Biomedical subjects

C N Chesterman

Publications and source records attributed to C N Chesterman.

At least 73 records · Page 4Linked to original sources

PubMed1994

Identification of possible inhibitory reactive centers in thrombospondin 1 that may bind cathepsin G and neutrophil elastase.

Thrombospondin 1 is a multidomain trimeric glycoprotein from platelets and a variety of normal and transformed cells of both mesenchymal and epithelial origin, which functions in cell adhesion and cell-cell interactions. We have recently shown that human thrombospondin 1 binds and inhibits the neutrophil enzymes, neutrophil elastase [Hogg, P.J., Owensby, D.A., Mosher, D.F., Misenheimer, T.M., & Chesterman, C.N. (1993a) J. Biol. Chem. 268, 7139-7146] and cathepsin G [Hogg, P.J., Owensby, D.A., & Chesterman, C.N. (1993b) J. Biol. Chem. 268, 21811-21818]. One mole of thrombospondin 1 trimer binds 3 mol of neutrophil elastase or up to 6 mol of cathepsin G, with site-binding dissociation constants around the nanomolar range, and the enzymes have been shown to interact with thrombospondin 1 in the vicinity of the calcium-binding type 3 repeats. None of the protein modules in this region, or within the whole thrombospondin 1 molecule, have previously been implicated in the inhibition of proteinases. We noted that there are two stretches of eight amino acids each in the human thrombospondin 1 type 3 repeats, residues 735-742 and 794-801, that have striking similarity to a reactive-site consensus sequence derived from selected members of the Kazal and Streptomyces subtilisin inhibitor families. Synthetic peptides corresponding to the putative P5 through P4' residues of both proposed reactive centers interacted efficiently with the active site of cathepsin G and were competitive inhibitors of the fibronectin-degrading and platelet-activating activities of this enzyme, while only the peptide corresponding to residues 793-801 efficiently interacted with the active site of neutrophil elastase and competitively inhibited its fibronectin-degrading activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

Quantitation of platelet derived growth factor receptors on cells from human arterial segments.

The study of receptor expression in tissue containing multiple cell types presents a two-fold problem. Firstly, in situ studies are difficult to perform quantitatively and secondly, enzymatic disruption of the primary tissue results in the loss of cell surface receptors which may or may not be resynthesised. In vitro culture of cells following isolation may also alter receptor expression. To circumvent these problems, we have devised a reproducible non-enzymatic method for releasing endothelial and smooth muscle cells from human arterial segments and separating the mixture into constituent cell populations, in order to facilitate semi-quantitative receptor investigation by ELISA. The method involves the mechanical disruption of small artery segments and passage through progressively smaller sieves, resulting in a mixed population of single cells. Magnetic beads covalently attached to identifying lectins or antibodies are then used in a multistep procedure to give highly enriched cell populations. The dissociation/enrichment steps can be modified to select other cell types which may be present in the primary tissue, such as macrophages. We have used these preparations for locating and semi-quantitating PDGF and EGF receptors on the constituent cells of human umbilical artery preparations by ELISA using monoclonal receptor antibodies.

Antibodies, Monoclonal↗

PubMed1994

Plasma P-selectin is increased in thrombotic consumptive platelet disorders.

P-selectin is a 140-kD protein found in the alpha-granules of platelets and the Weibel-Palade bodies of endothelial cells that on cell activation is expressed on the cell surface and also secreted into the plasma. The secreted form of P-selectin, like plasma P-selectin, differed from platelet membrane P-selectin in that its molecular mass was approximately 3 kD lower under reducing conditions. Both the secreted and plasma forms of P-selectin contained cytoplasmic sequence as determined by Western blot analysis with an affinity-purified rabbit anti-P-selectin cytoplasmic peptide antibody. We have measured plasma P-selectin and beta-thromboglobulin (beta TG) concurrently in (1) patients with consumptive thrombotic disorders, including disseminated intravascular coagulation (DIC), heparin-induced thrombocytopenia (HIT), and thrombotic thrombocytopenic purpura (TTP)/haemolytic uremic syndrome (HUS); (2) patients with idiopathic thrombocytopenic purpura (ITP); and (3) healthy controls. Patients with DIC, HIT, and TTP/HUS, but not ITP, had significantly elevated plasma P-selectin and beta TG levels when compared with their age-matched healthy controls. The increased plasma P-selectin and beta TG in patients with thrombotic disorders were likely to be the result of in vivo platelet and endothelial cell damage or activation. We also found that avoidance of veno-occlusion and other tedious measures customarily taken during blood collection and sample preparation to prevent in vitro platelet activation did not affect plasma P-selectin assay results. In addition, plasma P-selectin levels were not influenced by the presence of renal failure or heparin administration. These results indicate that plasma P-selectin may be a useful new marker for thrombotic diseases.

Adult↗

PubMed1994

Structural basis for the extracellular retention of PDGF A-chain using a synthetic peptide corresponding to exon 6.

Alternative splicing of PDGF A-chain exon 6 yields two species that differ by a highly conserved cationic carboxyl-terminus consisting of 18 amino acid residues (A194-211). Previous findings have demonstrated that a synthetic peptide representing A194-211 binds to cultured cells and interferes with the binding and biological activity of several polypeptide growth factors. We now demonstrate that the peptide, which binds to the extracellular matrix in a specific and glycosaminoglycan-dependent manner, can also inhibit the binding of basic FGF to the matrix. The cellular retention signal encoded by exon 6 accounts for differences in the mitogenic responsiveness to conditioned media from Chinese hamster ovary cells transfected with the cDNA for the long and short splice forms of the PDGF A-chain. That the PDGF A-chain exon 6 product may share anionic binding sites with a matrix-resident polypeptide growth factor in the extracellular matrix suggests a role for A194-211 in the storage of PDGF bearing this sequence.

Amino Acid Sequence↗

PubMed1994

Antiphospholipid antibodies and thrombosis.

Antiphospholipid antibodies are a diverse group of immunoglobulins initially thought to have specificity to phospholipid epitopes. It is apparent that autoimmune anticardiolipin antibodies require a serum cofactor beta-2-glycoprotein I (beta 2GPI) for their binding to phospholipids. Lupus anticoagulant also may bind to phospholipids by beta 2GPI or by prothrombin. The description of binding to protein-phospholipid epitopes may explain several perplexing features of these antibodies both in vitro and in vivo. Antiphospholipid antibodies have a well-established association with clinical disease--in particular thrombosis, thrombocytopenia and recurrent fetal loss. The mechanism of the predisposition to thrombosis seen with these antibodies is poorly understood. It has been suggested that they may cause endothelial dysfunction by causing increased tissue factor expression, by inhibiting prostacyclin secretion or by inhibiting fibrinolysis. Various platelet-activating activities have also been described. The evidence that antiphospholipid antibodies promote thrombosis by effects on endothelium or platelets is inconclusive. Inhibition of protein C activation, or of activated protein C action, has been demonstrated in vitro. A poor correlation between thrombosis in vivo and these inhibitory effects has been found. Beta-2-glycoprotein I has been identified as a cofactor for binding to phospholipid by thrombogenic anticardiolipin antibodies. That beta 2GPI may be a natural anticoagulant of importance remains to be proved. Inhibition by antiphospholipid antibodies of this anticoagulant function could explain the propensity to thrombosis seen in association with these antibodies.

Antibodies, Anticardiolipin↗

PubMed1993

Thrombospondin 1 is a tight-binding competitive inhibitor of neutrophil cathepsin G. Determination of the kinetic mechanism of inhibition and localization of cathepsin G binding to the thrombospondin 1 type 3 repeats.

Thrombospondin 1 was recently shown to bind to and inhibit the activity of neutrophil elastase (Hogg, P. J., Owensby, D. A., Mosher, D. F., Misenheimer, T. M., and Chesterman, C. N. (1993) J. Biol. Chem. 268, 7139-7146). This finding led us to question whether thrombospondin 1 also binds and inhibits the other major serine proteinase of neutrophils, cathepsin G. In a competitive binding assay, cathepsin G bound to thrombospondin 1 reversibly and saturably with a dissociation constant in the low nanomolar range. The kinetic mechanism of inhibition of cathepsin G activity by thrombospondin 1 was determined using the synthetic cathepsin G substrate, Suc-Ala-Ala-Pro-Phe-p-nitroanilide, and is consistent with hyperbolic tight-binding inhibition in which thrombospondin 1 binds cathepsin G and the Michaelis cathepsin G-substrate complex and weakens, but does not abolish, the efficiency of hydrolysis of Suc-Ala-Ala-Pro-Phe-p-nitroanilide. In the presence of 2 mM calcium ions, 2.9 +/- 0.4 mol of cathepsin G interacted with 1 mol of thrombospondin 1 trimer with a site-binding constant of 7.0 +/- 3.5 nM, which reduced the efficiency of hydrolysis of Suc-Ala-Ala-Pro-Phe-p-nitroanilide 8.5 +/- 1.4-fold. A lower limit for the on rate constant of 5 x 10(6) M-1 S-1 was established. The affinity of binding and stoichiometry for the interaction between cathepsin G and thrombospondin 1 was enhanced in the absence of calcium ions. In the presence of EDTA, 5.3 +/- 0.5 mol of cathepsin G interacted with 1 mol of thrombospondin 1 with a site-binding constant of 2.1 +/- 1.6 nM, implying the existence of two binding sites for cathepsin G on each subunit of thrombospondin 1, one or both of which is variably exposed and sensitive to calcium ions. Thrombospondin 1 protected fibronectin from cleavage by cathepsin G and blocked cathepsin G-mediated platelet aggregation. In summary, the binding of cathepsin G to thrombospondin 1 is tight, reversible, and close enough to the active site of cathepsin G to perturb the interactions of a small synthetic substrate and exclude a macromolecular protein substrate and platelets. Using defined proteolytic fragments and different conformers of thrombospondin 1, the binding sites for cathepsin G have been localized to the thrombospondin 1 type 3 repeats.

Amides↗

PubMed1993

Anticardiolipin antibodies block the inhibition by beta 2-glycoprotein I of the factor Xa generating activity of platelets.

Antiphospholipid antibodies, defined either by lupus anticoagulant (LA) activity or positive anticardiolipin immunoabsorbent assay (ACA) are associated with a predisposition to thromboses, recurrent fetal loss or thrombocytopenia. The mechanisms for these predispositions remain undefined. We have enriched immunoglobulin fractions from two patient plasmas to obtain antibodies with LA activity but no ACA, or conversely, with ACA positivity but no LA, in order to investigate in vitro characteristics which might explain a thrombotic propensity. beta 2-glycoprotein I (beta 2-GPI), the plasma cofactor required for ACA binding to negatively charged phospholipid, has previously been shown to inhibit prothrombinase generation in the presence of activated platelets (8). We now report that beta 2-GPI, at physiological concentrations, inhibits the generation of factor Xa in the presence of activated gel-filtered platelets. Further, ACA interferes with this inhibition, resulting in protracted, unopposed factor Xa generation. This interference with beta 2-GPI, a natural anticoagulant component of plasma, is potentially prothrombotic. LA immunoglobulins behave differently and inhibit factor Xa generation in a manner similar to beta 2-GPI. These findings provide the basis for a previously unsuspected mechanism for thrombosis in patients with aPL.

Adult↗

PubMed1993

Thrombospondin is a tight-binding competitive inhibitor of neutrophil elastase.

Thrombospondin, a glycoprotein of three identical disulfide-bonded subunits, is a constituent of platelet alpha-granules and a variety of normal and transformed cells and binds to cell surfaces and becomes incorporated into extracellular matrix. It has been implicated in processes such as wound healing and tumor growth and metastasis. In addition, thrombospondin was shown recently to be an inhibitor of the fibrinolytic enzyme, plasmin. In the cause of studying the effects of thrombospondin on other serine proteinases, we found that thrombospondin binds neutrophil elastase in an active-site-dependent manner and competitively inhibits the activity of the enzyme. In a competitive binding assay, neutrophil elastase bound to thrombospondin with a dissociation constant of 17 +/- 7 nM, expressed per mole of thrombospondin trimer, or 52 +/- 20 nM, expressed per mole of thrombospondin subunit. In kinetic studies of the inhibition of the amidolytic activity of neutrophil elastase by thrombospondin, 2.7 +/- 0.3 mol of elastase interacted with 1 mol of thrombospondin trimer with a site-binding constant of 57 +/- 13 nM. Lower limits for the on rate constant of 5 x 10(6) M-1 s-1 and off rate constant of 0.27 s-1 were established. Affinity of binding of neutrophil elastase to thrombospondin was sensitive to ionic strength and calcium ions. Thrombospondin was cleaved by neutrophil elastase, but the site(s) of the limited cleavage are independent of the competitive inhibition of elastase activity by thrombospondin. Neutrophil elastase inactivated with phenylmethylsulfonyl fluoride did not compete with active elastase for binding to thrombospondin, implying that a functional active site is important for the interaction of elastase with thrombospondin. Thrombospondin protected fibronectin from cleavage by neutrophil elastase. In summary, the binding of neutrophil elastase to thrombospondin is tight, reversible, and close enough to the active site of elastase to exclude small synthetic tripeptidyl p-nitroanilide substrates and macromolecular protein substrates. Two potential reactive centers that may be involved in binding elastase have been identified in the calcium-binding type 3 domains of thrombospondin. Neutrophil elastase is the enzyme primarily responsible for degrading and solubilizing connective tissue during inflammatory processes. These findings suggest a previously unsuspected mechanism for regulation of elastase activity at inflammatory sites.

Amino Acid Sequence↗

PubMed1993

Uses of heparin.

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Drug Administration Schedule↗

PubMed1993

Beta 2-glycoprotein I is a requirement for anticardiolipin antibodies binding to activated platelets: differences with lupus anticoagulants.

Antiphospholipid (aPL) antibodies are of major interest not only because the lupus anticoagulant (LA) causes an inhibition of in vitro blood coagulation, but also because the presence of aPL antibodies confers a risk of thrombosis. The inhibition of in vitro phospholipid-dependent coagulation (LA) is thought to be caused by the binding of LA to procoagulant phospholipid surfaces, thus impeding the clotting process. Another class of aPL antibodies are those originally described to be directed against negatively charged phospholipids, in particular cardiolipin (ACA). ACA are usually directed against a complex antigen consisting of negatively charged phospholipid and a plasma protein, beta 2-glycoprotein I (beta 2-GPI). Further, there is antibody heterogeneity even within individual patients so that ACA and LA are separable using physicochemical techniques such as ion exchange chromatography and chromatofocusing. Using such techniques we have enriched Ig fractions for LA and ACA from two patient plasmas. The majority of Ig with LA activity had a pI of 7.2 to 7.3 whereas ACA had a pI of 5.0 to 5.2. Using these enriched fractions labeled with [125I]-iodine we have shown that LA binds to platelets in a specific and saturable manner. Binding is dependent on thrombin activation. [125I]-ACA behaves differently. Like LA, binding is specific and dependent on thrombin activation but in this case requires the presence of beta 2-GPI. ACA, in the presence of beta 2-GPI, competes for binding with LA suggesting the same or contiguous site. There is no cross-reactivity of these antibodies with GPIIb/IIIa and the most likely binding site is phospholipid. In neither case does LA nor ACA have an effect on thrombin-induced release of serotonin or beta-thromboglobulin nor do they affect platelet aggregation induced by a number of agonists. This antibody binding may play an etiological role in thrombocytopenia associated with aPL, but does not explain thrombosis on the basis of hyperaggregability or increased platelet release.

Adult↗

PubMed1993

Increased expression of platelet IgG Fc receptors in immune heparin-induced thrombocytopenia.

Our previous finding that heparin-dependent antibodies in heparin-induced thrombocytopenia (HIT) bind to platelets via platelet IgG Fc receptors (FcRs) prompted this study. Platelet FcRs in 16 patients with HIT, 23 control patients, and 42 normal subjects were studied. Patients with HIT had substantially increased platelet FcRs during the acute illness. Those who suffered serious thrombotic complications or died shortly after diagnosis had significantly more FcRs per platelet than those with milder disease. Consistent with their increased FcRs, platelets of patients with HIT showed increased aggregation reactivity to aggregated IgG and heparin-dependent antibodies. Platelet FcRs in patients with HIT remained elevated for 1 to 3 months after the acute illness then stabilized to a mean value not significantly different from either control group. The increased expression of FcRs on HIT platelets and their increased reactivity to heparin-dependent antibodies may contribute to the pathogenesis of thrombocytopenia and thrombosis in HIT.

Aged↗

PubMed1993

Hematologic correlates of left atrial spontaneous echo contrast and thromboembolism in nonvalvular atrial fibrillation.

OBJECTIVES: This study examined the relation between left atrial spontaneous echo contrast, hematologic variables and thrombo-embolism in patients with nonvalvular atrial fibrillation. BACKGROUND: Left atrial spontaneous echo contrast is associated with left atrial stasis and thromboembolism in patients with nonvalvular atrial fibrillation. However, its hematologic determinants in patients with nonvalvular atrial fibrillation are unknown. METHODS: Clinical, hematologic and echocardiographic variables were prospectively measured in 135 consecutive patients with nonvalvular atrial fibrillation undergoing transesophageal echocardiography. RESULTS: Patients with left atrial spontaneous echo contrast (n = 74, 55%) had an increased fibrinogen concentration (p = 0.029), platelet count (p = 0.045), hematocrit (p = NS) and left atrial dimension (p = 0.005). Multivariate analysis showed that left atrial spontaneous echo contrast was independently related to hematocrit (odds ratio = 2.24, p = 0.002), fibrinogen concentration (odds ratio = 2.08, p = 0.008) and left atrial dimension (odds ratio = 1.90, p = 0.004) but not platelet count. It was also associated with left atrial thrombus (n = 15, p = 0.001) and with recent embolism (n = 40, p < 0.001). In 40 clinically stable outpatients without previous embolism, left atrial spontaneous echo contrast was significantly related to hematocrit (p = 0.005), fibrinogen concentration (p = 0.035) and left atrial dimension (p = 0.029) but not to coagulation factor VII, D-dimer, erythrocyte sedimentation rate, platelet count, plasma beta-thromboglobulin, plasma glycocalicin or glycocalicin index. CONCLUSIONS: Left atrial spontaneous echo contrast in patients with nonvalvular atrial fibrillation is independently related to hematocrit, fibrinogen concentration and left atrial dimension, indicating a relatively hypercoagulable state in addition to stasis. These findings support the hypothesis that left atrial spontaneous echo contrast is due to erythrocyte aggregation. Hematologic factors may contribute to its association with thromboembolism.

Aged↗

PubMed1993

Accumulation of PDGF B and cell-binding forms of PDGF A in the extracellular matrix.

PDGF is a powerful mitogen initially identified within platelets, but also shown to be produced by a wide variety of cell types. PDGF is encoded on two separate genes. These give rise to three polypeptides, PDGF B and two forms of PDGF A (SA and LA), resulting from alternative splicing of the PDGF A gene primary transcript. We report that in CHO cells transfected with PDGF gene constructs and producing moderate levels of PDGF homodimers, much of the PDGF LA and B produced, but little if any SA, is found in the matrix laid down beneath the cells. Immunoreactive PDGF in cells, and in matrix below expressing cells, was visualized by laser confocal microscopy. Western blotting of protein in matrix extracts, cell extracts, and secreted into the growth medium was used to demonstrate that the range of PDGF A polypeptides seen in the matrix was overlapping with those reported previously to be cell associated in cell types such as NIH3T3 and COS 7. This suggests that attachment to matrix or cell surface may be alternative fates for these polypeptides, with fate dependent on the characteristics of the producing cells. Immunoreactive PDGF A and B could be partially released by incubation of matrix material with heparin but not with other glycosaminoglycans. Digestion of matrix with chondroitin ABC lyase but not heparitinase or collagenase displaced some PDGF from its attachment sites. The results indicate attachment of PDGF to matrix proteoglycans, at least partly through the glycosaminoglycan moieties, and perhaps to additional components. The significance of matrix deposition for PDGF action is discussed.

Animals↗

PubMed1993

Identification of IgG Fc receptor type II on human megakaryoblastic cell lines (MEG-01 and UT-7).

In this study, Fc gamma receptors (Fc gamma R) on the cells of human megakaryoblastic leukaemia cell lines (Meg-01 and UT-7) were investigated. Binding of the anti-Fc gamma RII monoclonal antibody (MoAb) IV. 3 but not anti-Fc gamma RI MoAb (32.2), anti-Fc gamma RIII MoAb (3G8) nor control murine IgGs to Meg-01 and UT-7 cells was demonstrated by immunocytochemical staining and flow cytometric analysis. There was specific and saturable binding of 125I-labelled IV.3 Fab fragments to the megakaryoblasts but no binding of 125I-labelled 32.2 Fab nor 3G8 Fab fragments. MoAb IV.3 immunoprecipitated a 40 kD protein (Fc gamma RII) from detergent cell lysates of surface radiolabelled Meg-01 and UT-7 cells. More importantly, IV.3 Fab almost completely inhibited the binding of 125I-labelled human aggregated IgG to the Meg-01 cells. In contrast, neither 32.2 Fab, 3G8 Fab nor Fab fragments of the control murine IgGs were inhibitory. These results indicate that only Fc gamma R Type II is expressed on megakaryoblasts in these two cell lines.

Antigen-Antibody Reactions↗

PubMed1993

Clinical analysis of 125 patients with the lupus anticoagulant.

A retrospective analysis of 125 consecutive lupus anticoagulant (LA) positive patients and 125 age, sex matched lupus anticoagulant negative controls is reported with the aims of defining further the clinical spectrum of disease, determining at-risk subgroups and management strategies. There was no significant difference in the incidence or pattern of complications in those with systemic lupus erythematosus (SLE) and related disorders, and those without SLE. Venous thromboembolism, immune thrombocytopenia, foetal loss, depression and hypertension were statistically more common in the LA group than in the control group. In contrast to previous reports, children aged ten years or less with the LA developed significantly more complications than controls. Patients with the LA secondary to drugs also developed complications, a finding which is also at variance with previous reports. There was a significant difference in the outcome of arterial disease (p < 0.04) and venous thromboembolism (p < 0.001) when long term anticoagulation was part of the treatment regimen.

Adolescent↗

PubMed1992

Effects of heparin and endothelial cell growth supplement on haemostatic functions of vascular endothelium.

Heparin in combination with endothelial cell growth factor (ECGF) affects physiological responses and growth of human umbilical vein endothelial cells (HUVEC). We have examined the effect of heparin, crude ECGF (endothelial cell growth supplement [ECGS]), or both on the basal and thrombin challenged output of metabolites by HUVEC. The supernatant and/or cell lysate was assayed for released prostacyclin, von Willebrand factor, tissue plasminogen activator, plasminogen activator inhibitor and thrombospondin. Heparin modified release of all these metabolites when in combination with ECGS, and in general these responses were the opposite of those generated by inflammatory mediators such as interleukin-1. It has been postulated that heparin acts by potentiating the effect of ECGF, but heparin inhibited thrombospondin release and enhanced that of von Willebrand factor in the absence of ECGS, while ECGS alone inhibited release of plasminogen activator inhibitor. Thus, under our experimental conditions it would appear that heparin and crude ECGF can affect HUVEC independently of one another.

6-Ketoprostaglandin F1 alpha↗

PubMed1992

Synthetic peptides representing the alternatively spliced exon of the platelet-derived growth factor A-chain modulate mitogenesis stimulated by normal human serum and several growth factors.

Alternative splicing results in two distinct forms of the platelet-derived growth factor (PDGF) A-chain that differ by a hydrophilic carboxyl terminus consisting of 18 amino acids (A194-211). The functional significance of this region is unclear. Previous results indicate that a radioiodinated tyrosinated synthetic peptide corresponding to A194-211 binds specifically, saturably, and with low affinity to a large population of sites on Balb/c 3T3 and several other cell lines (Khachigian, L. M., Owensby, D. A., and Chesterman, C. N. (1992) J. Biol. Chem. 267, 1660-1666). In this paper, we report that (Y)A194-211 and A194-211 can modulate the cellular proliferative response to normal human serum and several individual polypeptide growth factors as a consequence. When these peptides were coincubated separately with whole serum, PDGF, epidermal growth factor, or fibroblast growth factor, DNA synthesis was inhibited in a dose-dependent fashion and maximally by 200 microM peptide. In addition, both peptides could attenuate the stimulation of cell division by serum and PDGF. Peptides with similar charge or length failed to modify the level of proliferation. These observations were not due to cytotoxicity. Synthetic peptides such as (Y)A194-211 and A194-211 that influence cellular proliferation may be useful in modulating the cellular response to selected growth factors.

3T3 Cells↗

PubMed1992

A tyrosinated peptide representing the alternatively spliced exon of the platelet-derived growth factor A-chain binds specifically to cultured cells and interferes with binding of several growth factors.

Platelet-derived growth factor (PDGF) is a potent mitogen for cells of mesenchymal origin. Alternative exon splicing is responsible for two forms of the PDGF A-chain which differ at the carboxyl terminus by a highly basic region consisting of 18 amino acids. To clarify the function of the region, we synthesized an octadecapeptide corresponding to this extension (A194-211), incorporated a tyrosine residue at the amino terminus, and used the radioiodinated construct in binding studies with Balb/c3T3 cells and a variety of human cell lines. 125I-(Y)A194-211 bound specifically, reversibly, saturably, and with low affinity to a large population of binding sites on these cells. In addition, (Y)A194-211 markedly reduced the binding of its parent protein, 125I-PDGF-AAL, to its receptor. (Y)A194-211 also attenuated the binding of epidermal growth factor and several other isoforms of PDGF, but did not interfere with the binding of transferrin to its receptor. These observations were not due to competitive binding of peptide directly to known receptors for the respective growth factors, but was likely due to interaction of (Y)A194-211 with extracellular glycosaminoglycan. Thus, A194-211 may represent an additional heparin binding domain on mature PDGF-AAL, and as an isolate, is capable of modulating interactions between several potent growth factors and their respective receptors.

3T3 Cells↗