Human Platelet-derived Growth Factor PURIFICATION AND RESOLUTION INTO TWO ACTIVE PROTEIN FRACTIONS*

Human platelets secrete a factor that stimulates cul- tured human cells to initiate DNA synthesis and to divide. This human platelet-derived growth factor (PDGF) has been purified -100,000-fold into two equally active homogeneous fractions, PDGF I (Mr 2: 31,000) and PDGF I1 (Mr = 28,000). The amino acid compositions of each are similar, highly basic, and show 18 half-cystine residues. Both PDGF I and I1 are glycoproteins, but differ in their carbohydrate compo- sitions. The data suggest that PDGF 11 may be a proteolytic cleavage product of PDGF I but do not rule out that the proteins may be separate but very similar gene products. Purified PDGF is active in stimulating DNA synthesis at 0.2 ng/ml.

Human platelets secrete a factor that stimulates cultured human cells to initiate DNA synthesis and t o divide. This human platelet-derived growth factor (PDGF) has been purified -100,000-fold into two equally active homogeneous fractions, PDGF I (Mr 2: 31,000) and PDGF I1 (Mr = 28,000). The amino acid compositions of each are similar, highly basic, and show 18 half-cystine residues. Both PDGF I and I1 are glycoproteins, but differ in their carbohydrate compositions. The data suggest that PDGF 11 may be a proteolytic cleavage product of PDGF I but do not rule out that the proteins may be separate but very similar gene products. Purified PDGF is active in stimulating DNA synthesis at 0.2 ng/ml.
Fibroblasts and smooth muscle cells cultured in vitro require serum for normal growth. Without serum, cell growth stops in the Go/G1 phase of the cell cycle. Human serum, when added back to growth-arrested fibroblasts, restores DNA synthesis and cell proliferation. The major activity stimulating DNA synthesis and cell growth in human serum originates from blood platelets and is called the platelet-derived growth factor (1-12). While the precise physiological role of PDGF' is unknown, it may be essential for wound healing, for tissue repair, and in the development of atherosclerosis (13). PDGF is obtained from frozen/thawed plateletrich plasma, or from platelets after exposure to thrombin (14, 15). PDGF is mitogenic for arterial smooth muscle cells, mouse fibroblast cell lines, glial cells, and skin fibroblasts (9, We report the purification of PDGF to near homogeneity. PDGF activity has been separated into two distinct protein fractions of molecular weight 31,000 (PDGF I) and 28,000 (PDGF 11). Each protein has been shcwn to be a glycoprotein of slightly different amino acid and carbohydrate composition.
* This research was supported by grants CA22409, HL14147, and HL22119 from the United States Public Health Service. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18

10, 16-20).
Each protein contains a large number of disulfide bonds, providing substantial stability to the unreduced protein, Previous reports on the purification of PDGF have appeared, but limited quantities of purified material have made assessment of the degree of purity difficult in the preparations obtained (21, 22). Large scale purifications have not been reported and the chemical composition of the protein is not known. Furthermore, as noted by Heldin (22), a broad distribution of PDGF has been noted previously in isoelectric focusing (14), gel fitration (14), and SDS-polyacrylamide gel electrophoresis (22). This apparent heterogeneity may be a result of inadequate separation of PDGF I and I1 in previous studies because of the small amounts of materials available. PDGF is known to be cationic (PI 9.8-10.2), to have M, of 25 to 32,000, and to lose mitogenic activity by exposure to trypsin or by reduction (21-24).

EXPERIMENTAL PROCEDURES
Methods-Protein concentration was determined by absorbance at 280 nm, by the Bio-Rad protein microassay (25), or by the method of Lowry et al. (26). Amino acid analyses were performed according to the methods of Spackman et al. (27). Half-cystine residues were determined as cysteic acid in separate analyses after performic acid oxidation and hydrolysis (28). Neutral and amino sugars were determined by gas-liquid chromatography following derivatization as described previously (29). Polyacrylamide gel electrophoresis in SDS was carried out as described by Laernmli (30). Liquid-phase preparative isoelectric focusing was run in an LKB Ampholine column 8101 (110 ml).
Mitogenic activity was measured by the PDGF-dependent incorporation of [methyl-3H]thymidine into trichloroacetic acid-precipitable material by mouse 3T3 fibroblasts (5). One unit of PDGF activity is that activity with a net incorporation of 10,000 dpm in excess of the control culture. All quantitative determinations were carried out in the linear range of the assay (to a net of 5 units or 50,000 dpm).

RESULTS
The assay (see "Methods") of PDGF is linear from -0-20 n g / d of purified PDGF (data not shown). The assay can detect PDGF concentrations as low as 0.1 ng/ml, and has been reproducible within approximately 10% during the course of this work.

Properties of the Platelet-derived Growth
Factor 8897 The preparation of platelet-derived growth factor which was obtained from Blue-Sepharose was directly subjected to the preparative isoelectric focusing. column (1.5 X 90 cm). A purification of -lo5-fold has been achieved, with a recovery of activity -10% (Table I). Fig. 1 illustrates the elution pattern of PDGF from Bio-Gel P-100. The protein peak is asymmetrical. By comparison with protein standards, the activity elutes with a molecular weight -27-33,000. Fig.  1 also shows the results of SDS-slab gel electrophoresis of each active fraction taken from the Bio-Gel P-100 column. PDGF activity is localized to two overlapping protein peaks. Fractions containing only one protein, at each end of the activity peaks, were collected, lyophilized, and have been characterized further. Fig. 2 shows the SDS-slab gel electrophoretic pattern of the two separate PDGF active fractions. The two fractions are cleanly separated from each other and each protein appears homogeneous. PDGF with a molecular weight of -31,000 and PDGF with a molecular weight of =28,000 (SDS-gel electrophoresis) were designated PDGF I and PDGF 11, respectively. PDGF I and I1 were extracted from the SDS gels and shown to have biological activity correlating precisely with the protein-staining bands, although recoveries of activity are quite low, averaging -10%. All other areas of the gels have been tested and found to be    The amino acid compositions of PDGF I and of PDGF I1 are nearly identical (Table 11) showing a striking predominance of basic amino acids, consistent with the isoelectric point of the intact protein (21). Each protein contains three tyrosine residues and 18 half-cystine residues. Preliminary evidence suggests all half-cystine residues are present in the disulfide form.
Both PDGF I and PDGF I1 stain positively with periodic acid-Schiff reagent. Direct carbohydrate analysis was performed, using gas-liquid chromatography of the hydrolyzed proteins (Table 111). PDGF I and I1 have significant amounts of covalently bound carbohydrate; -7% of PDGF I is estimated to be neutral and amino sugars. Significant differences are found in the carbohydrate analysis of PDGF I and PDGF   (-) (2 pl/assay, 1:IO dilution) of the samples was analyzed and plotted as above. Shown in the inset is the protein-staining pattern of SDS-polyacrylamide gels after electrophoresis of fractions containing peak PDGF activity. I1 was achieved. PDGF I and I1 appear either to have an identical isoelectric point or to migrate together, held by forces resistant to 4 M urea. This latter possibility is supported by preliminary results of gel filtration under nondenaturing conditions, which suggest that nondenatured PDGF migrates with an approximate molecular weight of 60,000.

DISCUSSION
Human PDGF has been purified approximately 100,000fold, with an apparent recovery of -10%. We now report that the purified protein may be separated into two separate protein peaks, designated PDGF I ( M , = 31,000) and PDGF I1 (M, -28,000). PDGF I and PDGF I1 appear to have approximately equal mitogenic potency. Both proteins are strongly basic, as reflected in the isoelectric point at 10.2 and in the strong predominance of basic amino acids in the amino acid composition analysis. The proteins contain only three tyrosyl residues and 18 half-cystine residues. Preliminary results suggest the half-cystine residues are all in disulfide linkage. This large number of disulfide bonds is unusual in growth factors and most likely explains the extreme stability of PDGF to heat denaturation.
The platelet-derived growth factor is shown also to be a glycoprotein, as demonstrated by positive periodic acid-Schiff staining of PDGF I and I1 in SDS gels and by direct analysis of hydrolyzed samples of isolated PDGF I and 11. Covalently bound carbohydrate has not been reported previously, perhaps because of the limited quantities of PDGF available for analysis.
The amino acid analyses were calcualted using an estimated molecular weight of 31,000 for both PDGF I and PDGF I1 to emphasize the striking similarities between the two proteins (Table 11). These data suggest either that PDGF I1 is the proteolytic cleavage product of PDGF I, or that PDGF I and PDGF I1 are separate gene products, or that the two proteins were subject to different post-translational processing.
Our results suggest that the two proteins isolated are the PDGF activity first reported by Ross et al. (9) and subsequently by Antoniades et al. (21) and by Heldin et al. (22). The present results now identify two separate PDGF activi-ties, establish that PDGF I and PDGF I1 are glycoproteins of different carbohydrate compositions, and provide an amino acid composition of the two proteins. Previously published purifications (21, 22) do not agree on all the properties of PDGF, especially on the apparent molecular weight of PDGF. Broad bands of PDGF were found in gels. Analysis of gel patterns in these works suggest that with the detection methods used and with the limited quantities of PDGF available for study, the broad distribution of PDGF may reflect the two forms of PDGF we have purified and separated.
Note Added in Proof-During the initial submission of this manuscript, Heldin et al. (Heldin, C.-H., Westermark, B., and Wasteson, A. (1981) Biochem. J. 193, 907-913) presented the amino acid composition of PDGF as purified previously (22). Their preparation was not resolved into separate protein components. Their amino acid composition was similar to ours except our results show 18 halfcystine residues compared to 11 found in their analysis.