Comparison of platelet-derived growth factor prepared from release products of fresh platelets and from outdated platelet concentrates

Vol. 116, No. 3, 1983

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 809-816

November 15, 1983

COMPARISON OF PLATELET-DERIVED GROWTH FACTOR PREPARED FROM RELEASE PRODUCTS OF FRESH PLATELETS AND FROM OUTDATED PLATELET CONCENTRATES C.N, Chesterman*~, T. Walker*, B. Grego+, K. Chamberlain*+, MoT.W. Hearn+ and F.J. Morgan+ *Melbourne University Department of Medicine and +St. Vincent's School of Medical Research, St. Vincent's Hospital, Fitzroy, Victoria, Australia, 3065

Received September 8, 1983 Platelet-derived growth factor was isolated from the release products of washed, human platelets and from freeze-thawed outdated platelet concentrates. On the basis of sodium dodecyl sulphate polyacrylamide gel electrophoresis and amino acid sequence determination we conclude that platelet-~derived growth factor released from platelets by the agonist thrombin (EC 3.4.4.13) is structurally similar to that isolated from lysed platelets and from platelet concentrates stored for more than 72 hr at room temperature.

Platelet-derived growth factor (PDGF) is the principal mitogen in serum which stimulates quiescent connective tissue cells in culture to initiate protein and DNA synthesis ( i ) .

Recent evidence suggests that PDGF is a basic

protein of about M r 30,000, composed of two polypeptide chains (Mr 13,00014,000 and about 16,000-17,000) joined by disulphide bonds (2,3).

Although

broad agreement with these proposals has been reached by others (4,5,6), other molecular weight forms of PDGF have been isolated (Mr 27-33,000) and their heterogeneity has been the subject of discussion (5-9).

Outdated platelet

concentrates are commonly used as the starting point for the preparation of PDGF or

alternatively lysed, washed platelets (2,3,7,9).

Leakage of platelet

granule components into the plasma during storage and the presence of plasma and platelet proteases are among the factors which may result in molecular weight heterogeneity of PDGF preparations. ~To whom correspondence should be addressed. Present address: University of New South Wales Department of Medicine, The St. George Hospital, Kogarah, Sydney, N.S.W., Australia, 2217. Abbreviations used: Platelet derived growth factor (PDGF), prostaglandin EI (PGE1) , sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), reversed phase high performance liquid chromatography (RP-HPLC).

0006-291X/83 $1.50 809

Copyright © 1983 by Academic Press, Inc. All rights o f reproduction in any form reserved.

VOI. 116, No. 3, 1983

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

To determine the characteristics of PDGF as it is released from platelets in vivo we have prepared PDGF from the thrombin release products of washed, freshly collected human platelets. outdated platelet concentrates.

Simultaneously we have prepared PDGF from Our results suggest that PDGF is released

from platelets as two distinct molecular weight species in accordance with the findings of Deuel et al. who used PDGF isolated from outdated platelets (5).

Our amino acid sequence studies are consistent with the proposal that

PDGF is released from platelets as a protein comprised of two nonidentical subunits linked by disulphides (8,9). 2. 2.1

MATERIALS AND METHODS Prepara__tion of PDGF from the release products of fresh platelet concentrates Platelet rich plasma was prepared from whole blood collected into I/5 volume of 0.07M citric acid, 0.1M trisodium citrate, 0.11M dextrose and centrifuged at 250 g for 15 minutes at 4°C within 4 hours of collection. To inhibit release during preparation, PGE I (Sigma) l~g/ml was added to the platelet rich plasma and the platelets pelleted 5y centrifugation. The platelets were resuspended in Tyrode's buffer with PGE 1 l~g/ml and washed three times at 4°C. The platelets were aggregated with bovine thrombin (EC 3.4.4.13) (Parke Davis, 0.I NIH-U/ml) in the presence of aprotinin (Trasylol, Bayer, i0 KIU/ml) and o the aggregated platelets centrifuged at 4 C at 2200 g fo¥ 5 minutes. Supernatant release products from 400 units of platelets were pooled and dialysed against 0.01M phosphate buffer at pH 7.0 and PDGF prepared essentially as described by Heldin et al. (2) using ion exchange chromatography with CMSephadex C-50 (Pharmacia) and absorption to Cibaeron Blue-Sepharose (Pharmacia). The active material was eluted with 50% ethylene glycol, dialysed against 0.1M acetic acid, lyophylised and applied to a column of Sephadex G-75 equilibrated in IM acetic acid. 2.2

Preparation of PDGF from outdated platelet concentrates Platelet-derived growth factor was purified from 55 litres of outdated platelet concentrates by the method of Deuel et al. (5) except that the final molecular exclusion chromatography was carried out using Sephadex G-75 in IM acetic acid. F3 Incorporation oft H]-thymidine into BALB/C 3T3 cells Quiescent 3T3 cells (obtained from Commonwealth Serum Lab., Melbourne) were maintained in 5% plasma-derived serum in Dulbeccor!~ modified Eagle's medium for two days before stimulation to incorporate ~ HJ-thymidine (185GBq/ mmol, Amersham) into DNA following a 60 minute3incubation with PDGF at 37°C. The cells were harvested at 40 hours and the [ Hj-thymidine incorporated into trichloracetic acid precipitable material was counted. 2.3

2.4

High PerfOrmanCe Liquid Chromatography HPLC was carried out using a Du Pont model No. 850 gradient elution system equipped with a variable wavelength detector. Solvents were HPLC grade and buffers prepared as previously described (i0). 2.5

Isolation of Protein from SDS Polyacrylamid e Gel SDS-PAGE was carried out according to Laemmli (ii). Elution of protein from the gel was carried out as described by Hunkapiller et al. (12). 2-Mercaptoethanol and sodium thioglycolate were omitted from the procedure. The migration of the protein was identified by staining of a lateral track with Coomassie brilliant blue.

810

Vol. 116, No. 3, 1 9 8 3

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

2.6

Amino Acid Sequence D e t e r ~ n a t i o n Automated amino acid sequence determinations on unreduced preparations of PDGF were performed using a Beckman 890C Sequencer with a straight-walled cup, using Polybrene as a carrier and 0.1M Quadrol. Quadrol buffer (Beckman) was repurified before use (G.S. Begg, B. Leslie and F.J. Morgan, unpublished) and the other reagents and solvents purified as previously described (13). Thiazolinones were treated with 20% trifluoraeetic acid at 80°C for 15 minutes under nitrogen, and the resulting PTH amino acids analysed by HPLC (HewlettPackard I084B) using the method of Zimmerman et al. (14). 3.

RESULTS

3.1

Purity of PDGF preparation s Analytical

Methods)

RP-HPLC revealed that neither the final Sephadex G-75 (see

preparation

from thrombin release products nor the preparation

from outdated platelet concentrates were homogeneous for chromatography

conditions).

(see fig. I and legend

The retention time of peak mitogenic

activity varied between 76 and 80 minutes for these preparations

depending

on their isolation history and handling conditions. Micro-preparative

RP-HPLC of the PDGF preparation

from release products

of fresh platelets yielded an active peak which was resolved into two bands on SDS-PAGE, with M r 31,000 and 27,500 respectively with almost equal staining

100

E ¢:

o c m D. o

o~

mm 50

4) ¢)

o w~

.O I,. 0 W .O

0

40

50

Volume

60

ml

Figure i. About 2.5mg PDGF prepared from outdated platelet concentrates (see Methods) was subjected to reversed phase HPLC. Chromatographic conditions were as follows: column, Merck Lichrosorb C_,dp i0 (250x4mm ID); flow rate, 0.Sml/min; linear 90 min gradient (indicated by the heavy ruled line) from 0 to 100% B where solvent A was HgO - 0.1% trifluoracetic acid and solvent B was 50% H20 - 50% isopropanol: -0.1% trifluoracetic acid. One ml fractions were collected and assayed for mitogenic activity after evaporating the isopropanol and subsequent lyophilization. The position of peak mitogenic activity is shown by the solid bar.

811

Vol. 116, No. 3, 1983 intensity

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(fig. 2a).

preparation resulted

Electrophoresis in the

under reducing conditions of this

appearance of at least four stained bands with

the major components being between M r 14,000 and 15,000, and with two minor components of M

r

18,000 and 13,000 (fig. 2b).

The fraction containing peak mitogenic activity from preparative RP-HPLC of the outdated platelet PDGF preparation showed at least three minor bands of protein staining on SDS-PAGE apart from the two major bands which migrated at M

r

31,000 and 29,000 respectively.

and eluted electrophoretically

These two bands were excised together

from the gel as described

(see Methods).

The

major mitogenic product from this procedure was shown to migrate as a band of M

r

weight

29,000 on SDS-PAGE with a minor staining band of higher molecular (see fig. 2c).

discrete bands of M

r

Reduction of this preparation resulted in two 15,000 and 13,500 (fig. 2d).

A ...~.

B i~

C

D

ill

i~ i! i! i~~ i¸ ~

i!i

i

31,000 27,500

- - -

~

~15,0 0 0 ~13,5OO

Figure 2. SDS polyacrylamide gel electrophoresis of preparations of PDGF prepared from platelet release products, (a) unreduced (b) reduced; and PDGF prepared from outdated platelet concentrates (c) unreduced and (d) reduced. Samples were boiled for 2 min in 1.25% SDS and eleetrophoresed for ~16 hr at 10ma in 12.5% slab gel (l.Smm) with 5% stacking gel. For samples electrophoresed under reducing conditions, 0.125% 2-mercaptoethanol was added to the sample buffer. The gels were silver-stained using the method of Wray et al (15) with minor modifications. MW standards were bovine serum albumin (68,000), ovalbumin (43,000), carbonic anhydrase (29,000) goose egg lysozyme (21,000) and hen egg lysozyme (14,300).

812

Vol. 116, No. 3, 1 9 8 3

3.2

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Amino Acid SequenCe Determination PDGF from both platelet release products and from outdated platelet

concentrates

(200-300 pmole) were subjected to amino acid sequence analysis.

One major amino terminal sequence was common to both preparations and predominated over other minor sequences

(fig. 3).

PDGF from fresh platelet release products revealed evidence of a second underlying

sequence being about 30% of the total (fig. 3a).

This second

sequence was also present at lower levels (25% of the major sequence) PDGF from outdated platelet concentrates

in the

along with the presence of minor

sequences.

4.

DISCUSSION Our approach to prepare PDGF from the release products of freshly

collected platelets had two objectives:

firstly to use a physiological

starting material and secondly to avoid artefacts related (>72 hr) storage of platelets at room temperature, maintaining

concentrates

for clinical use.

to the long term

the usual practice

The proteolytie

for

inhibitor

aprotinin was added to the buffer during platelet release to further minimise the possibility of proteolysis. The procedure described in this communication resulted

in a PDGF

preparation active in the mitogenic assay at less than Ing/ml shown) and composed of forms of M in our laboratory

r

31,000 and 27,500.

(5,8) have shown a similar pattern and the two forms

have been assigned PDGF-I and PDGF-II respectively

(7,8).

from the release products of fresh platelets.

the pattern on reductive

with three major polypeptide others

(5).

in the molecular weight of the subunit components of

PDGF is notable in the preparation In particular

Earlier preparations

(16) and PDGF isolated from outdated platelets both here

and in other reports

Heterogeneity

(results not

SDS polyacrylamide

bands evident,

gel electrophoresis,

is similar to that reported by

Johnsson et al (7) have suggested that the size heterogeneity

may only involve one subunit

(molecular weight

18,000).

Waterfield et al (9) have isolated the same peptide

813

Subsequently

(designated Peptide II)

Vol. 116, No. 3, 1983

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

120! G L U T A M I C

A

ACID

ALANINE

PROLINE

VALINE

1 00

8ol ~.,J 6oi \ / ~ \ ~ 4o

\/

I\:

20

1 20

LEUCINE

ISOLEUCINE

THREONINE

50

100

"\.

80

,,"I, 6o a.

SERINE

40 30

/\/v--

20!

20 10

~

_._•/~.~--..

"''2~'--"

B

1 20

MAJOR SEQUENCE:

SER-ILE-GLU-GLU-ALA-VAL-PRO-ALA-VAL-

MINOR SEQUENCE:

THR-LEU- X -

X

-LEU-THR°ILE -

GLUTAMIC ACID

ALANINE

VALINE

' ISOLEUCINE

LEUCINE

SERINE

PROLINE

100 SO J

~ 4o 2O

120 1OO

SO

SO

4O

~ eo

SO

0

40

0.

2O

/ L.,/--

THREONINE

2O ....

,o

t\.~.j,

MAJOR SEQUENCE:

SER-ILE-GLU-GLU-ALA-VAL-PRO-ALA-VAL-

MINOR SEQUENCE:

THR-LEU-ALA-X

-LEU-THR-ILE-

Figure 3. Sequence analysis of unreduced PDGF isolated from thrombin release products of fresh platelets (a) and from outdated platelet concentrates (b). The recovery of the PTH amino acids was quantified by measuring peak heights from the HPLC analyses (see Methods)• Each point represents the yield of the PTH amino acids from sequential cycles• Due to the presence of double peaks in the analysis of the PTH amino acids, the yield data for serine and threonine have been estimated from peak maxima. An identical major sequence was determined in a less clearly defined minor sequence ( 30% in the and 25% in the outdated platelet PDGF). Further apparent in the preparation from outdated platelet

both preparations with release product PDGF underlying sequences are concentrates•

using an alternative HPLC procedure and demonstrated that both the 18,000 and the 14,500 molecular weight polypeptide chains exhibit size heterogeneity presumably associated with post-translational modification. Despite the apparent differences differences in proportions

in the subunit composition and the

of PDGF-I and PDGF-II in our two preparations

814

Vol. 116, No. 3, 1983

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

there is unequivocal consonance in the major amino terminal sequence of the two preparations with less certain agreement in the subsidiary sequence detected.

If PDGF is composed of two non-identical polypeptide chains the

explanation for the substantially lower yield of the second subunit sequence may reside in its greater susceptibility to proteolytic cleavage during processing, release or preparation.

The primary sequence obtained in the

present study is identical to that designated la by Antoniades and Hunkapiller (8) and peptide II by Waterfield et al (9) and the secondary sequence may represent a mixture of the chains termed 2a and 2b (8) or the peptides I, III and IV (9). Our results are consistent with the proposals (7-9) that size heterogeneity of PDGF preparations and the derived subunits is

likely to be due to

truncation of the carboxy-terminal regions of two distinct subnnit polypeptides in addition to the amino terminus of one of those polypeptide chains.

It

does not exclude the possibility of differential glycosylation of the polypeptide chains ( 5 ) .

Perhaps more importantly the results support the

notion that the active mitogen released into the surrounding milieu by platelets stimulated by the physiological agonist thrombin, is structurally similar to that isolated from platelets following prolonged storage as platelet concentrates. ACKNOWLEDGEMENTS We acknowledge the expert technical assistance of Miss F. Lambrou and Mr. R. Owe-Young. Fresh platelet concentrates were the gift of St. Vincent's Hospital Blood Bank (Dr, B. Rush) and the Red Cross Blood Transfusion Centre, Melbourne (Dr. K. McGrath). Dr. R.E.H. Wettenhall provided helpful advice in the preparation of the manuscript which was typed by Miss Joan Osbourne. The work has been supported by grants from the National Health and Medical Research Council of Australia and equipment grants from the Ian Potter Foundation, the Utah Foundation and Du Pont Instrument Division (Wilmington, Delaware). REFERENCES (i) Ross, R. & Vogel, A. (1978) Cell 14, 203-210. (2) Heldin, C-H, Westermark, B., Wasteson, ~.(1979) Proc. Natl. Acad. Sci. USA. 76, 3722-3726. Biochem. J. 193, (3) Heldin, C-H, Westermark B, Wasteson, %. (1981) 907-913. (4) Antoniades, H.N., Scher, C.D., Stiles, C.D. (1979) Proc. Natl. Acad. Sci. USA. 76, 1809-1813. (5) Deuel, T.F., Huang, J.S., Proffitt, R.T., Baenziger, J.U., Chang, D., Kennedy, B.B. (1981) J. Biol. Chem. 256, 8896-8899.

815

Vol. 116, No. 3, 1983 (6) (7) (8) (9)

(i0) (ii) (12) (13) (14) (15) (16)

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Raines, E.W., Ross, E. (1982) J. Biol. Chem. 257, 5154-5160. Johnsson, A., Heldin, C-H~ Westermark, B., Wasteson, ~. (1982) Biochem. Biophys. Res. Comm. 104, 66-74. Antoniades, H.N. and Hunkapiller, M.W. (1983) Science 220, 963-965. Waterfield, M.D., Scrace, G.T., Whittle, N., Stroobant, R., Johnsson, A., Wasteson, A., Westermark, B., Heldin, C-l{., Huang, J.S. and Deuel, T.F. (1983) Nature 304, 35-39. Hearn, M.T.W. and Grego, B. (1981) J. Chromatogr. 203, 349-363. Laemmli, U.K. (1970) Nature (Lond.) 227, 680-685. Hunkapiller, M.W., Lujan, E., Ostrander, F. and Hood, L.E. (1983) Methods in Enzymology 91, 227-235. Begg, G.S., Pepper, D.S., Chesterman, C.N., Morgan, F.J. (1978) Biochemistry 17, 1739-1744. Zimmerman, C.L., Appella, E. and Pisano, J.J. (1976) Anal. Biochem. 75, 77-85. Wray, W., Boulikas, T., Wray, V.P. and Hancock, R. (1981) Anal. Biochem. 118, 197-203. Chesterman, C.N., Walker, T., Chamberlain, K. and Morgan, F.J. (1982) Proc. 6th Int. Cong. on Atherosclerosis, Berlin, pp. 410-415.

816