Specific Activities of Bovine Thrombin and Thrombin Components

Summary

Bovine thrombins (Gel-T) isolated from bioactivated crude prothrombin, obtained from Holstein, Jersey and Hereford single animal plasmas, occur in two distributions: either T₁, T₂ and T₃ as 1:2:1 (Type I), or T₁ and T₂ as 1:1, with T₃ absent or present to the extent of 5% of total Thrombin (Type II). Since components differentially denature during procedures used to fractionate, examinations of Gel-T were conducted so that statistical procedures could be used to evaluate particular variances. The variance due to the Gel-T preparative procedure and Gel-T intrinsic specific activity yield a coefficient of variance (S_c) of 0.55%. Thus 95% of single animal Gel-T specific activities fall within 1.1% of the average value. There are no significant differences between animals of different breed, or of bloods drawn from the same animal at different times. Evidently, Gel-T represent a reproducible reference standard. Analysis of distributions shows that the intrinsic specific activities of thrombin components are identical to within 1 %, and are approximately 1.0 NIH unit per 6.0 × 10⁻⁴ absorbance unit (3,250 ±450 NIH units per mg). The clotting time (τ) - thrombin concentration (T) relation, log τ = — ã log T + C, is examined. Over the range of τ from 15 to 60 sec it is shown that a T series can be reproduced to within 1 % in thrombin concentration. The variances in the assay system are then due almost entirely to variance in the assay substrate, fibrinogen. These are an S_c of 1.7% for τ̄ between sets of tubes containing dilute fibrinogen (τ-tubes) from one lot of Stock-F, and S_c of 12% between τ̄ for sets of τ-tubes from Stock-F prepared from different lots of Armour Fraction-I, and an S_c of 1.9% for ã̄( =0.679) for sets of T-tubes, obtained either from one Stock-F or Stock-F obtained from different lots of Fraction-I. Errors in several procedures for determining unknown thrombin concentrations are examined.

^* Special Fellow of the National Institutes of Health and of Biophysics Training Grant GM 00778. A portion of this work was submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Massachusetts Institute of Technology, 1969.

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