Heparin Cofactor II (HCII) is a plasma serine protease inhibitor synthesized mainly in the liver. Circulating freely in blood plasma, it serves as a key regulator of the coagulation cascade. Unlike antithrombin, HCII selectively suppresses thrombin rather than multiple clotting proteases. Its inhibitory activity remains weak under physiological conditions; binding to glycosaminoglycans such as heparin sodium induces conformational rearrangement of HCII, exposing its reactive site loop to rapidly trap thrombin. HCII limits excessive fibrin generation and prevents pathological thrombosis, maintaining the balance between hemostasis and anticoagulation in the vasculature. HCII binds Hep-Na via positively charged residues, triggering structural shift to accelerate thrombin inhibition. Thus a functional ELISA assay was conducted to detect the interaction of recombinant rat HCII and Hep-Na. Briefly, HCII was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μL were then transferred to Hep-Na-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-HCII pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody for 1h at 37℃, wells were aspirated and washed 5 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50 μL stop solution to the wells and read at 450/630nm immediately. The binding activity of recombinant rat HCII and Hep-Na was shown in Figure 1, the EC50?for this effect is 2.56μg/mL.