Formation of ¹²⁵I-thrombin-HCII complexes ex vivo. (A) Plasma was obtained from HCII^+/+ mice 5 to 60 minutes after intravenous injection of 10 mg/kg porcine skin dermatan sulfate (DS) (lanes 5-8). The plasma samples were incubated for 2 minutes with ¹²⁵I-thrombin (T), and formation of ¹²⁵I-thrombin-HCII complexes (T-HCII) was assayed by SDS-PAGE and autoradiography. A sample containing only ¹²⁵I-thrombin is shown in lane 1. The thrombin-HCII complex was identified by incubating ¹²⁵I-thrombin with purified mouse HCII (5 μM) (lane 2) or with mouse plasma (P) (lane 4) in the presence of 50 μg/mL dermatan sulfate. ¹²⁵I-thrombin was also incubated with mouse plasma in the absence of dermatan sulfate (lane 3). Radioactive bands above T-HCII probably represent complexes of thrombin with α2-macroglobulin; bands below T-HCII probably represent thrombin-antithrombin or partially degraded complexes. (B) ¹²⁵I-thrombin-HCII bands from the gel in panel A were cut out, and the radioactivity was measured in a gamma counter.