Abstract
An assay for thrombin is presented wherein thrombin-catalyzed hydrolysis at Arg-Aα-16 to release fibrinopeptide A (FPA) from fibrinogen is measured using high-performance liquid chromatography (HPLC). In this assay one thrombin unit (TU) is defined as that amount of thrombin that will release half of the FPA in one min from one ml of a solution of >90% clottable normal human fibrinogen (≤0.35 μM) at 37°C, pH 7.4, F/2 0.15. One TU is equivalent to ∼ 0.1 NIH unit of thrombin and ∼ 1 pmol of pure human thrombin. At 37°C, pH 7.4, and plasma levels of fibrinogen of 3 mg/ml, one TU will catalyze the release of 3.6 nmol FPA min -1. Variability in fibrinogen samples which produce dramatic differences in clotting time assays with the same sample of thrombin, produce little or no variation in the catalytic assay for TU. The assay for TU obviates the need for maintenance of a thrombin reference standard.
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