Multiple Bovine Thrombin Components
Robert D. Rosenberg 1 and David F. Waugh 1
From the
1 From the Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
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. T2 and T6 were isolated essentially in pure form. Specific activities and component abundances of fractions revealed that T1, T2, and T3 have significantly higher activities than T4, T5, and T6. Thrombins from 60 single animal plasmas contained only T1, T2, and T3, in two distribution types: I, having T1, T2, and T3 in a ratio of 1:2:1, or II, having T1 and T2 as a 1:1 ratio, with T3 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, T1 and T2 may first appear at a ratio of 1:2. An attractive model is as follows. Two prothrombins, P1 and P2, are present in equal amounts. P2 yields T2, and P1 yields T1 and T3, but not sequentially. As the animal ages, the pathway to T3 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 T4, T5, and T6 are derived from T1, T2, and T3 as a result of peptide cleavages in the larger fragment.
Submitted on February 27, 1970
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