Effects of cv-3988, an antagonist of platelet-activating factor (paf), on washed rabbit platelets

THROMBOSIS RESEARCH 41; 211-222, 1986 0049-3848/86 $3.00 t .OO Printed in the USA. Copyright (c) 1986 Pergamon Press Ltd. All rights reserved.

EFFECTS

OF

CV-3988, AN ANTAGONIST OF PLATELET-ACTIVATING (PAF), ON WASHED RABBIT PLATELETS

Charles

FACTOR

P. Cox

Veterans Administration Medical Center and University of Oklahoma Health Sciences Center, Oklahoma City, OK.

U.S.A. (Received 2.7.1985; Accepted in revised form 22.9.1985 by Editor A.P. Fletcher) ABSTRACT Platelet-activating factorfl-O-alkyl-2-acetyl-Sn-glyceroAGEPC) is a small, extremely 3-phosphorylcholine or CV-3988, a unique strucpotent phospholipid mediator. has recently been introduced as tural analogue of AGEPC, a selective antagonist of AGEPC-induced In vitro platelet activation and 1~ viva systemic hypotension. At concentrations greater than 5 x lo-' MI CV-3988 itself induced responses in washed rabbit aggregation and secretion CV-3988 inhibited AGEPC-induced platelet platelets. activation at concentrations as low as lo-' M, but also collagen and blocked platelet activation induced by calcium ionophore A23187 at concentrations between 10D6 M The mechanism of inhibition, however, did and 10m5 M. not depend on increased levels of intracellular 3',5'In fact, CV-3988, (CAMP). cyclic adenosine monophosphate like AGEPC itself, appeared to lower CAMP levels in washed rabbit platelets.

INTRODUCTION recently has 1-O-alkyl-2-acetyl-u-glycero-3-phosphorylcholine been identified as the structure of platelet-activating factor This potent (PAF) obtained from several mammalian cells (1,2). phospholipid mediator is also referred to as acetyl glyceryl names PAF-acether. both phosphorylchollne (AGEPC) and ether denoting the unique structural characteristics of the molecule Numerous structural analogues of AGEPC have been (Figure la). prepared by altering the length of the alkyl chain, by making

Key

Words:

AGEPC,

platelet

activation, 211

CAMP

212

CV-3988 AND PLATELET ACTIVATION

Vol. 41, No. 2

substitutions for the acetyl group and the polar head group* and All of these analogues by altering the stereospecificity (3-11). although greatly have varying degrees of biological activity, dIminished from that of the natlve AGEPC, which functions in Terashita and co-workers (12) have concentrations. picomolar unique AGEPC analogue, rnc-3-(N-n-octadescribed a recently 2-thiazolioethyl phosphate, decylcarbamoyloxy)-2-methoxypropyl deslgnated CV-3988 (Figure lb). They demonstrated that CV-3988 AGEPC-mediated aggregation in rabbit antagonized specifically (PRP), and prevented AGEPG-induced platelet-rich plasma the one-clip hypertensive rats. systemic hypotension in one-kidney, Terashita and colleagues have subsequently demonstrated that CV3988 inhibited the _in vitrq binding of C3 HIAGEPC to platelets (13) prevented from guinea rabbits and humans and pigs, in rats (14). We endotoxin-induced systemic hypotension in viva now report that CV-3988 prevents AGEPC-induced aggregation and secretion in [3Hlserotonin-labeled washed rabbit platelets by a mechanism which Is independent of increased levels of intracellular CAMP.

~2C-o-_(CH2),5_,,-CH3

nc3

--- %

I

0

CH

N3C-o-Cn H c-s

---Hco~ocn 2

I

+ -N(a,),

raz

2-CH2-N

0

A:

Platelet-Actlvatlng

FIG.

1

An:

8:

I c=cn H

cv-3988

Structure of platelet-activating factor (l-0-alkyl-2-acetyl-qn-glycero-3-phosphorylcholine) is shown in Panel A; structure of CV-3988 (rk(;-3-N-n-octadecylcarbamoyloxy)_2 -methoxypropyl 2-thiazolioethyl phosphate) is shown in Panel B.

MATERIALS

source of laboratory libitum.

\

0

Factor

+/

AND METHODS

Female New Zealand White rabbits were used as the platelets. Animals were maintained on a standard chow (Purina Mills, St. Louis, MO) and water &j

Platelet dissolved in

Activatou: AGEPC phosphate-buffered

(Bachem, saline

Inc., (PBS)

Torrance, CA)‘ was containinng 2.5

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CV-3988 AND PLATELET ACTIVATION

213

mg/ml bovine serum albumfn. Calcium fonophore A23187 (free acid) was purchased from Calbfochem-Behrfng (San Diego, CA) and dfssolved in dfmethylsulfoxfde at 500 ug/ml and diluted In PBS immediately before use. Soluble calf-skin collagen (Worthington NJ) was dfluted in PBS immediately before Bfochemfcal~~ Freehold, use. CV-3988: A pure sample of the AGEPC antagonist CV-3988 was generously provided by Dr. M. Nfshfkawa (Takeda Chemical Industrfes, Ltd., Osaka, Japan) and prepared according to his fnstructfons. A stock solution of CV-3988 (10'3 M) was prepared in physiologic saline and alfquots were stored at -200 C. For each assay* an alfquot was warmed to 560 C to re-dissolve the serf al Ten-fold and adjusted to pH 7.2 with 0.1 N NaOH. drug, dflutfons were prepared in PBS, pH 7.2, Immediately before use. Although CV-3988 was practically insoluble in water at room temperature, dflutfons of CV-3988 prepared by this method did not precipitate out of solutfon during the course of the experiments. a,: MI) was immediately

Prostaglandfn dissolved in before use.

Pharmaceutfcals, Kalamazoor I, (Upjohn PBS absolute and diluted In ethanol

Buffers: All chemicals used for the preparation of washing purchased from Sigma Chemfbuffers were reagent grade or better, All buffers were prepared fresh daily in cals (St. Loufs, MO). as previously described (15). double-glass-dfstflled water,

Platelet:

platelets maintained bioassay.

prepared were at 37' C In

a

as 5%

Washed C3Hlserotonfn-labeled rabbit described (151, and previously CO, atmosphere until used in the

The aggregatfon and Platelet Q#xg.aaud Secretfqn : secretion studies were based on the turbfdfmetrfc method of Born were Washed platelets (120 ul of 1.25 x 10' platelets/ml) (16). diluted with 480 pl of placed In sflfconfzed cuvettes and modfffed Tyrodes solution containing 1.0 mM calcfum, yfeldfng a ffnal concentratfon of 2.5 x lo* platelets/ml in a volume of The platelet suspensions were continuously stfrred at 600 ul. In a model DP247E dual-channel 1300 rpm and warmed at 37 'C (lo-’ to CV-3988 platelet aggregometer (Sfenco, Morrison, CO.). M1 or PGIp (4.7 x lo-’ to 2.9 x 10v5 M) was added to the 1o-5 cuvette and stirred with the platelets for 60 set, at whfch time a 100 ul background sample was removed and the stimulus (AGEPC, A231871 was added. Aggregation was continuously collagen or seconds after monitored on a strip-chart recorder, and sixty a 100 ul alfquot was removed to determine addfng the stimulus, These alfquots were stimulus-induced secretion of C3Hlserotonfn. centrifuged for 60 set at 12,000 x g (Model 5414 Mfcrofuge, NY) and 50 pl of the supernatants Brfnkman Instruments, Westbury, were mfxed with 5 ml Readysol v-HP liquid scfntfllatfon cocktail CA) and counted in a liquid scfn(Beckman Instruments, Irvine, The total amount of C3Hlserotonfn available counter. tfllatfon for secretion was determined by lysing random cuvettes of plateThe stimulus-specific lets with 50 ul of 2.5% Trfton X-100. from each cuvette was calculated as: release of C 3Hlserotonfn

Vol. 41, No. 2

CV-3988 AND PLATELET ACTIVATION

214

C3HJserotcnin= ~f~slUlu5-~Jaz&lnulus seaetial { cpnfor TtltcnX-lOO-b&gmmdcpnfarTtlt~X-100 X (

Qt.&_ Final volune of Tritcn X-lOOcu&te Firralv"luneofsunplecw

x100 I

Control platelets were pre-treated with the respective diluents and assayed in parallel with the fnhibitorfor CV-3988 and PGI,, Platelet aggregation responses were assessed treated platelets. at 60 set post-stimulus for AGEPC and A23187 and 120 set postthe aggregation stimulus for collagen, using the height of tracings (in cm) as measured from the lowest point (bottom of The percent inhibition of platelet aggregation shape change). and secretion responses was calculated as: [(control response inhibited response) / control response1 x 100.

In separate studies, CAMP levels were platelet CAMP levels: determined in similar aliquots of washed rabbit platelets incubated for 60 set in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine or IBMX (1O-3 M) + CV-3988 (10 -6 to

lo-'

M)

or

IBMX

(10 -3 Ml+

PGI,

(2.9

X

10m5

Ml.

Platelets were lysed by the addition of 1 ml of ice-cold 1 N HCl and sonicated for 30 sec. The lysed and sonicated platelets were centrifuged for 2 min at 12,000 x g and the supernatants were retained and storedn at -20 O C for subsequent CAMP determination using the Gammaflo radioimmunoassay system (17).

RESULTS The effect of CV-3988 on washed rabbit platelets in the absence of platelet stimuli is shown in Figure 2. At concentrations less that 10m5 MI CV-3988 alone had no effect on washed rabbit 10e5 M CV-3988 consistently induced a shape change, platelets. although there was no secretion of C3Hlserotonin associated with and the platelets did not aggregate. At a final this response* concentration of 5 x 10-S M or greater, however, CV-3988 induced both shape change and aggregation (Figure 2a). The aggregation response consisted of a 30-45 set lag period followed by a rapid aggregation with almost complete clarification of the platelet suspension. As shown in Figure 2b, the aggregation responses were also associated with the release of C3Hlserotonin, which increased over time in a non-linear mannerr reaching a maximal secretion between 5 and 10 min. Aggregation tracings for AGEPC (2 x 10-10 MI, collagen (5Oug /ml) and A23187 (O.lug/ml) in the presence of 10vg to lo- 5 M CV-3988 are shown in Figures 3, 4 and 5 (respectively), along with the corresponding data for C3+llserotonin secretion. Although CV-3988 effectively antagonized platelet aggregation and secretion induced by 2 X 10 -lo M AGEPC over the range of 1OB8 to 10S5 M, CV-3988 also inhibited platelet activation induced by collagen and A23187. At low5 M CV-3988, aggregation and secretion induced by 50 ug/ml Both collagen were inhibited by 100% and 60X, respectively. were aggregation and secretion 0.1 pg/ml A23187 induced by

Vol. 41, No. 2

CV-3988 AND PLATELET ACTIVATION

215

I

012345 TIME (mid

FIG

2

SAMPLE

TIME POST-8TIYULU8

(min)

At sufficiently high concentrations, CV-3988 Itself induces both aggregation (left-hand panel) and secretion of I:%lserotonln (right-hand panel) in CV-3988 was washed rabbit platelets. added at Time 0 and aggregatlon and secretion were monitored for 10 mfn.

The concentration of CVcompletely blocked by 1(r5 M CV-3988. 3988 required for 50% inhibition of C $11 serotonln secretlon 5 X 1O-6 M for collagen and 3 (1C50) was 1.5 X lo- ' M for AGEPC, The ICSO for platelet aggregation was A23187. x 10 -6 M for M for collagen and 5 X 10-6 M for AGEPC, 2.5 X 10m7 1o-7 M for A23187. effect of 5 X 10m7 M CV-3988 As shown In Figure 6, the inhibitory the amount of AGEPC used to could be overcome by fncreasing The secretory response was almost stimulate the platelets. M AGEPC and the aggregation response completely restored at 10S7 The ability of CVwas completely restored by 5 x 10-g M AGEPC. 3988 to inhibit both platelet aggregation and secretion induced by 2 X lO-'O M AGEPC in a dose-related manner was compared with depicted in Figure 7, show that CVThe results, that of PGI,. 15 times more effective than PGI, at 3988 was approximately and approximately 20 times Inhibiting AGEPC-mediated aggregation. C~tilserotonfn AGEPC-mediated preventing more effective at platelet activation by Increasing secretion. Since PGI, Inhibits we studied the effects of graded intracellular levels of CAMP, The platelets. doses of CV-3988 on CAMP levels In washed rabblt

216

CV-3988 AND PLATELET ACTIVATION

Vol. 41, No. 2

0 AGQREGATlOn -SECRETION

012345

(min)

TIME

FIG 3

__-

v,

CV-3960

Inhibition of AGEPC-induced platelet activation by CV-3988. Platelets were pretreated with CV-3988 or PBS for 1 min before the addition of AGEPC (2 x 10 -lot41 at Time 0. Platelet aggregation and secretion were monitored for 5 min.

16 14

i

I

12.

OAQQREQATIOW

0 lo-'u

*SECRETION

10 loo8 10‘%A 6

4

1 I

f

ii

9!

f

/

0123456

TIME (min)

FIG 4

--

1

* la%

Sx16'M

lcetvl

cv-3988

Collagen-induced platelet activation is blocked by CV-3988. 50 pg/ml collagen was added at Time 0 and aggregation and secretion were monitored for 6 min.

1tPM

CV-3988 AND PLATELET ACTIVATION

Vol. 41, No. 2

217

0 AGGREGATION . SECRETION

16%

i23456 TIME

FIG

5

ltrsl

(mid

5x16&

la%

1Q6U

CV-3988

Inhibition of A23187-induced platelet activation following pretreatment of washed rabbit platelets with CV-3988. Calcium ionophore A23187 (0.1 I.tg/ml) was added at Time 0 and aggregation and secretion were monitored for 5 min.

1 ooo AGGREGATION . SECRETION

80-

60-

40-

20-

0.1

1 I I 0.2 0.3 0.5

1 1 AGEPC

FIG 6

t 5

10

7 100

x lo-@M

Inhibitory effects of CV-3988 (5 x lo-' M1 on the activation of washed rabbit platelets was overcome by increasing the concentration of AGEPC used to stimulate the platelets.

CV-3988 AND PLATELET ACTIVATION

218

Vol. 41, No. 2

INHIBITOR

FIG

7

Inhibition of aggregation (o----o) and secretion (1 induced by 2 X lo-" M AGEPC in the presence of PG12 or CV-3988. Concentrations producing 50% inhibition (IC sd are shown in the upper left corner.

shown in Table 1, are quite results, interesting. All reaction mixtures contained sufficfent IBMX (10 -3 M) to allow measurable amounts of CAMP to accumulate. A relatively large dose of AGEPC did not significantly alter platelet CAMP levels, nor (10 -8 M), did 10F6 M to lo-' M CV-3988. PG& (2.9 X 10 -' M), however, increased CAMP levels almost SO-fold. This PGI, -Induced Increase In intracellular CAMP was markedly attenuated by both AGEPC (10 -' M) as well as by the AGEPC antagonist CV-3988. Because CV-3988 Itself suppressed the PGIz-induced elevation of platelet CAMP levels# it was not possible to determine whether CV-3988 antagonized or enhanced the effect of AGEPC on the PGI2-induced Increase In platelet CAMP levels.

DISCUSSION Various structural analogues of AGEPC have been prepared and evaluated by different investigators (3-111, until the but Introduction of CV-3988 (121, there have not been any published reports of selective antagonlsts of AGEPC based on structural analogues. Terashlta and co-workers (12) reported that this unique structural analogue of AGEPC inhibited AGEPC-lndyced aggregation in rabbit PRP over the range of 1O-6 M to 3 X loMI but did not affect aggregation induced by adenoslne diphosphate (10 uM)r arachldonic acid (80-1OOU M), collagen (lo-30 Ug/ml) or calcium ionophore A23187 (3 PM). Furthermore, they found that in

Vol. 41, No. 2

CV-3988 AND PLATELET ACTIVATION

Table -OF

AND

219

1

CV-3Q88

ON

PLATFIFT

=*"P

’ EVEu .

&

ibitor&&lmuli Control 10-O M AGEPC 2.9 X lo-' M ffi1

2.16 + 0.08 1.76 It0.06 102.65 + 4.24

lo-" M CV-3988 lo-' M CV-3988 1O-6 M (Y-3988

2.04 If. 0.11 1.90 +-0.07 2.60 + 0.07

2.9 X 1O-5 M FGI + lo-*M AGEPC

62.15 +-1.78

2.9 X 1O-5 M f%I + 10-4M Cf3988 2.9 X lo-' M FGI + lo-'M CV3988 2.9 X lo-' M I%1 + 10-6M CV3988

12.74 + 0.90 44.95 +-1.49 87.49 + 2.26

’ CAMP:

pmol/1.25 X 10 * platelets,

their system specifically approximately

whfch PGEl, fncreasing by 80 times more

mean It S.E.M.

inhibits platelet aggregation CAMP levels (181, platelet potent than CV-3988.

nonwas

Using washed rabbit platelets, we found that CV-3988 (1O-8 M to 10-5M) blocked platelet activation induced by 2 X 1O-1o M AGEPC between our observations tICso = 2.5 X 10 -' Ml. The discrepancies and those of Terashita alt nl (12) may be partially explained by Terashita and codifferences in the bioassay systems used: plasma) whereas we used workers used PRP (4.5 X 10' platelets/ml Because rabblt washed platelets (2.5 X lO'platelets/ml buffer). an acetyl-hydrolase which specifically degrades plasma contains the half-life of AGEPC in plasma is about 30 sec. AGEPC (19-211, Thus PRP requires approximately 100-200 times more AGEPC than (1) to achieve the similar numbers of washed rabbit platelets and consequently requires same degree of platelet activation, CV-3988 to competitively block the higher concentrations of AGEPC. Our findings that CV-3988 had a significant agonist effect at concentrations greater than 10-S M and also blocked collagen and A23187 stimulated responses are more difficult to explain on the basis of differences between assay systems, since we obtained complete inhibition of aggregation and secretion responses at M concentrations of CV-3988, whereas they reported lO_'M to 1r6 no agonists effects of CV-3988 and no inhibition of collagen or It is posslble that Interactions A23187 at 3 X lo-' M CV-3988. between CV-3988 and albumin In PRP, similar to those observed may limit the effectiveness of between AGEPC and albumin (221, a degradation product of CV-3988 Alternatlvel~~ CV-3988 in PRP. in the dflutions of CV-3988 used In these studies, and not CVThis might be responsible for our observations. 3988 itself, since these results were obtained latter possibility Is unlikely, using dlfferent platelet preparations and dilutions consistentlyr

220

CV-3988 AND PLATELET ACTIVATION

Vol. 41, No. 2

Our observatlon that CV-3988 did not increase CV-3988. and in fact may lower CAMP, suggests that platelet CAMP levels, and CV-3988 on collagen calcium effect of inhibitory the ionophore A23187 is not due to a non-specific elevation of as that which is associated with PG12 CAMP such platelet mediated inhibition of a wide variety of platelet stimuli.

of

ACKNOWLEDGEMENTS The author thanks his Section Chief, Dr. Sami I. Said, for his Anna Zambelli, Teresa assistance, encouragement and editorial Long and Marlene McVey for preparing the manuscript and Suzie Carter for preparing the illustrations. Supported by Medical The author is a Research Funds from the Veterans Administration. Fellow of the Puritan-Bennett Foundation. Parker 8. Francis

REFERENCES

1.

PINCKARD, R.N., L.M. McMANUS, D.J. HANAHAN and M. HALONEN.: Immunopathology of acetyl glyceryl ether phosphorylcholine (AGEPC). In: Immunocolqqv of the lung H.H. Newball, ted.) Marcel Dekker, Inc., New York, 1983, pp 73-107.

2.

BENVENISTE, an factor:

“Fd”:

Bier

Paltavy 3;5-376.

J. and B.B. VARGAFTIG.: ether lipid with biological teds.1

Biom.e’

Academic

Press,

Platelet-activating activity. In: Ether . H.K. Mangold and 4iWua Inc., New Yorkr 1983, pp

3.

GOETZL, E.J., C.K. DERIAN, A.I. TAUBER and F.H. VALONE.: l-O-hexadecyl-2-acyl-Ill_9lycero-3-phosNovel effects of phorylcholine mediators on human leukocyte function: Delineation of the specific roles of the acyl substftuents. Res Coauoun g&:881-888, 1980.

4.

HANAHAN, D.J., P.G. MUNDER, K. SATOUCHI, L.M. McMANUS R.N. PINCKARD.: Potent platelet stimulating activity enantiomers of acetyl glyceryl ether phosphorylcholfne its methoxy analogues. Piochem Biophys Res Cou:183188, 1981.

5.

HEYMANS, F., E. MICHEL, M.-C. BORREL, B. WICHROWSKI, J.-J. GODFROID, 0. CONVERT, E. COEFFIER, M. TENCE aqd 3. H NMR BENVENISTE.: New total synthesis and high resolution spectrum of platelet-activating factor, its enantlomer and racemlc mixtures. -em Blopbvs Acta 666:230-237, 1981.

6.

SATOUCHI, K., R.N. PINCKARD, L.M. McMANUS and D.J. HANAHAN.: Modification of the polar head group of acetyl glyceryl ether phosphocholine and subsequent effects upon platelet activation. J Biol Chem 756:4425-4432, 1981.

and of and

Vol. 41, No. 2

CV-3988 AND PLATELET ACTIVATION

221

7.

TENCE, M.r F. MICHEL, E. COEFFIER, J. POLONSKY, J.-J. GODFRDID and J. BENVENISTE: Synthesis and biological activity some structural analogs of platelet-activating factor of (PAF-acether). Agents and Actions 11:558-559r 1981.

8.

E.A. CRESS, T.-C. LEE, B. MALONE, J.R. SURLES, BLANK, M.L., C. PIANTADOSI, J. HADJU and F. SNYDER.: Structural features of platelet activating factor (l-alkyl-Z-acetyl-u1-glycero-3phosphocholine) required for hypotensive and platelet serotonfn responses. Res Commun Chem Path01 P-1 38:320, 1982.

9.

HADVARY, P. and H.R. BAUMGARTNER.: Activation of human rabbit blood platelets by synthetic structural analogs platelet-activating factor. Th Res 34:143-156, 1983.

10.

MOSCHIDIS, M.C., C.A. DEMOPOULOS KRITIKOU.: and L.G. Phosphono-platelet activating factor. I. Synthesis of l-Ohexadecyl-2-0-acetyl-glyceryl-3-~2-trimethylammonium-methyl~phosphonate and its platelet activating potency. Chenr Lfoids 35:87-92, 1983.

11.

O'FLAHERTY, J.T., W.L. SALZER, S. COUSART, C.E. MCCALL, C. PIANTADOSI, J.R. SURLES, M.J. HAMMET and R.L. WYKLE.: comparative studPlatelet-activating factor and analogues: ies with human neutrophils and rabbit platelets. Req Cammun Chem Path01 Pharmacol 39:291-309, 1983.

12.

TERASHITA, Z.-I., S. TSUSHIMA, Y. YOSHIOKA, H. INADA and K. NISHIKAWA.: CV-3988: A specific of platelet-activating factor (PAF). Life-:

and of

NOMURA, Y. antagonist

1975-1982,

1983. 13.

Inhibition by TERASHITA, Z.-I., Y. IMURA and K. NISHIKAWA.: CV-3988 of the binding of I: HI-platelet activating factor .viol 34: 1491-1495, 1985. (PAF) to the platelet.

14.

Y. IMURA, K. NISHIKAWA and S. SUMIDA.: Is TERASHITA, Z.-I., platelet activating factor (PAF) a mediator of endotoxin shock 7 Eur J Pharmacol 109:257-261, 1985.

15.

COX, C.P., J. LINDEN and S.I. SAID.: VIP elevates cyclic AMP (CAMP) levels and inhibits _in vitro activation induced by platelet-activating factor &ade:.1:325-328, 1984.

16.

BORN, G.V.R.: diphosphate and

17.

BROOKER, G.r completely 194.:270-276,

18.

TATESON, J.E., S. prostacyclin (PGX) platelets. P?andins

Aggregation its reversal.

of blood platelets Nature 194:927-929,

L.L. TERASAKI and M.G. PRICE.: radioimmunoassay automated 1976.

platelet platelet (PAF).

by adenosine 1962.

The Gammaflo, a system. Science

MONCADA and J.R. VANE.: on cyclic AMP concentrations 13:389-397, 1977.

Effects of in human

222

CV-3988 AND PLATELET ACTIVATION

Vol. 41, No. 2

19.

WARDLOW COX, M.L. and FARR, R.S., C.P. of an acid-labile factor Preliminary studies that inactivates platelet-activating factor. Wthal:318-330, 1980.

20.

COX, K.E. WARDLOW, C.P. MENG and D.E. FARR, R.S., M.L. GREENE.: Human serum acid-labile factor is an acylhydrolase inactivates factor. platelet-activating that Fed Prc&z &2:3120-3122, 1983.

21.

COX, C.P., M.L. WARDLOW, K.E. MENG, FARR.: Substrate specificity of acylhydrolase that inactivates AGEPC. pp. 299-304, 1983.

22.

HOPPENS, C.M., PINCKARD.: R.N. activation phosphorylcholine.

J.C. by

D.E. the m.s,

R. JORGENSEN.: in human sera Clin Imm

GREENE and phosphatide

R.S. 2-

LUDWIG, R. CASTILLO, L.M. McMANUS and Albumin modulation of rabbit platelet 1-O-hexadecyl-2-acetyl-a-glycery7-3fed Proc 42:693A, 1983.