Heparan sulfate (HS), a highly sulfated polysaccharide attached to protein cores to form HS proteoglycans (HSPGs), abundantly expresses in the vasculature, liver and lung. HS structure, which is determined by temporal and spatial expressed serial HS biosynthesis enzymes, is not homogenous and provides variable binding sites for ligands to modulate various biological functions.Endothelial HS, containing a unique high affinity antithrombin (AT) binding pentasaccharide motif (HSAT), was believed to be responsible for nonthrombogenic property of endothelium by enhancing AT anticoagulant activity. However, mouse genetic studies determined a normal level of HSAT is not essential for hemostasis. Currently, it remains unknown if HS plays any role in hemostasis in vivo. In chapter 2 study, we demonstrated that endothelial HS plays an anti-thrombotic role in hemostasis by testing tail bleeding time test, lipopolysaccharide-induced microthrombosis model and the FeCl3-induced acute carotid artery thrombosis model. Mechanistically, we determined that endothelial HS interacts with exocytosed tissue plasminogen activator (tPA) and plasminogen (Plg) from circulation to form a HS-tPA-Plg ternary complex thereby accelerating tPA-mediated Plg activation on endothelial cell surface, playing a profibrinolytic function in hemostasis. In liver, circulating tPA is quickly internalized and cleared by hepatocytes, kupffer cells and liver sinusoidal endothelial cells via low density lipoprotein receptor related protein- 1 (LRP1) and mannose receptor. Membrane HSPGs are cell surface endocytic receptors to internalize ligands and were reported in association with LRP1 to mediate factor VIII uptake and clearance. In chapter 2 study, we demonstrated that endothelial HS binds tPA. Currently, it is unknown if liver HS associates with LRP1 to mediate tPA clearance. In chapter 3 study, we demonstrated that HS from hepatocytes but not endothelial cells and kupffer cells promotes tPA clearance. Mechanistically, HS sequestrates tPA at the hepatocyte surface and interacts with LRP1 to present tPA to LRP1 and also acts as an independent endocytic receptor to mediate tPA uptake. The severe acute respiratory syndrome (SARS)-like coronavirus disease (COVID-19) is caused by SARS-CoV-2 and has been a serious threat to global public health with limited treatment. Cellular HS has been found to bind SARS-CoV-2 spike protein (SV2-S) and cooperate with cell surface receptor angiotensin-converting enzyme 2 (ACE2) to mediate SARS-CoV-2 infection of host cells. In chapter 4 study, we determined that host cell surface SV2-S binding depends on and correlates with host cell surface HS expression. The higher binding affinity of SV2-S G614 mutant to heparin and upregulated HS expression may be one of the mechanisms underlying the higher infectivity of the SARS- CoV-2 G614 variant and the high vulnerability of lung cancer patients to SARS-CoV-2 infection, respectively. Host cell infection by SARS-Cov-2 wildtype and G614 variant pseudovirus and increased infection caused by upregulated HS expression can be effectively blocked by heparin lyase and heparin, and possibly surfen and heparin derivatives.