Heparin cofactor II is prepared from fresh frozen plasma by a modification of the procedure of Griffith, et al., (9). Purity is ≥95% by SDS PAGE and activity is determined based on thrombin inhibition in the presence of dermatan sulfate. Inhibition of Thrombin by Heparin Cofactor II: Thrombin (IIa), heparin (HEP) and heparin cofactor II (HCII) interact to form a ternary complex via a random order bireactant mechanism. Heparin subsequently dissociates from the complex as thrombin forms a covalent complex with the heparin cofactor II. Heparin cofactor II (HCII) (heparin cofactor A, antithrombin BM, dermatan sulfate cofactor, human leuserpin-2) is a single chain glycoprotein (Mr=65,600, 10% carbohydrate) member of the serine protease inhibitor (serpin) family. Amino acid sequence analysis of heparin cofactor II reveals 3 potential N-glycosylation sites and 2 sulfated tyrosines located in two internal repeats of 7 residues located near the NH2-terminal. The plasma concentration is estimate