THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 258, No. 3, Issue of February 10. pp. 2010-2013. 1983 Printed in U. S.A

A Role of Calcium-activated Phospholipid-dependent Protein Kinase in Human Platelet Activation COMPARISON OF THROMBIN AND COLLAGEN ACTIONS*

(Received for publication, August 10, 1982)

Kimihiko Sane$, Yoshimi Takai, Junji Yamanishi, and Yasutomi Nishizuka From the Department of Biochemistry, Kobe University School of Medicine, Kobe 650, and the Department of Cell Biology, National Institute for Basic Biology, Okazaki 444, Japan

In human platelets stimulated by thrombin and col- lagen, diacylglycerol is rapidly produced from phos- phatidylinositol. Concurrently, an endogenous protein having a molecular weight of about 40,000 (40K protein) is phosphorylated, and serotonin is released. These reactions are all inhibited by a prior treatment of plate- lets with prostaglandin El, dibutyryl cyclic AMP, so- dium nitroprusside, or with 8-bromo-cyclic GMP, which are known as potent inhibitors for platelet activation. Ca2+-activated phospholipid-dependent protein kinase (protein kinase C) preferentially phosphorylates 40K protein. As judged by fingerprint analysis, the sites in 40K protein that are phosphorylated during the platelet activation appear to be identical with those phospho- rylated by protein kinase C in a purified cell-free sys- tem. 12-O-Tetradecanoylphorbol-13-acetate, which di- rectly activates protein kinase C by substituting for diacylglycerol, stimulates 40K protein phosphorylation and release reaction without inducing diacylglycerol formation. Tetracaine, which inhibits protein kinase C by competing with phospholipid, blocks 40K protein phosphorylation and serotonin release without inhibit- ing the receptor-linked diacylglycerol formation. The results indicate that thrombin and collagen activate platelets in almost similar mechanisms and that protein kinase C may lie on a common pathway which leads to the release of serotonin. However, analysis with indo- methacin indicates that the role of thromboxane A2 appears to be more predominant for the action of col- lagen, and it is suggestive that this arachidonate me- tabolite activates platelets in an analogous mechanism to thrombin.

It has been reported that in platelets both thrombin and collagen induce rapid phosphorylation of two endogenous

* This investigation has been supported in part by the research grants from the Scientific Research Fund of the Ministry of Educa- tion, Science and Culture, Japan (1979-1982), the Intractable Diseases Division, Public Health Bureau, the Ministry of Health and Welfare, Japan (1982), a Grant-in-Aid of New Drug Development from the Ministry of Health and Welfare, Japan (1979-1982), the Yamanouchi Foundation for Research on Metabolic Disorders (1977-1982), and the Mitsuhisa Cancer Research Foundation (1981). The data are taken in part from the dissertation that will be submitted by K. Sano to Kobe University School of Medicine in partial fulfillment of the requirement for the degree of Doctor of Medical Science. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicat.e this fact.

$ To whom correspondence should be addressed at Department of Biochemistry, Kobe University School of Medicine, Kobe 650, Japan.

proteins having approximate molecular weights of 40,000 and 20,000 (1) and that this protein phosphorylation is associated with release of dense body constituents such as serotonin (2). 20K protein’ is myosin light chain, and a specific calmodulin- dependent protein kinase is responsible for this reaction (3). Another series of studies has revealed that phosphatidylino- sitol turns over rapidly in response to thrombin as well as collagen (4-6). In general, this phospholipid turnover is initi- ated by the action of phospholipase C which is specific for phosphatidylinositol. The primary products of this receptor- linked reaction are identified as diacylglycerol and inositol phosphate (partly recovered as inositol 1,2-cyclic phosphate (7)). Recent studies in this laboratory have postulated a function of this diacylglycerol in the activation of Ca2’-acti- vated phospholipid-dependent protein kinase that is referred to as protein kinase C (8-11). Diacylglycerol is normally almost absent from membrane but is transiently produced from phosphatidylinositol in a signal-dependent manner (5) . Thus, in an earlier report (12), it has been suggested that in thrombin-activated platelets protein kinase C may be respon- sible for the phosphorylation of 40K protein. Extending these observations, the present report will describe more direct evidence that this protein kinase may play a role of crucial importance in the platelet activation not only by thrombin but also by collagen. A slight difference of the responsibilities of thromboxane A2 for the actions of thrombin and collagen will also be briefly described.

EXPERIMENTAL PROCEDURES’

RESULTS

40K Protein Phosphorylation by Protein Kinase C“40K protein, which was purified partially from human platelets, served as a preferable substrate for a homogenous preparation of protein kinase C. This phosphorylation reaction was de- pendent on the simultaneous presence of Ca”, phospholipid, and diacylglycerol (Fig. 1). The faint band shown with a small

imate molecular weight of 20,000; 40K protein, protein having ap- I The abbreviations used are: 20K protein, protein having approx-

proximate molecular weight of 40,000, TPA, 12-0-tetradecanoyl- phorbol-13-acetate; EGTA, ethylene glycol bis(p-aminoethyl ether)N,N,N‘,N’-tetraacetic acid; SDS, sodium dodecyl sulfate.

* Portions of this paper (including “Experimental Procedures,” part of “Results,” Figs. 1 to 5, and Tables I and 11) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Be- thesda, MD 20814. Request Document No. 82M-2188, cite the au-

photocopies. Full size photocopies are also included in the microfilm thors, and include a check or money order for $5.60 per set of

edition of the Journal that is available from Waverly Press.

2010

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Protein Phosphorylation in Platelet Activation 201 I

WOW was due to the labeling of the enzyme molecule itself. It was noted that this autophosphorylation reaction again required Ca2+, phospholipid, and diacylglycerol. Acid hydrol- ysis of the radioactive 40K protein resulted in the formation of phosphoserine but not phosphothreonine. The enzyme did not react with tyrosyl residue in itself nor in any substrate proteins tested including 40K protein and histone fractions.

Next, the radioactive 40K protein, which was phosphoryl- ated by a homogeneous preparation of protein kinase C in vitro or by stimulation of intact platelets with either thrombin or collagen in vivo, was isolated and purified by SDS-poly- acrylamide gel electrophoresis. Then, the radioactive three samples of 40K protein were subjected to tryptic digestion followed by fingerprint analysis under the conditions specified under “Experimental Procedures.” The autoradiographs thus prepared were very similar, and almost identical results were obtained for thrombin and collagen (Fig. 2).

DISCUSSION

It has been suggested earlier (12) that protein kinase C is activated in platelets upon stimulation by thrombin and is responsible for the phosphorylation of 40K protein which is apparently related to release of serotonin. This assumption has been based primarily on the observations that the throm- bin-induced formation of diacylglycerol accompanies 40K pro- tein phosphorylation and that the addition of this protein kinase to crude platelet homogenates enhances the phospho- rylation of 40K protein. The results presented in this report provide more direct evidence that protein kinase C is ipso facto responsible for the phosphorylation of 40K protein. In particular, the sites in 40K protein that are phosphorylated in a purified cell-free system appear to be identical with those phosphorylated in intact platelets. Experimental results ob- tained using an activator and inhibitor of protein kinase C are also compatible with the conclusion that this enzyme activa- tion is most likely linked to the receptor-mediated breakdown of phosphatidylinositol to diacylglycerol (see Miniprint). In fact, human platelets contain a large quantity of this enzyme, and the enzymatic activity is more than 20 times higher than that of cyclic AMP-dependent protein kinase as assayed with calf thymus H1 histone as a common phosphate acceptor (12). Cyclic nucleotide-dependent protein kinases appear to be inhibitory for platelet activation presumably through phos- phorylation of distinct proteins as proposed previously (30- 32).

The present studies also seem to establish that collagen shows a mode of action very similar to that of thrombin and that protein kinase C may lie on a common pathway which eventually leads to the release of serotonin. Nevertheless, the response of platelets to indomethacin observed during the action of collagen is slightly different from that observed during the action of thrombin as described in the Miniprint and also as pointed out by other workers (33). Namely, in the thrombin action diacylglycerol formation, 40K protein phos- phorylation and serotonin release are not significantly affected by this drug, whereas in the collagen action these three reactions are profoundly inhibited particularly when small quantities of collagen are employed. Presumably, only a lim- ited number of platelets which adhere to collagen fiber may be activated directly, and thromboxane A2 which is derived from the phospholipid degradation may subsequently cause a cascade of the activation of a large number of nonadherent platelets. If this is the case, the results presented in this report

imply that thromboxane A2 induces platelet activation in a manner analogous to thrombin. The detailed causal relation- ship between 40K protein phosphorylation and serotonin re- lease remains to be explored.

Acknowledgments-We are grateful to S. Nishiyama and K. Ya- masaki for their skillful secretarial assistance.

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5. 6.

7.

8.

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14. 15.

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18. 19. 20.

21.

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23. 24. 25.

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27.

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32.

33.

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Haslam, R. J., and Lynham, J. A. (1977) Biochem. Biophys. Res.

Hathaway, D. R., and Adelstein, R. S. (1979) Proc. Natl. Acad.

Lloyd, J. V., and Mustard, J. F. (1974) Br. J. Haematol. 26,243-

Rittenhouse-Simmons, S . (1979) J. Clin. Znuest. 63, 580-587 Bell, R. L., and Majerus, P. W . (1980) J . Biol. Chem. 255, 1790-

1792 Dawson, R. M. C., Freinkel, N., Jungalwala, F. B., and Clarke, N.

(1971) Biochem. J. 122, 605-607 Takai, Y., Kishimoto, A,, Iwasa, Y., Kawahara, Y., Mori, T., and

Nishizuka, Y. (1979) J. Biol. Chem. 254,3692-3695 Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mori, T.,

Nishizuka, Y., Tamura, A., and Fujii, T. (1979) J. Biochem.

Takai, Y., Kishimoto, A,, Kikkawa, U., Mori, T., and Nishizuka,

Kishimoto, A., Takai, Y., Mori, T., Kikkawa, U., and Nishizuka,

Kawahara, Y., Takai, Y., Minakuchi, R., Sano, K., and Nishizuka,

Mustard, J . F., Perry, D. W . , Ardlie, N. G., and Packham, M. A.

Glynn, I. M., and Chappell, J. B. (1964) Biochem. J . 90, 147-149 Folch, J., Lees, M., and Sloane Stanley, G. H. S. (1957) J. Biol.

Chem. 226,497-509 Rouser, D., Kritchevsky, G., and Yamamoto, A. (1967) in Lipid

Chromatographic Analysis, Vol. 1, pp. 99-162, Marcel Dekker, Inc., New York

Kikkawa, U., Minakuchi, R., Takai, Y., and Nishizuka, Y. (1982) Methods Enzymol., in press

Costa, J. L., and Murphy, D. L. (1975) Nature 255, 407-408 Laemmli, U. K. (1970) Nature 227, 680-685 Bligh, E. G., and Dyer, W . J. (1959) Can. J . Biochem. Physiol.

Vitiello, F., and Zanetta, J . P. (1978) J. Chromatogr. 116, 637-

Needleman, P., Minkes, M., and Raz, A. (1976) Science 193, 163-

Pires, E. M. V., and Perry, S. V. (1977) Biochem. J. 167, 137-146 Beemon, K., and Hunter, T. (1978) J. Virol. 28, 551-566 Hunter, T., and Sefton, B. M. (1980) Proc. Natl. Acad. Sci. U. S.

Criss, W. E., Yamamoto, M., Takai, Y., Nishizuka, Y., and Morris, H. P. (1978) Cancer Res. 38, 3532-3539

Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193,265-275

Castagna, M., Takai, Y., Kaibuchi, K., Sano, K., Kikkawa, U., and Nishizuka, Y. (1982) J . Biol. Chem. 257, 7847-7851

Mori, T., Takai, Y., Minakuchi, R., Yu, B., and Nishizuka, Y. (1980) J. Biol. Chem. 255,8378-8380

Haslam, R. J., Davidson, M. M. L., Davies, T., Lynham, J. A,, and McClenhaghan, M. D. (1978) Adu. Cyclic Nucleotide Res. 9,533-552

Takai, Y., Kaibuchi, K., Matsubara, T., and Nishizuka, Y. (1981) Biochem. Biophys. Res. Commun. 101, 61-67

Takai, Y., Kaibuchi, K., Sano, K., and Nishizuka, Y. (1982) J. Biochem. (Tokyo) 91,403-406

Kinlough-Rathbone, R. L., Packham, M. A., Reimers, H.-J., Ca- zenave, J.-p., and Mustard, J. F. (1977) J. Lab. Clin. Med. 90, 707-719

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2012 Protein Phosphorylation in Platelet Activation Supplement to:

*A Role of Calcium-activated, Pho*pholipid-dependent Protein Kinase in Human Platelet ACtaYation: Comparison of Thrombin and Collagen Actions*

by

Kimihiko Sano. Yoshimi Takai, Junji Yamanishi. and Yasutomi Nishizuka

EXPERIMENTAL PROCEDURES

healthy voluntez. Platelet-rich plasm and vashed pldtelet(~ were prepared by the method of nustard et al. (13) except that the first and second wsshins

Materials and Chemicals- id-citrate dextrose blood was obtained from

fluids contained 20 g ~ r ~ t a g l a n d i n Ei and no calcium. Isotopes were pui- Chased from the fOllOvinq sources: carrier-free H332P04, Japan Radioisotope Association. Tokyo: 12-14clserotonin (58 m€i/-l), The Radiochemical Centre, mmersham: 13Hl arachidonic acid (78.2 Ci/-l) and high specific activity of Iv-~~PIATP (2,600 Ci/-l), New England Nuclear, Bo11ton. Bovine throbin and 5011aqen were the products of MOChida Pharmaceutical CO. and Hormon-Chemia. respeitively. Trybsin (type I) and bovine v-globulin were obtained from signa.

cyclic M P and 8-bromo-cyclic GMP were purchased from Boehringer Manheim. Prostaglandin El was a generous gift from On0 Pharmaceutical co.. Dibutyryl

Tetracaine hydrochloride vas donated by Kyorin Pharmaceutical CO.. Fatty acid-

diolein, heparin, and dimethylsulfoxide were the products of Nakazai Chemicals free bovine berum albumin was purchased fiom Uiles. Sodium nitropruseidi,

Ltd.. Thromboxane 02 was the product of Funakoahi Pharmaceutical Co.. TPA WaB

the product of P. Bor'chert, Eden Prairie, Uinnasota. IV-~~PIATP was prepared by the method of Glynn and Chappell ( 1 4 ) . A mixture of phospholipids was ex- tracted from bovine brain by the method of FOlCh et a l . 115) and fractionated on a silicic acid column as described by Rouser e F a K 1161. Protein kinase C was purified to homogeneity from the soluble fracTiEi of rat brain a* described I l l ) . Other materials and chemicals were obtained from comer~ial IIOUIE~S.

- - -.- Purification of 4OK Protein-All procedure. were carried out at 0-4.C. Hashed platelets (3 x IO'" cells) were disrupted by Sonication using a Kontes Sonifier, Model K881440. for 90 S in 2 mI Of 20 111 TCiS/HCI a t pH 1.5 Contain- ing 5 mM ECTA. 2 nW EDTA. 0.011 leupeptin, 2 mM ~enylmethyl~ulfony1 fluoride

60 Ein at 100.000 x g, and the sup-rnatant ( 2 ml, I4 mg Of protein) was sub- 10 mM 2=mercaptoeZmol, and 0.25 M sucrose. -The sonicate was centrifuged fo;

iected to a Seohsdex c-100 c o l m (80 x 1.4 c m l eauilibrated vith 20 111 Tris/ ~~

HC1 at pH 1 . 5 containing 2 111 EDTA, 2 mM BGTA. and 5 mM 2-msrCaptOethanOl. Elution was carried Out Wit lT the Same Kffar, and fr&hnS Of 2 m1 each were collected. An aliquot Of each fraction Was Subjected to SDS-polyacrylamide slab gel electrophoresis to identify 4OK protein under the conditions described bel-. 40K protein appeared in FraEtiOnS 31 through 40. These fractions (20 ml, 2.6 mg of protein) were collected, diluted 5 times with cold water, and then subjected to a DE-52 column (5 x l o ) which was equilibrated with 20 .II TrisjHCl at pH 7.5 containing 0.5 nW EDTA 0 5 111 EGTA and 1 Oll 2-mercaptor

vas carried out with a 24-ml linear concentration gradient Of NaCl I O to 0.4 E) ethanol. After the column was wasred wit; 4 6 mrof th; eame biiffer, elution

peared in Fractions 9 through 12. Those fractions ( 4 ml, 0.5 mg of protein) in the same buffer. Fractions of 2 m l sash were collected. 4OK protein ap-

were pool& and used for the nubsequent studies. This preparation of 401 pro- teln was free from endogenous protein kinases, protein phosphatases, and other

201 pure as estimated by SDS-polyacrylamide gel electrophoresis. interfering materials and phosphate acceptors. This preparation was roughly

-

Prwarati;; of Radioactive Pl~tel~ts-Pl.telet-ZiCh plasma was incu- bated w i t h 12- Clssrotonin ( 5 0 nCi/mI of platelet-rich plsma) or 13Hlarachi-

donis acid in a fatty acid-free bovine serum albumin solution was prepared by donic acid (1.0 pCi/ml of platelet-rich P1.md at 37.C for I h. ['Hlarachi-

the method of Rittenhouse-simens 15). Hashed platelets were labelled with 3 2 ~ i by the method of Haelam aod ~ynham 12). The radioactive platelet. were finally suspended at 5 x 10' cslls/ml in a Tyrode's solution containing 138 111 NaC1, 2.7 + KC1, 12 E NaHC03, 0.36 + N W 2 W 4 , 2 UqCI2, and 5.5 + glG0.e.

AnalYain of Protein Phosphorylation and serotonin Release-A suspn- a i m of platelets (1.8 . I ) , Which .)ere prelabelled w i t h either 32Pi or I2-l'CI serotonin, was stirred at 37.C in an aggragmster, Bryeton Mnufacturinp Ltd., and various drugs and stimulants 10.2 m1) were added with stirring as indicated

~ ~~

and mixed V i t h 25 p l Of 16.58 formalin for analysis of serotonin release or in each experiment. After appropriate InteNaIs, 2 5 0 4 aliquOt. W e r e removed,

vith 125 pl Of an SDS-Stop Solution (98 SDS containing 6% 2-mercaptoethanol,

rylation. or measuring serotonin release, the mixture was centrifuged for 40 158 glycerol, and 186 DVI Tris/HCl a t pH 6.7) for analysis Of protein phospho-

s at 12,000 x g, and the radioactivity in the supernatant wan determined (18). Fox analysls of phosphoprotelna, the mixture w a ~ Wiled in a water bath for 3 mln. A 60-111 aliquot (about 30 pg Of protein) Was subjected to SDS-polyacryl- amide slab gel electrophoresis as described by "11 ( 1 9 ) . The gel Vas

and exposed to a Kodak Royal X-mat film to prepare an autoradiograph. The stained With CODmaSsie brilliant blue, dried On a Hhatm~n NO. 1 filter paw=,

relative intensity of each band was quantitated by measuring the absorbance at 430 ryll using a Shlmadzu dual-wavelength chromatoqqram *canner, nodel cs-910.

which were labelled with 13Hlarachidonic acid was incubated an dessribd above, and vdr~ous drugs and stimulants were added as indicated in each experiment. The reaction (1.0 mll was terminated by the addition of 3.75 ml of chloroform/ methanol 11:2), and lipids were directly extracted from the platelets by the method of Bligh and Dyer 120). The sample was subjected to thin layer chroma-

ether/ethsnol/NH, (50:40:2:0.1) to isolate diocylglycerol 15). or using a sol- tography on a Silica gel G plate using a solvent system of benrene/diethyl

vent system of methyl acetare/l-prop~nol/chloroform/m~th~~~l/O.251 KC1 125:25:

by I2 vspour, scraped off from the plate into vials, and the radioactivity was 25:10:9) to separate various phoapholipids 121). Various lipids were detected

determined.

Analysis 0A Diacylglyfer01 and Other Lipide-A suspension Of platelets

acid were lncubata as descrlbed above. and the reaction was stopped by the addition of 0 . 2 m l of 1.5 N formic acid. Radioactive arachidonic acid meta- bolites were extracted twice each time w i t h 10 m l of ethyl acetate, applied t o a Sillca gel G plate, and developed with chloroform/mEth.nol/~~~ti~ acid/watsr 190:8:1:0.81 (221. Thromboxane 82 was identified bv an authentic marker and

Throml?oxsne 82 Deter.in~tion-Pl~telerll latmlled with 13Hlar.chidonic

Its radioactivity was determined as described 122).

Phos howlation of 4OK Protein-The reaction mixture 1250 pl) con- tain& 5 :mol Of TrisTiiCl-itpH, 1.25 wol of magnesium acetate, 12.5 -1 Of lV-32PlATP 15 x lo6 cpm/mll. 100 mol Of CaCl2. 20 pg Of phospholipid, 0.6 pg of diolein, 5 pg of 4OK protein, and 0.1 pg of protein kinase C. Since the preparations of 4OK protein and protein kinase C both contained 3.4 x 10" EGTA in their stock solutions. the free Ca2+ Concentration in the complete

method of P i m a and Perry 123). After incubation f o r 3 min a t 30'c. the rea=- incubation mixture was expected to be atout 6 x U as estimated by the

tion was terminated by placing the tube in ethanol at -80.C. After lyophilira- tion. the sample vas subjected to SDS-polyacrylamide slab gel electrophoresis and an autoradiograph was prepared.

covered from wlvacrvlamide oel. was m d e bv the method of Baemon and Hunter ing

" . . of this wlution uned was approximately 2 mW0.1 cm2 gel (1.5 m thick). The

overnight at 37-C. The gel fragments were removed by Centrifugation. After homogenate was made 58 in 2-mercaptoethsnol, boiled for 5 min, and then shaken

the addition of 25 pg/ml of bovine v-globulin as a carrier, the protein was precipitated by adding trichloroacetic acid (final, 208). followed by standing for 4 h a t 4.C. After centrifugation the pellet was washed once vith ethanol at -1O.C. and then vith ethanol/ether I I : l ) at -10-C. The dried residue was dxssolved in I50 p l of chllled performic acid and incubated for 2 h at 4-c. This performic acid was prepared by mixing 308 H202 and 988 formic acid I1:9),

diluted Vlth water, frozen, and lyophllired. The oxidized protein was treated followed by standing for 1 h at r- temperature. The protein solution was

vlth 30 pg of trypsin in 0 . 5 ml of 50 @j NH.HCO, for 18 h at rmm temperature. Then, 20 pg of trypsin vas supplemented and incubation was continued for ad- ditional 4 h. The digest was diluted vith water, frozen, and lyophilized. These procedures were repeated twice more. The final residue was dissolved in 20 p l of butanot/pyridine/~cetic acid/vater (2:1:1:18). An aliquot was spat- ted on a cellulose-coated thin layer plate. Electrophoresis was carried out at 1.000 V for 30 mi" at 4.C Using a Pharmacia flat bed apparatus, Uodel FBE 3000. in the DFeSence Of the same buffer Solution. The OeDtides Were then

. -

chroiatoqraphid to the second dimension wIth butsnol/pyridine/aoeti~ acid/ Water (32.5:25:5:20) as a solvent, and an autoradioqraph was prepared.

w ~ * dE;z;~ ;; Ph~phor:Ytf.~~"~o:':d~.,13:cr4~i~~:%p~~Z~~~~~~in The HC1 was removed-under reduced pressure, and the acid hydrolysates were analyzed by electrophoresis on a cellulose-coated thin layer plate (251 and also by column Chromatoqmphy 126). Under the conditions employed, phospho- serine, phosphothreonine, and phosphotyrosine were separated from one another.

HE1 d h

determined uelng a Packard Tri-Carb liquid scinti1l;tion ;pectrometer, nodel 3320. Protein was determined bY the method of Loyrv et al. (211 with bovine

Other Procedures-The radioactivity of 32P- "C- and 'H-s-les was

b&

2%

RESULTS

1 2 1 3 4 5 6 7

Fig. 1. Phomphorylation Of 40K protein by protein kinase C In E. +he phosphorylation of 40K protein by protein kinase C was carria Out as described under 'EXPERIIIWTAL PROCEDURES- except that either Cs2+, phospholipid, diolein, Or protein kinase C Was omitted as indicated. For the analyeis Of the phos- phorylation of 40K protein in intact platelet., the platelets, which were .~ labelled with 32Pi, vere (Itimulated a t 37.C by t h e d i n for 1 min. Lane- 1 and 2 in vivo phosphorylationi Lanes 3 through 7 in vitro phosphorylation Lane 1; iiTM thrombin8 Lane 2,- thrombin: L&T3..1nus protein kinas; C i

complete system. - Lane 4. minus phosphomd: e 5. minus d i m n : e 6, minus CaCl2; E 7.

I L r

A 0

0

0 4 0

B 0

0

0 i

Fig. 2. AUtOradiographs of tryptic digeats of 4OK protein phoephorylated in in

or collagen. The phosphorylation of 40K protein by protein kinase C in vitro virro system by protein kinase C and in intact platelets stimulated by thrombiii was carried out as described in the legend to Pig. 1. The p h o s p h o r y l ~ i ~ ~ O K protein in intact platelets was carried out as described in the legend to Fig. 1 except that the platelets Were stimulated by either thrombin for I min or collagen for 2 min. The reaction was terminated by the additlon of SDS-atop s o l ~ t i o n , and then an aliquot was subjected to SDS-polyacrylamide slab gel electrophoresis. The radioactive 40K protein was extracted from the gel. di- gested with trypsin, and subjected to cellulose-coated thin layer plate elec- trophoresls in the horizontal dimension (negative pole left: positive pole

under *EXPERIUENTAL PROCEDURES-. Arrowheads indicate the Origin.' A In Vitro right1 followed by ascending chromatography in v e r t i c a l dlmsnsion'as described

phosphorylation; E, & vivo phosphorylation induced by thrombin; $,-hyLr phosphorylation inducedFcollagen.

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Protein Phosphorylation in Platelet Activation 2013

then stimulated

0.25 640 0.76 4.870

0 . 2 8 650

0 26 630

0 . 2 8 0.7s 6,050

700

0.54 I , 600

0.49 2.040

None 0 Collagen 14.040

4 Collagen * Sodlw nitr~~r~~slde 5,630

I10 1.090

5.23 600 0.85 7.150

370 0.30 2,010

directly activates proteln kinase C I n the presence of both Ca2+ and phoapho- lipid (181. IC in a180 shown chat several ph~8pholipld-lnteracti"g drug8 svch

phospholipid but not with dlacylglycerol 1 2 9 1 . In parallel wrch thelle observe- &= tetracaine profoundly lnhlbit protem kinase C activation by conweting with

protein phasphorylatrm as wall a8 BeTotOnin release without dlacylglycerol tione. the e*perlnante with intact platelets indzcated that TPU induced 40K

was relatively poor, although (OK prOLeln Was maximally phOBphOrylated 11s ab- formation (Tabla 111. However, the extent of serotonin release elicited by TPA

I ' ( I

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K Sano, Y Takai, J Yamanishi and Y Nishizukaplatelet activation. Comparison of thrombin and collagen actions.

A role of calcium-activated phospholipid-dependent protein kinase in human

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