* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Glycoprotein IIIa Is Phosphorylated in Intact Human
Survey
Document related concepts
Cytokinesis wikipedia , lookup
Biochemical switches in the cell cycle wikipedia , lookup
List of types of proteins wikipedia , lookup
Magnesium transporter wikipedia , lookup
Histone acetylation and deacetylation wikipedia , lookup
P-type ATPase wikipedia , lookup
Protein (nutrient) wikipedia , lookup
Protein moonlighting wikipedia , lookup
Protein structure prediction wikipedia , lookup
Signal transduction wikipedia , lookup
G protein–coupled receptor wikipedia , lookup
Transcript
From www.bloodjournal.org by guest on June 18, 2017. For personal use only. Glycoprotein IIIa Is Phosphorylated in Intact Human Platelets By Leslie V. Parise, Anne B. Criss, Lisa Nannizzi, and Mark R. Wardell The glycoprotein Ilb-llla complex (GP Ilb-llla) is a multifunctional transmembrane protein on platelets. Its most completely described function is as a fibrinogen receptor that mediates platelet aggregation, but it is also involved in clot retraction, signal transduction, calcium transport, and other events. However, the mechanisms that regulate the functions of GP Ilb-llla during platelet activation are largely unknown. One possible mechanism is phosphorylation, since several other receptors are regulated by this process. We found that GP Illa, but not GP Ilb, was phosphorylated in 32P-labeled platelets, predominantly on threonine residues. Furthermore, GP llla phosphorylation increased four- fold in platelets activated with thrombin or phorbol 12myristate 13-acetate, but not at all in platelets treated with prostacyclin, an inhibitor of platelet activation. The thrombin-induced increase in phosphorylation was inhibited by pretreating platelets with prostacyclin or with staurosporin, a specific protein kinase C inhibitor. Thus, there is an increase in the level or turnover of phosphate on GP llla during platelet activation, most likely involving protein kinase C. This phosphorylation may regulate some aspect(s) of GP Ilb-llla function. 0 1990 by The American S o c i e t y of Hematology. G mechanism by which activation-induced changes in G P IIb-IIIa function could be regulated. LYCOPROTEINS IIb A N D IIIa are abundant integral membrane proteins on platelets that exist as Ca2+-dependent heterodimer complexes, termed glycoprotein (GP) IIb-IIIa.'~*Glycoprotein IIb, the a-subunit (molecular weight [mol wt] 140,000), consists of a heavy chain (mol wt 120,000) disulfide-linked to a light chain (mol wt = 25,000); G P IIIa, the @-subunit,is a single polypeptide of mol wt i~ 105,000.2Recent cloning and sequencing studies have established that G P IIb-IIIa is a member of a large family of adhesive receptors, the integrins (reviewed in reference 3). These studies also predict that G P IIIa and the light chain of G P IIb each contain a membrane-spanning region near its carboxy t e r m i n ~ s , ~a . ~short cytoplasmic domain (20 and 41 amino acids, respectively), and a much larger extracellular component. The G P IIb-IIIa complex contributes to several platelet functions, most of which are acquired during platelet activation. For example, it acquires the ability to bind fibrinogen,6>' thereby mediating platelet aggregation during thrombus formation. In addition, G P IIb-IIIa is believed to mediate subsequent clot retraction by bridging the extracellular fibrin network to the contractile apparatus of the latel let.^,^ The complex also plays a role in signal transduction in stimulated platelets; for example, in tyrosine-specific protein phosphorylation" and in N a + / H + exchange across the plasma membrane." Furthermore, G P IIb-IIIa contributes to platelet adhesion to the subendothelium" and acts as a passive Ca2+tran~p0rter.I~ Although G P IIb-IIIa has multiple functions, the mechanisms that regulate them are largely unknown. Since phosphorylation regulates the functions of other receptors, such as the @-adrenergic, insulin, and epidermal growth factor receptors (reviewed in reference 14), we investigated whether any components of the G P IIb-IIIa complex are phosphorylated. The present study shows that G P IIIa is phosphorylated, primarily on threonine residues, and that the phosphorylation or turnover of phosphate increases dramatically in thrombin- or phorbol 12-myristate 13-acetate (PMA)activated platelets. The agonist-induced phosphorylation is inhibited by prostacyclin (PGI,), an inhibitor of the intracellular events leading to platelet a ~ t i v a t i o n . 'In ~ addition, evidence is provided that G P IIIa phosphorylation in platelets is catalyzed by protein kinase C (PKC). This study clearly implicates phosphorylation of G P IIIa as a potential - - Blood, Vol 75,No 12 (June 15). 1990:pp 2363-2368 MATERIALS AND METHODS Platelet preparation. Human platelets were obtained on the day of use and washed as previously described16 but in the absence of PGI, and EDTA, and were resuspended to 1 to 2 x lo9platelets/mL in buffer A (12 mmol/L NaHCO,, 138 mmol/L NaCl, 5.5 mmol/L glucose, 2.9 mmol/L KCI, 10 mmol/L HEPES, pH 7.4). The platelet suspension was incubated with 1 mCi of H,[32P]04(ICN Biomedicals, Inc, Irvine, CA)/mL of platelets at room temperature for 30 minutes, sedimented at 730g for 10 minutes, and resuspended to 0.7 to 0.8 x 10' platelets/mL in buffer A with 0.36 mmol/L NaH,P04 H,O. The platelets were then incubated at 37OC without stirring for the times and at the concentrations of agents indicated in the figure legends. The concentrations of agents were chosen on the basis of their ability to induce either maximal aggregative or inhibitory responses. The concentrations of PGI, were chosen according to their ability to inhibit platelet aggregation induced by 0.1 U of thrombin/mL. These concentrations varied with platelets from different donors and ranged from 60 to 370 nmol/L. The actual phosphorylation experiments with thrombin and PMA were done with activated, aggregation-competent platelets but without stirring and, therefore, without aggregation per se, so that GP IIb-IIIa could be quantitatively and reproducibly immunoprecipitated. Immunoafinity chromatography. Approximately 1.2 x 1O9 platelets were incubated at 37OC with platelet agonists, antagonists, or appropriate buffers, as indicated in the figure legends and text. The incubations were terminated by diluting the sample with an From the Gladstone Foundation Laboratories for Cardiovascular Disease, Cardiovascular Research Institute. University of California at San Francisco, San Francisco, CA; and ihe Department of Pharmacology and Centerfor Thrombosis and Hemostasis, School of Medicine, University of North Carolina, Chapel Hill, NC. Submitted November 8, 1989; accepted February 27. 1990. Supported by FIRST Award No. R29HL38405 and Grant No. 1989-91-A-07 from the American Heart Association-NC aflliate (L.V.P.). Address reprint requests io Leslie V. Parise. PhD. Department of Pharmacology, CB #7365, Faculty Laboratory Ofice Building, University of North Carolina, Chapel Hill. NC 27599-7365. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C.section 1734 solely to indicate this fact. 0 1990 by The American Society of Hematology. 0006-4971/90/7512-0027$3.00/0 2363 From www.bloodjournal.org by guest on June 18, 2017. For personal use only. PARSE ET& 2364 q u a l volume of icecold lysis buffer (2% Triton X-100.20 mmol/L Tris. 2 mmol/L sodium metavanadatc. 40 mmol/L molybdic acid. 80 mmol/L sodium pyrophosphate. 4 mmol/L EGTA. 0.2 mmol/L trifluoperazine. 0.2 mmol/L leupeptin. 2 mmol/L phcnylmethylsulfonyl fluoride. 40 mmol/L KH,PO,. pH 7.2). For platelets treated with thrombin. 0.2 U of hirudin/mL was also included in the lysis buffer. The lysate was antrifuged for I5 minutes a t 31.OOOg to sediment the platelet cytoskeleton. Glycoprotein Ilb-llla was purified from the supernatant by preclearing on a 0.2-mL glycine-Sepharose column (glycine 1 I mg/mL] covalently coupled to CNRr-activated Sepharose ISigma. SI Louis. MO])followed by adsorption onto a 0.4-mL column of A,A.-Scpharosc (AIAI is a specific anti-GP Ilb-ll la monoclonal antibody" supplied by Dr Joel Bennett. and was coupled at I mg/mL of CNBr-activated Sepharox). The column was washed with 23 column volumes of buffer R ( I50 mmol/L NaCI. O.I%Triton X-100.0.02%NaN,. ZOmmol/LTris.pH 7.4).TheGP Ilb-llla was eluted with a buffer of 0.1%Triton X-100. 2 mmol/L CaCI,. 0. I mol/L glycine, pH I I .3. and each fraction was collected into 0.5 volume of neutralization buffer (0.1% Triton X-100. 2 mmol/L CaCI,. I mol/L Tris. pH 6.6). Glycoprotein Ilb-llla was precipitated from I mL of each fraction with trichloroacetic acid (I2'X-).resuspendedinZIOpLofbufferR.andmixcdwith95pLofa modified Laemmli sample buffer (32.3% glyarol Ivol/vol]. 6.5% sodium dodecyl sulfate (SDS) [wt/vol]. 0.01% bromphenol blue Iwt/vol]. 0.04 N NaOH. 0.2 mol/L Tris [final concentration. using I mol/L stock Tris. pti 6.8)) with or without 16%8-mercaptoethano1 (vol/vol). and heated a t loooC for 5 minutes before being subjected to electrophoresis.'' Gels for autoradiography were dried immediately; the proteins were neither fixed nor stained. Imnrunopncipirarion. For immunoprecipitation. I mL of lysate was centrifuged at 12.800g for 15 minutes at 4%. and the supernatant was added to 50 pL of glycine-Sepharose (resuspended I : I in buffer B) and nutated at 4% for I hour. The lysate was again antrifuged at 1 2 . 8 0 0 ~for I minute to sediment the glycineSepharose. The lysate supernatant was then added to 50 p L of the anti-GP Ilb-llla monoclonal antibody. A,&, coupled to Sepharose. and nutated at 4% for 3 hours. The h,A&pharosc was xdimented as described above. and the supernatant was discarded. The A,&Sephanrse was washed with an additional 1.25 mL of buffer R and rescdimented. The CP Ilb-llla was released by muspending the - -1GPllb- 2 3 r A,A,-Scpharosc in 0.25 mL of the pH 11.3 elution buffer and recovering the supernatant. Phmphwmino acid analysis. Glycoprotein Ilb-llla was ilated by immunoaffinity chromatography a s described above. exthat I .5 to 2 x IO' lyscd platelets and a 0.8 to I mL A,A,-Sepharosc column were used. Approximately 2.4 mL of the immunoafinitypurified GP Ilb-llla was precipitated with trichloroacetic acid ( 1 2%). redissolved with 400pL of a modified Laemmli sample buffer ( 10.9% glycerol [vol/vol]. 2.2% SDS Iwt/vol]. 5.5% 8-mercaptoethanol [vol/voll. 0.002% bromphenol blue [wt/vol]. 0.14 N NaOH. and 0.07 mol/L Tris [final concentration. using I mol/L stock Tris. pH 6.81). and heated to loooC for 5 minutes.'" After electrophoresis on a preparative 7.5% gel. GP llla was electrocluted at 100 V for 3 hours into IS mmol/L ammonium carbonate and 0.1% SDS (wt/ vol). pH 8.2. To remove the SDS. the electroeluted protein was precipitated with IS volumes of I% (vol/vol) HCI in acetone at -20% for 2 hours. and the precipitated GP llla was pellcted by low-speed antrifugation. The pellet was washed in HCl/acetone and ramtrifuged. and the acetone-supernatant was discarded. The washed pellet was dissolved in 6 N HCI. sealed in vacuo. and hydrolytnd for 2 hours at I IOOC. The hydrolysates were dried over solid NaOH in vacuo. dissolved in 80 pL of electrophoresis buffer ( I % pyridine Ivol/vol]. 10% glacial a a t i c acid [vol/vol]. pH 3.5). and then electrophoresed along with phosphoserine. phosphothnw nine. and phosphotymine standards on Whatman / I chromatography paper for 1.25 hours at 3 kV in the electrophoresis buffer described above. The p i t i o n s of the standards were determined after staining with 0.2% ninhydrin (wt/vol) in acetone. and the "P-labeled amino acids were identified after autoradiography. RESULTS To determine whether GP Ilb or GP llla was phosphorylated in intact human platelets, washed platelets were labeled with H,['*PJO, and lyscd with an icc-cold bufTcr containing Triton X-I00 and inhibitors of phosphatase and kinase activity. After removal of the cytoskeleton. GP Ilb-llla was purified from the lysate by immunoafinity chromatography with A2A, a monoclonal antibody that recognizes the GP Ilb-llla complex." The purified material was examined on 4 5 ' I . L Fbl. M " t h o f p b @ o q h d II I I Caomassie 32p I 1251 3ip I 1251 QPnbtrom t" I-WWMQ+ p w w OP nb-llb from "P-(.kkd plat.kn was olutrophorosod am 7.6% SDS-PO)Y.cry(.mido gds run undu rodudng llanos 1 through 3)or nonrodudng conditions llama 4 and 6). Lano 1. g d s t a i d with Coocruuk Brilliant Bluo; b m2 and 4. autoradiograms showing that OP Ilk. but not GP Ilb. m s phosphorylatod; lanos 3 and 6, autoradiograms of '%I.a(od t3P Ilb and OP Ilb standards. imolatad as proviously dastxibod." -0th Itb was oborvod t o bo phor9hwy(.tod rogmrdloas of whothor it m s i h t o d from control platdots or plat+ lots t r u t o d with thrombin, M A , or PO4 (Fig 2): tho aramplos in knos 2 and 4 aro from P M A - t r u t o d pbtolots. From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 2365 G P l l l ~PHOSPHORYLATION IN HUMAN PLATELETS SDS gels by Coomassie staining and autoradiography (Fig 1). The purified material was essentially homogeneous (Fig 1, lane 1). Only one component of the complex was phosphorylated. This was identified as G P IIIa (lanes 2 and 4) on the basis of its co-electrophoresis with iodinated G P IIIa (lanes 3 and 5) from the purified G P IIb-IIIa complex. The possibility that G P IIIa was phosphorylated only as a consequence of detergent lysis of platelets was eliminated by two experiments. First, "P-labeled platelets were lysed in the presence of excess unlabeled adenosine triphosphate ( A T P 0.5 mg/mL); the unlabeled ATP would be expected to inhibit phosphorylation of G P IIIa that might occur after lysis and the release of cytoplasmic [32P]ATP.No inhibition of G P IIIa phosphorylation by excess unlabeled ATP was observed (data not shown). Second, when unlabeled platelets were lysed in the presence of [32P]ATP,no phosphorylation of G P IIIa was observed (data not shown). Thus, phosphorylation occurs before lysis, most likely on the cytoplasmic domain of G P IIIa. To determine whether G P IIIa underwent increased phosphorylation when platelets were activated, "P-labeled platelets were exposed to the agonists thrombin or PMA. Thrombin caused a 4.1-fold increase in G P IIIa phosphorylation within 3 minutes, followed by a steady decrease (Fig 2A). The time course of G P IIIa phosphorylation was similar to other events that occur under similar conditions during platelet activation, eg, the expression of platelet fibrinogenbinding activity." Further, the fold-increase was comparable with that of other platelet proteins whose functions are regulated by phosphorylation (see Discussion). As shown in Fig 2B, PMA, an agent that directly activates PKC," caused an increase in G P IIIa phosphorylation similar to that induced by thrombin (4.3-fold over the control value a t 5 minutes). This was also followed by a decrease. The decrease in phosphorylation shown in Fig 2A and B was specific for G P IIIa; it did not occur with total precipitated phosphoproteins (data not shown). Therefore, the decrease probably represents a loss of phosphate from G P IIIa, rather than an exchange with unlabeled phosphate. The changes in G P IIIa phosphorylation shown in Fig 2 were also apparent when G P IIIa phosphorylation was measured in separate experiments as the amount of '*P incorporated per milligram of G P IIIa. In those experiments, 32P-labeled platelets were treated for 3 minutes with thrombin or 5 minutes with PMA. After lysis, relatively large amounts of G P Ilb-IIIa were isolated from these platelets by immunoaffinity chromatography. The milligram amount of GP IIIa was quantitated from Coomassie-stained gels versus a standard curve of G P IIIa on the same gels, and the amount of 32P was quantitated by densitometry of G P IIIa on autoradiograms. When the results were expressed as the amount of 32P/mg of G P IIIa, we found that thrombin induced a 2.5-fold increase and PMA induced a 4.5-fold increase in G P IIIa phosphorylation relative to the control. The responses of platelets to agonists are inhibited by agents such as PGI, that elevate cyclic adenosine monophosphate (AMP) levels within the cytosol. The basis for the inhibition is presumably related to the phosphorylation of certain platelet proteins by cyclic AMP-dependent protein Thrombin 80 401 >- m L, , c 0.5 1 r Control # 3 5 10 3 6 Control 401 9) ""1 -0.5 1 3 5 1'0 TIME (minutes) Fig 2. Time course of "P incorporation into GP llla in intact platelets. Washed, "P-labeled platelets were incubated at 37°C with either (A) human thrombin (0.1 UlmL: 50 U/mL stock in 0.75 mol/L NaCI, 8.3 mmol/L polyethylene glycol BOOO, 10 mmol/L Tris. pH 7.4). ( 6 )PMA (10 pmol/L; 5 mmol/L stock in dimethylsulfoxide), or (C) PGI, (60nmol/L; 0.14 mmol/L stock in 0.1 mol/L NaCI. 50 mmol/L Tris, pH 12) for the indicated times. Control samples for each treatment were incubated with the buffer alone. Upon completion of each incubation, the platelets were lysed with ice-cold lysis buffer and GP Ilb-llla was isolated by immunoprecipitation (see Materials and Methods). The relative amount of "P incorporated into GP llla was quantitated by densitometry of autoradiograms of 7.5% polyacrylamide gels containing the immunoprecipitated and reduced glycoproteins. Densitometrywas done under conditions of linear exposure of the autoradiogram. The data shown are representative of two (A) or three (B and C) experiments per treatment. Each data point is en average of duplicate samples. kinase.,' When resting platelets were treated with PGI,, G P IIIa phosphorylation was not induced (Fig 2C), suggesting that cyclic AMP-dependent protein kinase was not responsible for phosphorylating G P IIIa. In a separate experiment, PGI, did inhibit the thrombin-induced phosphorylation of GP IIIa, indicating that the agonist-induced phosphorylation is on the stimulus-response pathway of platelets. (In this experiment, pretreatment of platelets with PGI, (370 nmol/ L) for 1 minute before the addition of thrombin (0.1 U/mL) 7.5% (SE, n = 3) inhibition of the resulted in a 72% thrombin-induced increase.) To determine which amino acid(s) was phosphorylated, 32P-labeled GP IIIa was electroeluted from a preparative SDS-gel, partially hydrolyzed with acid, and subjected to high-voltage paper electrophoresis under conditions that separated P-serine, P-threonine, and P-tyrosine. Regardless of whether platelets were treated with PMA, thrombin, or From www.bloodjournal.org by guest on June 18, 2017. For personal use only. PARIS ET AL 2366 b u l k (ie. control). threonine was observed to be the major "P-labcled amino acid residue (Fig 3). A low level of serine phosphorylation was alsoobserved (Fig 3. lanes I and 3). but no tyrosine phosphorylation was apparent. Rccause PMA directly activates PKC and induces GP l l l a phosphorylation. we investigated whether this enzyme participated in the thrombin-induced phosphorylation of GP I 1 la. since thrombin is a physiologic platelet activator.'? I n thcsc experiments. platelets were pretreated with staurosprin. an inhibitor of PKC." The concentration of staurosprin ( I pmol/l.) used was a minimum value. selected because i t was just high enough to maximally inhibit the thrombin-induced phosphorylation of P47. a known substrate of PKC in platclcts" (Fig 4A). Phosphorylation of P47 was inhibited e,- -.' 1 2 - Thrombin PMA 3 P-serP-TtU- P-Tyt- Control Fig 3. odd analpis of t3P ))(. hd.1.dtrom " P - ~ a k k d piatekts. a m o t o i n IIIJ trom = ~ - t a k t e dp ~ ~ ~ o was subjected to partial amino acid hydrolysis followed by highvoltage paper electrophoresis u n d a conditions that separated P-serine I P - S r ) . P-threonine IP-Thr), and P-tyroshe (P-Tyr). The positions of those amino acids were determined by ninhydrin staining of unlabeled phosphory(.ted standards that had been added to each sample before elmxrophoresis. Autoradiography of the papor electrophoretogram showed that threonine was the major phosphorylated amino add from contrd. thrombin-treated. or PMA-treated platelots. P, inorganic phosphate. under conditions in which many other platelet proteins remained phosphorylated. even at relatively high concentrations of staurosprin (Fig 4A. inset). supporting the relative specificity of this inhibitor. This finding contrasts with results obtained with sphingosine. another inhibitor of PKC": at a concentration that minimally inhibited P47 phosphorylation. sphingosinecauscd the disappearance of many other phosphorylated bands in a total platelet lysate (data not shown). When '-'P-labeled platelets were pretreated with staurosporin. the thrombin-induced phosphorylation of GP I 1 la was completely inhibited (Fig 4R)-cvidencc that PKC phosphorylatesGP I1l a in thrombin-stimulated platelets. DISCUSSION This study shows that GP l l l a of the GP I l b - l l l a complex is phosphorylated. primarily on threonine residues. in intact human platelets. Activation of platelets with the physiologic agonist thrombin dramatically increases this phosphorylation. whereas pretreatment of platelets with PGI,. an inhibitor of platelet activation. inhibits this increase. The agonistinduced increase in GP l l l a phosphorylation appears to be mediated by PKC. Thus. the activation-induced phosphorylation or turnover of phosphate on GP l l l a is a potential regulatory mechanism for one or more of the many functions associated with the GP I l b - l l l a complex in platelets. Four lines of evidence suggest that PKC is the likely mediator of GP l l l a phosphorylation. ( 1 ) Thrombin and PMA. which inducc GP l l l a phosphorylation. are known to activate PKC in platelets." (2) The relatively specific inhibitor of PKC. staurosprin (which has been used to identify PKC-mediated events in completely inhibits the thrombin-induced phosphorylation of GP I l l a . (3) PGI,. which activates the cyclic AMP-dependent protein kinase. docs not induce GP l l l a phosphorylation. eliminating this enzyme as a possible candidate. (4) The amino acid residues that become phosphorylated are threonine (and. to a lesser extent. serine). and PKC is a serine-threonine-specific entyme.-" The putative cytoplasmic domain of GP l l l a does contain two threonine residues that are especially likely targets for PKC-mediated phosphorylation: residua 758 and 762 (see reference 4 for the complete amino acid sequence of GP llla).""' Precisely which residuc(s) is phosphorylated remains to be determined. Several lines of evidence suggest that the agonist-induced phosphorylation of GP l l l a has the potential to regulate GP I l b - l l l a function in platelets. First. the time course of the increased phosphorylation is comparable with the time course of certain changes in function related to GP I l b - l l l a : for example, fibrinogen binding to platelets.'' tyrosine-specific protein phosphorylation in thrombin-stimulated pla~clets.'~ and the redistribution of GP I l b - l l l a on the surface of t s activated platelets." Second. the magnitude of the change in GP l l l a phosphorylation is roughly comparable with that of other platelet proteins whose functions are affected by phosphorylation. Some examples are the 2.5-fold increase in myosin light chain phosphorylation in thrombin-treated platelets." which enables actin to cause myosin-ATPase activation": the twofold to threefold increase in phosphorylation of actin-binding protein in prostaglandin E,-treated From www.bloodjournal.org by guest on June 18, 2017. For personal use only. GPIIIA PHOSPHORYLATION IN HUMAN PLATELETS 2367 Fig 4. Inhibition of thrombin-stimulated P47 and GP IIla phosphorylation by staurosporin. (AI UP-labeled platelets w e r e treated for 10 minutes a t 37°C with increasing concentrations of staurosporin to determine the minimum concentration that maximelly inhibited P47 phosphorylation. The inset shows t h e phosphorylation pattern of whole platelet lysates from UP-labeled platelets. N o t e the number of proteins that remain phosphorylated a t concentrations of staurosporin that maximally inhibit P47 phosphorylation. The graph showa the amounts of P47 phosphorylation in t h e presence of increasing concentrations of staurosporin, as determined by densitometry. Based on this experiment, a concentration of 1 pmol/L staurosporin was selected for further study. (B) UP-labeled platelets were pretreated for 10 minutes a t 37°C with 1 pmollL staurosporin or solvent alone (dimethylsulfoxide) followed by thrombin (0.1 U/mLl for 3 minutes. The broken line indicates the basal level of phosphorylation in untreated platelets (23.7%). Staurosporin inhibited the thrombin-induced phosphorylation by 104.6% 1 6.6% (SE, n = 4). platelets t h a t is reported t o stabilize this protein against calpain hydrolysis": a n d t h e 1.4-fold increase in G P Ib, phosphorylation t h a t inhibits actin polymerization.'' Third. agonist-induced phosphorylation w a s inhibited by PGI,. a n agent t h a t also inhibits agonist-induced changes in platelet function. W h e n platelets a r e activated. GP I l b - l l l a is converted from a form that c a n n o t bind extracellular fibrinogen into a form t h a t can." In addition, undefined activation-induced events cause t h e intracellular d o m a i n of t h e G P Ilb-llla complex lo bind lo the cytoske1eton.x The critical imporlance of these changes in t h e function of GP I l b - l l l a is exemplified by Glanzmann's thrombasthenia. a hereditary bleeding disorder. PhtClCtS from these patients have either lOW amOUntS Of or functionally a b n o r m a l G P Ilb-llla,'x~'" resulting in t h e absence of agonist-induced platelet aggregation o r clot r e traction. Despite t h e importance of agonist-induced changes in G P I l b - l l l a , very little is known a b o u t t h e intracellular mechanisms t h a t regulate them. The present study shows an activation-induced chemical modification of G P l l b l l l a . Further studies are necessary t o assess t h e consequence of G P llla phosphorylation on t h e numerous functions associated with G P llb-llla. ACKNOWLEDGMENT We thank Dr Joan Fox for many hclprul suggalions: Kerry Humphrey for manuscript preparation; A] Averbach and Sally Gullat1 Seehafer for editorial assistance; and Charles Benedict for graphics. wc also thank Dr Joel Bcnnett (University of Pennsylvania) and Dr John Fenton I ] (New York State Department o f H a ] t h . Albany, NY) for their generous gifts ofA:A,and human thrombin. rcspectivcly. REFERENCES I . Kunicki TJ. Pidard D. Rosa J-P. Nurden AT: The formation of Ca . '-dependent complexa of platelet membrane glycoproteins Ilb and Ilia in solution as determined by crossed immunoelectrophorcsis. Blood 58:268. I98 I 2. Jcnnings LK. Phillips DR: Purification ofglycoprotcins Ilband I I I from human platelet plasma membranes and characteri7ation of a calcium-dependent glycoprotein Ilb-Ill complex. J Biol Chcm 257:10458. 1982 3. Phillips DR, Charo IF, Parise LV. Fitzgerald LA: The platelet membraneglycoprotcin Ilb-llla complcx. Blood 71:831. 1988 4. Fitzgcrald LA. Steincr B, Rall S C Jr. Lo S-S, Phillips DR: Protein sequence of endothelial glycoprotein llla derived from a CDNA clone. Identity with platelet glycoprotein Ilia and similarity to "integrin." J Biol Chem 262:3936. 1987 5 . Poncz M. Eisman R. Hcidcnrcich R. Silver SM. Vilaire G. Surrey S. Schwartz E. Bennett JS: Structure of the platelet membrane glycoprotein Ilb. Homology to the Q subunits of the vitronectin and fibronectin membrane receptors. J Biol Chem 26223476. 1987 6. Bennett JS. Vilaire G:Exposure of platelet fibrinogen receptors by ADP and epinephrine. J Clin Invest 64:1393. 1979 7. Parise LV, Phillips DR: Reconstitution of the purified platelet fibrinogen receptor. Fibrinogen binding propertia of the glycoprotein Ilb-llla complex. J Biol Chem 260:10698. 1985 8. Phillips DR. Jennings LK, Edwards HH: ldcntification of membrane proteins mediating the interaction of human platelets. J Cell Biol86:77. 1980 9. Cohen I. Gerrard JM. White JG: Ultrastructureofclots during isometriccontraction. J Cell Biol93:775. 1982 10. Ferre]l JE Jr, Martin GS: Tyrosincespecific protein phosphorylation is regulated by glycoprotein Ilb-llla in platelets. Proc Na11 Acad Sci USA 86:2234. 1989 1 1 . Banga HS. Simons ER. Brass LF. Rittenhouse SE: Activation of phospholipasa A and C in human platelets exposed to From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 2368 epinephrine: Role of glycoproteins IIb/IIIa and dual role of epinephrine. Proc Natl Acad Sci USA 83:9197, 1986 12. Weiss HJ, Hawiger J, Ruggeri ZM, Turitto VT, Thiagarajan P, Hoffmann T: Fibrinogen-independent platelet adhesion and thrombus formation on subendothelium mediated by glycoprotein IIb-IIIa complex at high shear rate. J Clin Invest 83:288, 1989 13. Rybak ME, Renzulli LA, Bruns MJ, Cahaly DP: Platelet glycoproteins IIb and IIIa as a calcium channel in liposomes. Blood 72:714, 1988 14. Sibley DR, Benovic JL, Caron MG, Lefkowitz RJ: Regulation of transmembrane signaling by receptor phosphorylation. Cell 48:913, 1987 15. Moncada S, Gryglewski R, Bunting S, Vane JR: An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation. Nature 263: 663, 1976 16. Fox JEB, Phillips DR: Role of phosphorylation in mediating the association of myosin with the cytoskeletal structures of human platelets. J Biol Chem 257:4120, 1982 17. Bennett JS, Hoxie JA, Leitman SF, Vilaire G, Cines DB: Inhibition of fibrinogen binding to stimulated human platelets by a monoclonal antibody. Proc Natl Acad Sci USA 80:2417,1983 18. Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 227:680, 1970 19. Marguerie GA, Plow EF: Interaction of fibrinogen with its platelet receptor: Kinetics and effect of pH and temperature. Biochemistry 20:1074, 1981 20. Sano K, Takai Y, Yamanishi J, Nishizuka Y: A role of calcium-activated phospholipid-dependent protein kinase in human platelet activation. Comparison of thrombin and collagen actions. J Biol Chem 258:2010, 1983 21. Best LC, Martin TJ, Russell RGG, Preston F E Prostacyclin increases cyclic AMP levels and adenylate cyclase activity in platelets. Nature 267:850, 1977 22. Hanson SR, Harker LA: Interruption of acute plateletdependent thrombosis by the synthetic antithrombin D-phenylalunylL-prolyl-L-arginyl chloromethyl ketone. Proc Natl Acad Sci USA 85:3184, 1988 23. Tamaoki T, Nomoto H, Takahashi I, Kat0 Y, Morimoto M, Tomita F Staurosporine, a potent inhibitor of phospholipid/Ca' dependent protein kinase. Biochem Biophys Res Commun 135:397, 1986 24. Kawahara Y, Takai Y,Minakuchi R, Sano K, Nishizuka Y: Phospholipid turnover as a possible transmembrane signal for protein phosphorylation during human platelet activation by thrombin. Biochem Biophys Res Commun 97:309,1980 25. Hannun YA, Loomis CR, Merrill AH Jr, Bell RM: Sphingosine inhibition of protein kinase C activity and of phorbol dibutyrate binding in vitro and in human platelets. J Biol Chem 261:12604,1986 26. King WG, Rittenhouse S E Inhibition of protein kinase C by staurosporine promotes elevated accumulations of inositol trisphos- PARISE ET AL phates and tetrakisphosphate in human platelets exposed to thrombin. J Biol Chem 264:6070, 1989 27. Nishizuka Y: The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature 308:693, 1984 28. Turner RS, Kemp RE, Su H, Kuo J F Substrate specificity of phospholipid/Ca2+-dependent protein kinase as probed with synthetic peptide fragments of the bovine myelin basic protein. J Biol Chem 260:11503,1985 29. Hassell TC, Kemp BE, Masaracchia RA: Nonmuscle myosin phosphorylation sites for calcium-dependent and calcium-independent protein kinases. Biochem Biophys Res Commun 134:240, 1986 30. OBrian CA, Lawrence DS, Kaiser ET, Weinstein IB: Protein kinase C phosphorylates the synthetic peptide ARG-ARG-LYSALA-SER-GLY-PRO-PRO-VAL in the presence of phospholipid plus either Ca2+ or a phorbol ester tumor promoter. Biochem Biophys Res Commun 124:296,1984 31. Ferrari S, Marchiori F, Borin G, Pinna LA: Distinct structural requirements of Ca2' /phospholipid-dependent protein kinase (protein kinase C) and CAMP-dependent protein kinase as evidenced by synthetic peptide substrates. FEBS Lett 184:72, 1985 32. Isenberg WM, McEver RP, Phillips DR, Shuman MA, Bainton DF: The platelet fibrinogen receptor: An immunogoldsurface replica study of agonist-induced ligand binding and receptor clustering. J Cell Biol 104:1655, 1987 33. Fox JEB, Say AK, Haslam RJ: Subcellular distribution of the different platelet proteins phosphorylated on exposure of intact platelets to ionophore A23 187 or to prostaglandin E,. Possible role of a membrane phosphopolypeptide in the regulation of calcium-ion transport. Biochem J 184:651,1979 34. Adelstein RS, Conti MA: Phosphorylation of platelet myosin increases actin-activated myosin ATPase activity. Nature 256:597, 1975 35. Chen M, Stracher A: In situ phosphorylation of platelet actin-binding protein by CAMP-dependent protein kinase stabilizes it against proteolysis bycalpain. J Biol Chem 264:14282, 1989 36. Wardell MR, Reynolds CC, Berndt MC, Wallace RW, Fox JEB: Platelet glycoprotein Ib, is phosphorylated on serine 166 by cyclic AMP-dependent protein kinase. J Biol Chem 264:15656, 1989 37. Plow EF, Marguerie GA: Induction of the fibrinogen receptor on human platelets by epinephrine and the combination of epinephrine and ADP. J Biol Chem 255:10971, 1980 38. Phillips DR, Jenkins CSP, Liischer EF, Larrieu M-J: Molecular differences of exposed surface proteins on thrombasthenic platelet plasma membranes. Nature 257599, 1975 39. Nurden AT, Rosa J-P, Fournier D, Legrand C, Didry D, Parquet A, Pidard D: A variant of Glanzmann's thrombasthenia with abnormal glycoprotein IIb-IIIa complexes in the platelet membrane. J Clin Invest 79:962, 1987 40. Fitzgerald LA, Leung B, Phillips DR: A method for purifying the platelet membrane glycoprotein IIb-IlIa complex. Anal Biochem 151:169, 1985 From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 1990 75: 2363-2368 Glycoprotein IIIa is phosphorylated in intact human platelets LV Parise, AB Criss, L Nannizzi and MR Wardell Updated information and services can be found at: http://www.bloodjournal.org/content/75/12/2363.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved.