Cyclic AMP and platelet prostaglandin synthesis

PROSTAGLANDINS

CYCLICAMPANDPLATELET PROSTAGLANDIN SYNTHESIS Jonathan M. Gerrard, Janet D. Peller, ThomasP. Krick, and James G. White Departments of Pediatrics and Biochemistry University of Minnesota Minneapolis, Minnesota 55455

ABSTRACT

The present study has investigated the influence of agents which elevate intracellular levels of endogenous platelet adenosine 3’5’cyclic monophosphate (cyclic AMP), and the effect of the exogenous cyclic AMPanalog, dibutyryl cyclic AMP, on the conversion of 14Carachidonic acid by washed platelets. Prostaglandin El (PGBl) , PGBl with theophylline, or dibutyryl cyclic AMPincubated with washed platelets prevented arachidonic acid induced platelet aggregation, but had no effect on the conversion of arachidonic acid to 12L-hydroxy5,8,10, 14-eicosatetraenoic acid fHBI’B), 12L-hydroxy-5,8,10 heptadecatrienoic acid (HHT), or thromboxane B2. Ultrastructural studies of the platelet response revealed that agents acting directly or indirectly to increase the level of cyclic AMPinhibited the action of arachidonic acid on washed platelets and prevented internal platelet The influence of PGBl with contraction as well as aggregation. theophylline, and dibutyryl cyclic AMPon the throtiin induced release of 14C-arachidonic acid from platelet membranephospholipids was also investigated. These agents were found to be potent inhibitors of the thrombin stimulated release of arachidonic acid from platelet phospholipids, due most likely to an inhibition of platelet phospholipase A activity. The results show that dibutyryl cyclic AMPand agents which elevate intracellular cyclic AMPlevels act to inhibit platelet activation at two steps 1) internal contraction and 2) release of arachidonic acid from platelet phospholipids. ACKNOWLEDGEMBNTS The authors are grateful to Dr. J.E. Pike, The Upjohn Ccnnpany, for supplying PGEl, to Dr. C.C. Sweeley for his initial help with the GC-massspectrometric detection of HBTE,HHI and thromboxane B2 and This work was to B. Seaquist for excellent technical assistance. supported by grants HL-11880, AM-06317, HL-06314, CA-12607, CA-08832, Dr. Gerrard is the and CA-11996 from the U.S. Public Health Service. recipient of a fellowship grant fram the Minnesota Heart Association.

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INTRODLJCI’IW In the last ten years it has become apparent that cyclic AMPplays a critical role in platelet physiology. Many agents which inhibit platelet activation elevate intracellular platelet cyclic AMPlevels either by stimulating platelet adenylate cyclase or by inhibiting phosphodiesterase (l-4). Day and Holmsen (5) postulated in 1971 that cyclic A?? inhibited platelet aggregation by affecting the distribution of intracellular calcium. White in 1974 16) nrovided evidence for such an effect by demonstrating that agents wh&& ‘act by elevating cyclic AMPlevels were potent inhibitors of platelet aggregation stimulated by the calcium ionophore A23187. Recently further support for the thesis that cyclic AMPmodulates intracellular calcium has come from studies by George and associates who have shown that cyclic AMPcan stimulate the uptake of calcium by a platelet membranefraction in a fashion analogous to the cyclic AMPstimulated calcium uptake into smooth muscle sarcoplasmic reticulum (7). An alternative hypothesis for the effect of cyclic AMPon platelet function has recently been proposed by Malmsten et al (8). Their observations suggest that raised intracellular cyclic AMPlevels act by inhibiting platelet prostaglandin synthesis. In the preset investigation, we have studied the influence of agents which elevate endogenous platelet cyclic AMP levels, and the cyclic AMPanalog dibutyryl cyclic AMP, added exogenously on platelet prostaglandin synthesis and platelet phospholipase A activity in order to further evaluate the mechanismof the inhibitory action of cyclic AMPon platelets, MATERIALS AND METHODS

A) General: Platelets for the present study were prepared from blood draiZiiZinorma1 humanvolunteers after informed consent, anticoagulated with 3.8% trisodium citrate, $3 6.5, in a ratio of 1 part anticoagulant to 9 parts blood, and centrifuged at 200 g for 20 minutes to separate platelet rich plasma (C-PRP). Prostaglandin El was kindly provided by Dr. J.E. Pike of the Upjohn Company. 14C arachidonic acid was purchased from NewEngland Nuclear, dibutyryl cyclic adenosinemonophosphate from the Sigma Chemical Company, and theophylline from Schwarz-Mann.

B) . Studies of the Conversion of 14C Arachidonic Acid by Plate-

lets: For study of the transformation or 14C arachidonic acid by was= platelets, the C-PRPwas centrifuged at 1000 g for 10 minutes at 4’C in the uresence of 1% EDI’A. and then resusDended in Hanks’ balanced salt solution (HBSS), pH 7.4, without calcium and magnesium. The procedure was then repeated twice more at which time the platelets were resuspended in HBSScontaining 0.5 mMCaC12at a concentration of 2 x 108 platelets/ml. To evaluate the effect of agents which elevate platelet cyclic AMPon the transformation of arachidonic acid, 1 ml samples of washed platelets were incubated with 3 uMPGEl together with 2 mMtheophylline, 1 mMdibutyryl cyclic AMF or buffer @SS) alone for 5 minutes. 3.3 @I 14C arachidonic acid (specific activity 28 mCi/fi made up as a sodium salt in .OIM Tris buffer pH 7.4 was then added to the platelets stirred on the agregometer. After three minutes the samples were added to extraction vials

JULY 1977 VOL. 14 NO. 1

PROSTAGLANDINS

containing 10 ml absolute ethanol. Samples were then dilutedwith 12 mls H20, acidified with lN HCLto a pH of 3 and extracted into diethyl ether. The ether was dried by addition of ?4gSO4,and then transferred to another vial. The MgS04was washed twice with diethyl ether to remove any arachidonic acid or metabolites and the ether samples were pooled. Recovery of arachidonic acid and metabolites using this procedure was greater than 90%. The arachidonic acid and metabolites were then methylated using diazomethane, and separated on a thinlayer chromatogram (silica gel G) using a solvent system containing the organic layer of 100:100:50 isooctane:water:ethyl acetate. The thin layer plates were then scanned on a thin layer radiochromatogram scanner and the conversion of arachidonic acid to product quantitated as described previously (9,lO). Further identification of the products was obtained by performing gas chromatography-mass spectrometric (GC-M5) analysis on samples repared similarly using 164 nanomoles arachidonic acid and 1 x 101g platelets in 4 mls. For the GC-MS studies the trimethylsilyl (‘l’Y4S)derivatives of the methyl esters of the metabolites of arachidonic acid were prepared. Following preparation of the methyl ester derivatives, the samples were dried in a vial under nitrogen and equal volumes of pyridine (Mallinkrodt) and N, Nbis (trimethylsilyl) triflumacetamide (BSTFA) (Supelco Inc) were added. The vial was then sealed under nitrogen and heated to 12O’C for 15 minutes. Additional experiments were carried out by chromatography of the methylated products using a second solvent system, the organic layer of 88:80:40:2 ethyl acetate, water, isooctane, acetic acid . Identi fication of the thrcmboxane B2 on the latter TLCplate was also ascertained using GC-MS. One further experiment was also conducted in which 2 x 109 platelets treated with 1 I&I dibutyryl cyclic AMPor buffer were then stirred with 33 nanomoles arachidonic acid. At the end of 5 minutes 15.8 nanomoles of ricinoleic acid was added as an internal standard and the samples extracted and derivatized as described earlier. Production of AA, HER, HHTand thromboxane B2 was then quantitated using GC-m. C). Electron Microscopy: Studies of platelet ultrastructure were performed by fixing samples exposed to control or experimental conditions in 0.1% glutaraldehyde in White’s saline, followed by 3% glutaraldehdye and then subsequently 1% osmic acid in Zetterquist’s buffer. Samples were dehydrated, embedded and sectioned according to our usual procedures and studies using a Philips 301 electron microscope (10). Studies of the Release of 14C Arachidonic Acid from PlateIet Phosphollplcls: The procedure of Bills and Silver m was used to evaluate the thrombin stimulated release of arachidonic 14C arachidonic acid was incubated acid from platelet phospholipids. with platelets in C-PRPfor 2 hours at 37’C. Following incubation, free arachidonic acid was removed by washing the platelets. The cells were pelleted at 4°C in the presence of 1% EDNA,and resuspended in HBSScontaining 3% albumin. The procedure was repeated, and the platelets resuspended in HBSScontaining 3% albumin and 2.5 n&lCaC12. The washed platelets were then preincubated with buffer control, B) .

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41

PROSTAGLANDINS 1 ug/ml PGEl plus theophylline, or dibutyryl cyclic AMPfor 5 minutes and then stirred with buffer alone or thrambin, 0.5 units/ml. After stirring for 5 minutes, the samples were extracted into chloroform/ methanol according to the procedure of Bills, Smith, and Silver. Recovery using the extraction procedure was greater than 92%. Following extraction, the organic phase was dried under vam and the phospholipids were separated from the free arachidonic acid and metabolites using silicic acid column chromatography (11). The elution profiles for a phospholipid, phosphatidyl choline, for 14C arachidonic acid and for a prostaglandin, 3&I-PGElare shown in Table I. The release of 14C TABLEI. Elution Pattern for the Silicic Acid ColumnUsed to Separate Free Arachidcmic Acid and Metabolites frm Platelet Phospholipids Per cent of Compoundin Each Fraction Fraction III Fraction II Fraction I 14C Arachidonic Acid

97%

2.8%

0.4%

3H-PGEl

1.6 %

97%

1.4%

Phosphatidyl Choline*

0.5%

0.0%

99.5%

* Measured as per cent of phosphorous in each fraction using the method of Bartlett for determining phosphorous (12). arachidonic acid from the platelet phospholipids was calculated by determining the per cent of total radioactivity in fractions I and II containing free arachidonic acid and met,abolites and subtracting the values obtained when buffer alone or inhibitor alone was added to The conversion of arachidonic acid to metabolites was the platelets. assessed using thin layer chromatography of the methylated derivatives as described earlier, followed by confirmatory GC-MS. Statistical comparison of results was ma& using the Students T test. RESULTS 1)

The Influence of Cyclic AMPon Platelet Prostaglandin Synthesis: Addition of3 V 14C arachidonic acid to washed platelets initiated platelet aggregtion and was accompanied by conversion of the arachidonic acid to 12L-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE), 12L-hydroxy-5,8,10-heptadecatrienoic acid (HHT), and thromboxane B2 (Figure 1). Addition of 28 $! indomethacin to the platelets prior to the addition of the radiolabelled arachidonic acid blocked aggregation and prevented conversion to HHTand thrcnnboxaneB2. In contrast, addition of 3 @l PGEl plus 2 mMtheophylline, or 1 &I dibutyryl cyclic AMPprevented arachidonic acid induced platelet aggregation but had no discernable effect on platelet prostaglandin and thromboxane synthesis (Table II).

42

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PROSTAGLANDINS

The Influence of Cyclic AMP on Platelet Aggregotion and Platelet Prostoglondin Synthesis

WASHED PLATELETS +

COllPlETE

WASHED

,I

Me-A1

\

!J\

/ \

1

NONE

1

‘?I

WASHED FIATELETS

0-d

I 15

10

0

5

Distance from Origin

’

MINE

I

IN00n;THAClN + AA

1

km)

v

The influence of PGEl ccPnbinedwith theophylline on the trans ormation of 14C arachidonic acid by washed platelets, and a canparison with the effect of indomethacin. PGEl 3 $I together with theophylline (2 nM) prevented platelet aggregation but had no effect on the transformation of arachidonic acid. Indomethacin, in contrast, prevented aggregation and also prevented transformation of arachidonic acid to the active prostaglandin endoperoxides and thromboxane A2 as shown by the absence of HHTand thromboxane B2 (TxB2), the products of these active ccpnpounds

.

To ensure that we were not measuring conversion of the arachidonic acid to a polar derivative which was not thromboxane B2, we chromatographed the material from the thromboxane B2 region on a second thin layer chromatography system using the organic phase of 88:80:40: 2 ethyl acetate, water, isooctane, acetic acid. More than 90% of the radioactivity in the thromboxane B2 region frcnn the first TLC separation was in one band on the second TLC system with an RF of 0.41. GCMS analysis of the methyl ester-trimethylsilyl (ME-‘MS) derivative of the material in this region of the thin layer plate is shown in Figures 2 and 3. Selective ion monitoring of the ions at m/e 256, m/e 301, and m/e 217, prominent in the mass spectrum of the ME-‘INS derivative of thromboxane B2, revealed their presence in the major peak on the gas-chromatogram tracing. The pattern was identical in the sample from the platelets treated with arachidonic acid whether or not PGEl and theophylline had been added prior to the arachidonic acid to prevent aggregation (Figure 2).

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PROSTAGLANDIJVS

TABLEII. The Influence of CyclicAw on the Conversion of 14C Arachidonic Acid (14C-AA) to Metabolites by WashedPlatelets Per centof Product* AA -

HHT

m2

19.6 +0.9 -

19.3 +3.2

35.6 t2.4

25.6 to.9 -

;;g!m3fl

23.0 t1.0

16.6 t1.0

36.9 +3.5 -

23.5 +3.1

WashedPlatelets +pCE1 3T.!M + Theophylline 2 IN t 14C-AA

20.5 +1.6 -

17.9 to.5 -

37.0 to.6

24.6 +1.7 -

WashedPlatelets + DibutyrylCyclicAMP 1 mM t 14C-Aq

20.0 t1.8

22.5 to.7 -

34.6 t1.2

22.8 to.7 -

WashedPlatelets + 14c-AA WashedPlatelets

datapresentedhere * Mean + SE of 4 replicates.The experimental are fiYom one of threeexperiments whichshowedsimilarresults.

TIC T)m/r301 e ,o f m/c 256 = -0 m/c 217 L

1.0

I

I

I

2.0

3.0

4.0

Time (minutes)

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JULY 1977 VOL. 14 NO. 1

PROSTAGLANDINS

B 7o.m

TIC SO.000 3OW 10,000 II 1200

o m/c 301 al E ‘g m/t 256 = = n/e 217

400 mE 0 3750 2500 1250

E

2cG Klca 0

E

I I.0

I

I

I

2.0

3.0

4.0

Time (minules)

Figure 2. Selective ion monitoring of the MesSi derivative of the methylester of the material in the region of the thin layer chromatogram corresponding to throtioxane B2, and obtained following addition of arachidonic acid to A) washed platelets or B) washed platelets preEqual proportions of material extreated with PGEl and theophylline. tracted from the TLCplates were injected. TIC = Total ion current. Column: 1% OV-1 at 24O’C.

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PROSTAGLANDINS

B

Figure 3. Mass spectrum of the Me3Si derivative of the methyl ester of thromboxane B2 obtained by addition of arachidonic acid to A) washed platelets and B) washed platelets pretreated with PGEl and theophylline. Confirming evidence that the major peak seen on the gas-chromatogram tracing was the ME-lMS derivative of thromboxane B2 was obtained from the complete mass spectrum of this compound. The mass spectrum showed a molecular ion at m/e 600, a base peak at m/e 256 and further ions at m/e 585, 529, 510, 439, 420, 366, 323, 201, 295, 225, 217 and 173 as described previously by Hamberg and Samuelsson (9). The mass spectnrm of the compound isolated from platelets treated with PGEl and theophylline prior to addition of arachidonic acid was identical to the compound from platelets treated with arahidonic acid alone (Figure 3). To provide additional confirmation of our thin layer chromatographic quantitation one further step was taken. We quantitated using computer assisted GC-MSwith ricinoleic acid added as an internal Again, as with the TLC quantitation, there was no signifistandard. cant influence of 1 mMDcAMPon the transformation of AA by platelets in spite of the fact that the DcAMPcompletely inhibited AA induced aggregation (Table III). The result confirms the lack of any effect of agents which elevate intracellular platelet cyclic AMPon the platelet enzymeswhich convert arachidonic acid to HETE, HHT and thranboxane B2.

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TABLEIII. The Effect of DcAXPon Platelet Metabolism of AA as Measured Using ComputerAssisted GC-MS AA (nanomoles)

HEIE (nanomoles)

HHT ThramboxaneB2 (nanomoles) (nanomoles)

Washed Platelets 2.99” + 33 nanomoles AA -+O.33

17.55 -+1.57

3.43 +0.49 -

9.67 -+0.47

WashedPlatelets +lmVDc.AMP 2.39 + 33 nanomoles AA -+0.34

18.00 -+1.65

3.82 -+0.40

9.87 -+0.34

*

Results are expressed as mean + SE of four replicates. 2)

The Influence of Cyclic AMPon Platelet Ultrastructural aanges Stimulated by Arachidonic Acid: Our experiments suggested that agents which elevate intracellular platelet cyclic MP levels do not interfere with platelet prostaglandin synthesis, and therefore, must act to prevent platelet aggregation by another mechanism. We used platelet ultrastructural studies to probe the mechanism of inhibition of cyclic AMPon arachidonic acid induced aggregation of washed platelets. Electron microscopic study of samples of the washed platelets alone or after incubation or stirring with buffer, PGEl, PGEl and theophylline, or dibutyryl cyclic AMPrevealed discoid cells similar to those in platelet rich plasma except that more pseudopods were present. Addition of arachidonic acid to unstirred samples of such platelets at 37“C produced a concentration dependent internal platelet contraction similar to that seen with the calcium ionophore A23187 03) or PGG2(14,lS). At low concentrations (0.3-l. 7 us) early stages in the movementof granules to the center of the platelet surrounded by an encircling band of microtubules and contractile microfilaments was seen. At higher concentrations (3.3- 33 the internal contraction had progressed to the point uV where many cells were degranulated and contained a central mass of contracted microfilaments. When arachidonic acid was added to stirred samples of washed platelets, the internal changes were similar, and in addition the platelets were aggregated, developing the close platelet-platelet association characteristic of aggregates formed after exposure to A23187, PGG2or collagen (13-15). Incubation of the platelets with 3 $l PGF$or3 @I PGEl plus 2 mMtheophylline, or with 1 mMdibutyryl cyclic AMPprior to addition of the ara&donic acid prevented both aggregation and internal contraction. 3) The Influence of Cyclic AMPon Phospholipase A Activity: Addition oi thrombin to platelets in which the phospholipids had been prelabelled with arachidonic acid resulted in 14.4% release of the labelled arachidonic acid and in aggregation of the platelets (Figure 4) *

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PROSTAGLANDINS

Platelet

Per Cent Release of

Cyclic AMP Phasphalipase

and Activity

10

15

“C-Arochidonic Acid from Plotelet Phospholipids El

Per Cent Aggregation 5o

I

5 25

0 PLAT$LETS

PLATELETS

PLATELETS

Pi+’

c&P

.L

THEOWYLLINE

I

0

THR&IN

T”R&SIN

I41

/7J

The influence of PGBl with theo hylline and dibutyryl cyclic iziiE%i e thrombin stimulated release of f 4C arachidonic acid from platelets with prelabelled phospholipids, and on throtiin induced

aggregation. The release of labelled fatty acid is shown as the mean + SE. The numbers in brackets below each column indicates the number of experiments performed. PCEl with theophylline significantly inhibited release of 14C arachidonic acid (p < 0.01) and so did dibutyryl cyclic AMP(p < 0.05). Released arachidonic acid was converted to HETE(34%)) HKI (19%)) and thromboxane B2 (16%) as determined by thin layer chromatography (mean of 3 experiments). GC-MSwas used to confirm the presence of the cornpounds and the relative amount of HBTBand HHT. Addition of PCE and theophylline or dibutyryl cyclic AMPto the platelets prior to ai-dition of the arachidonic acid resulted in marked inhibition of phospholipase A activity as measured by release of labelled arachidonic acid from A secondary consequence of inhibition of the platelet phospholipids. phospholipase was the absence of detectable HBTB,HHTand thromboxane B2 production. It is of interest in spite of inhibition of phospholipase A activity, thrcmbin was still able to induce considerable aggregation of platelets pretreated with 1 IN dibutyryl cyclic AMP. At this concentration of dibutyryl cyclic AMPits effect on the platelet phospholipase A activity is, therefore, somewhatselective in that it can inhibit the enzyme activity with relatively little inhibitory activity on thrambin stimulated aggregation. DISCUSSION The present investigation has evaluated the role of agents known to elevate platelet cyclic AMP, and the cyclic AMPanalog, dibutyryl

48

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PROSTAGLANDINS

cyclic AMP, on platelet prostagladin synthesis and on platelet phospholipase A activity. We have found that these potent inhibitors of platelet aggregation can act at two different steps to inhibit platelet activation. PGEl or PGEl plus theophylline or dibutyryl cyclic RIP can prevent platelet aggregation of washed platelets stimulated by arachidonic acid without inhibiting platelet prostaglandin and throbboxane synthesis. The result suggests that cyclic AMPcan act to inhibit platelet activation at a step after synthesis of prostaglandin G2 and thromboxane A2. Secondly? we have shown that cyclic AMPcan inhibit at the level of phosphollpase activation, the step prior to synthesis of prostaglandins. Demonstration of cyclic AMPinhibition of platelet activation at the step after synthesis of PGG2and thromboxane JQ fits well with the recent demonstration that agents which elevate intracellular cyclic AM levels and dibutyryl cyclic AMPcan inhibit platelet aggregation stimulated by PGG2(14,15, 16). Indeed, the earlier ultrastructural investigation into the inhibition of PGG2by agents which elevated intracellular cyclic AMPlevels yielded results which parallel very closely the findings reported in the present study. PGEl, PGEl and theophylline, or dibutyryl cvclic AMPnot only inhibited platelet aggregation but also inhibited the internal contractile process stimulated by PGG2. The effect of cyclic AMPon the response of platelets to PGG2resembled closely ultrastructural studies of the inhibition of the calcium ionophore, A23187 (13,14). Inhibition by cyclic AMPmay be related to activation of a calcium pumpwhich removes calcium from the cytoplasm of the platelet thereby inhibiting the activation of the platelet contractile proteins by calcium (5,6,7). The present investigation has demonstrated that agents which elevate intracellular cvclic AMPand dibutvrvl AMPare uotent inhibitors of thrombin stimulated release of arachidonic acid from&platelet phospholipids . The release of arachidonic acid from platelet phospholipids probably involves a balance between two enzyme activities phospholipase A2 which cleaves the fatty acid from the phospholipids and an acyl transferase which puts the fatty acid back on the phospholipid. Thus, in theory, cyclic A?@ could be acting to inhibit phospholipase A activity or to stimulate acyl transferase activity. The present experiments do not differentiate between these two possibilities. However, since platelet phospholipase A activity has been shown sensitive to the calcium concentration (16)) the result suggests a unifying theory for the action of cyclic AMPon platelet function through modulation of calcium availability. Thus, stimulation of calcium uptake into an intracellular store by cyclic AMPprobably acting through stimulation of a protein kinase may inhibit platelet activation by removing 1) the calcim necessary for phospholipase A activity and 2) the calcium required to activate the platelet contractile proteins. The findings of the present study contrast with the results of Malmsten et al (8) who found that dibutyryl cyclic AMPdid inhibit conversion of arachidonic acid to thrcanboxaneB2. We have no adequate explanation for the discrepancy! though we note that Minkes et al (18) have just published findings which correlate well with the results presented here.

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PROSTAGLANDINS

1.

Marquis, N.R., R.L., Vigdahl and P.A. Tavomina: PlateletAggregation. I. Regulationby cyclicAM? and pmstaglandin El. Biocbem. Biophys.Pees.Gmmnm. 36:965,1969.

monophosphate 2. Salzman,E.W. and L. Levine: Cyclic 3'5'-adenosine in human blood platelets. II. Effect ofN6-Zl-0-dibutyrylcyclic 3'5'-adenosine monophosphateon platelet function. J. C1i.n.Invest. 50:131,1971. 3. Xlls, D.C.B. and J.B. Smith: The influenceon platelet aggregation of drugs that affect the accumilationof adenosine3'5'-cyclic monophosphatein platelets. Biochem.J. 121:185,1971. 4. Haslam, R.J.: Roles of cyclicnucleotidesin plateletfunction. Biochemistryand Pharmacologyof Platelets. CIBA Foundation Symposium35:121, 1975. 5. Day, H.J. and H. Holmsen: Conceptsof the blood plateletrelease reaction. Ser. Haemat. 4:3, 1971. 6. White, J.G., G.H.R. Fao, and J.M. Gerrard: Effects of the ionophore,A23187 on blood platelets.I. Influenceon aggregation and secretion. Am. J. Path. 77:135,1974. 7. George,J.N., R. Kaser-Glanzmann, M. Kakabova,and E.F. Luscher: Adenosine3'5'-cyclicmonophosphate(c&p) stimulationof active calciumtransportby a me&rane fractionfrom hunan platelets: a possible controllingmechanismof platelet function. Blood 48: 1007, 1976. 8. Malmsten,C., E. Granstrom,and B. Samuelsson: CyclicAMP inhibits synthesisof prostaglandinendopemxide (PGG2)in human 68:569, 1976. platelets. Biochen.Biophys.Res. Cm. 9. Hamberg,M. and B. Samuelsson: Prostaglandinendopemxides: Novel transfomationsof arachidonicacid in human platelets. Pmt. Natl. Acad. Sci. U.S.A. 71:3400,1974. 10. Gerrard,J.M., J.G. White, G.H.R. Fao, and D. Taunsend: Localization of plateletprostaglandinproductionin the plateletdense tubularsystem. Am. J. Path. 83:283,1976. 11. Bills, T.K., J.B. Smith, and M.J. Silver: titabolismof [14C] arachidonicacid by hunan platelets. Biochimicaet Biophysics Acta 424:303,1976. 12. Bartlett,G.R.: Phosphorousassay in column chromatography. J. Biol. them. 234:466,1959. 13. Gerrard,J.M., J.G. White, and G.H.R. Rae: Effect of the ionophore A23187 on blood platelets. II. Influenceon ultrastructure. Am. J. Path. 77:151,1974. J.M., J.G. White, and D. To.nwznd: Pmstaglandin G2 and 14. Ger-rard, platelet contraction. Pmstaglandins 11:470, 1976. 15. Gerrard,J.M., D. Tarnsend,S. Stoddard,C.J. Witkop and J.G. White: The influenceof prpstaglandinG2 on plateletultrastructureand platelet secretion. Am. J. Path. 86:99, 1977. 16. Miller, O.V. and R.R. Gonnan: Modulationof plateletcyclic nucleotidecontentby PGEl and the prostaglwdin endoperoxide PGG2. J. CyclicNucleotideResearch2:79, 1976. 17. Derksen,A. and P. Cohen: Patternsof fatty acid release fron endogenoussubstratesby human platelets,homogenatesand membranes. J. Biol. Chem. 250:9342,1975. 18. Minkes,M., N. Stanford,?4.M-Y. Chi, G.J. Roth, A. Raz, P. Needleman and P.W. Majems: Cyclic adenosine3'S'-monophosphate inhibits the availabilityof arachidonateto prostaglandinsynthetase in hunan plateletsuspensions. J. Clin. Invest. 59:449,1977. Received

50

l/27/77 - Approved

5/17/77

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