Assessment of the role of heparan sulfate in high molecular weight kininogen binding to human umbilical vein endothelial cells.
The assembly and activation of the kinin forming system components on human umbilical vein endothelial cells (HUVEC) have been studied in great detail. Proteins such as gC1qR, cytokeratin-1 and u-PAR have been identified to be responsible for Zn2+-dependent binding of high molecular weight kininogen (HK) to HUVEC. Heparan sulfate has also been shown to have a major role in Zn2+-dependent binding of HK to the endothelial cell line, Ea.hy 926. In this study, we have analyzed the possible contribution of heparan sulfate to high molecular weight kininogen binding to HUVEC using multiple approaches. The presence of heparan sulfate on HUVEC was analyzed by staining with an antibody specific for heparan sulfate. Incubation of the cells with bacterial heparinases removed the heparan sulfate from the cell surface to the level seen with a control antibody, however, the Zn2+-dependent binding of HK was not affected. Further, blocking of heparan sulfate with a specific antibody to heparan sulfate even after digestion with heparinases did not reduce HK binding whereas antibodies to the proteins gC1qR and cytokeratin-1 consistently reduced the binding of HK to the endothelial cells. The binding intensities of FITC-labeled HK were similar in heparinase-treated and -untreated HUVEC. The rate of kallikrein formation by the assembly of factor XII, HK and PK were similar in both heparinase-treated and non-treated HUVEC. All of these data indicate that heparan sulfate does not contribute significantly to HK binding to HUVEC.
Fernando
L P
LP
Department of Medicine, Division of Pulmonary and Critical Care, Allergy and Clinical Immunology and Konishi-MUSC Institute for Inflammation Research, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
Fernando
A N
AN
Joseph
K
K
Kaplan
A P
AP
D000906:Antibodies / Q000494:pharmacology; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism; D019763:Heparin Lyase / Q000494:pharmacology; D006497:Heparan Sulfate / Q000502:physiology*; D006801:Humans; D007610:Kallikreins; D007633:Keratins / Q000502:physiology; D007700:Kinetics; D019679:Kininogen, High-Molecular-Weight / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000502:physiology; D011485:Protein Binding; D011951:Receptors, Complement / Q000502:physiology; D014471:Umbilical Veins; D015032:Zinc
D000906:Antibodies; D019763:Heparin Lyase; D006497:Heparan Sulfate; D007610:Kallikreins; D007633:Keratins; D019679:Kininogen, High-Molecular-Weight; D008562:Membrane Glycoproteins; D011951:Receptors, Complement; D015032:Zinc; C032402:complement 1q receptor