeman, C,, Bnhlcr, F.R. and Nclsa~n, M.T. t1~181 Natut~ 334, 703-705, 36 Suldan, Z. and Brass, L.F. (1~1) Blood 781 2887-2893. 37 Phillips, D.R., Charo, I.F., parise, L.V. and Fitzgerald, I-A. (1988) Blond 71,831-g43. 3S Bansa, H.S., $imons, B,R, Brass. L.F. and Ratenhouse, S.E. t1986) pix3c.Natl. Acad. Sc[. USA 83, 9197-9201. 39 Swean, J.D., Blair, I.A., Cragoe. E.J. and Limbird. L.I:, tl'~Ss) J. Biol. Chem. 261, 8660-8665, 4{I SweaR, J~D,, Connolly, T,M,~ CraSo¢, E,.L and Lirahird, L.IL ( 1g~161J. Biol. Chera, :261,8667-8673. 41 Lanza, F.~ Wolf~ D, Fox, C.F., Kicffer, N., Seyer, J.M., Fried, V.A., Coaghlin, $.R., Phillips, D.R. and Jeanings. LK. l199D J. Biol. Chem. 266, lQ638-10645. 42 noucheix, C., nenoit, P., Ftachet, P., Billard, M., Worthinl~lOn. R.E., Ga~non, J. and Uzan. G. t 19911J.Biol.Chem. 266,117-122. 43 Rends, F., Boaeheix, C., Lebi'¢1, MI. Bourdeau, N.. Benoit, P,, Maclour, J., Sofia, C. and Le~/-Toledano, S. 11987) Biochem. Biopbys. Res. Commun. 146, 1397-1404.
256 44 Ham. T~ Sumida, M., Yzsukawa, M., Walanabe, A~ Okuda, H. and Kobayashi, Y. ( l ~ ) BMod 75, lg87-109L 45 SJUlaS~, J.R., Scchaf=r, J.G, Tang, S-C., Ma~llls-Smlth. A. and S]law, A,R.E. (I989) J. BioL Chem. 264, 1~-289-t2293. 46 $11attil. $d, and Brass, L.F. (1987) J, Biol. Chcm. 2621 tJo2-11Xl0, 47 Ware, J.A. Smith, M. and Salzman, E.W. (198T) J. Clin. Invest. ~0, 2&7-27L 48 Phillips, D . R . Charo, I.!E aad Scarborough. R.M. (t991) Cell 65. 359-36--..
-
49 Fujimota, T . Fujimum, IL and Kuram(~,_~, A. (1991) J. Biol. Cltem. 266. 1637~-16375_ SO Chow. T.W_, Hellums~ J.D.~ Moak¢, J . L and Kroll, M.H. (1OO2) BlmvJ g0. 113-120. 5L K='olI, M,H., Claur¢, R,E,, Miller, & L 0990} Bit,chem. Biophys, Rcs. Commun. 17L,1252-t257.