Binding and endocytosis of heparin by human endothelial cells in culture

Binding of heparin and low molecular weight heparin fragments (CY 222, Mr range 1500-8000) to human vascular endothelial cells was studied. Primary culture of human umbilical vein endothelial cells and either 125I or 3H-labeled heparin or [125I]CY 222 were used. Slow, saturable and specific binding was found. No other tested glycosaminoglycan, excepting a highly sulfated heparan fraction, was able to compete for heparin binding. Two groups of binding sites for [3H]heparin could be distinguished: one with high affinity (Kd = 0.12 microM) and another with lower affinity (Kd = 1.37 microM) and a relative large capacity of binding (1.16 X 10(7) molecules/cell) was calculated. The Kd for unlabeled heparin, as calculated from competition experiments, was 0.23 microM. Much lower affinity was calculated for unlabeled low molecular weight heparin fragments CY 222 (Kd = 4.3 microM) from competition experiments with [125I]CY 222. The binding reversibility was only partial for unfractionated heparin. Even by chasing with unlabeled compound, a fraction of 25-30% was not dissociable from endothelial cells. This fraction was much lower if incubation was carried out at 4 degrees C. The addition of basic proteins (histones) to the incubation medium greatly enhanced the undissociable binding at 37 degrees C, but not at 4 degrees C. The undissociable fraction of heparin was not available to degradation by purified microbial heparinase. These results suggest that a fraction of bound heparin is internalized by the vascular endothelium.     

Endothelial binding sites for heparin. Specificity and role in heparin neutralization.

The specificity of endothelial binding sites for heparin was investigated with heparin fractions and fragments differing in their Mr, charge density and affinity for antithrombin III, as well as with heparinoids and other anionic polyelectrolytes (polystyrene sulphonates). The affinity for endothelial cells was estimated by determining I50 values in competition experiments with 125I-heparin. We found that affinity for endothelial cells increases as a function of Mr and charge density (degree of sulphation). Binding sites are not specific receptors for heparin. Other anionic polyelectrolytes, such as pentosan polysulphates and polystyrene sulphonates, competed with heparin for binding to endothelial cells. Fractions of standard heparin with high affinity for antithrombin III also had greater affinity for endothelium. However, these two properties of heparin (affinity for antithrombin III and affinity for endothelial cells) could be dissociated. Oversulphated heparins and oversulphated low-Mr heparin fragments had lower anticoagulant activity and higher affinity for endothelial cells than did their parent compounds. Synthetic pentasaccharides, bearing the minimal sequence for binding to antithrombin III, did not bind to endothelial cells. Binding to endothelial cells involved partial neutralization of heparin. Bound heparin exhibited only 5% and 7% of antifactor IIa and antifactor Xa specific activity, respectively. In the presence of 200 nM-antithrombin III, and in the absence of free heparin, a limited fraction (approx. 30%) of bound heparin was displaced from endothelial cells during a 1 h incubation period. These data suggested that a fraction of surface-bound heparin could represent a pool of anticoagulant.     

Urokinase binding and receptor identification in cultured endothelial cells.

Recombinant human single-chain urokinase (rscu-PA), two-chain urokinase (tcu-PA), and diisopropyl-fluorophosphate-treated tcu-PA (DFP-tcu-PA) bound to cultured human and porcine endothelial cells in a rapid, saturable, dose-dependent and reversible manner. Analysis of specific binding results in cultured human umbilical vein endothelial cells (HUVECs) gave the following estimated values for Kd and Bmax: 0.57 +/- 0.08 nM (mean +/- S.E.) and 188,000 +/- 18,000 sites/cell for 125I-labeled rscu-PA; 0.54 +/- 0.10 nM and 132,000 +/- 23,900 sites/cells for 125I-labeled tcu-PA; 0.89 +/- 0.14 nM and 143,000 +/- 30,300 sites/cell for 125I-labeled DFP-tcu-PA, respectively. Values for Kd were similar for primary and subcultured (six passages) HUVECs, but Bmax values were lower in subcultured HUVECs. Similar Kd values were found in cultured porcine endothelial cells; however, Bmax values varied depending on the endothelial cell type. All 125I-labeled urokinase forms yielded similar cross-linked approximately 110-kDa ligand-receptor complexes with cultured HUVECs, and 125I-labeled DFP-tcu-PA bound to a single major approximately 55-kDa protein in whole-cell lysates (ligand blotting/autoradiography), suggesting the presence of a single major approximately 55-kDa urokinase receptor in cultured HUVECs. The approximately 55-kDa urokinase receptor, isolated from several separate batches of cultured HUVECs (3-5 micrograms of protein, approximately 1 x 10(9) cells), by ligand affinity chromatography, exhibited the following properties: retained biologic activity as evidenced by its ability to bind 125I-labeled rscu-PA by ligand blotting/autoradiography and formation of a cross-linked 125I-labeled approximately 110-kDa rscu-PA-receptor complex; single-chain approximately 55-kDa protein, following reduction; complete conversion to and formation of a single major deglycosylated approximately 35-kDa protein, following treatment with N-glycanase.     

Physical-chemical interaction of heparin and human plasma low-density lipoproteins

This study characterizes the physical-chemical interactions of heparin with human plasma low-density lipoproteins (LDL). A high reactive heparin (HRH) specific for the surface determinants of LDL was isolated by chromatography of commercial bovine lung heparin on LDL immobilized to AffiGel-10. HRH was derivatized with fluoresceinamine and repurified by affinity chromatography, and its interaction with LDL in solution was monitored by steady-state fluorescence polarization. Binding of LDL to fluoresceinamine-labeled HRH (FL . HRH) was saturable, reversible, and specific; HRH stoichiometrically displaced FL . HRH from the soluble complex, and acetylation of lysine residues on LDL blocked heparin binding. Titration of FL.HRH with excess LDL yielded soluble complexes with two LDL molecules per heparin chain (Mr 13,000) characterized by an apparent Kd of 1 microM. Titration of LDL with excess HRH resulted in two classes of heparin binding with two and five heparin molecules bound per LDL and apparent Kd values of 1 and 10 microM, respectively. At physiological pH and ionic strength, the soluble HRH-LDL complexes were maximally precipitated with 20-50 mM Ca2+. Insoluble complexes contained 2-10 HRH molecules per LDL with the final product stoichiometry dependent on the ratio of the reactants. The affinity of HRH for LDL in the insoluble complexes was estimated between 1 and 10 microM. Insoluble LDL-heparin complexes were readily dissociated with 1.0 M NaCl, and their formation was prevented by acetylation of the lysine residues on LDL.     

Heparin stimulates fibroblasts growth induced by platelet derived growth factor

We have demonstrated that 125I unfractionated heparin binds to cultured human skin fibroblasts with a Kd of 1.16 10(-8) M and is internalized partly. A low molecular weight heparin fraction (PK 10169) competed (50%) with 125I unfractionated heparin, but to a lesser extent than cold unfractionated heparin (90%). When the cell proliferation was induced by pure PDGF, heparin markedly potentiated the fibroblast growth. Similar stimulation was observed when the growth was induced by FGF or EGF. Low molecular weight heparin enhanced the fibroblast proliferation induced by PDGF but to a lesser extent than unfractionated heparin. Chondroitin sulfate has no effect. PDGF did not modify the heparin binding on fibroblast cultures either at 4 degrees C or 37 degrees C and did not alter the process of heparin internalization. PDGF binding to the cultured fibroblast (Kd 10.1 +/- 3.4 10(-10) M) was not modified by the presence of heparin when studied at 4 degrees C or 37 degrees C.     

Demonstration of specific binding sites for pituitary adenylate cyclase activating polypeptide (PACAP) in rat astrocytes

The high and low affinity binding sites for PACAP were identified in rat astrocytes using [125I]PACAP27 as the labeled ligand. Scatchard analysis of displacement of the bound tracer by unlabeled PACAP27 indicated the existence of two classes of binding sites, with the dissociation constant (Kd) = 1.22 +/- 0.4 nM, the binding maximal capacity (Bmax) = 821 +/- 218 fmols/mg protein for the high affinity binding site, and Kd = 0.59 +/- 0.06 microM, Bmax = 563 +/- 12 pmols/mg protein for the low affinity binding site, respectively. The specificity of [125I]PACAP27 binding was tested using PACAP38 and peptides structurally related to PACAP, such as VIP, GHRF, PHI, secretin and glucagon. PACAP38 completely displaced the binding of [125I]PACAP27 and Scatchard analysis also indicated the presence of two classes of binding sites with similar Kd and Bmax to those for PACAP27. VIP and GHRF competed with [125I]PACAP27, but to a much lesser extent than unlabeled PACAP27 in binding. Other peptides tested did not displace the binding of [125I]PACAP27 at 10(-6) M.     

Characterization of receptors for vasoactive intestinal peptide solubilized from the lung

The zwitterionic detergent CHAPS was used to solubilize functional receptors for vasoactive intestinal peptide (VIP) from guinea pig lung. The solubilized receptors were resolved by high performance gel filtration in 3 mM CHAPS into two active fractions with apparent Stokes radii of 5.9 +/- 0.1 and 2.3 +/- 0.1 nm. The binding of 125I-VIP to the two receptor fractions was time-dependent, reversible, and saturable. Trypsin destroyed the binding activity of the receptor fractions, indicating their proteinic nature. Unlabeled VIP competitively displaced the binding of 125I-VIP to the 5.9-nm fraction (IC50 = 240 pM) and the 2.3-nm fraction (IC50 = 1.2 microM). Scatchard analysis indicated a single class of binding sites in each receptor fraction, with Kd values 300 pM and 0.97 microM for the 5.9- and 2.3-nm Stokes radii fractions, respectively. When the high affinity, 5.9-nm Stokes radius fraction was rechromatographed in 9 nM CHAPS, 46% of the binding activity eluted in the low affinity, 2.3-nm Stokes radius fraction, indicating that the latter is a product of dissociation of the high affinity receptor complex. GTP inhibited the binding of 125I-VIP to the high affinity complex but not the low affinity species. Scatchard plots of VIP binding by the high affinity receptors treated with GTP suggested the presence of two distinct binding sites (Kd 4.4 and 153 nM), compared to a single binding site (Kd = 0.3 nM) obtained in untreated receptors. The nonhydrolyzable GTP analog, guanyl-5'-yl-imidodiphosphate, inhibited VIP binding by the high affinity receptor fraction with potency nearly equivalent to that of GTP. These observations suggest that GTP-binding regulatory proteins are functionally coupled to the VIP-binding subunit in the high affinity receptor complex. The peptide specificity characteristics of the two receptor fractions were different. Peptide histidine isoleucine and growth hormone releasing factor, peptides homologous to VIP, were 87.5- and 22.9-fold less potent than VIP in displacing 125I-VIP binding by the high affinity receptor complex, respectively. On the other hand, growth hormone-releasing factor was more potent (22.7-fold) and peptide histidine isoleucine was less potent (31.3-fold) than VIP in displacing the binding by the low affinity species.     

Characterization of plasminogen binding to human capillary and arterial endothelial cells.

Phenotypic diversity of endothelial cells that line the various vascular spaces has been well established. However, it is not known if biochemical differences also exist, particularly in the numbers of receptors for plasma proteins. Equilibrium binding techniques were used to assess potential differences in the binding of 125I-labelled plasminogen to cultured human umbilical arterial endothelial cells and capillary endothelium, as compared with umbilical venous cells. The kinetic behaviour of plasminogen binding to all three types of cells was similar, with optimal binding occurring between 20 and 30 min of incubation. Binding of plasminogen to arterial, capillary, and venous cells was concentration dependent and reversible upon addition to excess unlabelled plasminogen. Scatchard analyses showed that artery, capillary, and venous endothelial cells all possess low affinity sites for plasminogen with Kd values of 0.30 +/- 0.07, 0.40 +/- 0.06, and 0.40 +/- 0.08 microM, respectively. Vein cells also possess an additional higher affinity binding site with a Kd of 0.07 +/- 0.01 microM, exhibiting a 6-fold greater affinity for plasminogen than the lower affinity sites on capillary and arterial endothelial cells. Assuming a stoichiometry of 1:1 for binding, the data indicate that arterial and capillary endothelial cells contain approximately 4.2 (+/- 0.9) x 10(6) and 4.1 (+/- 0.6) x 10(6) plasminogen receptors per cell. Venous cells contain both low and high density binding sites with 6.2 (+/- 0.8) x 10(6) and 12.4 (+/- 2.4) x 10(6) sites per endothelial cell. The presence of a higher affinity site on vein cells, but not on artery or capillary cells, may signal functional differences relating to fibrinolytic activity on the surface of these cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presence of high affinity receptor for interleukin-1 (IL-1) on cultured porcine thyroid cells

Using 125I-interleukin-1 beta (125I-IL-1 beta) as a ligand, a specific receptor of high affinity dissociation constant (1.1 +/- 0.2 x 10(-10) M) with binding sites (350 +/- 40/cell) for interleukin-1 beta (IL-1 beta) has been demonstrated on cultured porcine thyroid cells. IL-1 alpha almost equally cross-reacted with the receptor (Kd = 1.2 +/- 0.3 x 10(-10) M and 350 +/- 50 binding sites/cell). TSH, IL-2 and other peptide hormones did not inhibit the binding of 125I-IL-1 beta to thyroid cells. Crosslinking study revealed a major band (approximately 95 kD) with a corrected molecular mass of approximately 78 kD. Moreover, both IL-1 beta and IL-1 alpha stimulated prostaglandin E2 production of cultured porcine thyroid cells, although the potency of IL-1 alpha was slightly greater than that of IL-1 beta. These results suggest that IL-1 may be involved in the regulation of thyroid cell function.     

Biosyntheses of interstitial collagens and fibronectin by porcine aorta smooth muscle cells. Modulation by low-molecular-weight heparin fragments

The effect of low-molecular-weight heparin fragments (CY222) on the biosynthetic phenotype of porcine aortic smooth muscle cells (SMC) was investigated in vitro on overconfluent cell cultures. Addition of increasing concentrations of CY222 to the culture medium of early passage SMC resulted in a dose-dependent decrease of type III to type I collagen ratio without change in total collagen biosynthesis. In the same range of concentrations CY222 did not affect the biosynthesis of fibronectin. However, heparin fragments decreased the proportion of the freshly synthesized pericellular form of fibronectin with a concomitant increase of neosynthesized intracellular fibronectin, indicating an inhibitory effect of CY222 on fibronectin secretion. Our results demonstrate that the biosynthetic phenotype of SMC in vitro can be modulated by low-molecular-weight heparin fragments and confirm also that interactions between cells and extracellular matrix molecules can modify the biosynthetic pattern of mesenchymal cells.     

Thyrotropin binding to and adenylate cyclase activity of porcine thyroid plasma membranes.

Plasma membranes have been purified from porcine thyroid gland homogenate by discontinuous sucrose gradient centrifugation. The preparations contained specific binding sites for thyrotropin but not for luteinizing hormone or the beta subunits of thyrotropin and luteinizing hormone. Optimum conditions of 125I-labeled thyrotropin binding were pH 6.0-6.5 and 37 degrees C. Thyrotropin binding was reduced by divalent (Ca2+, Mg2+) and monovalent cations (Na+, K+, Li+), 50% inhibition being obtained at 10 mM and 50 mM respectively. Displacement curves of 125I-labeled bovine or porcine thyrotropin by the unlabeled hormone from three species was in the order of increasing concentrations (bovine greater than porcine greater than human) which is the order of decreasing biological activity of these hormone preparations in the assay in vivo in the mouse. The validity of the results was established by controlling that porcine membranes bound the native and the 125I-labeled hormones with equal affinity. A single type of high-affinity (Kd = 0.28 nM) binding sites was detected for bovine and porcine thyrotropins. In contrast, porcine plasma membranes bound human thyrotropin with a lower affinity (Kd = 70 nM). A good correlation was found at equilibrium and in the conditions of the cyclase assay, between receptor occupancy and adenylate cyclase activation for the three hormones.     

Binding and endocytosis of apo- and holo-lactoferrin by isolated rat hepatocytes.

We characterized binding and endocytosis of 125I-bovine lactoferrin by isolated rat hepatocytes. Iron-depleted (apo-Lf), approximately 30% saturated (Lf), and iron-saturated (holo-Lf) lactoferrin were used. At 4 degrees C, cells bound 125I-apo-Lf and 125I-holo-Lf with nearly identical apparent first order kinetics (t1/2 = approximately 42 min). Holo-Lf and apo-Lf competed with each other for binding. Hepatocytes bound lactoferrin optimally at pH greater than or equal to 7 but poorly at pH less than or equal to 6. Ca2+ (greater than or equal to 100 microM) enhanced Lf binding to cells, and holo-Lf remained monomeric with Ca2+ present as determined by gel filtration chromatography. With Ca2+, cells exhibited approximately 10(6) high affinity sites (Kd approximately 20 nM) and approximately 10(7) low affinity sites (Kd approximately 700 nM) for both apo- and holo-Lf. Without Ca2+, cells bound 125I-holo-Lf by the low affinity component only. EGTA and dextran sulfate together released greater than or equal to 90% 125I-Lf prebound at 4 degrees C, but individually removed separate populations of surface-bound 125I-Lf. Cells bound 125I-Lf in a Ca(2+)-dependent manner with dextran sulfate present. We conclude that the high affinity but not the low affinity sites require Ca2+; only the low affinity sites are dextran sulfate-sensitive. Neither transferrin nor asialo-orosomucoid blocked lactoferrin binding to hepatocytes. Some cationic proteins but not others inhibited lactoferrin binding. At 37 degrees C, hepatocytes endocytosed 125I-apo-Lf and 125I-holo-Lf similarly, and hyperosmolality (greater than 500 mmol/kg) blocked uptake by approximately 90%. These data support the proposal that hepatocytes regulate blood lactoferrin concentration by receptor-mediated endocytosis.     

Characterization of somatostatin binding sites in cytosolic fraction of rat intestinal mucosa

Specific binding sites for somatostatin have been characterized in cytosolic fraction of rat intestinal mucosa by using 125I-labelled Tyr11-somatostatin and a variety of physicochemical conditions. The binding depended on time, temperature and pH, and was reversible, saturable and specific. At apparent equilibrium, the specific binding of 125I-Tyr11-somatostatin was competitively inhibited by native somatostatin in the 1 nM-4 microM concentration range. Binding studies suggested the presence of two classes of binding sites: a class with high affinity (Kd = 0.07 microM) and low capacity (4.6 pmol/mg protein) and a class with low affinity (Kd = 1.05 microM) and high capacity (277 pmol/mg protein) at 25 degrees C. Somatostatin exhibited competitive inhibition of tracer binding, while neuropeptides such as neurotensin, substance P, Leu-enkephalin, and vasoactive intestinal peptide were ineffective. The presence of somatostatin binding sites in cytosolic fraction of intestinal mucosa, together with the known occurrence of somatostatin in D-cells and nerve endings in the small intestine, strongly suggest that this peptide may be involved in the physiology and physiopathology of intestinal epithelium.     

Acidic fibroblast growth factor is present in regenerating limb blastemas of axolotls and binds specifically to blastema tissues

The growth of regenerating limbs of amphibians depends upon proliferation of the blastema cells that accumulate beneath the epidermal cap. The epidermal cap is known to be mitogenic for the blastema cells. We have extracted a mitogenic activity from both the mesenchymal and epidermal (epidermal cap) components of cone stage blastemas which is retained on heparin-Sepharose and elutes with 1.15 M NaCl. This fraction stimulates neurite outgrowth of PC12 cells and [3H]thymidine incorporation into CCL 39 cells and is potentiated by heparin. The 2 M fraction was inactive. The heparin-Sepharose-purified growth factor cross-reacts with bovine acidic FGF polyclonal antibodies and shows a Mr of 16,000 on Western blots. Blastema membranes contain specific high affinity binding sites (Kd = 25 pM; capacity = 30 fmole/mg protein) and low affinity binding sites (Kd = 18 nM; capacity = 30 pmole/mg protein) for aFGF as revealed by Scatchard analysis. 125I-aFGF which is bound specifically by both the epidermal cap and mesenchyme of blastema frozen sections is displaced by an excess of unlabeled factor and inhibited by heparin. Heparinase treatment and 2 M NaCl washing which decreased the binding was fourfold more efficient for epidermal cap than for mesenchyme suggesting the presence of high affinity receptors in the latter tissue. The presence of aFGF (or a closely related molecule) in blastemas is consistent with our earlier results that showed stimulation of proliferation of cultured blastema cells by acidic or basic FGF or heparin alone. These results suggest the possibility that aFGF is stored in the epidermal cap during limb regeneration and that it stimulates the proliferation of the underlaying mesenchyme.     

Characterization of the binding of human tissue-type plasminogen activator to platelets

Cell surface binding sites for the constituent proteins of the fibrinolytic system may play a role in the localization and regulation of fibrinolysis. In the present study, specific binding of recombinant human tissue-type plasminogen activator (rt-PA) to human blood platelets was identified and characterized. 125I-labeled rt-PA was found to bind specifically, saturably, and reversibly to the surface of gel-filtered platelets, reaching equilibrium within 5 min at 22 degrees C. Scatchard analysis revealed a single class of binding sites. Unstimulated platelets bound 120,000 +/- 24,000 (mean +/- S.D.) molecules/platelet with an apparent Kd of 340 +/- 25 nM, whereas thrombin-stimulated platelets bound 290,000 +/- 32,000 molecules/platelet with an apparent Kd of 800 +/- 60 nM. Binding of 0.1 microM 125I-rt-PA was greater than 90% reversible by a 50-fold excess of unlabeled rt-PA. Binding was not inhibited by fibrinogen or single chain urokinase-type plasminogen activator, but plasminogen partially competed for binding of 125I-rt-PA to platelets (up to 40% displacement). These findings indicate that the platelet surface possesses a large number of specific, low affinity binding sites for t-PA and provide further evidence for the role of platelets in localization and regulation of fibrinolysis.     

Kinetics of radiolabelled (99mTc) heparin and low molecular weight heparin fractions CY 216, CY 222 in patients with uncomplicated myocardial infarction

Kinetics of plasmatic disappearance of radiolabelled (99mTc) heparin (Choay, Mr mean 15,000) and low molecular weight heparin (LMWH) fraction CY 216 (Choay, Mr mean 5,000) and CY 222 (Choay, Mr mean 4,000) was compared in 2 women and 8 men (aged 50-71, mean 65 years) with uncomplicated myocardial infarction. The three technetiated heparins were consecutively injected intravenously (67 nanomoles) to each of 10 patients, at intervals of 3-5 days, 14-28 days after acute cardiac onset. The plasma radioactivity was counted in blood samples collected within a period of 5 h. Radiolabelled heparin and LMWH fractions CY 216, CY 222 disappeared from plasma following a biexponential clearance curve with a fast and slow component reflecting the biodistribution (alpha) and elimination (beta) phase. The bioavailability values (AUC, t0.5 alpha, t0.5 beta) as well as distribution and elimination rates were similar for all three technetiated heparins. The bulk of injected 99mTc-heparin or LMWH fraction was rapidly distributed to the tissular compartment (t0.5 alpha = 13 min), whereas the radiocomplex remaining in the circulation was slowly eliminated with a half-time (t0.5 beta) of an average 320 min. Radioactivity eliminated from plasma was only partially (30-50%) excreted in the urine. The results indicate that after a low-dose intravenous administration LMWH fractions CY 216 and CY 222 maintain the pharmacokinetics properties of standard heparin, especially the rapid distribution to the tissular compartment.     

Galanin receptor and its ligands in the rat hippocampus

Receptors for the 29-amino-acid peptide, galanin, in membranes from the rat ventral hippocampus were examined using chloramine-T-iodinated porcine galanin as ligand. The equilibrium binding of 125I-galanin showed the presence of a high-affinity binding site (Kd = 1.91 +/- 0.40 nM). The concentration of the high-affinity-binding sites was 107 +/- 15 fmol/mg membrane protein. The on rate constant was estimated to be 2.6 +/- 0.1 M-1 min-1 at 37 degrees C. The affinity of rat galanin (differing in three amino acid residues from the porcine protein) was equal to that of porcine galanin. The 125I--galanin-binding site is a trypsin-sensitive membrane protein, which is heat-denaturated at 60 degrees C within 5 min. The effect of GTP and its analogs and of pertussis-toxin-catalyzed ADP-ribosylation on the binding of 125I-galanin suggest that the galanin receptor is coupled to an inhibitory G protein (Gi protein). 127I-galanin was shown to be a ligand with affinity equal to that of galanin in displacing 125I-galanin. The 125I-galanin-binding site in the ventral hippocampus recognizes as a ligand the tryptic fragments 1-20 and 21-29 of rat galanin and the synthetic fragments 12-29, 18-29 and 21-29 of porcine galanin. None of these afforded full inhibition of the binding of fragment 1-29 of 125I-galanin at a concentration of 1 microM.     

Evidence that binding to the carboxyl-terminal heparin-binding domain (Hep II) dominates the interaction between plasma fibronectin and heparin

We assessed the participation of the three known heparin-binding domains of PFn (Hep I, Hep II, Hep III) in their interaction with heparin by making a quantitative comparison of the fluid-phase heparin affinities of PFn and PFn fragments under physiologic pH and ionic strength conditions. Using a fluorescence polarization binding assay that employed a PFn affinity-purified fluorescein-labeled heparin preparation, we found that greater than 98% of the total PFn heparin-binding sites exhibit a Kd in the 118-217 nM range. We also identified a minor (less than 2%) class of binding sites exhibiting very high affinity (Kd approximately 1 nM) in PFn and the carboxyl-terminal 190/170 and 150/136 kDa PFn fragments. This latter activity probably reflects multivalent inter- or intramolecular heparin-binding activity. Amino-terminal PFn fragments containing Hep I (72 and 29 kDa) exhibited low affinity for heparin under physiologic buffer conditions (Kd approximately 30,000 mM). PFn fragments (190/170 and 150/136 kDa) containing both the carboxyl-terminal Hep II and central Hep III domains retained most of the heparin-binding activity of native PFn (Kd = 278-492 nM). The isolated Hep II domain (33-kDa fragment) exhibited appreciable, but somewhat lower (2-5-fold), heparin affinity compared to the 190/170-kDa PFn fragment. Heparin binding to the 100-kDa PFn fragment containing Hep III was barely detectable (Kd greater than 30,000 nM). From these observations, we conclude that PFn contains only one major functional heparin-binding site per subunit, Hep II, that dominates the interaction between heparin and PFn.     

Affinity and distribution of surface and intracellular hyaluronic acid receptors in isolated rat liver endothelial cells

125I-Hyaluronic acid (HA) uniquely modified only at the reducing end (Raja, R.H., LeBoeuf, R. D., Stone, G.W., and Weigel, P.H. (1984) Anal. Biochem. 139, 168-177) binds specifically to rat liver endothelial cells in suspension or in culture. About 67-85% of the HA binding sites in isolated cells in suspension and 50% in cultured cells were intracellular, since they were exposed after permeabilizing cells with digitonin. Specific 125I-HA binding at 4 degrees C varied from 60 to 80% for intact cells and from 70 to 90% for permeabilized cells. Freshly isolated permeabilized cells bound about 500,000 HA molecules/cell at saturation. Within 5 h of culture, however, total HA binding decreased to 250,000 molecules/cells and then remained constant for at least 36 h. Surface HA receptor activity was essentially the same on cultured cells or cells in suspension (approximately 10(5)/cell). Cultured cells had 1.8 x 10(5) fewer intracellular receptors/cell. The affinities of surface and intracellular receptors of cells in culture and in suspension were essentially the same. The average Kd, determined by equilibrium binding studies, was 5.8 +/- 2.8 x 10(-8) M (n = 12). Dissociation of bound 125I-HA from permeable cultured cells was rapid (t1/2 = 30.9 min;kappa off = 3.7 x 10(-4) s-1). A variety of carbohydrates had essentially identical effects on 125I-HA binding to surface or total cellular receptors in cells in culture or in suspension. Chondroitin sulfate and heparin competed almost as effectively as unlabeled HA for 125I-HA binding at 4 degrees C. Other saccharides including polygalacturonic acid, dextran, glucuronic acid, and N-acetylglucosamine competed poorly or not at all. We conclude that (i) the 125I-HA binding sites within liver endothelial cells are HA receptors, identical in affinity and specificity to those on the cell surface; (ii) the distribution of cellular HA receptors is similar to other receptor systems with about 50-80% being intracellular; (iii) the liver endothelial cell HA receptor recognizes several glycosaminoglycans; and (iv) the liver endothelial receptor is different in function and characteristics than the fibroblast HA receptor.