In vitro generation and measurement of murine soluble fibrin. A. Western Blotting. Murine plasma samples were treated with 0.02 U/ml thrombin and separated via gel electrophoresis under non-reducing (upper panel) and reducing (lower panel) conditions. Fibrin(ogen) was detected with a polyclonal anti-fibrinogen antibody. Lanes 1, 3: plasma without thrombin treatment. Lanes 2, 4: thrombin-treated plasma. Lane 1: Arrowhead points at undigested 340 kDa fibrinogen hexamer (αβγ)2. Lane 2: upper band depicts high molecular weight cross linked fibrin (XLF), lower band represents remaining undigested fibrinogen. Lane 3: Arrowhead points at fibrinogen monomers consisting of α- (64 kDa, not visible), β-(56 kDa) and γ- (47 kDa) chains. Lane 4: formation of α-chain dimers (α-α, weakly stained) and γ-chain dimers (γ-γ); lower band represents fibrinogen β- chains and remaining non cross-linked γ-chains. B. Agglutination test for soluble fibrin. The same samples as in A were evaluated with the SF agglutination test as described in the Methods section. 1: Untreated plasma samples show no agglutination. 2: Thrombin-treated plasmas agglutinated and gave a positive test result. C. Semiquantitative measurement of soluble fibrin. Murine plasma samples were treated with different amounts of thrombin (100 mU/ml, 50 mU/ml, 25 mU/ml, 10 mU/ml, 6 mU/ml and 0 mU/ml as indicated). For a direct comparison with the chromogenic assay shown in panel D, and in contrast to the regular agglutination test protocol, thrombin was added before dilution of the sample in Owren's buffer followed by incubation at 37°C for 10 min. The amount of thrombin used for activation of the plasma correlated with the agglutination reaction, thus allowing for semiquantitative evaluation of test results. D. Quantitative analysis of soluble fibrin by a chromogenic assay. Murine plasma samples were treated with different amounts of thrombin and absorption at 405 nm was correlated with the thrombin concentration used to induce soluble fibrin. There was a linear relationship beween 15 mU/ml and 50 mU/ml thrombin.