A chemically synthesized pentasaccharide, a specific ligand for AT III, and a synthetic, sulfated bis-lactobionic acid amide, a ligand for HC-II, were studied along with heparin to determine the relative contribution of AT III and HC II in the inhibition of protease activation in plasma. The global clotting assays (PT, APTT, Heptest, TT), amidolytic anti-IIa and anti-Xa assays, and defined biochemical systems supplemented with purified AT III and HC II were used in this study. In the plasma-based assays, heparin exhibited strong inhibition in both thrombin and Factor Xa-based assays, whereas pentasaccharide was only active in Factor Xa-based assays and lactobionic acid was only active in thrombin-based assays. In the AT III supplemented systems, heparin was able to inhibit strongly both Factor Xa and thrombin, while pentasaccharide could only inhibit Factor Xa. Lactobionic acid was ineffective at mediating its actions through AT III. In the HC II-mediated inhibition of thrombin, heparin and lactobionic acid both had strong inhibitory actions. Pentasaccharide was ineffective in this assay. All three agents failed to inhibit Factor Xa via HC II. These studies suggest that specific synthetic analogues of heparin such as the pentasaccharide and lactobionic acid can be used to study the relative contributions of AT III and HC II in the control of protease activation during thrombogenesis.