MATERIALS AND METHODS

Materials. Human HCII and human thrombin were prepared as previously described (Blinder et al., 1989). Thrombin was labeled with 125I by the chloramine T method to a specific activity of~ 1 X 106 cpm/pmol (Tollefsen & Blank, 1981). Bovine lung heparin and porcine skin dermatan sulfate were purchased from Sigma. The dermatan sulfate was treated with nitrous acid to remove traces of contaminating heparin (Teien et al., 1976). Polyethylene glycol) 8000 (PEG) was purchased from Sigma. Restriction enzymes were purchased from Promega and New England Biolabs. Oli-gonucleotides were synthesized in the Protein Chemistry Facility of Washington University. Chromogenic Assay of HCII Activity in Murine Plasma. Murine plasma (0-4 mL) was incubated with human thrombin (7 nM) and dermatan sulfate (200 Mg/mL) or heparin (67 Mg/mL) at room temperature in100 mL of 0.15 M NaCl, 0.02 M Tris-HCl, and 1 mg/mL PEG, pH 7.4 (TS/PEG buffer). Thrombin was added last to initiate the reaction. After 60 s, 500 mL of 100 mM tosyl-Gly-Pro-Arg-p-nitroanilide (Chromozym TH, Boehringer Mannheim) in TS/PEG buffer was added, and the absorbance at 405 nm was determined continuously for 100 s. The rate of change of absorbance was proportional to the concentration of active thrombin that remained in the incubation.