Fibrinogen and thrombin are required for aluminum nodule formation. (A) At 4 hours after Alhydrogel injection, nodules were collected and protein was eluted by boiling in SDS, separation by SDS-PAGE, and analysis with Coomassie staining. Lane 1, protein eluted from aluminum nodules; lane 2, normal mouse serum. (B) Plasma (0.1, 0.05, 0.025 μL), and proteins eluted from 4-hour Alhydrogel nodules (4 μg, 2 μg), were analyzed by the use of Western blot for fibrinogen under reducing conditions. Note that the fibrinogen bands in the plasma samples are probably compressed by albumin, which is very abundant but not visualized. (C) FibA KO mice were tested for the ability to form aluminum nodules in vivo compared with littermate controls. (D) FibA KO mice were injected with 1 mg of human fibrinogen 10 minutes before an injection of Alhydrogel. At 5 hours later, mice were evaluated for nodule formation. (E) Mice given 2.5 μg/mL warfarin in their drinking water for 7 days were injected with Alhydrogel and evaluated 5 hours later for the presence of aluminum nodules. (F) Mice were injected intraperitoneally with lepirudin 10 minutes before injection with Alhydrogel, then evaluated 5 hours later for the presence of aluminum nodules. All experiments included at least 3 mice per group and were performed at least 3 times, except panel D, which was performed twice.