The binding of heparin to the extracellular matrix of endothelial cells up-regulates the synthesis of an antithrombotic heparan sulfate proteoglycan.
Exposure of endothelial cells to heparin and other antithrombotic drugs specifically stimulates the synthesis of an antithrombotic heparan sulfate (HS). In the present work, biotinylated heparin (BiotHep) was used to characterize the binding site(s) of heparin responsible for the stimulus in HS synthesis on endothelial cells. No differences were observed between biotinylated and non-biotinylated heparin in their ability to increase the synthesis of HS. In kinetic studies the BiotHep showed fast, saturable and specific binding with an apparent K(D) of 83 nM to adherent cells and 44 nM to the extracellular matrix (ECM) in the absence of cells. By confocal and electron microscopy, BiotHep bound only to the ECM, co-localizing with fibronectin. The same pattern of binding to the ECM was observed using heparin conjugated with FITC or Alexa Fluor 488 in the presence or absence of fetal calf serum. However, after degradation of HS, heparin binds to the cell surface, indicating that endogenous HS possibly occupied the heparin binding sites. Analyses by flow cytometry and confocal microscopy of cells with non-associated ECM, showed labeling of the cell surface using syndecan-4 monoclonal antibody as well as wheat germ agglutinin, but no binding of heparin. Furthermore, the stimulation in HS synthesis is not elicited by heparin in the absence of ECM. These results indicate that the stimulus for the synthesis of HS does not require binding of the heparin to the cell surface, and the signaling may be mediated through the ECM.
Departamento de Bioquímica, Universidade Federal de São Paulo, São Paulo, SP, Brazil.
Edvaldo S
ES
Trindade
Constance
C
Oliver
Maria C
MC
Jamur
Hugo A O
HA
Rocha
Célia R C
CR
Franco
Rodrigo I
RI
Bouças
Thais R
TR
Jarrouge
Maria A S
MA
Pinhal
Ivarne L S
IL
Tersariol
Tiago C
TC
Gouvêa
Carl P
CP
Dietrich
Helena B
HB
Nader
D000818:Animals; D001665:Binding Sites; D019921:Biotinylation; D019556:COS Cells; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D005109:Extracellular Matrix / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D005343:Fibrinolytic Agents / Q000378:metabolism / Q000494:pharmacology*; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006493:Heparin / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D006834:Hydrazines; D007700:Kinetics; D008024:Ligands; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D011485:Protein Binding; D011509:Proteoglycans / Q000378:metabolism*; D011817:Rabbits; D015398:Signal Transduction / Q000187:drug effects*; D015854:Up-Regulation
C417561:Alexa 488 hydrazide; D005343:Fibrinolytic Agents; D005456:Fluorescent Dyes; D006834:Hydrazines; D008024:Ligands; D011509:Proteoglycans; C036090:heparin proteoglycan; D006493:Heparin; D016650:Fluorescein-5-isothiocyanate