Multiple Bovine Thrombin Components

Purified thrombins are isolated from Parke-Davis thrombin topical and from bioactivated crude prothrombins obtained from single animal plasmas. Six thrombin components are present in the former by gel electrophoresis at pH 8.9, ionic strength 0.16, and 30°, but interband protein is high. Ultracentrifugation and gel filtration studies show that, as either ionic strength or temperature is decreased, reversible thrombin self-association and interaction with the gel matrix increase. Electrophoretic mobility and resolution decrease, the latter to give a single diffuse band. Partial fractionation of the six components, numbered according to increasing anodal mobility, was accomplished by differential elution from cellulose phosphate. T 2 and T 6 were isolated essentially in pure form. Specific activities and component abundances of fractions revealed that T 1 , T 2 , and T 3 have significantly higher activities than T 4 , T 5 , and T 6 . Thrombins from 60 single animal plasmas contained only T 1 , T 2 , and T 3 , in two distribution types: I, having T 1 , T 2 , and T 3 in a ratio of 1:2:1, or II, having T 1 and T 2 as a 1:1 ratio, with T 3 absent or present at about 5% of total thrombin. Distribution type for pedigreed animals correlates ( a ) with age: below 8 months, only I was observed (5 animals); between 8 and 30 months, either I or II was observed (16 animals); and above 30 months, only II was observed (11 animals); and ( b ) with the two-stage clotting time of the original plasma, with the use of the corresponding serum to supply activators. Other correlations were not found. Component distribution is not a simple, genetically determined characteristic. During activation to produce Distribution I, T 1 and T 2 may first appear at a ratio of 1:2. An attractive model is as follows. Two prothrombins, P 1 and P 2 , are present in equal amounts. P 2 yields T 2 , and P 1 yields T 1 and T 3 , but not sequentially. As the animal ages, the pathway to T 3 is lost. Reduction and alkylation of thrombin from thrombin topical results in a dispersion of five fragments between molecular weight of ∼32,000 and ∼5,000. Thrombins from single animal plasmas yield only these two. Probably T 4 , T 5 , and T 6 are derived from T 1 , T 2 , and T 3 as a result of peptide cleavages in the larger fragment.