The Biosynthesis of Phosphatidylinositol

in Human Platelets

CHARLEST. LUCAS, FRANKL. CALL II, and WILLiAM J. WILLIAMs

From the Hematology Section, Department of Medicine, School of Medicine,University of Pennsylvania, Philadelphia, Pennsylvania 19104

A B S T R A C T Homogenates of human platelets canmediate the synthesis of phosphatidylinositol from myo-inositol and cytidine diphosphate diglyceride. The cyti-dine diphosphate diglyceride: myoinositol, phosphatidyltransferase activity is particulate-bound, and the highestspecific activity is found in the membrane fraction. Theproduction of phosphatidylinositol is decreased by sulf-hydryl-binding agents, and the addition of thiols to theplatelet homogenates increases the enzymatic activity.The reaction exhibits a pH optimum of 8.5-9.0. Divalentcations stimulate the reaction, and manganous chloridewas the most effective of those investigated. The Km ofthe enzyme for myoinositol is 0.27 mm, and the Km forcytidine diphosphate diglyceride is 0.53 mm. The enzy-matic activity of platelets isolated from patients withseveral diseases known to interfere with platelet clot-promoting function is similar to the enzymatic activityof platelets from normal donors.

INTRODUCTIONHuman platelets rapidly incorporate radioactive phos-phate, acetate, and glycerol into phospholipids (1-3).Phosphatidylinositol represents a small fraction of thephospholipids labeled with acetate and palmitate (2), butit is a significant proportion of the phospholipids labeledwith glycerol (3). Moreover, in experiments using radio-active phosphate, phosphatidylinositol is intensely la-beled both in vivo and in vitro (1). A selective increasein radioactive phosphate incorporation into phosphatidic

A preliminary report of this work has been published inabstract form: Call, F. L., II, C. T. Lucas, and W. J.Williams. 1969. J. Clin. Invest. 48: 13a.

Doctors Lucas and Call were formerly Trainees in He-matology under Training Grant AM-5228 from the NationalInstitute of Arthritis and Metabolic Diseases, U. S. PublicHealth Service.

Dr. Williams' present address is the Department ofMedicine, Upstate Medical Center, Syracuse, N. Y. 13210.

Received for publication 9 April 1970.

acid and phosphatidylinositol occurs in leukocytes dur-ing phagocytosis as well as in a number of cells duringsecretion (4-8). These data suggest that phosphatidyl-inositol production may be important in membrane in-tegrity and function.

Previous work in this laboratory has demonstratedthat human platelets can synthesize cytidine diphosphatediglyceride (CDP diglyceride) from cytidine triphos-phate and phosphatidic acid (9). When myoinositol waspresent during this reaction, CDPdiglyceride accumula-tion decreased, and myoinositol-14C was incorporatedinto a chloroform-soluble product. This suggested thatCDP diglyceride was the precursor of a lipid contain-ing myoinositol. Several laboratories (10-12) haveshown that the synthesis of phosphatidylinositol can oc-cur by the following reaction: CDP diglyceride + myo-inositol -> phosphatidylinositol + cytidine monophosphate(CMP). The studies reported in this paper demonstratethat human platelets contain the enzyme CDP-diglycer-ide: myoinositol, phosphatidyl transferase and charac-terize its properties.

METHODSSeparation of blood cells and preparation of homogenates.

Human platelets were prepared and washed as previouslydescribed (9). Platelets, leukocytes, and erythrocytes werecounted by routine techniques (13, 14).

Platelet pellets were resuspended to an estimated countof 2 X 109/ml in 0.25 M sucrose containing 10' M NaEDTAand 10' M2-mercaptoethanol adjusted to pH 7.0 with NaOH(subsequently referred to as buffered 0.25 M sucrose). Theplatelet suspension was frozen (solid C02) and thawed sixtimes, and the disrupted platelets were used as the enzymesource in most of the experiments.

Polymorphonuclear (PMN) leukocytes, lymphocytes, anderythrocytes were obtained from fresh whole blood anti-coagulated with heparin, using methods which minimizeplatelet contamination, and they were processed as previ-ously described (9). Except where indicated, all disruptedcells were stored at -20'C for no longer than 2 wk beforethey were assayed for enzymatic activity.

Preparation of CDP diglyceride. Phosphatidic acid was

The Journal of Clinical Investigation Volume 49 1970 1949

prepared from hen egg yolks, and it was reacted with cyti-dine-5'-monophosphomorpholidate in anhydrous pyridine toproduce CDP diglyceride (15). The CDP diglyceride waspurified by silicic acid chromatography and stored as theammonium salt in aqueous solution at - 200C (9). The cyti-dine: phosphorus: ester molar ratio was 1.0: 2.16: 2.0, andtwo thin-layer chromatographic systems showed one spot(9).

Preparation of phosphatidylinositol. Isolation of phos-phatidylinositol was initiated by aluminum oxide chroma-tography of a lipid extract of peas (16). Phosphatidylinositolwas eluted with chloroform: methanol (1: 1, v/v) containing12% HO. Chloroform and methanolic 0.15 N HCl wereadded in sufficient volume to yield two phases (17). Thelower chloroform phase containing the phospholipids waspassed through a short cellulose column to remove aminoacids (18). Final purification was achieved by chromatog-raphy on silicic acid (19). The phosphorus: ester: nitrogenmolar ratio of the phosphatidylinositol was 1.0: 2.15: 0.03.

Thin-layer chromatograms on plates of silica gel (slurredin 1 mMNa2CO3) developed with chloroform: methanol:acetic acid: H20 (25: 15: 4: 2, v/v) showed one spot with anR, of 0.52 (20). Two-dimensional thin-layer chromato-grams on plates of Silica Gel G developed with chloroform:methanol: H20 (65: 25: 4, v/v) and diisobutylketone: aceticacid: H20 (80: 50: 10, v/v) showed a single spot with re-spective R, values of 0.26 and 0.1 (21).

Following deacylation of the phosphatidylinositol withmethanolic NaOH (22), 96% of the phosphate was recoveredin the aqueous extract. The water-soluble product was iden-tified as D-glycerol-l- (L-myoinositol-l-hydrogen phosphate)(GPI) by its identical migration (R, 0.36) with an authenticsample of this substance on paper chromatograms developedwith methanol: formic acid: H20 (160: 26: 14, v/v (23) andits superimposition on the GPI peak during cochromatog-raphy on a column of Dowex 1-X10 formate (Dow ChemicalCo., Midland, Mich.) (10). Recovery of phosphorus wasgreater than 95%o by both methods, and no other phosphorus-containing substances were found.

Enzyme assay. The enzyme assay technique was similarto that employed by Carter and Kennedy (24). The reactionmixture was prepared in a 50 ml siliconized, conical-tippedglass tube, fitted with a ground-glass stopper, and maintainedin melting ice. A suspension of disrupted platelets, 0.2 ml,was added to the tube followed by 0.2 ml of CDPdiglyceridein Tris-HCl buffer [tris (hydroxymethyl) amino methane](Tris) and 0.05 ml of myoinositol-2-3H. The reaction mixturewas brought to 37°C, and 0.05 ml of manganous chloride wasadded. The concentrations of the reactants used are givenwith each experiment. Following incubation the reactionwas stopped by addition of 5 ml of methanolic 0.1 NHCl. 10 ml of chloroform was added, and the mixture waswashed three times with 20 ml of 2 M KCl. The radioactivityof an aliquot of the chloroform phase was determined aspreviously described (9).

Chemical analyses. Protein, lipid esters, phosphorus, ni-trogen, and cytidine were determined by previously de-scribed methods (9).

Materials.- Myoinositol was obtained from PfanstiehlLabs., Inc., Waukegan, Ill. D-glycerol-l- (L-myoinositol-l-hydrogen phosphate) was a product of Calbiochem, LosAngeles, Calif. Myoinositol-2-monophosphate was a productof Mann Research Labs., Inc., New York, and myoinositol-2-3H (SA 3.4 Ci/mmole) was obtained from New EnglandNuclear Corp., Boston, Mass. Cytidine-5'-monophospho-morpholidate was a product of Sigma Chemical Co., St.Louis, Mo.

RESULTSPreliminary experiments. Whenplatelets suspended in

buffered 0.25 M sucrose were incubated at 370C in the4presence of CDP diglyceride, myoinositol-2-3H, MnCl2,and Tris-HCl buffer, pH 7.5, radioactivity was foundin the chloroform extract indicating the synthesis of alipid product containing myoinositol. Platelets disruptedby repeated freezing and thawing were four times moreeffective in catalyzing this reaction.

Product identification. A reaction mixture identicalwith that described in Fig. 1, except that it contained 1mMmyoinositol-2-8H (SA 7.5 Ci/mole) and 1.2 mgof platelet protein in a final volume of 1.0 ml, was in-cubated at 370C for 60 min. Phosphatidylinositol 0.1mmole was added at the end of the incubation period, anda chloroform extraction was performed as outlined in theMethods section, except that the volumes were doubled.The chloroform phase was taken to dryness under a

stream of nitrogen, the residue was suspended in chloro-form, and aliquots were used for thin-layer chromatog-raphy and hydrolysis.

The chloroform extract was examined for phospha-tidylinositol by thin-layer chromatography as describedin the Methods section. The lipids were eluted from thegel (20), and the radioactivity was determined as previ-ously described (9). In two experiments 88% and 93%of the radioactivity migrated with the phosphatidylinosi-tol spot. The remaining radioactivity trailed the phospha-tidylinositol spot slightly in both systems, probably rep-resenting incomplete migration of the lipid.

An aliquot of the chloroform extract was taken to dry-ness, and the residue hydrolyzed in methanolic NaOHfor 10 min at 370C (22). After deacylation, the water-soluble products (containing 98% of the radioactivity)were examined by descending paper chromatography,using phenol: H20 (4:1, w/v) as the solvent, andascending chromatography, using n-propanol: NH40H:H20 (5: 4: 1, v/v) as the solvent (25, 26). Inositol phos-phate, myoinositol, and GPI had respective Rf values of0.05, 0.25, and 0.1 in the phenol: H20 system, and 0.35,0.5, and 0.6 in the n-propanol: NH4OH:H20 system.95% of the water-soluble radioactivity after alkalinehydrolysis migrated with GPI in both systems.

The lipid formed in the reaction mixture was also hy-drolyzed in 2 N HCl (19). The hydrolysis products were

chromatographed on paper using the same two solventsystems. In both systems 80% of the radioactivity mi-grated as inositol phosphate and 15% as myoinositol; thisagrees well with the data obtained by Hanahan andOlley using phosphatidylinositol isolated from beef liver(19).

Time course. The rate of formation of phosphatidyl-inositol was not linear over the time interval investigated

1950 C. T. Lucas, F. L. Call 11, and W. J. Williams

(Fig. 1). 60 min was chosen as the incubation period forthe remainder of the experiments.

Enzyme concentration. The enzymatic activity varieddirectly with the protein concentration of the disruptedplatelets up to 0.7 mg/0.5 ml of reaction mixture. Athigher protein concentrations the reaction rate deviatedfrom linearity.

pH optimum and effect of buffer salts. The enzymewas active over a wide range of pH (6.3-10.6) with anoptimal activity at pH 8.5-9.0 in 0.08 M Tris-HCl(Fig. 2). Variation of the concentration of Tris-HClfrom 0.04 to 0.32 M had no effect on phosphatidylinosi-tol synthesis, but lower concentrations buffered the reac-tion poorly with resultant loss of enzyme activity. Boricacid-NaOH and phosphate buffers both at 0.08 M caused60% and 34% inhibition of enzyme activity respectively(not illustrated).

Substrate and cofactors. In the absence of CDP di-glyceride, divalent cation, or enzyme there was no sig-nificant production of phosphatidylinositol (Table I).The affinity of the enzyme for CDPdiglyceride is shownin Fig. 3. The Km for CDP diglyceride was 0.53 mmwhen calculated from a Lineweaver-Burk plot of the re-ciprocal of the velocity vs. the reciprocal of the substrateconcentration, but it appears to be about 0.2 mmfrom theplot of the reaction rate against substrate concentration.The Km of the enzyme for myoinositol was 0.27 mmwhen the CDP diglyceride concentration was 0.92 mm(Fig. 4). Here the Km estimated from the plot of thereaction rate against substrate concentration agreed with

32

~24-

6

8

20 40 60 80 100rime (min)

FIGURE 1 The production of phosphatidylinositol in rela-tion to time. The reaction mixtures contained 0.6 mg ofprotein derived from platelets disrupted by repeated freezingand thawing, 0.1 M sucrose, 0.4 mm Na2EDTA, 4 mM2-mercaptoethanol, 0.92 mmCDP diglyceride, 3.6 mMmyo-inositol-2-8H (SA 0.7 Ci/mole), 4 mmMnCl2, and 0.08 MTris-HCl buffer, pH 8.5, in a total volume of 0.5 ml. Thereaction mixtures were incubated at 370C for the timeindicated.

*30

60 7.0 80 90 IQO 110pH

FIGURE 2 Effects of pH and buffer salts on phosphatidyl-inositol production. The conditions were identical with thosein Fig. 1 except that the reaction mixtures contained 0.47mg of platelet protein and were incubated for 60 min. Thebuffer salt and pH were varied as indicated, but the finalsalt concentration was always 0.08 M. X, imidazole-HCl;0, Tris-HCl; A, carbonate-bicarbonate; 0, glycine-NaOH.

that calculated from the Lineweaver-Burk plot. Thevelocity of the reaction using 3.6 mmmyoinositol was ap-proximately 85% of that achieved using myoinositolconcentrations up to 10 mM.

The synthesis of phosphatidylinositol was increased bydivalent cations, and MnCls was the most effective ofthose investigated. Using 0.92 mm CDP diglyceride,maximal enzyme activity occurred with 3 mmMnCl2.Higher concentrations of MnCls up to 12 mmwere notinhibitory. When 0.15 mmCDP diglyceride was used,maximal activity occurred with 0.8 mmMnC]6, and therewas no inhibition at MnCIh concentrations as high as 4mM. Addition of MnCla to the reaction mixture before ad-dition of the disrupted platelets caused precipitation ofthe CDPdiglyceride and a 90% decrease in phosphatidyl-

TABLE IEffect of Omission of Reagents from the Complete

Reaction Mixture*

Phosphatidyl-Omissions form inositolreaction mixture formed

mjumole/mgprotein/min

None 0.322Na2EDTA 0.3382-Mercaptoethanol 0.140CDPdiglyceride 0.001MnCl2 0.005Enzyme 0.0003

* The complete reaction mixtures were identical with thosedetailed in Fig. 1 except that they contained 0.4 mgof plateletprotein and were incubated for 60 min.

Biosynthesis of Phosphatidylinositol in Human Platelets 1951

Q5 ID is 2DCOPD(mM)

FIGURE 3 The production of phosphatidylinositol at varyingconcentrations of CDP diglyceride (CDPD). The conditionswere identical with those detailed in Fig. 1 except that theCDP diglyceride concentration was varied as indicated, andthe time of incubation was 60 min.

inositol production. MgCl2 could be substituted for MnCI2but was less effective. CDPdiglyceride production with 4mMand 40 mmMgCla was only 50% and 85% of thatachieved with 4 mmMnCls. CaC12, CUSO4, and ZnSOt,all at 4 mm, caused no significant increase in enzymaticactivity. 4 mmCaCl2, in the presence of 4 mmMnCl,produced a 33% decrease in enzymatic activity.

The addition of thiols increased enzymatic activity. Inthe absence of Na2EDTA, 4 mm, 10 mm, and 20 mM2-mercaptoethanol increased phosphatidylinositol pro-duction to 250, 330, and 340%, respectively. 0.5 mmand1.0 mMglutathione increased production to 112% and136%, respectively.

In the absence of added 2-mercaptoethanol para-chloromercuribenzoate (PCMB) 0.1 mmand 0.5 mmde-creased enzyme activity by 58% and 97%. 1.25 mmand10 mMiodoacetate caused 32% and 70% inhibition ofenzyme activity in the absence of 2-mercaptoethanol.2-mercaptoethanol reversed most of the inhibition pro-duced by both PCMBand iodoacetate.

In the absence of 2-mercaptoethanol Na2EDTA 0.4mmincreased enzymatic activity by 40%. This concen-tration of Na2EDTA had no effect in the presence of4.0 mm 2-mercaptoethanol. Na2EDTA concentrationshigher than 1 mmwere inhibitory.

Temperature effects. The production of phospha-tidylinositol was investigated at 4, 25, 37, 50, and 600C.The enzyme activity was greatest at 370C, and all otherexperiments reported were performed at this temperature.When disrupted platelets were heated to 1000C for 5 min,all enzyme activity was lost. Storage of the disruptedplatelets at 4VC for 7 days resulted in a 95% loss of en-zyme activity. Storage of - 20'C resulted in no lossof activity after 7 days, but there was a 40% decrease inactivity after 1 month.

Miscellaneous observations. There was no marked in-hibition of the reaction by addition of either of the reac-tion products. 3.3 mmand 6.6 mmCMPproduced an11% and a 26% decrease in phosphatidylinositol produc-tion. 0.1 mM, 0.4 mm, and 2.0 mmphosphatidylinositolcaused, respectively, an 8%, 12%, and 20% decrease inthe reaction rate.

The enzymatic activity was not increased by severaldetergents. Tween-80 0.1 mg/ml and Trition X-100 2mg/ml had no significant effect on phosphatidylinositolproduction. Tween-80 1 mg/ml, sodium cholate 4 mg/ml, and Cutscum 2.5 mg/ml, respectively, caused 29%,29%, and 88% inhibition of enzyme activity. The stud-ies with sodium cholate and Cutscum were complicatedby incomplete removal of myoinositol-2-'H from thechloroform extract, and the phosphatidylinositol produc-tion was corrected by appropriate controls.

Attempts to solubilize the enzymatic activity by soni-cation or by treating the platelet particles with organicsolvents or detergents was unsuccessful. Precipitation ofthe platelet particles with acetone or methanol: H20(2: 1, v/v) resulted in more than 90% loss of enzymeactivity.

0.1 M NaCl, 0.04 M KCI, 0.04 M NH4Cl, and 1 mmand4 mMpotassium fluoride had no effect on the reactionrate. Albumin 0.4 mg/ml and 2.0 mg/ml decreased phos-phatidylinositol production by 2.5% and 21%.

Enzymatic activity in platelet subcellular fractions.Washed platelet pellets were homogenized as describedby Marcus, Zucker-Franklin, Safier, and Ullman (27),and fractionated by differential centrifugation as previ-ously described (p). A similar homogenate was used toprepare "membranes" and "granules," as described by

Myoinositol OM)

FIGURE 4 The production of phosphatidylinositol at varyingconcentrations of myoinositol. The conditions were identicalwith those detailed in Fig. 1 except that the myoinositolconcentration was varied as indicated (SA 2.1 Ci/mole whenmyoinositol concentrations were 0.125-0.5 mmand 0.7 Ci/mole when concentrations were 1.5-10 mM). The time ofincubation was 60 min.

1952 C. T. Lucas, F. L. Call II, and W. J. Williams

Marcus et al. (subcellular fractions were not examinedby electron microscopy). The platelet subcellular frac-tions were washed once and resuspended in sufficientbuffered 0.25 M sucrose to make the final protein con-centration 1-2 mg/ml. The 100,000 g pellet and the"membranes" contained the greatest specific activity(Table II). The 100,000 g supernate had no significantenzymatic activity and did not contain inhibitors whenmixed with the other fractions.

Enzymatic activity in other blood cells. Lymphocytesand PMNleukocytes disrupted by freezing and thawingcatalyzed the conversion of myoinositol-2-3H into achloroform-soluble product. The reaction rate forlymphocytes was 0.153 mnmole/mg of protein per min or0.918 m/Amole/108 cells per min. The values for PMNleukocytes were 0.178 mumole/mg protein per min or1.246 memole/10' cells per min. Erythrocyte membranesdid not catalyze the conversion of myoinositol-2-3H intoa chloroform-soluble product. These results indicatethat less than 1% of the activity present in the plateletpreparations could be attributed to contamination byother cell types.

Enzymatic activity in platelets from normal subjectsand patients. Platelets from 12 normal subjects dis-rupted by repeated freezing and thawing (protein con-centration 1.2-2.4 mg/ml) catalyzed the production of0.35-0.55 mtmole of phosphatidylinositol per mg of pro-tein per min. The platelets of five uremic patients (bloodurea nitrogen greater than 90 mg/100 ml), two patientswith polycythemia vera, and one patient with Glanz-mann's thrombasthenia had enzymatic activity in thesame range.

TABLE ITEnzyme Activity in Platelet Subcellular Fractions*

Phosphatidyl-inositol

Platelet fraction formed

mpmolc/mgprotein/min

Unfractionated 0.3061000 g pellet 0.22112,000 g pellet 0.416100,000 g pellet 1.446100,000 g supernate 0.003Membranesl 1.320Granulest 0.402

* The conditions of the reactions were identical with those de-tailed in Fig. 1 except that 0.2-0.5 mgof protein derived fromhomogenized platelets was used, and the time of incubationwas 60 min.$ These subcellular fractions were isolated on continuoussucrose gradients as described by Marcus, Zucker-Franklin,Safier, and Ullman (27).

DISCUSSIONThe incorporation of myoinositol into phosphatidylinosi-tol was first demonstrated by Agranoff, Bradley, andBrady using guinea pig kidney mitochondria (28).While investigating the increase in phosphatidylinosi-tol synthesis caused by cytidine nucleotides, they isolateda liponucleotide which appeared to be CDP diglyceridesince the water-soluble products of alkaline hydrolysiswere CMPand occasionally cytidine diphosphate glyc-erol. They speculated that this compound might functionas a precursor of phosphatidylinositol. Paulus and Ken-nedy synthesized CDP diglyceride and characterized anenzyme in chicken liver microsomes which catalyzed theconversion of CDPdiglyceride and myoinositol to phos-phatidylinositol and CMP (10). Although the enzymewas relatively specific for myoinositol, two inososescould also release CMP from CDP diglyceride at aslower rate. In the presence of saturating concentrationsof manganous chloride (2 mM) some myoinositol wasincorporated into phosphatidylinositol in the absence ofcytidine nucleotides, and evidence that this involvedan exchange reaction was presented. Similar observa-tions have been reported during the study of phospha-tidylinositol synthesis in guinea pig brain microsomes(12, 29). In guinea pig pancreas homogenates and brainmicrosomes, albumin was necessary for optimal enzy-matic activity (11, 12).

The data presented here demonstrate that humanplatelets can mediate the production of phosphatidylinosi-tol from myoinositol and CDP diglyceride. No incor-poration of myoinositol-2-8H into a chloroform-solubleproduct was observed in experiments in which CDPdi-glyceride was omitted. It therefore appears that no ex-change reaction occurs with platelet preparations. TheKm of the enzyme for CDP diglyceride was 0.53 mmwhen calculated from the Lineweaver-Burk plot, but itwas about 0.2 mmwhen estimated from the plot of thereaction rate against substrate concentration. Similardiscrepancies between the Kmestimated in these two wayshave been reported in other systems involving lipidemulsions (30) and remain unexplained. The Kmof 0.53mMfor CDPdiglyceride is similar to those reported forguinea pig pancreas microsomes and brain microsomeswhen CDP-dipalmitin was used as a substrate. The fattyacid composition of the CDP diglyceride used in theseexperiments was not determined. Since it was syn-thesized from hen egg phosphatidylcholine, it would beexpected to be a mixture of highly unsaturated, long-chained fatty acids (31, 32). The Km might be differentfor CDPdiglyceride with specific fatty acid composition.Benjamins and Agranoff have demonstrated this phe-nomenon in guinea pig brain microsomes (12). Similarconsiderations apply to the specific activity of the en-zyme as discussed below. The Km of the enzyme for

Biosynthesis of Phosphatidylinositol in Human Platelets 1953

myoinositol is 0.27 mm, a value close to the Km formyoinositol (0.2 mM) found for the enzyme present inchicken liver microsomes, but distinctly less than theKm of the enzyme (1.5 mM) in brain and pancreas mi-crosomes. The pH optimum of 8.5-9.0 is slightly higherthan that described for the enzyme in other mammaliantissues (pH 7.5-8.6). The enzyme in platelet "mem-branes" has a specific activity which is about one-halfthe specific activity of the enzyme in pancreas micro-somes, and only 6% of the specific activity of the en-zyme in guinea pig brain microsomes. The high specificactivity in brain microsomes is partially due to the use ofthe substrate CDP-didecanoin, for which the enzyme hasa faster reaction rate and a lower Km (1.7 X 10-4 M)than the other CDPdiglycerides (12). The marked ef-fect of thiol compounds and sulfhydryl-binding agents onthe enzymatic activity in platelets has not been demon-strated in other mammalian tissues.

We have previously demonstrated that platelets cansynthesize CDP diglyceride from phosphatidic acid andcytidine triphosphate. The enzyme characterized in thisreport could then convert the CDPdiglyceride to phos-phatidylinositol. These data provide further evidencethat the rapid incorporation of radioactive phosphorusinto phosphatidylinositol represents synthesis of thisphospholipid at least in part and is not due solely to anexchange phenomenon. The activity of the enzyme wasnormal in platelets from patients with diseases known tobe characterized by abnormal platelet function (uremia,polycythemia, Glanzmann's thrombasthenia). The roleof this biosynthetic pathway in platelet metabolism re-mains to be elucidated.

ACKNOWLEDGMENTSThis work was supported in part by Hematology TrainingGrant AM-5228 from the National Institute of Arthritis andMetabolic Disease, U. S. Public Health Service, and theC. W. Robinson Foundation.

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