Porcine Platelet-derived Growth Factor Research Articles

Participation of phospholipase D and α/β-protein kinase C in growth factor-induced signalling in C3H10T1/2 fibroblasts

We have studied phospholipase D (PLD) activation in relation to protein kinase C (PKC) and the involvement of PLD in extracellularly regulated kinase 1 (MAPK) (ERK1) activation and c- fos mRNA expression in C3H/10T1/2 (Cl8) fibroblasts. In these cells, the PLD activity was significantly increased by porcine platelet-derived growth factor (PDGF-BB), phorbol 12-myristate 13-acetate (PMA), and epidermal growth factor (EGF). PLD activation by PDGF-BB and PMA, but not EGF, was inhibited in Cl8 cells expressing the HAβC2-1 peptide (Cl8 HAβC2-1 cells), with a sequence (βC2-1) shown to bind receptor for activated C kinase 1 (RACK1) and inhibit c-PKC-mediated cell functions [Science 268 (1995) 247]. A role of α-PKC in PLD activation is further underscored by co-immunoprecipitation of α-PKC with PLD1 and PLD2 in non-stimulated as well as PMA- and PDGF-BB-stimulated Cl8 cells. However, only PKC in PLD1 precipitates was activated by these agonists, while the PKC in the PLD2 precipitates was constitutively activated. The c- fos mRNA levels in Cl8 cells increased more than 30-fold in response to either PDGF-BB, EGF, or PMA. Approximately 60% inhibition of this increase in c- fos mRNA levels was observed in Cl8 HAβC2-1 cells. Formation of phosphatidylbutanol (PtdBut) at the expense of phosphatidic acid (PtdH) in the presence of n-butanol inhibited ERK1 activation and c- fos mRNA expression in PDGF-BB-treated Cl8 cells. ERK activation by PMA was unaffected by n-butanol in Cl8 cells but almost abolished by n-butanol in Cl8 HAβC2-1 cells, showing that ERK activation by PMA is heavily dependent on PKC and PLD1. In contrast, ERK activation by EGF in both cell types was not sensitive to n-butanol. These results indicate (1) a role of a functional interaction between the RACK1 scaffolding protein and a αPKC-PLD complex for achieving full PLD activity in PDGF-BB- and PMA-stimulated Cl8 cells; (2) PLD-mediated PtdH formation is needed for optimal ERK1 activation by PDGF-BB and maximal increase in c- fos mRNA expression. These findings place PLD as an important component in PDGF-BB- and PMA-stimulated intracellular signalling leading to gene activation in Cl8 cells, while EGF does not require PLD.

Porcine model of proliferative vitreoretinopathy with platelets

PURPOSE. To develop an experimental model of proliferative vitreoretinopathy (PVR) in the pig, and determine the efficacy of platelet-derived growth factor (PDGF) compared with different platelet plasma concentrates in its development. METHODS. Animals were divided into four groups of 12 pigs each. Groups 1, 2, and 3 underwent four 3-mm-long retinotomies, a partial mechanical vitrectomy, and six transconjunctival retinal cryoapplications and were injected intravitreally with, respectively, 0.2 ml of platelet rich plasma, 0.2 ml of a solution containing 200 ng of porcine PDGF, and 0.2 ml of platelet concentrated plasma. Group 4 received only an intra-vitreal injection of 0.2 ml of porcine PDGF. RESULTS. In Group 1, retinal detachments (RDs) developed in six eyes (50%) (two eyes, total RDs; four, extensive RDs). In Group 2, focal RDs developed in six eyes (50%). In Group 3, 11 eyes (92%) developed Rds (six eyes, total RDs; three, extensive RDs, two, focal RDs). Group 4, did not develop lesions. Statistically significant differences were found between Group 3 and the other groups. Group 2 RDs were associated with the presence of vitreoretinal membranes but there were no signs of PVR. In Groups 1 and 3, signs of anterior PVR, posterior PVR, and retinal holes with rolled edges were observed. CONCLUSIONS. We have developed a model of PVR in the pig, the retina of which more closely resembles that of humans. Platelet plasma more effectively contributed to the development of an experimental model of porcine PVR than 200 ng of PDGF. The efficacy depends on the platelet concentration of the plasma. These results suggest that other growth factors and plasma components may interact synergistically with PDGF in the pathogenesis of PVR.

Autocrine growth stimulation of human meningioma cells by platelet-derived growth factor.

The authors have previously shown that meningioma-derived conditioned medium profoundly stimulates the in vitro proliferation of meningioma cells. In this paper, self mitogenic agents found in the conditioned medium-autocrine growth-stimulatory factors actually secreted by human meningioma cells-are characterized as proteins related to the B chain of platelet-derived growth factor (PDGF) and possibly to the A chain of PDGF as well. The addition to conditioned medium of a neutralizing antibody against PDGF-BB caused a significant inhibition of the conditioned medium-stimulated DNA synthesis in all three meningioma cultures studied. A similar neutralizing effect was observed with an anti-PDGF-AA antibody in one meningioma culture studied. Gel filtration chromatography of concentrated conditioned medium from two different meningiomas using a Sephadex G-100 column revealed similar profiles from both conditioned media with a major peak of mitogenic activity against meningioma cells at a molecular weight (M(r)) of approximately 32 to 36 kD, accompanied by a minor peak at approximately 22 kD. The major peak mitogenic activity was significantly reduced by addition of an anti-PDGF-BB antibody. Western blot analysis of protein extracts from five meningioma specimens was performed using a monoclonal antibody against the B chain of PGDF, and a major band of PDGF-B immunoreactivity was detected at an M(r) of approximately 19 kD in all five meningiomas under both reducing and nonreducing conditions. Exogenous human and porcine PDGFs both exhibited a significant dose-dependent stimulation of DNA synthesis in two of three and three of five meningioma cultures examined, respectively. Although not all meningiomas investigated proved to share the biological activity associated with PDGF and these results may be preliminary, it seems that the autocrine growth-stimulatory loop established by PDGF-B-related molecules plays an important functional role in meningioma cell proliferation.

Inhibition by Rapamycin of PDGF- and bFGF-Induced Human Tenon Fibroblast Proliferation in Vitro

Fibrosis of subconjunctival tissues is a major cause of bleb failure following glaucoma filtration surgery. The aim of the present investigation was to demonstrate the effect of Rapamycin, a clinically relevant macrolide antibiotic with potent immunosuppressive properties, on human Tenon fibroblast proliferation induced by platelet-derived growth factor and basic fibroblast growth factor. Primary Tenon fibroblast cultures were derived from patients undergoing trabeculectomies or routine cataract extractions. Rapamycin was added in concentrations of 0.1-100 ng/ml with or without 3-30 ng/ml of porcine platelet-derived growth factor or of human recombinant basic fibroblast growth factor. Two days after treatment, the cells were examined and counted. The results were expressed as the percent of cell growth in treated culture relative to its untreated control. Rapamycin was not cytotoxic at any of the concentrations tested. Inhibition of platelet-derived growth factor-induced Tenon fibroblast proliferation occurred with all doses of Rapamycin, the most marked effect being observed with 30 ng/ml (60% inhibition, p < 0.001). In contrast, optimal inhibition of basic fibroblast growth factor-induced proliferation was only 37% (p < 0.01), achieved with 10 ng/ml of the peptide. Rapamycin potently inhibits platelet-derived growth factor-induced fibroblast proliferation in vitro without any apparent cytotoxicity. It may eventually prove to be a useful adjunct to glaucoma filtration surgery.

Quantitative Analysis of Polymerase Chain Reaction Products Using Biotinylated dUTP Incorporation

A method for relative quantitation of specific mRNA species by polymerase chain reaction (PCR) has been developed by using the incorporation of biotinylated dUTP. Transferred biotinylated PCR products gave a sensitive colorimetric signal which could be quantitated by video analysis. In the exponential phase of amplification, the linearity and reproducibility of reverse transcription and PCR demonstrated the same efficiency of cDNA synthesis and PCR for the two target genes, ICAM-1 and β-actin, and allowed the normalization of ICAM-1 expression. These results suggested that in the exponential phase of amplification a relative quantitation of mRNA could be determined. We used this approach to analyze the different effects of TNF-α, IL-1β, and purified porcine platelet-derived growth factor stimulations on ICAM-1 expression in smooth muscle cells.

PDGF‐receptor protein tyrosine kinase activity in carotid artery is enhanced by injury and inhibited in vivo by tyrphostin RG 13291

AbstractTyrphostins (TKI) are synthetic low molecular weight compounds which inhibit in vitro platelet‐derived growth factor (PDGF) mitogenesis by blocking PDGF receptor protein tyrosine kinase activity (PDGF R‐PTK). To determine whether TKIs act by this mechanism in vivo, a rat model of vascular injury was used. Rat carotid arteries were injured by implantation of a tygon cuff machined so that 1 mm of each end touched the artery. PDGF R‐PTK was measured ex vivo by incubation of the excised denuded artery with porcine PDGF followed by homogenization and Western blotting with antiphosphotyrosine/antiPDGF‐R antisera. In arteries from control rats, PDGF produced a maximal 2‐fold increase in R‐PTK at 120 ng/ml. In cuffed arteries, PDGF stimulated R‐PTK in a dose‐related manner after one day of injury; R‐PTK remained elevated for 4–5 days post injury (6‐fold increase in R‐PTK activity at 180 ng/ml). A similar response to PDGF was also evident in contralateral uncuffed arteries. Intraperitoneal injection (+ 2 h) of TKI RG 13291 inhibited PDGF R‐PTK of cuffed artery with an IC50 value of ∽ 2 mg/kg and of the uncuffed artery with IC50 value of ∽ 30 mg/kg. It is concluded that TKIs are potential in vivo inhibitors of injury‐induced PDGF R‐PTK activity and may be useful as inhibitors of PDGF‐dependent effects such as proliferation. © 1993 Wiley‐Liss, Inc.

Effects of growth factors and hormones on basal and FSH-stimulated inhibin production by porcine granulosa cells in vitro

The effect of several growth factors, protein and steroid hormones on follicle stimulating hormone (FSH)-stimulated and basal inhibin secretion by mature porcine granulosa cells (g-cells) in culture was examined in order to elucidate the putative role of growth factors and hormones in the regulation of inhibin secretion by porcine g-cells in vitro. Cells were incubated with the respective hormones over a timespan of 0-144 h and immunoreactive inhibin was measured with a radioimmunoassay against porcine inhibin. Epidermal growth factor (EGF) and human transforming growth factor type beta (TGF-beta) decreased basal and gonadotrophin-stimulated inhibin and progesterone in a dose-dependent manner. In the absence of insulin, insulin-like growth factor type I (IGF-I) caused a 4-fold enhancement of basal inhibin secretion, but inhibin secretion was elevated only to 20% above control in the presence of 500 nM insulin. Porcine platelet-derived growth factor (PDGF) had no significant effect on basal or FSH-induced inhibin secretion by g-cells. In addition, neither gonadotrophin-releasing hormone (GnRH) nor prolactin (PRL), arginine vasopressin (AVP) and oxytocin affected basal or FSH-stimulated inhibin release by porcine g-cells. Oestradiol caused a slight but significant (P less than 0.01) rise of basal inhibin production (158% of control) in the last 2 days of culture (96-144 h) and the effect of androstenedione on basal (158% of control) and FSH-stimulated (140% of control) inhibin release (P less than 0.01) was also only visible on Days 4-6 of culture. In contrast to androstenedione and oestradiol, progesterone did not show any effect during 6 days of culture in a dose range of 10(-5) to 10(-9) M. Like steroids, prostaglandin E2 (PGE2) had a stimulatory effect on basal inhibin production (250% of control) by porcine g-cells, visible on Days 3-6 of culture, but an inhibitory effect on FSH-stimulated release (less than 40% of control). Over all the experiments with different hormones and growth factors, tested in varying doses and over a time span of 0-144 h, there was a strong correlation between progesterone and inhibin secretion by g-cells (0-48 h = 0.78; 48-96 h = 0.92; 96-144 h = 0.92). These results suggest that EGF, TGF-beta, IGF-I, oestradiol and androstendione as well as PGE2 have para- and/or autocrine modulatory effects on basal and FSH-stimulated inhibin secretion by mature porcine g-cells in vitro and further demonstrate that the secretion of the proteohormone inhibin and the steroid progesterone are closely related.

Autoregulation of MeWo metastatic melanoma cell growth: characterization of intracellular (FGF, MGSA) and secreted (PDGF) growth factors.

MeWo melanoma cells (clone LC1) secrete a potent mitogenic activity susceptible to reinitiate DNA replication in quiescent rodent fibroblasts (CCL39, NRK-49F, NIH-3T3) but not in BHK-21 kidney cells. This activity appears to be closely related to platelet-derived growth factor (PDGF) based on 1) its cationic nature, heat and acid resistance, but sensitivity to reducing agents; 2) its apparent molecular weight (33 kDaltons) as estimated by Biogel filtration, once dissociated from binding proteins by mild acidic treatment; 3) its weak affinity for heparin; and 4) its ability to compete with 125I-PDGF for binding to human and rodent fibroblasts, and to be recognized by anti-PDGF antibodies. Although MeWo cells coexpress the PDGF-A and PDGF-B (c-sis) chain gene transcripts, the secreted product shows reactivity on CCL39 fibroblasts more compatible with the PDGF-BB than with the PDGF-AB isoform. MeWo cell lysates contain activities that bind moderately and strongly to heparin-Sepharose, being eluted with 1.0 and 2.0 M NaCl, respectively. The latter may correspond to basic fibroblast growth factor (basic FGF), consistent with the expression of basic FGF gene mRNAs. The former has not been fully characterized and is probably not the product of the acidic FGF gene. In addition, MeWo cells react positively with the FB2 AH7 antibody, thus indicating that they elaborate material similar to melanoma growth-stimulating activity (MGSA). MeWo cells proliferate in serum-free medium in a cell-density-dependent fashion, both in liquid and semisolid cultures. Their division is modestly enhanced by basic FGF and by human and porcine PDGF but not by the factors that they release. However, the absence of demonstrable 125I-PDGF binding sites on MeWo cells, in conjunction with their lack of sensitivity to suramin growth inhibition, suggests that the secreted PDGF does not act as an autocrine factor. Instead, the autonomous proliferation of MeWo melanoma cells may result from the concerted action of basic FGF and MGSA, which are mostly cell-associated.

Identification and Structural Analysis of the A Type Receptor for Platelet-derived Growth Factor: Similarities with the B type receptor

Abstract Binding analyses using 125I-labeled platelet-derived growth factor (PDGF)-AA and PDGF-BB were used to identify a clonal human glioma cell line (U-343 MGa 31L) which expresses the A type but not the B type receptor for PDGF. The glioma cells were devoid of a B type receptor transcript, and immunoprecipitation with an antiserum raised against a B type receptor peptide rendered no signal. Similar analyses using human foreskin fibroblasts, which express both A and B type PDGF receptors, revealed a B type PDGF receptor-specific 5.5-kilobase pair mRNA in a Northern blot experiment, and 160,000 and 180,000 molecular weight components upon immunoprecipitation. A second antiserum, raised against purified porcine PDGF receptor preparations, was reactive with Mr 140,000 and 170,000 components in the U-343 MGa 31L cells, as well as in human fibroblasts. In addition, this antiserum precipitated the Mr 160,000 and 180,000 components from the fibroblasts. Exposure of cells to PDGF-AA, as well as to PDGF-BB, induced an increased rate of degradation of the Mr 170,000 component in the clonal glioma cells and in fibroblasts. The Mr 180,000 component in fibroblasts was degraded only when cells were exposed to PDGF-BB. This allowed the identification of the Mr 170,000 component as the cell surface expressed form of the A type receptor for PDGF. A structural relatedness between the A and B type PDGF receptors was furthermore indicated by similarities in peptide patterns, after limited proteolytic digestion.

Temporal relationship between inositol polyphosphate formation and increases in cytosolic Ca2+ in quiescent 3T3 cells stimulated by platelet-derived growth factor, bombesin and vasopressin.

We determined the temporal relationship between the formation of inositol phosphates and increase in cytosolic [Ca2+] elicited by bombesin, vasopressin and platelet-derived growth factor (PDGF) in quiescent Swiss 3T3 cells. These responses were measured under identical conditions. Bombesin caused a rapid increase in inositol 1,4,5-trisphosphate which coincided with the increase in cytosolic [Ca2+]. This was followed by a slower but marked increase in inositol 1,3,4-trisphosphate and inositol-bisphosphate. Vasopressin elicited a similar sequence of events. In sharp contrast, highly purified porcine PDGF induced increases in cytosolic [Ca2+] and inositol 1,4,5-trisphosphate that were temporally uncoupled: detectable inositol polyphosphate formation occurred after Ca2+ mobilization from intracellular stores. The same temporal dissociation was observed when a recombinant v-sis product was used instead of porcine PDGF. However, PDGF was as effective as bombesin in stimulating the formation of inositol phosphates after 5-10 min of incubation. The data suggest that PDGF increases cytosolic [Ca2+] via a different signal transduction pathway from that utilized by bombesin and vasopressin. These findings have important implications for understanding the signal transduction pathway activated by PDGF.

Characterization of two monoclonal antibodies reactive with the external domain of the platelet-derived growth factor receptor.

Two monoclonal antibodies against the receptor for platelet-derived growth factor (PDGF) were obtained by immunizing mice with pure PDGF receptor preparations derived from porcine uterus. The antibodies, denoted PDGFR-B1 and PDGFR-B2, both bound to the external domain of the receptor, as demonstrated by indirect immunofluorescence and binding of 125I-labeled antibodies to intact human fibroblasts. Both antibodies precipitated pure 175-kDa 32P-labeled autophosphorylated porcine PDGF receptor as well as a Mr 175,000 glycoprotein from metabolically labeled cells. The monoclonal antibodies did not inhibit binding of 125I-PDGF to human fibroblasts and did not stimulate these cells to undergo mitosis. Both antibodies induced clustering and down-regulation of their antigen. However, this resulted in only a partial loss of cell surface binding sites for PDGF itself, consistent with the conclusion that the monoclonals recognized only one of two or several receptors for PDGF. Clustering and down-regulation were not seen when the cells were incubated with monovalent Fab' fragments of the PDGFR-B2 antibody. The antibodies also stimulated autophosphorylation of pure PDGF receptor, and PDGFR-B2 was shown to stimulate phosphorylation of phosphofructokinase, an exogenous substrate for the PDGF receptor kinase. High concentrations of PDGFR-B2 antibody, or Fab' fragments thereof, failed to enhance the PDGF receptor kinase activity, compatible with the possibility that dimerization was of importance in the antibody-stimulated kinase activity of purified PDGF receptors.

Ca2+-mobilizing actions of platelet-derived growth factor differ from those of bombesin and vasopressin in Swiss 3T3 mouse cells.

Addition of the mitogenic peptides bombesin and vasopressin to quiescent Swiss 3T3 mouse cells increased the cytosolic Ca2+ concentration without any measurable delay. In contrast, there was a significant lag period (16 +/- 1.2 s) before platelet-derived growth factor (PDGF) increased cytosolic Ca2+ concentration. This lag was not diminished at high concentrations of either porcine or human PDGF. Similar results were obtained in 3T3 cells loaded with quin-2 or fura-2. The differences in the effects of bombesin, vasopressin, and PDGF on Ca2+ movements were also substantiated by measurements of 45Ca2+ efflux and of cellular 45Ca2+ content. Activation of protein kinase C by phorbol esters inhibited Ca2+ mobilization induced by either bombesin or vasopressin. In contrast, phorbol esters had no effect on PDGF-induced cytosolic Ca2+ concentration increase or acceleration of 45Ca2+ efflux. Finally, bombesin and vasopressin caused a rapid increase in the production of inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate, whereas PDGF, even at a saturating concentration, exerted only a small effect. These results indicate that the signal transduction pathways activated by PDGF that lead to Ca2+ mobilization can be distinguished from those utilized by bombesin and vasopressin.

Activity of human, bovine and porcine platelet-derived growth factor in a radioreceptor assay with human placental membrane protein

Radioiodinated human platelet-derived growth factor ( 125I-PDGF), and unlabeled human (Hu), bovine (Bo), and porcine (Po) PDGF were used to study PDGF receptors in human term placental membrane preparations. The binding of 125I-Hu-PDGF was inhibited by unlabeled preparations. Half-maximal occupancy of the binding sites occurred at 7–9 nM of Hu-, Bo- or Po-PDGF. Scatchard analysis of the binding data indicated a single class of receptors having a K d value of about 1.1–1.2 × 10 −10 M. Thus, the sensitive radioreceptor assay does not detect any species specificity of PDGF.

4] Purification of fibroblast-derived growth factor

This chapter focuses on the purification process of fibroblast-derived growth factor (FDGF). FDGF is a potent polypeptide mitogen secreted into the growth medium by a simian virus 40 (SV40) transformed hamster fibroblast cell line (SV28). Studies with partially and highly purified FDGF have established that its physical and biological properties, which are strikingly similar to those of human and porcine platelet derived growth factor (PDGF) and FDGF is immunologically related to human PDGF. Although the production of PDGF-like molecules by a number of cell types in culture has been reported, including cells transformed by simian sarcoma virus whose sis oncogene encodes the B chain of human PDGF, the purification of these factors has proven difficult because of their cationic and hydrophobic properties and their presence in conditioned media at low levels.

Highly purified fibroblast-derived growth factor, an SV40-transformed fibroblast-secreted mitogen, is closely related to platelet-derived growth factor.

Fibroblast-derived growth factor (FDGF), a polypeptide secreted by an SV40-transformed baby hamster kidney cell line (SV28), was purified approximately 1000-fold from SV28-conditioned medium. FDGF, which gave a single band on SDS-polyacrylamide gel electrophoresis, is a hydrophobic and cationic protein of apparent mol. wt. 31 000 containing disulphide-linked polypeptides. This factor is positive in Western blots using human platelet-derived growth factor (PDGF) anti-serum. FDGF is a potent mitogen for Swiss 3T3 cells; half-maximal stimulation of DNA synthesis was achieved at a concentration of approximately 1 nM, comparable with those for human and porcine PDGF. FDGF inhibits EGF binding to Swiss 3T3 cells, as does PDGF. The coincidence of the physical, biological and immunological characteristics of FDGF and PDGF strongly suggests that they are closely related in structure.

Platelet-derived growth factor and multiplication-stimulating activity II, but not multiplication-stimulating activity III-2, stimulate [3H]thymidine and [35S]sulphate incorporation by fetal rat costal cartilage in vitro.

The actions of partially purified porcine platelet-derived growth factor (PDGF) and highly purified multiplication-stimulating activity (MSA) II and MSA III-2, which are somatomedins, were investigated on the incorporation of [3H]thymidine and [35S]sulphate by fetal rat costal cartilage in vitro. This was compared with their effects in the presence of 1% fetal calf serum (FCS) on the uptake of thymidine by growth-arrested fetal rat fibroblasts. Platelet-derived growth factor at concentrations of 0.21-21 micrograms/l enhanced the incorporation of both isotopes by fetal cartilage in the presence of 1% FCS, but had an inconsistent action on thymidine uptake and no significant action on sulphate uptake in serum-free medium. Platelet-derived growth factor promoted thymidine uptake by growth-arrested, isolated fetal rat fibroblasts. Multiplication-stimulating activity II (10-100 micrograms/l) stimulated the uptake of thymidine and sulphate by fetal cartilage in medium containing 1% FCS but had no consistent action in serum-free medium, although MSA II and PDGF had a synergistic effect on thymidine uptake in the absence of serum. Multiplication-stimulating activity III-2 had no consistent action on thymidine or sulphate incorporation by fetal cartilage in either serum-free or serum-supplemented medium. However, the same preparation of MSA III-2 stimulated the uptake of [3H]thymidine into fetal rat fibroblasts with a half-maximal response at a concentration of 5-10 micrograms/l. The results identify PDGF as a possible mitogenic agent for fetal rat connective tissues in vitro and show a differential sensitivity of fetal cartilage to MSA peptides.

Ionic responses rapidly elicited by porcine platelet-derived growth factor in Swiss 3T3 cells.

Addition of porcine platelet-derived growth factor (PDGF) to quiescent cultures of Swiss 3T3 cells caused a marked, dose-dependent stimulation of Na+ influx and Na-K pump-mediated 86Rb+ uptake. Porcine PDGF (a single component in SDS polyacrylamide gels) stimulated ion fluxes to the same maximal extent as partially purified preparations, and exhibited half-maximal effect at 6 ng/ml (2 X 10(-10) M). Maximal effect was achieved at 30 ng/ml (10(-9) M). In the presence of insulin, PDGF elicited mitogenesis at comparable concentrations. PDGF stimulated ion uptake in a time-dependent fashion; maximal effect was obtained after 5 min of exposure to the growth factor. PDGF stimulates Na+ influx via an amiloride-sensitive pathway, suggesting that PDGF enhances the activity of a Na+/H+ antiport system. In accordance with this possibility, the mitogen caused an increase of intracellular pH by 0.15 pH units, as judged by the steady-state distribution of labelled 5,5-dimethyloxazolidine-2,4-dione (DMO). Porcine PDGF stimulated E-type prostaglandin synthesis and cAMP accumulation but these events could be dissociated from the stimulation of the ionic fluxes, which was detected within minutes and was not blocked by indomethacin. It is suggested that PDGF elicits multiple signals to stimulate cell proliferation in 3T3 cells.

Partial purification and characterization of porcine platelet-derived growth factor (PDGF)

Platelet derived growth factor (PDGF) has been partially purified from porcine platelets. Purification steps included heparin-agarose chromatography of the material released by thrombin-stimulated washed porcine platelets and Blue-Sepharose chromatography. Preparative isoelectric focusing showed that isoelectric point of porcine PDGF is at pH 10.0–11.0 and elution experiments from sodium dodecyl sulfate (SDS) polyacrymlam de gels indicated that its molecular weight is close to 30 kD. The immunoglobulin fraction prepared from anti-human PDGF serum inhibited the mitogenic activity of porcine PDGF. These experiments suggest a homology of porcine and human PDGF. Porcine platelet factor 4 and porcine platelet basic protein were devoid of significant mitogenic activity.