HEPARIN BINDING TO ENDOTHELIAL CELLS POTENTIATES THROMBINSTIMULATED PGI2 PRoDUCTION

1987 ◽  
Author(s):  
T Bårzu ◽  
P Molho ◽  
R Cariou ◽  
G Tobelem ◽  
J Caen
Keyword(s):  
Binding Sites ◽  
Umbilical Vein ◽  
Primary Cultures ◽  
Low Molecular Weight ◽  
Heparin Binding ◽  
Serum Free Medium ◽  
Free Medium ◽  
Specific Mechanism ◽  
High Affinity ◽  
Stimulation Of

We studied the consequences of hepari n (H) binding to endothel iaI cells (EC) on their basal and thrombin-stimulated PGI2 production. Primary cultures of human umbilical vein EC were incubated with different H concentrations, in serum free medium for 5 hrs. The amount of 6-keto-pGF1α was measured in the medium after 5 hrs with an enzyme-linked inmunoassay. At all concentrations used (0.75 to 75 μg/ml) H did not alter the 5-hour basal production of PGI2 (control, 10.1 α 1.4 ng/106 cells; H 15 μg/m1 ; 10.8 ± 2.7 ng/106 cells). Basal or thrombin (0.1 U/m])-rtimulated (10 min) pGI2 production was then determined using EC bearing only bound heparin. The low, unstimulated PGI2 release (0.412 ± 0.04 ng/106 cells) was not significantly changed in the presence of bound H, but the thrombin-stimulated release was potentiated.The KD for H binding to EC is 2.5 μg/ml. Thus at 3 μg/ml, half maximal saturation of binding sites and maximal potentiation of thrombin action were achieved. This concentration of bound H shifted the dose-response curve of thrombin induced PGI2 production to the left. Similar effect was obtained with half maximal saturating concentration of low molecular weight H (CY 222). Neither arachinodate nor LC4-induced PGI2 release were modified by H binding to EC, suggesting that potentiation is specific to thrombin. Since bound H was shown to not modify the high affinity thrombin binding to EC, the potentiating effect of bound H could related rather to interference with the specific mechanism of thrombin-stimulation of PGI2 production.

High Affinity Binding Sites for Activated Protein C and Protein C on Cultured Human Umbilical Vein Endothelial Cells

1994 ◽  
Vol 72 (03) ◽  
pp. 465-474 ◽  
Author(s):  
Neelesh Bangalore ◽  
William N Drohan ◽  
Carolyn L Orthner
Keyword(s):  
Binding Sites ◽  
Protein C ◽  
Umbilical Vein ◽  
Activated Protein C ◽  
Affinity Binding ◽  
Cell Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Gla Domain ◽  
Umbilical Vein Endothelial Cells

SummaryActivated protein C (APC) is an antithrombotic serine proteinase having anticoagulant, profibrinolytic and anti-inflammatory activities. Despite its potential clinical utility, relatively little is known about its clearance mechanisms. In the present study we have characterized the interaction of APC and its active site blocked forms with human umbilical vein endothelial cells (HUVEC). At 4° C 125I-APC bound to HUVEC in a specific, time dependent, saturable and reversible manner. Scatchard analysis of the binding isotherm demonstrated a Kd value of 6.8 nM and total number of binding sites per cell of 359,000. Similar binding isotherms were obtained using radiolabeled protein C (PC) zymogen as well as D-phe-pro-arg-chloromethylketone (PPACK) inhibited APC indicating that a functional active site was not required. Competition studies showed that the binding of APC, PPACK-APC and PC were mutually exclusive suggesting that they bound to the same site(s). Proteolytic removal of the N-terminal γ-carboxyglutamic acid (gla) domain of PC abolished its ability to compete indicating that the gla-domain was essential for cell binding. Surprisingly, APC binding to these cells appeared to be independent of protein S, a cofactor of APC generally thought to be required for its high affinity binding to cell surfaces. The identity of the cell binding site(s), for the most part, appeared to be distinct from other known APC ligands which are associated with cell membranes or extracellular matrix including phospholipid, thrombomodulin, factor V, plasminogen activator inhibitor type 1 (PAI-1) and heparin. Pretreatment of HUVEC with antifactor VIII antibody caused partial inhibition of 125I-APC binding indicating that factor VIII or a homolog accounted for ∼30% of APC binding. Studies of the properties of surface bound 125I-APC or 125I-PC and their fate at 4°C compared to 37 °C were consistent with association of ∼25% of the initially bound radioligand with an endocytic receptor. However, most of the radioligand appeared not to be bound to an endocytic receptor and dissociated rapidly at 37° C in an intact and functional state. These data indicate the presence of specific, high affinity binding sites for APC and PC on the surface of HUVEC. While a minor proportion of binding sites may be involved in endocytosis, the identity and function of the major proportion is presently unknown. It is speculated that this putative receptor may be a further mechanisms of localizing the PC antithrombotic system to the vascular endothelium.

Novel concatameric heparin-binding peptides reverse heparin and low-molecular-weight heparin anticoagulant activities in patient plasma in vitro and in rats in vivo

Blood ◽  
2004 ◽  
Vol 103 (4) ◽  
pp. 1356-1363 ◽  
Author(s):  
Barbara P. Schick ◽  
David Maslow ◽  
Adrianna Moshinski ◽  
James D. San Antonio
Keyword(s):  
Molecular Weight ◽  
Low Molecular Weight Heparin ◽  
Tandem Repeats ◽  
Factor Xa ◽  
Low Molecular Weight ◽  
Heparin Binding ◽  
High Affinity ◽  
Binding Peptides

Abstract Patients given unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH) for prophylaxis or treatment of thrombosis sometimes suffer serious bleeding. We showed previously that peptides containing 3 or more tandem repeats of heparin-binding consensus sequences have high affinity for LMWH and neutralize LMWH (enoxaparin) in vivo in rats and in vitro in citrate. We have now modified the (ARKKAAKA)n tandem repeat peptides by cyclization or by inclusion of hydrophobic tails or cysteines to promote multimerization. These peptides exhibit high-affinity binding to LMWH (dissociation constant [Kd], ≈ 50 nM), similar potencies in neutralizing anti–Factor Xa activity of UFH and enoxaparin added to normal plasma in vitro, and efficacy equivalent to or greater than protamine. Peptide (ARKKAAKA)3VLVLVLVL was most effective in all plasmas from enoxaparin-treated patients, and was 4- to 20-fold more effective than protamine. Several other peptide structures were effective in some patients' plasmas. All high-affinity peptides reversed inhibition of thrombin-induced clot formation by UFH. These peptides (1 mg/300 g rat) neutralized 1 U/mL anti–Factor Xa activity of enoxaparin in rats within 1 to 2 minutes. Direct blood pressure and heart rate measurements showed little or no hemodynamic effect. These heparin-binding peptides, singly or in combination, are potential candidates for clinical reversal of UFH and LMWH in humans.

Human endometrial epithelial cells grown on collagen in serum-free medium. Estrogen responsiveness and morphology

1991 ◽  
Vol 125 (1) ◽  
pp. 101-108 ◽  
Author(s):  
Bertil G. Casslén ◽  
Michael J. K. Harper
Keyword(s):  
Epithelial Cells ◽  
Primary Cultures ◽  
Collagen Matrix ◽  
Human Endometrium ◽  
Serum Free Medium ◽  
Free Medium ◽  
Endometrial Stromal Cells ◽  
Prolonged Incubation ◽  
Serum Free ◽  
Endometrial Epithelial Cells

Abstract. The aim of the study was to explore the possibility of using human endometrial epithelial cells in serum-free culture as a sensitive assay for hormonal effects on the human endometrium. Glands were isolated following enzymatic digestion of the endometrial tissue and plated on a collagen matrix. The epithelial cells were grown in either medium containing serum or in supplemented serum-free medium. No morphologic difference was found between cells grown in these two media for up to 5 days, using either light or scanning electron microscopy. Secretion of prostaglandin F2α (PGF2α) in response to estradiol was not lower in serum-free medium than in medium containing serum for the first 2 days of culture, whereas secretion declined after prolonged incubation in the serum-free medium. This response to estradiol was clearly dose-dependent, and it was further enhanced by addition of arachidonic acid, the precursor for prostaglandin synthesis, to the medium. Co-culture of endometrial stromal cells did not influence the secretion of PGF2α by epithelial cells. We conclude that the secretion of PGF2α from primary cultures of human endometrial epithelial cells grown on collagen in serum-free medium can be used for a limited period as an assay of estrogenic effects on the human endometrium.

Role of external Na+ and cytosolic pH in agonist-evoked cytosolic Ca2+ response in human platelets

1994 ◽  
Vol 267 (6) ◽  
pp. C1543-C1552 ◽  
Author(s):  
M. Kimura ◽  
K. Nakamura ◽  
J. W. Fenton ◽  
T. T. Andersen ◽  
J. P. Reeves ◽  
...  
Keyword(s):  
Binding Sites ◽  
Human Platelets ◽  
Thrombin Receptor ◽  
Free Medium ◽  
Amidolytic Activity ◽  
Cytosolic Ph ◽  
Amino Acid Peptide ◽  
High Affinity ◽  
Inositol 1,4,5 Trisphosphate

The role of external Na+ in agonist-evoked platelet Ca2+ response is poorly understood. This was explored in this study. Removal of external Na+ decreased both cytosolic Ca2+ mobilization and external Ca2+ entry, induced by thrombin but not by ADP or vasopressin. That external Na+ regulates thrombin activities was demonstrated by 1) Na+ dependency of the amidolytic activity of thrombin, 2) inhibition of thrombin binding to the high-affinity binding sites in Na(+)-free medium, and 3) attenuation of thrombin-induced inositol 1,4,5-trisphosphate production in Na(+)-free medium. Moreover, Ca2+ response to the thrombin receptor 6-amino acid peptide was independent of external Na+. The role of external Na+ in modifying agonist-evoked Ca2+ response through activation of Na+/H+ antiport and cytosolic alkalinization was then explored. Cytosolic alkalinization by monensin or NH4Cl enhanced thrombin, ADP, and thimerosal-induced external Ca2+ entry. Thimerosal-induced acceleration of external Ca2+ entry was diminished by the inhibition of Na+/H+ antiport. Thus external Na+ enhances thrombin activities, and cytosolic pH mediates store-regulated external Ca2+ entry. However, Na+/H+ antiport activation is not essential for agonist-evoked Ca2+ mobilization and external Ca2+ entry.

Activation of endothelin ETB receptors increases glomerular cGMP via an L-arginine-dependent pathway

1992 ◽  
Vol 263 (6) ◽  
pp. F1020-F1025 ◽  
Author(s):  
R. M. Edwards ◽  
M. Pullen ◽  
P. Nambi
Keyword(s):  
Nitric Oxide ◽  
Guanylate Cyclase ◽  
Binding Sites ◽  
Soluble Guanylate Cyclase ◽  
Maximum Effect ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
High Affinity ◽  
Dependent Pathway ◽  
Stimulation Of

The effects of endothelins (ET) on guanosine 3',5'-cyclic monophosphate (cGMP) levels in intact rat glomeruli were examined. ET-3 produced a rapid approximately fivefold increase in cGMP levels with the maximum effect occurring at 1 min. The ET-3-induced increase in cGMP accumulation occurred in the absence and presence of 3-isobutyl-1-methylxanthine. ET-1, ET-2, ET-3, and the structurally related toxin, sarafotoxin S6c, all increased glomerular cGMP levels in a concentration-dependent manner and with similar potencies (EC50 approximately 15-30 nM). The L-arginine analogue, N omega-nitro-L-arginine (L-NNA), reduced basal levels of cGMP and also totally inhibited ET-induced increases in cGMP as did methylene blue, an inhibitor of soluble guanylate cyclase. The effect of L-NNA was attenuated by L-arginine but not by D-arginine. The stimulation of cGMP accumulation by ET-3 was dependent on extracellular Ca2+ and was additive to atriopeptin III but not to acetylcholine. The ETA-selective antagonist, BQ 123, had no effect on ET-3-induced formation of cGMP. Glomerular membranes displayed high-affinity (Kd = 130-150 pM) and high-density (approximately 2.0 pmol/mg) binding sites for 125I-ET-1 and 125I-ET-3. ET-1, ET-3, and sarafotoxin S6c displaced 125I-ET-1 binding to glomerular membranes with similar affinities. BQ 123 had no effect on 125I-ET-1 binding. We conclude that ET increases cGMP levels in glomeruli by stimulating the formation of a nitric oxide-like factor that activates soluble guanylate cyclase. This effect of ET appears to be mediated by activation of ETB receptors and may serve to modulate the contractile effects of ET.

scholarly journals Stimulierung von Kulturen embryonaler Rattenzellen durch eine Proteinfraktion aus fötalem Kälberserum / Stimulation of Embryonic Rat Cells in Culture by a Protein Fraction Isolated from Fetal Calf Serum

1971 ◽  
Vol 26 (10) ◽  
pp. 1045-1048 ◽  
Author(s):  
Dieter F. Hülser ◽  
Werner Frank
Keyword(s):  
Cell Cycle ◽  
Culture Medium ◽  
Fetal Calf Serum ◽  
Surface Membrane ◽  
Calf Serum ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Membrane Potential Difference ◽  
Stimulation Of

Normal embryonic rat cells incubated in serum-free medium accumulate in G1-phase of the cell cycle. On addition of a growth-stimulating protein isolated from fetal calf serum they are triggered to proceed through the cycle, and they resume DNA-synthesis 15 to 20 hours later. In this paper it is demonstrated that the surface membrane potential difference (PD) decreases immediately after changing serum-free medium against culture medium containing either calf serum or the isolated serum protein; the original PD is restored 2 to 3 hours later. Serumprotein without growthstimulating activity does not affect the PD.A permanent rat cell line which grows independently of serum also has been tested. The PD of these cells is not significantly influenced by calf serum.

scholarly journals Mechanism of thrombin binding to endothelial cells

Blood ◽  
1983 ◽  
Vol 61 (2) ◽  
pp. 368-372 ◽  
Author(s):  
PT Bauer ◽  
R Machovich ◽  
P Aranyi ◽  
KG Buki ◽  
E Csonka ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Binding Sites ◽  
Antithrombin Iii ◽  
Affinity Binding ◽  
Heparin Binding ◽  
High Affinity Binding ◽  
Aortic Endothelial Cells ◽  
High Affinity ◽  
Lysine Residues ◽  
Two Populations

Abstract The interaction of human alpha-thrombin with mini-pig aortic endothelial cells was studied using 125I-labeled enzyme. Equilibrium between bound and free thrombin was attained within 1 min, and the Klotz-Hunston equations indicated two populations of binding sites. Approximately 30,000 sites/cell belonged to the high-affinity class with a Kd of about 3 x 10(-8) M. Modification of two lysine residues of thrombin with pyridoxal 5′-phosphate (PLP2-thrombin) destroyed the high- affinity binding and about three-fourths of the low-affinity bindings. When the lysine residue of thrombin involved in heparin binding was protected with heparin against chemical modification (PLP-thrombin), the modified enzyme remained similar to the native one with respect to cellular binding, with some loss of low-affinity binding only. Heparin, in a tenfold molar excess to enzyme, inhibited the binding of the native as well as the PLP-thrombin, whereas it did not influence the interaction between PLP2-thrombin and the cell. Since heparin might interfere with both the enzyme and the cell, the binding of heparin to endothelial cells was also examined. The results revealed that 3H- heparin also bound to cells. This binding was characterized by a Kd of 3 x 10(-7) M, approximately 10(6) sites/cell. Furthermore, thrombin bound to endothelial cells was released by antithrombin III. On the basis of these and other data in the literature, a model is proposed for the mechanism of the binding of thrombin to endothelial cells.

Sign in / Sign up

Export Citation Format

Share Document