In vitro synthesis of low molecular weight proteins in human platelets: Absence of labelled release products

THROMBOSIS RESEARCH 36; 619-631, 1984 0049-3848/84 $3.00 + .OO Printed in the USA. Copyright (cl 1984 Pergamon Press Ltd. All rights reserved.

INVITRO

SYNTBESIS

OF LOWWOLECULARWEIGHTPF6Yl'EINS

ABSENCE

INHUMANPLATELETS:

OF LABELLED RELEASE PRODUCTS

T.Shaw(a), C.N.Chesterman(b) and F.J.Worgan(c) Department of Medicine, University of Welbourne(a,b) and St. Vincent's School of Medical Research(c), Victoria, 3065, Australia. Present addresses: (a) Department of Wicrobiology, LaTrobe University, (b) Department of Medicine, University Victoria, 3003, Australia: of New South Wales, St. George Hospital, N.S.W., 2217, Australia.

(Received 4.5.1984; Accepted in revised form 13.9.1984 by Editor B.G. Firkin) AESTRACT

Isolated human blood platelets incubated at 37'C in Fikr~ incorporated labelled amino acids into compounds which included some low molecular weight (<80KDa) proteins, as determined by autoradiography after sodium dodecyl sulphate-polyacrylamide gel reducing (SDS-PAGE) under conditions. Nonelectrophoresis inhibited dialysable plasma both uptake and factor(s) by Actinomycin D, was incorporation, which although unaffected inhibited partly by Chloramphenicol and almost completely by Puromycin and Cycloheximide, results which confirm that synthesis is directed by pre-existing mRNAs, some of which is mitochondrial. Assuming that the ml?NA coding for proteins which are truly "platelet specific" must be present in megakaryocyte cytoplasm, we investigated the possibility that such RNA may be sufficiently its translation stable for to continue in platelets. Although leakage from platelet alpha-granules and cytoplasm during incubation was negligible and platelets retained their secretory potential we were unable to detect radiolabelled proteins in thrombin-released material after incubation. we conclude that either alpha-granule proteins are not synthesised in platelets or their megalcaryocyte progenitors, or that their mFlUAs become degraded by the time platelets reach the peripheral circulation. Alternatively, the mechanism which concentrates these proteins in granules does not function in circulating platelets.

Several

studies

Key words:

[l-4] established

platelet-specific

that da DQXQ protein synthesis

proteins,

619

can occur

protein synthesis, Percoll.

620

PROTEIN SYNTHESIS IN PLATELETS

Vo1.36, No.6

in platelets, despite the absence of a nucleus, rough endoplasmic reticulum, or even significant numbers of free ribosomes. Booyse and Rafelson [3], who were unable to detect RNA synthesis in isolated platelets in W, predicted that this ability must be due to the presence of stable mRNA inherited from megakaryocytes, a view which they were later able to support [4] with experimental evidence. However, this conflicts with other studies [a-8] suggesting that DNA-dependent RNA, and even DNA synthesis, attributed to mitochondrial activity, can be detected in platelets. Early reports [3,4,9,10] provisionally identified the major products of platelet protein synthesis as contractile proteins, specifically actin and myosin, usually on the basis of co-recovery with unlabelled tracers, but the identity of other products has never been investigated. Since platelets are anucleate, protein synthesis in the platelet cytoplasm must be directed by stable mlWAs [4]. If the low molecular weight alpha-granule proteins platelet factor 4 (PF4), beta-thromboglobulin (BTG) and platelet-derived factor the growth are truly (PDGF) "platelet-specific", their mRNAs must be transcribed exclusively from megakaryocytic DNA, a possibility which seems increasingly probable in the light of recent evidence [ll-163. If so, it is also possible that these mRNA8 are stable and that with sufficiently sensitive methods, their translation may be detectable in platelets. We have studied the uptake and incorporation of labelled amino acids into TCA-precipitable material in isolated platelets with the aims firstly of optimising incorporation &I y&r~ under conditions which allow platelets to retain their secretory ability, and secondly of determining whether any of the products of & apyp synthesis are secreted proteins.

m: Analytical grade chemicals were used. Sources of other material8 were: radiochemicals - AstershamAustralia P/LI Aquas01 II New England Nuclear! Percoll and Sephadex - Pharmacia (south Seas) Ltd., cell media - Cormnonwealth Serum Laboratories, Melbourne; culture aprotinin *VTrasylol** injection, Bayer, imipramine Gem-y; hydrochloride - "Tofranil" injection, Ciba-Geigy, Auetralia. Reagent grade thrombin was kindly donated by Dr. D. Aronson of the F.D.A. Bureau of Biologicals, Bethesda, U.S.A.. B : Except where specified otherwise all procedures were performed aseptically at room temperature. Plasticware was used throughout. Platelets were isolated from citrate-anticoagulated venous blood obtained from healthy adult donors who had not ingeated compounds known to affect platelet function during the previous ten days and who had cell counts within the normal range. Platelet rich plasma (PRP) was prepared by centrifugation at aproximately 250 xg for 10 min. Alternatively, two cycles of Percoll density gradient centrifugation were used to isolate platelets from plasma proteins and other blood cells. These preparations are referred to as Percoll-waahed platelets (PWP). Details of their preparation have recently been published elsewhere 1171. To assess the extent to which PWP had been washed free of plasma proteins,

Vo1.36, No.6

PROTEIN SYNTHESIS IN PLATELETS

621

added to 125I-human fibrinogen injection, (code IM.43P, ~100 Ki/mg) was samples of donor blood to give a gamma count of aproximately lo6 cpm/ml; recovered cell suspensions were subsequently counted for 1251 activity. PPP was prepared by pelleting the residual PlateletPIBP;Plaamam in some experiments cells after PRP isolation at 5,000 x g for 20 min j changes 1 ml aliquote were dialyaed overnight at 4'C against two 1 litre of phosphate buffered saline. using a Red and white blood cell counts were obtained GUlCounts: Coulter *lSg' counter; platelet counts were estimated using a Coulter model Cell counts ZF particle counter which had a 50 x 60 $I sampling aperture. in suspensions for labelling were verified by phase contrast microscopy. Eagle's Minimum Essential I.abellincrQfProteinsSvnthesiaed&lY&rQI Medium (NEM) buffered to pH 7.4 with 10 1164 EEPES (N-2_hydroxyethyllabelled amino acid(s) piperaeine-N '-2-ethaneeulphonic acid) containing and lacking the appropriate unlabelled amino acid(s) was added to PRP or PWP. The resulting suspensions were incubated at 37'C with occasional gentle agitation in humid 5%COz:95% air. Inhibitors of protein synthesis, if used, were added 15-30 min before the addition of label which was either a acid mixture (Amersham code TRE.440), tritiated amino L-14,5- ?+J]leucine (61 Ci/mmol; code TRE.170), or L-[35S]methionine (>600 Ci/ mmoi ; At various times after addition of label, code SJ.204). platelets were either pelleted then released as described below, or washed twice with 5 vol of ice-cold ETP (EDTA-theophylline-Prostaglandin El [17]), then pelleted. Drained ETP pellets were lysed in one volume of 0.1% SDS (Sodium dodecyl sulphate). Small aliquots of SDS-lysate were removed for assay of [3H] or r3%] (uptake), and an equal volume of 20% TCA (trichloroacetic acid) containing cold amino acids was added to the remainder. Precipitates were allowed to settle at 4'C overnight before being washed repeatedly with cold 5% TCA to eliminate free label, then When TCA was added, Percoll assayed for [ k] or E3%J incorporation. co-precipitated with PWP proteins, but did not interfere with radioassays. 0.45 ml aliquots of platelet AaareaometrvmReleaseStudies: suspensions were stirred in an aggregometer at 37'C. After 1 min, 0.05 ml buffer with or without effecters of release or aggregation were added. After a further l-10 min , 0.1 ml aliquots were pipetted into ice-cold ETP to arrest release and platelets pelleted by centrifugation. In some experiments, cells were preincubated 15-30 min at 37'C with 0.1 uCi/ml 5-hydroxy[G-3B]tryptamine creatine sulphate (code TRA.223j 500 mCi/nunol)~ in these experiments ETP included 2uM imipramine hydrochloride to prevent reuptake. Supernatents were assayed for LDB (lactate dehydrogenase) and BTG as described previously [17], and for radioactivity as described below.

-timProducts:

After incubation, platelets in PRP were processed as described in results; PWP were pelleted and resuspended in 0.1 volume of REM containing excess (1OmM) cold amino acids, aprotinin (100 KIU/ml), Ca++(lmM) and thrombin (l-2 U/ml) (final concentrations), and incubation continued a further lo-15 min at 37' C. Cells were then pelleted and the supernatent removed and either diluted with an equal volume of distilled water, or dialysed overnight at 4' C against 1Dmu phosphate, 1OmM Nacl, p?I 7.0, before application to a 15 x 1 cm column of CM sephadex ~50. The column was washed with the same buffer until the absorbance at 260 nm approached zero, when bound material was eluted with

622

PROTEIN SYNTHESIS IN PLATELETS

Vo1.36, No.6

a 0.1-1.0 M Nacl gradient and 5 ml fractions collected for assay. Samples (volume
sill-and

Platelet suspensions following Dlateletreeovervr recovery from Percoll (PWP) were preferable to PRP on the following grounds. The recovery of platelets was 09.6f 6.7% (x f SD, n=4) in PWP compared to 62 & 7.4% in PRP. Leuko e contamination was similar, 15 f cF platelets for PWP. S/lo6 platelets for PRP and 14 f 6/10 When 12'1 fibrinogen was added to whole blood prior to preparation only 6.1 f 0.6% counts remained with the PWP indicating low plasma protein contamination.

ti ra agid~r When PI@ was incubated with [ ]-amino acid mixture, and samples removed at intervals and processed as described above, uptake of label was rapid, but radioactivity began to disappear from cells after about three hours, a phenomenon which was paralleled by changes in the amount of label incorporated into Loss of label was not due to cell TCA-precipitable material (Fig 1). lysis, since leakage of LDH was never significantly above background (plasma) levels2 maximum leakage of STG represented only about 2% of the total, which was stable throughout the incubation period (data not shown).

lo uci/ml --l-T--

Incorporation of (3H] into EffectsQfInhibitarsQfProteinSvntheeisl TCA precipitable material in PRP was assayed after 2 hr incubation under specific conditions described above, with and without inhibitors. Results, which are presented in Table 1, confirm that synthesis is de ~ppo and dependent on pre-existing mRNA, most of which appears to be cytoplasmic. Chloramphenicol inhibited incorporation by about 30%.

Vol.36,

No.6

PROTEIN

IN PLATELETS

SYNTHESIS

623

l!mIal Effect

of Inhibitors on Protein Synthesis in PRS ------

_____-_~_I____~-----~~--~~~~-~~ Inhibitor

Incorporation

and final concentration

(%)

100

None Cycloheximide

10 mM

Puromycin

1mM

Chloramphenicol

5 m&l

Actinomycin

D

14i5 6f3 61&9 91i6

10 UM

zcable 1:

Inhibitore were added 15-30 minutes before addition of labelj incubation was for 2 hr. Results, which are relative to incorporation without inhibitor, are mean8 f SD's for at least 3 experiments using different donors.

3

I

0

2

4

HOURS

I

4

6

8

INCUBATION

-1 Time-course of uptake and incorporation (TCA ppt) of [3H]-amino acids into platelet proteins in PRP. Data are plotted aa means f SD's.

624

PROTEIN SYNTHESIS IN PLATELETS

Vo1.36, No.6

PRP from the same donors was Reswneetithrombinafter-: incubated for 2 hr as described above. After incubation,plateletswere

pelleted and resuspendedin the same Volume of autologousPPP, and samples removed for aggregometry (see methods). In a duplicate set of experiments, [%I-serotonin replaced C&i]-amino acids. Release was calculated as a percentageof the total after correctingfor background which never exceeded 5%. [3H]-serotonin and ETG release was dose-dependent,but no dose dependentrelease of [3H]-aminoacids could be detected (Fig 2A). LDH assays (results not shown) indicatedthat no cell lysis occurred at any thrombin concentration. PRP was incubated for 2 hr with [*J-amino acids as above and release inducedby thrombin. The serum recoveredafter standingovernightat 4OC was diluted with an equal volume of distilled water and chromatographed on CM-Sephadex(see methods). Results of a typical experimentare presented in Pig 2B. In this, and nine similar experiments, control columns in which label was added to platelet-freeserum showed similar elution patterns,but when small peaks of radioactivity co-eluting with BTG or PP4 were pooled, lyophilised, redissolvedand incubatedunder the conditionsused for RIAs (see above),

ChromatoaraDhvQfreleaeeoroductsr

significant radioactivity was not precipitated by the appropriate antisera. These results suggested that some [JR]-amino acids were non-specifically bound to serum proteins.

I

2A

THROMBIN

CONCENTRATION

(U/ml)

28

2

30

AZ Response of platelets in PRP to thrombin after incubation with [3R]-labelled serotoninor [3H]-aminoacids (means f SD's for 4 experimentswith differentdonors). Br Typical result of chromatographing thrombin-released material from platelets prelabelledwith [3H]-amino acids on CM-Sephadex, using a salt gradient in 10 mM phosphatebuffer pH 7.0 (see Methods).

625

PROTEIN SYNTHESIS IN PLATELETS

Vo1.36, No.6

Low platelet concentrations were at first thought to of PWP to thrombin of the apparently slow response be the cause stimulation (Fig 3A). Closer inspection showed that clumping and settling of any small aggregates which foraed in response to aggregating agents was inhibited because the density of the suspending medium was cloee to that Further investigation revealed that PWP underwent of the aggregates. although rapid release when st%mulated by thrombin (Fig 3B) and that, PWP secretion was dose-related (Pig 3C). PWP relatively insensitive, suspensions failed to respond to even high concentrations of RSstocetin or and resuspended in PFP, normal but when diluted, pelleted, ATP, aggregatory response was restored (not shown).

!zbrmun-:

‘Ii-Serotonin

38 1

BTGH ‘H-Serotonin

0

/ 1

I

I

,

,

4

5

c++

M2lNUTE3S

012345

10

MINUTES

012

5 U/ml

10

THROMBIN

A: Aggregometer trace from PWP suspension of c.1.5 x lo8 platelets/mli time of thrombin addition or buffer (control) arrowed. Bz Time-course of release from PWP induced by 2 U/ml thrombin; 8amples removed to PTP at times indicated. C: pwp response to different doses of thrombin; samples taken 5 min after thrombin addition. (Results in B and c are means f SD's for 4 experiments using different donors.)

UDtakeMdincorwrationQfL3H1-rsinoacidsinperpt

Eaving established the functional capacity of PWP, incorporation of labelled amino acids into platelet proteins was re-examined in a series of experiments using [35S]-methionine or [3H]-leucine in place of the labelled amino acid mixture in order to avoid probable non-specific binding observed

626

PROTEIN SYNTHESIS IN PLATELETS

Vo1.36, No.6

mixture in order to avoid probable non-specific binding observed In PWP,3which were essentially free of contaminating plasma previously. proteins the rate of [‘HJ-leucine incorporation remained almost linear over an 8 hr period, plateauing between 4 and 8 hr. The specific activity of the recovered TCA precipitate was directly proportional to the available labelled leucine, and was dramatically reduced in the presence of 20% autologous plasma (Fig 4A). Kinetics of [35S]-methionine incorporation were similar. The effect of added unlabelled leucine and of different concentrations of dialysed PFP was investigated in subsequent experiments, in which the incubation time was 2 hr and [ HI-leucine W&belled leucine at concentrations greater concentration was 1 Ki/ml. than about 0.1 m&lcompeted with labelled leucine for uptake, resulting in a precipitous decrease in the specific activity of recovered XA Dialysed PFP produced similar dose-dependent precipitates (Fig 4B). inhibition of both uptake and incorporation (Fig 4C). Further experiments in which [3H]-leucine was added to PFP established that dialysis reduced the concentration of free amino acid by a factor of about 104-fold (results not shown). Using PWP preparations, attempts were again made to by detect incorporation of label into thrombin-releasable products Results were similar to those aggregometry and column chromatography. obtained using PRP.

0

48

4A

T

20.-

&i 60

I- a-E 0

3 E

B 00.-

a0

P

0

2

1 HOURS

3

INCUBATION

4

I! 0

10-s lo-’ lo-’ 10-z

LEUCINE

MOLARITY

!

!

!

!

0 2 4 6 8 PFP CONCENTRATION

A: Uptake and incorporation (TCA ppt) of C3H]-leucine in PWP. The inhibition of incorporation caused by addition of PFP (20% of shoy. B: Inhibition is also fanal conentration) uptake and incorporation by added unlabelled [+I]-leucine adding different leucine (means of duplicates). Cr Effect of concentrations of dialysed PFP on Uptake and incorporation. (Results in A and C are means f SD's for 4 experiments using different donorsr in C, poopwere washed once and resuspended in =.)

!

10 1%)

627

PROTEIN SYNTHESIS IN PLATELETS

Vo1.36, No.6

SDS-PAC;EQfL35Smm:

Autoradiography of dried gels following SDS-PAGE of reduced and denatured platelet lysates revealed about eight major, and several minor bands of radioactivity after 2 hr Approximate 2568of the major bands are given in Pig 5B. MWe incubation. of products ~60 KDa could not be accurately estimated in this By&em. Addition of chloramphenicol dramatically inhibited incorporation into all but 3 products with MWs in the range 60-60 KDa (Pig 5A, track b) and addition of 20% (final concentration) dialysed autologous PPP inhibited incorporation fairly uniformly, although a product with MW about 56 ma (indicated by arrowhead) appeared relatively unaffected (Pig 5, track c). major 15% gels resolved low molecular weight components well but radioactive products were not detected in the region c 30 KDa (compare CBB-stained tracks in Pig 5 with autoradiographs).

a

SDS-PAGE b c

16

cm FROM TOP OF 0 14 12 10

GEL 6

4

2

:--.._ _... z- .

--1ck

..--

T-.

.

-

68

M.

,_.. -12.3-

-*

5A ARG

CBB

15

20

30 40 M.W. (APPROX.)

A: SDS-PAGE of radiolabelled platelet lysates. PWP were incubated 2 hr with 20 ,,ci/ml [35S]-xethionine and then processed as described in methods. 'a';b*, and 'c' are autoradiographs) 'd' and 'e', which are CBB-stained, show a PWP lysate (d) and MW markers (e) from the saxe gel. PWP in 'b' and 'c' were treated identically to 'a* except that the incubation media contained 5 III%! chloramphenicol (b) and 20% added PPP (c) [final concentrations]. B: Densitometer scans of the autoradiographs shown in 5A. Estimated MWs axe indicated1 the major band (arrowed) is presumed to correspond to platelet actin.

60 80 KDa.

628

PROTEIN SYNTHESIS IN PLATELETS

Vo1.36, No.6

Platelets have long been known capable of de DQYQ protein synthesis, presumed to be due to their inheritance from megakaryocytes of stable mRNA(s) [l-4,6-10], but even in more recent studies [26,37], the nature of the aythesised products has never been fully investigated. Since platelets are reasonably be expected that anucleate, it may platelet-specific proteins are synthesised in megakaryocytes and packaged into alpha-granules for subsequent release from circulating platelets. Identification of the site(s) of synthesis of these proteins would greatly facilitate their study by leading the way to isolation of their ml?NAs and ultimately, cloning of cDNAs. When these studies commenced, strong immunological evidence supported the megakaryocytic origin of PP4 [U-15] B!IG [11,12,14] and possibly PMjp 1161, although these reports did not preclude the possibility that their they synthesis elsewhere and that occurred were taken up by that megakaryocytes. In addition, there were two reports which suggested high molecular weight alpha-granule proteins, factor VZII antigen [ls] and factor XIII [ZO], are eynthesised in megakaryocytes. More recently, Rye and co-workers [14] have produced the first direct evidence for the megakaryocytic origin of an alpha-granule protein by demonstrating that megakaryocytesynthesise a protein which co-purifies enriched cultures with PF4. Because of the difficulties inherent in isolating pure megakaryocytea, and the possibility that mRNAs for secreted platelet-specific proteins may not only originate in megakaryocytes but may also be stable, we undertook a re-investigation of & ~QYQ protein synthesis in platelets uaing experimental conditions under which platelets retained both alpha-granule constituents and the capacity to undergo release. BTG was selected as the main alpha-granule marker because of its relative abundance [Zl]. We optimised conditions for in yifra incorporation and maintenance of alpha-granule integrity by isolating platelets in gradients of Percoll, eliminated plasma which minimised leukocyte contamination and virtually protein. The latter factor was found to be important because the presence of low concentrations inhibited of plasma factor(s) uptake and incorporation and caused apparent breakdown of labelled products after a short time lag. This phenomenon, which has also been reported by Plow 12211 seems unlikely to have been due to free plasma amino acids, firstly because of the low concentrations of free amino acids (0.008-0.57 mW)[23] in plasma and secondly because dialysed plasma produced the same effect. Although, after incubation, platelets responded to low doses of thrombin by release of 60-902 of alpha-granule components without undergoing lysia, we were unable to detect labelled BTG or PF4 amongst the released material, even using sensitive RIAs. However our failure to detect material does not provide labelled proteins in released conclusive evidence that platelets are incapable of their synthesis and it remains possible that these factora are synthesised in platelet cytoplasm, but that the organellar machinery necessary for their transport and packaging into alpha-granules in readiness for secretion is unavailable. Were this the case, then it should still be possible to detect low molecular weight labelled compounds corresponding to secreted platelet-specific proteins in platelet Although cytoplasm. low MW compounds were detected by

629

PROTEIN SYNTHESIS IN PLATELETS

Vo1.36, No.6

autoradiography of platelet lysatee, they were not major products and be breakdown products or non-secretory proteins.

may

The 42 EDa band we presume to be platelet actin, since there is good is synthesised in both platelets [3,9,10] and that it evidence megakaryocytes [24]. It is worth mentioning that although actin is not a mitochondrially-coded protein [ZS], its synthesia appears to be inhibited mitochondrial protein by chloramphenicol, a specific inhibitor of because chloramphenicol interferes with synthesis. This may be protein for cytoplaamic required mitochondrial energy production synthesis. We have, as yet, made no attempt to identify the higher MW components. Data presented here confirm other reports [l-10,26,27] that platelet8 are capable of & m protein synthesis which is virtually independent of RNA synthesis and is partly mitochondrial. Whilst unable at this stage to provide further insight into the biosynthesis of "platelet-specific" proteins, we have described a convenient plasma-free system whereby platelet protein synthesis and release may be simultaneously studied in w.

TS was supported by a grant from the National Heart Foundation of Australiaj this work was also partly supported by a grant from the National Health and Medical Research Council of Australia to CNC and PJM. The authors are grateful to Prof. J.S. waid for access to facilities at LaTrobe University.

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4.

BDOYSE,F.H. and RAFBLSON,M.E.Jr. Stable mRBA in the contractile protein in human platelets. Biochim w 188-190,

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PROTEIN SYNTHESIS IN PLATELETS

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7.

8.

AGAM,G.,

BESSLER,H. by human platelets.

11.

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RABELLINO,E.M.,

m

BioDhva.

RNA synthesis BEfa 521, 41-48, 1976.

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platelet mitochondrial Biomedicine 22, 66-69, 1977.

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in cellular

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AGAM,G. and DJA.LDE'ITI,M.Inhibition of human protein

9.

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iL Q&L

Paehol. s,

1227-1231,

betaand

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SCRDMAN,H.A., mvINE,S.P., BECKSTEAD,J.H. and Immunocytochemical localisation of platelet-specific D.P. proteins in human platelets and megakaryocytes and their secretion in response to thrombin stimulation. ~ m BipL 91, 404a (abstr. 23062), 1981. HUVERSON,P.S.,

RAINTON,

15.

RYO,R.,

=,A.,

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16.

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Measurement Of the MARTIN,J.P., SFiAW,T., HEGGIE,J. and PEt?IN~,D.G. and its relationship to volume. density ’ of platelets human Br, L m s, 337-352, 1983.

18.

cleavage of structural proteins during the assembly _I,U.K. 222, 680-685, 1970. the head of the bacteriophage T4. m

19.

NACHMAN,R.,

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and JAFPR,E.A.

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McDONAGX,R.P.

and MCDDWAGCJ.

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In:Progreas in Haemostasis and XAPLAN,lC.L. Beta-thromboglobulin. T.A.Sl?ABT (Ed.) New York: Grune and Stratton, Thrombosis, Vo1.5. 1960, pp.153-176.

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The free amino acids of human STBIN,W.Ii. and MOORB,S. ~ B~QJ.. Q&~R 211, 915-926, 1954.

24.

synthesis of actin by NACBWAN,R., IBVINB,R. and JAPFB,E. formation with guinea complex megakaryocytes. Pig Biachm. BioDhvs B m, 91-105, 1976.

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ANDERSON,L. Identification of mitochondrial proteins and some of their precursors in two-dimensional electrophoretic maps of human cells. w Nat&_ &a&+_ a U.S.A. ZB, 2407-2411 1981.

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influencing the in yifrp protein Haamostae pZ, 666-679, 1979. blood

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and