Negative Feedback Regulation of Human Platelets via Autocrine Activation of the Platelet-derived Growth Factor a-receptor*

Transcription

1 THE JOURNAL OF Blo~oc~ciu. CHEMISTRY 1994 by The Americn Society for Biochemistry nd Moleculr Biology, Inc. Vol. 269, No. 19, Issue of My 13, pp , 1994 Printed in U.S.A. Negtive Feedbck Regultion of Humn Pltelets vi Autocrine Activtion of the Pltelet-derived Growth Fctor -Receptor* (Received for publiction, Jnury 3, 1994, nd in revised form, Mrch 7, 1994) Flemming S. VssbotnlOn, Ole Kristin HvnenPII, Crl-Henrik Heldinl, nd Holm HolmsenO From the $Ludwig Institute for Cncer Reserch, Biomedicl Center, S Uppsl, Sweden nd the Deprtment of Biochemistry nd Moleculr Biology, University of Bergen, N-59 Bergen, Norwy Humn pltelets contin pltelet-derived growth fctor (PDGF) in their -grnules which is relesed during pltelet exocytosis. We show by immunoprecipittion nd 12'I-PDGF binding experiments tht humn pltelets hve functionlly ctive PDGF -receptors, but not P-receptors. The PDGF -receptor (PDGFR-) ws identified s 1'7-kD glycosylted protein-tyrosine kinse s found in other cell types. Stimultion of pltelets with.1 unit/ml thrombin resulted in significnt increse (2-&fold) of the tyrosine phosphoryltion of the PDGFR-, s determined by immunoprecipittion with phosphotyrosine ntiserum s well s with PDGFR- ntiserum. The observed thrombin-induced utophosphoryltion of the PDGFR- ws inhibited by the ddition of neutrlizing monoclonl PDGF ntibody. Thus, our results suggest tht the pltelet PDGFR- is stimulted in n utocrine mnner by PDGF secreted during pltelet ctivtion. Preincubtion of pltelets with PDGF inhibited thrombin-induced pltelet ggregtion nd secretion of ATP + ADP nd j.3-hexosminidse. Thrombin-induced pltelet ggregtion ws lso reversed when PDGF ws dded 3 s fter thrombin stimultion. Inhibition of the utocrine PDGF pthwy during pltelet ctivtion by the PDGF ntibody led to potentition of thrombin-induced P-hexosminidse secretion. Thus, the PDGFR- tkes prt in negtive feedbck regultion during pltelet ctivtion. Our demonstrtion of PDGF -receptors on humn pltelets nd its inhibitory function during pltelet ctivtion identifies new possible role of PDGF in the regultion of thrombosis. Pltelet-derived growth fctor (PDGF)' is potent mitogen nd chemotctic fctor for mesenchymlly derived cells (reviewed in Ref. 1). Receptors for PDGF hve recently lso been found on other cell types, e.g. cpillry endothelil cells (2, 3) nd neuronl cells (4). Severl observtions suggest tht PDGF my be involved in disorders with excessive cell prolifertion, such s cncer, therosclerosis, nd chronic inflmmtory conditions (reviewed in Ref. 5). Evidence hs been presented sug- * This work ws supported in prt by the Blix' Foundtion. The costs of publiction of this rticle were defryed in prt by the pyment of pge chrges. This rticle must therefore be hereby mrked "duertisernent" in ccordnce with 18 U.S.C. Section 1734 solely to indicte this fct. 9 Supported by grnt from the Norwegin Cncer Society. To whom correspondence should be ddressed Dept. of Biochemistry nd Moleculr Biology, University of Bergen, N-59 Bergen, Norwy. I( Supported by grnt from the Norwegin Reserch Council for Science nd Humnities. The bbrevitions used re: PDGF, pltelet-derived growth fctor; GFP, gel-filtered pltelets; PBS, phosphte-buffered sline; PDGFR, pltelet-derived growth fctor receptor(s1; mab 6Dl1, monoclonl ntibody ginst PDGF gesting norml physiologicl roles of PDGF during wound heling, plcentl growth, erly embryogenesis, s well s during glil cell differentition (5). Structurlly, PDGF is disulfide-linked dimer composed of two homologous polypeptide chins denoted A nd B (1). The PDGF isoforms, PDGF-AA, -AB, nd -BB, medite their effects by binding to two protein-tyrosine kinse receptors (PDGFR), denoted the - nd P-receptors (6-8). Lignd binding induces receptor dimeriztion nd utophosphoryltion, which correltes with ctivtion of the receptor kinses (9-11). The -re- ceptor binds both PDGF A- nd B-chins with high ffinity, wheres the P-receptor binds only the PDGF B-chin. Both receptor types medite stimultion of DNA synthesis nd mitosis (1). Interestingly, the PDGF P-receptor nd -receptor hve gonistic nd ntgonistic effects, respectively, on the chemotctic response of humn fibroblsts (12, 13). Humn pltelets contin ll three PDGF isoforms, minly s PDGF-AB heterodimers (14, 15). PDGF is n -grnule component (16) nd is secreted during the pltelet exocytosis induced by thrombin, collgen, or ADP (17). Humn pltelets hve been shown previously to bind lz5i-pdgf, nd the ddition of exogenous PDGF ws found to modulte collgen-induced pltelet responses (18). We hve therefore exmined the possibility tht humn pltelets hve functionl PDGF receptors tht cn be ctivted by PDGF secreted during pltelet ctivtion. Our results demonstrte the presence of PDGF -recep- tors (PDGFR-) but not PDGF p-receptors in humn pltelets. Activtion of the pltelets by thrombin resulted in ctivtion nd tyrosine phosphoryltion of PDGFR- in n utocrine mnner. The observed utophosphoryltion of the PDGFR- ws independent of exogenous PDGF nd the utocrine receptor ctivtion ws inhibited by the ddition of neutrlizing PDGF ntibodies. Moreover, the ddition of exogenous PDGF or PDGF ntibodies inhibited or potentited thrombin-induced pltelet responses, respectively. MATERIALS AND METHODS Cell Culture nd Pltelet Isoltion"AG1518 humn foreskin fibroblsts, pssge 15-3 (Humn Genetic Mutnt Cell Repository, Cmden, NJ), were cultured in Egle's minimum essentil medium supplemented with 1% fetl clf serum, 1 units/ml penicillin, nd 5 pdml streptomycin in humidified 5% CO,, 95% ir t 37 "C. Pltelets from humn blood, nticogulted with cid citrte dextrose, were trnsferred by gel-filtrtion (Sephrose CL-2B; Phrmci LKB Biotechnology Inc.) into clcium-free Tyrode's buffer (ph 7.3) contining.2% bovine serum lbumin (Miles Lbortories) nd 5 mm glucose s described previously (19). The pltelet concentrtion in gelfiltered pltelets (GFP) ws then djusted to 3.5 x 1' cellslml s mesured by Coulter counter. Porcine PDGF ws prepred s described (2). Antibodies-The monoclonl PDGF ntibody mab 6Dll (subclone 6Dll El, kindly provided by Dr. Inger Hgen, Center for Industril Reserch, Oslo, Norwy), neutrlizes the biologicl ctivity of PDGF-BB nd -AB (21), nd lso cross-rects with PDGF-AA (22). The rbbit ntiser PDGFR-3 nd PDGFR-7 were rised ginst peptides in

2 13875 Antithrombotic Activity of the PDGF -Receptor stimultion: FIG.1. H u m n pltelets hve functionlly ctivepdgf -receptors.humn pltelets (left pnel) or fibroblsts (right pnel)were incubted withporcine PDGF or recombinnt humn PDGF-BB (1ng/ml) or vehicle. Cell lystes were immunoprecipittedwithcontrolntiserum (Ctr.)or ntiserum specific for the PDGF - or P-receptors ( nd P, respectively), nd the beds were subjected to n in vitrokinsessy by incubtion with [y-"piatp; smples were nlyzed by SDS-gel electrophoresis followed by exposure using PhosphorImger. The PDGF -receptor (17 kd) nd 6-receptor (18 kd) bnds re indicted by rrows, to the left nd to the right, respectively. n E 8 ntiserum: Ctr. r porclne PDGF 1 r PDGF-BE 1 P Ctr. P Ctr. P c - 97 humn pltelets humn fibroblsts - V v v 5 6 n m m I 4 LL (3 n vl'- thrombin (.1 U/ml) 2 N competition with: - AA BB FIG.2. Cross-competition for binding of lz5i-pdgf-bbto hum n pltelets. The bindingof '"I-PDGF-BB to humn pltelets in the bsence (-1 or presence of 2 ng/ml unlbeled PDGF-AA (AA) or PDGF-BB (BB) t for 2 h t "C ws determined. The dt represent totl cell-ssocited ''I rdioctivity -c 1 S.D., n = 4. the C termini of the murine PDGFR-P (23) nd the humn PDGFR- (8), respectively. Therbbitphosphotyrosinentiserum(nti-PY) FIG.3. Immunostining of PDGF on resting nd thrombin-c(24) ws ffinity-purified using phosphotyrosine-sephrose CL-4B tivted pltelets. Humn pltelets were incubted with vehicle or.1 unitlml thrombin, stined with the monoclonl nti-pdgf ntibody column. '2,51-PDGF-BBBinding Experiments-Humn recombinnt PDGF- 6Dl1, nd visulized with fluorescein isothiocynte-conjugted rbbit BB expressed in yest ws lbeled with lz5i, ccording to the Bolton- nti-mouse ntibodies. Resting pltelets were lso stined with 6Dll Hunter procedure, to specific ctivities of 12,-23, c p d n g (25). fter preincubtion of the ntibodies with 4 ng/ml PDGF-BB (control ). Aliquots of.5 ml of GFP were incubted with 1 ng/ml '"I-PDGF-BB with or without 2 ng/ml unlbeled PDGF-AA or PDGF-BB for 2 h on CL-4B (Phrmci LKB Biotechice. The pltelets were then centrifuged for 1 min t 2,8 x g nd incubtion time). Protein A-Sephrose wshed four times withice-cold PBS (136.7 mm NCl, 2.7 mm KCI, 13.1 nology Inc.) ws used to collect the immune complexes. The immunolysis nd once with 2 mm mm N,HPO,, 1.5 mm KH,PO,) contining 1mg/ml bovine serum lbu- precipittes were wshed four times in buffer Tris-HCI (ph 7.5), 15 mm NC1. Kinse ssys wereperformed in 35 pl min,.9 mm CCI,, nd.5 mm MgCI,. The pltelet pellets were from cut of 2 mm HEPES (ph7.5). 1 mhl MnCI,, 1mhl dithiothreitol, contining the Eppendorf,tube nd the ''.'I rdioctivitywsmesuredin 1 pci of [y-:"piatp (Amershm; specific ctivity, 3 Ci/mmol) for 3 y-counter. Immunohistochemistry-GFP (.5 ml) ws incubted with or without min t room temperture (26). Immune complexes were eluted by het.1 unitlml thrombin for 6 s t 37 "C nd fixed by ddition of 5 mm ing the beds t 95 "C for 4 min in 35 pl of 2 x smple buffer (4%SDS, formldehyde contining 4mM EDTA. The pltelets were trnsferred to 2 8 mercptoethnol,.2 M Tris-HCI (ph 8.8),.5 M sucrose, 5 mht EDTA, slides coted with.1% polylysine. The PDGF ntibodymab 6Dll ws.1% bromphenol blue). Smples were subjected to SDS-gel electrodded to prllel sections overnighton ice. To ssure the specificity of phoresis in 7.5% crylmide gels; gels were then fixed, dried, nd n6dl1, the ntibody solutions were preincubted overnight on ice with or lyzed by PhosphorImger (Moleculr Dynmics)for mesurement of the reltive mount of rdioctivity in the receptor bnds. without 4ng/ml PDGF-BB. The slides were then wshed in PBS nd incubted with secondry fluorescein-conjugted rbbit nti-mouse im- Pltelet Aggregtion nd Secretion-GFP were pretretedwith PDGF, PDGF ntibodies, or vehicle for 6 s t 37 "C before incubtion munoglobulin (dilution 1:3 in PBS) for 2 h. In Vitro Immune Complex Kinse Assy-Humn pltelets nd sub- with thrombin(.4-.3 unitlml finl concentrtion)or vehicle for 6 s. confluent humn fibroblsts weresolubilized in lysis buffer (.5M NCI, After 6-s incubtion with thrombin, smpleswere withdrwn for sub.2 M Tris-HC1 (ph 7.4),.5% Triton, 1%Trsylol, 1 mm phenylmeth- sequent nlysis. Pltelet ggregtion ws mesured in Pyton dul ylsulfonyl fluoride, 1 mm dithiothreitol) for 2 min t 4 "C. The lystes chnnelggregtion module connected tolinertwo-chnnelrewere then centrifuged t 1, x g for 15 min, nd the superntnts corder nd quntified by the initil increse in light trnsmission. were subjected to immunoprecipittions with PDGF receptor specific Dense grnule secretion ws determined s the extrcellulr ppernce of ADP plus ATP, mesured by luciferin-lucifersemethod (27). ntiserum, ffinity-purified nti-py, or control preimmune serum (2 h

3 13876 Antithrombotic Activity A of the PDGF -Receptor 35 - T FIG. 4. Autocrine ctivtion of PDGF -receptors during pltelet ctivtion. Humn pltelets were preincubted with recombinnt PDGF-BB (PDGF) or 6Dll ntibodies (monoclonl nti-pdgf) for 1 min before incubtion with thrombin for 5 min t 37 "C. Cell lystes were immunoprecipitted with nti-phosphotyrosine (A) or PDGFR- ntiserum (B) nd subjected to in vitro kinse ssy by incubtion with [y32platp, before nlysis by SDS-gel electrophoresis nd utordiogrphy. A, the reltive rdioctivity of the PDGF -receptor bnds s mesured by PhosphorImger nlyses fter SDS-gel electrophoresis. Brs illustrte? 1 S.D. B, immunoprecipittion of treted pltelets (left pnel) nd control fibroblsts (right pnel) with PDGFR- ntiserum. The reltive rdioctivity of the -receptor bnds (rrow) is indicted on the bse line. The dt re representtive of three different experiments T preincubtion: ctr. PDGF nti-pdgf ctr. ctr. thrombin U/ml: ctr B preincubtion: PDGF monoclonl r nti-pdgf 1 thrommn Ulml: Ctr. Ctr Mr (X163 1 I"-+ PDGF Ctr _r rltivr rdioctlvlty (x of Ch): humn pltelets humn fibroblsts Secretion of cid hydrolses from lysosomes ws mesured fluorometri- probbly due to dimeriztion of the receptors by the receptor clly by the extrcellulr concentrtion of P-hexosminidse (28). ntiserum (26). Notbly, the 32P content of the PDGF -receptor RESULTS from pltelet lystes incresed pproximtely 2% on incubtion with porcine or recombinnt humn PDGF-BB (Fig. 1). Humn Pltelets Hve PDGF - but Not P-Receptors- Thus, resting humn pltelets hve PDGF -receptors which Humn pltelets hve been shown previously to bind "'Icn be ctivted by exogenous PDGF. PDGF (18). To determine whether pltelets hve functionl To investigte further the presence of PDGF receptors on PDGF receptors, humn pltelets, or humn fibroblsts s positive control, were incubted with porcine PDGF, recombihumn pltelets, cross-competition experiments with nnt PDGF-BB (1 ng/ml), or vehicle for 3 min on ice. Cell PDGF-BB nd unlbeled PDGF-AA or -BB (2 ng/ml) were lystes were immunoprecipitted with PDGF receptor-specific performed. Approximtely 4% of the totl pltelet-ssocited ntiser or preimmune serum, wshed, nd incubted with "'1 rdioctivity ws specific binding of '"I-PDGF-BB. [y-32p]atp before SDS-gel electrophoresis. From both humn PDGF-AA ws found to completely block the specific binding of pltelets nd humn fibroblsts the PDGFR- ntiserum pre- '"I-PDGF-BB (Fig. 2). Thus, in greement with the in vitro cipitted 17-kD 32P-phosphorylted component correspond- kinse ssy (Fig. 11, the binding experiments showed n exing to the mture glycosylted PDGF -receptor (Fig. 1, rrows clusive presence of the PDGF -receptor on humn pltelets. to the left 1. The PDGF P-receptor ws identified in the humn Relocliztion of PDGF to the Pltelet Membrne fter Pltefibroblsts s 18-kD protein (Fig. 1, rrow to the right) but let Activtion-PDGF is secreted during pltelet ctivtion (17) ws not found in the pltelets. In greement with previous nd my thus bind to the membrne PDGF -receptors fter observtions, the PDGF receptor bnds contined reltive externliztion. We therefore performed immunohistochemicl high mount of rdioctivity even in the bsence of lignd, stinings of resting or thrombin-ctivted pltelets with

4 Antithrombotic Activity of the PDGF -Receptor monoclonl ntibody (mab 6Dll) ginst PDGF (21). The mab 6Dll gve grnulr stining of the resting pltelets (Fig. 31, consistent with the known locliztion of PDGF in the -grnules. In contrst, the thrombin-ctivted pltelets ppered in clusters, nd the PDGF-positive stining ws loclized extrcellulrly to the pltelet membrne (Fig. 3). The grnulr stining of resting pltelets ws blocked by preincubtion of the ntibody with 4 ng/ml of PDGF-BB (Fig. 3). Autocrine Activtion of the PDGF -Receptors during Pltelet Activtion-The ssocition of PDGF to the membrne of ctivted pltelets my indicte tht the endogenous pltelet PDGF binds nd stimultes the PDGF -receptor in n utocrine mnner fter relese from the -grnule. To exmine this.- -w possibility, pltelets were preincubted with PDGF-BB (1 E ng/ml), mab 6Dll nti-pdgf (5 pg/ml), or vehicle for 1 min, nd then thrombin (.5 nd.1 unit/ml) or vehicle ws dded m - U for further 5 min t 37 "C. For comprison, humn fibroblsts nv I-2 were incubted in prllel with or without PDGF. The cells + were lysed, immunoprecipitted with phosphotyrosine nti- rno 3 serum (nti-py, Fig. 4A) or with PDGFR- ntiserum (Fig. 4B) x nd incubted with [-y-32p]atp before SDS-gel electrophoresis. The reltive mount of rdioctivity in ech receptor bnd ws n determined by PhosphorImger. As shown in Fig. 4A, PDGF Q incresed the tyrosine phosphoryltion of component in pltelets of the sme size s the PDGF -receptor by pproximtely c 25%. Interestingly, thrombin lso incresed the phosphoryl-.- -w tion of the puttive PDGF -receptor in dose-dependent mn-," L O ner; t.1 uniffml thrombin incresed the phosphoryltion by +,o 524% of control. The notion tht the phosphorylted component m + immunoprecipitted with nti-py ntiserum ws identicl to.lc x the PDGF -receptor ws supported by the finding tht similr :be results were obtined by immunoprecipittion with PDGFR- I ntiserum (Fig. 4B 1. Moreover, the thrombin-induced phospho- Q 1 I I ryltion of the PDGFR-, s immunoprecipitted with nti-py s well s PDGFR- ntiserum, ws completely inhibited by thrombin (U/ml) preincubtion of the pltelets with neutrlizing PDGF ntibodies (Fig. 4). This strongly suggests tht the ctivtion of the FIG. 5. Inhibition of thrombin-induced pltelet ctivtion by PDGFR- during thrombin-stimultion ws due to utocrine PDGF. Humn pltelets were preincubted with vehicle (open circles) or porcine PDGF (closed circles) for 6 s before incubtion with different ctivtion by endogenous PDGF secreted from the pltelet concentrtions of thrombin. Aggregtion ws mesured in Pyton -grnules. Thrombin lso incresed the phosphoryltion of the ggregtion module (A), nd the smples were nlyzed for ATP plus PDGFR- in pltelets preincubted with PDGF (dt not ADP (B) nd P-hexosminidse (C) secretion fter 6 s t 37 "C. The shown) dt re representtive of six seprte experiments mde from different donors; burs illustrte PDGF Znhibits Thrombin-induced Pltelet Activtion-The S.E. The secretion is presented s percent secretion of totl pltelet content. effects of ctivtion of the PDGF -receptor by exogenous PDGF on thrombin-induced pltelet ggregtion nd secretion were exmined. Pltelets were preincubted with porcine negtive feedbck mechnism by inhibiting pltelet ctiv- PDGF or vehicle for 6 s nd further with different concentr- tion. Pltelets were preincubted with PDGF ntibodies for 6 tions of thrombin (-.3 uniuml), smples were then nlyzed s before thrombin incubtion (.1-.2 uniffml), nd secretion for secretion from dense grnules or lysosomes fter 6 s. As shown in Fig. 5, PDGF inhibited both thrombin-induced ggreof P-hexosminidse ws mesured. The thrombin-induced lysosoml secretion ws significntly potentited by the PDGF gtion nd pltelet secretion ofatp plus ADP from dense grn- ntibodies; t.3-.4 uniffml thrombin, the mab 6Dll inules from lysosomes t thrombin concentrtions less thn.1 uniffml. Similr effects of PDGF on pltelet secretion ws lso observed fter 3- nd 6-s incubtion with thrombin (dt not shown). Since PDGF is relesed from ctivted pltelets, the effect of PDGF on thrombin-induced ggregtion ws lso studied by ddition of PDGF before or fter thrombin (.1 unit/ml) incubtion. PDGF ws found to cresed the secretion by 6% (Fig. 7A). Pltelets were lso preincubted with different concentrtions of PDGF or PDGF ntibodies before incubtion with.5 unit/ml thrombin. As shown in Fig. 7B, exogenous PDGF-BB t 1 ng/ml inhibited secretion by 5%. In contrst, the PDGF ntibodies (2 pg/ml) incresed the thrombin-induced secretion by more thn 2%. Thus, the utocrine PDGF pthwy during reverse the pltelet ggregtion when dded s 3 fter incub- pltelet ctivtion most likely function in negtive feedbck tion with thrombin (Fig. 6). regultion of pltelet ctivtion. Neutrlizing PDGFAntibodies Potentite Secretion-The bove results showed tht DISCUSSION exogenous PDGF inhibited pltelet ctivtion nd tht the PDGF -receptor ws ctivted n in utocrine mnner during Pltelet gonists like thrombin, collgen, nd epinephrine stimulte tyrosine phosphoryltion of severl proteins in plte- thrombin stimultion. The bility of the PDGF neutrlizing ntibodies to inhibit the utocrine PDGF pthwy (Fig. 4) prompted us to investigte whether this pthwy functions s lets, suggesting tht such phosphoryltion is involved in pltelet ctivtion (29-31). However, cloning of the membrne receptors of these gonists hve shown tht neither of them

5 13878 A Antithrombotic Activity of the PDGF &-Receptor - 3 sec 3 sec M Thrombln PDGFINCI PDGFINsCI Thrombln PDGF PDGF FIG. 6. Reversion of thrombin-induced ggregtion by PDGF. Pltelets were incubted with 1 ng/ml porcine PDGF or vehicle (NC1) 3 s prior (A) or fter ( E) stimultion with.1 unit/ml thrombin. The ggregtion ws mesured in Pyton dul chnnel ggregtion module. contin cytoplsmic tyrosine domin (321, nd the tyrosine phosphoryltion in pltelets hs so fr been recognized s secondry to ctivtion ofpp6"" (33) nd Src-relted proteintyrosine kinses like pp6fyn nd ~ ~ 6(34, 2 ~ 35). ' The ~ present identifiction of PDGF -receptors in humn pltelets is thus the first demonstrtion of the presence of tyrosine kinse receptor in these cells. The receptor ws identified by immunoprecipittion s 17-kD component corresponding in size to the mture glycosylted protein s found in other cell types (8). Only PDGF -receptors nd not P-receptors were found, s verified by receptor binding studies with cross-competition of lz5i-pdgf-bb by high doses of unlbeled PDGF-AA. The PDGFR- tyrosine kinse of resting pltelets ws stimulted by the ddition of PDGF. Interestingly, thrombin significntly incresed the utophosphoryltion of the PDGFR- independent of exogenous PDGF, s determined by immunoprecipittion with nti-py s well s PDGFR- ntiserum (Fig. 4). This effect of thrombin ws completely inhibited by preincubtion of the pltelets with PDGF neutrlizing ntibodies. Thus, the observed thrombin-induced phosphoryltion of the PDGF -receptor ws most likely due to the relese of endogenous PDGF from -grnules, rther thn secondry phosphoryltion of the PDGF -receptor by n intrcellulr signl trnsduction pthwy ctivted by thrombin. In fct, immunohistochemicl stining showed tht the PDGF-positive compo- nent ws trnsferred fter thrombin ctivtion from grnulr intrcellulr pttern to circulr stining in nd round the pltelet membrne. Moreover, thrombin hs previously been found to induce tyrosine phosphoryltion of protein components of kd, corresponding in size to the PDGF -receptor (3, 31). The concept of utocrine-positive feedbck during pltelet ctivtion is well estblished; pltelet secretion of ADP nd serotonin from dense grnules, secretion of -grnule components like PF 4, fctor VIIUvon Willebrnd, thrombospondin, nd fibrinogen, s well s prostnoid formtion from liberted rchidonte, hve ll been shown to mplify the pltelet response (36). However, pltelet grnule components tht function in negtive feedbck pthwy hve not been reported. Previous work hs shown tht ddition of PDGF inhibits certin responses in resting (37) nd collgen-ctivted pltelets (18). We found tht exogenous PDGF (5-1 ng/ml) inhibited thrombin-induced pltelet ggregtion nd secretion t physiologicl doses of less tht.1 uniffml of thrombin. In fct PDGF ws ble to reverse thrombin-induced ggregtion when dded 3 s fter thrombin stimultion, suggesting tht rpid nd efficient inhibitory pthwy is initited by the PDGFR-. The thrombin (U/ml) pg/rnl Mob 6D11 1 I ng/ml PDGF FIG. 7. Effects of PDGF nd PDGF ntibodies on thrombininduced P-hexosminidse secretion. A, pltelets were preincubted with PDGF ntibodies or vehicle for 6 s t 37 "C before incubtion with different concentrtions of thrombin for 2 min t 37 "C. The secretion of P-hexosminidse in the bsence of PDGF ntibodies ws set t 1%. E, pltelets were preincubted with different concentr- tions of PDGF (closed circles) or PDGF ntibodies (open circles) before incubtion with.5 uniffml thrombin. The dt re representtive of three different experiments performed in triplicte, the brs illustrte 1 S.D. 6

6 bility of the PDGF-neutrlizing ntibodies to inhibit the thrombin-induced ctivtion of the PDGFR- llowed us to study the functionl significnce of this utocrine loop during pltelet ctivtion. We found tht the PDGF ntibodies significntly potentited the thrombin-induced lysosoml secretion of P-hexosminidse. Dose-response studies showed ththe thrombin-induced secretion ws inhibited or potentited by preincubtion with exogenous PDGF or PDGF ntibodies, respectively. It is therefore likely tht our observed utocrine ctivtion of the PDGFR- elicits negtive feedbck pthwy during pltelet ctivtion. The inhibitory signl pthwy of the PDGFR- in ctivted pltelets remins to be identified. Severl signl trnsduction molecules which interct with the PDGF receptors hve been identified, including phospholipse C-y (381, the GTPse ctivtion protein of Rs (39, 41, the regultory subunit (~85) of phosphtidylinositol 3 kinse (41, 421, nd members of the c-src fmily kinse (43), s well s the dptor molecules Nck (44) nd Grb2 (45). Interestingly, phospholipse C-y, GTPse ctivtion protein, phosphtidylinositol 3 -kinse, nd the Src proteins re present in humn pltelets (32) nd re reported to be tyrosine phosphorylted during pltelet ctivtion; it is thus possible tht certin of these molecules re involved in the inhibitory PDGF pthwy. The PDGF -receptor hs been shown to inhibit the chemotctic response induced by the PDGF P-receptor in humn fibroblsts (12, 13); the moleculr mechnism behind this effect is not known. Since pltelet ctivtion nd chemotxis both involve cell shpe chnges, it is possible tht similr components in pltelets nd fibroblsts, respectively, re involved in these inhibitory signl trnsduction pthwys. Secretion of PDGF from pltelets, endothelil cells, smooth muscle cells, nd ctivted mcrophges represent source of PDGF generlly recognized s importnt for chemotxis nd cell growth in therosclerotic lesions (reviewed in Ref. 46). Our observtions suggest tht thrombosis nd therosclerosis re regulted by PDGF in complex mnner. Elevted levels of PDGF my prevent pltelet ggregtion, s well s inhibit chemotxis by ctivtion of the PDGF -receptor (12, 13) of smooth muscle cells (47). Production of nitric oxide (48) nd prostglndin I, (49) on endothelil surfces inhibit pltelet ctivtion. In ddition, secretion of PDGF from endothelil cells (5) could tke prt in the ntithrombogenic mechnisms of the vessel wll. 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