The glycocalyx is located on the apical surface of vascular endothelial cells which line the lumen. When vessels are stained with cationic dyes such as Alcian blue stain, transmission electron microscopy shows a small, irregularly shaped layer extending approximately 50–100 nm into the lumen of a blood vessel.  Another study used osmium tetroxide staining during freeze substitution, and showed that the endothelial glycocalyx could be up to 11 μm thick. It is present throughout a diverse range of microvascular beds (capillaries) and macrovessels (arteries and veins).  The glycocalyx also consists of a wide range of enzymes and proteins that regulate leukocyte and thrombocyte adherence, since its principal role in the vasculature is to maintain plasma and vessel-wall homeostasis.  These enzymes and proteins include:
*Endothelial nitric oxide synthase (endothelial NOS)
*Extracellular superoxide dismutase (SOD3)
*Angiotensin converting enzyme
*Antithrombin-III
*Lipoprotein lipase
*Apolipoproteins
*Growth factors
*Chemokines
The enzymes and proteins listed above serve to reinforce the glycocalyx barrier against vascular and other diseases.  Another main function of the glycocalyx within the vascular endothelium is that it shields the vascular walls from direct exposure to blood flow, while serving as a vascular permeability barrier.  Its protective functions are universal throughout the vascular system, but its relative importance varies depending on its exact location in the vasculature.  In microvascular tissue, the glycocalyx serves as a vascular permeability barrier by inhibiting coagulation and leukocyte adhesion.  Leukocytes must not stick to the vascular wall because they are important components of the immune system that must be able to travel to a specific region of the body when needed.  In arterial vascular tissue, the glycocalyx also inhibits coagulation and leukocyte adhesion, but through mediation of shear stress-induced nitric oxide release.  Another protective function throughout the cardiovascular system is its ability to affect the filtration of interstitial fluid from capillaries into the interstitial space.
The glycocalyx, which is located on the apical surface of endothelial cells, is composed of a negatively charged network of proteoglycans, glycoproteins, and glycolipids. Along the luminal surface of the vascular glycocalyx exists an empty layer that excludes red blood cells.