Thrombin activation rate constant: One-stage chromogenic assay for the extrinsic system

Some properties of a one-stage, chromogenic clotting assay using tissue thromboplastin as an activator are described. The chromogenic assay uses S-2238 as substrate and estimates thrombin by the rate of absorbance produced. Under assay conditions, it was observed that the log (thrombin or absorbance/min) is linear with time. The linearity appeared to extend from zero thrombin up to maximum thrombin concentration for all concentrations of each plasma type examined including factor V and factor VII deficient plasmas. Since the linear slope, b, appears to be a measure of the rate of thrombin production, it was called the Thrombin Activation Rate Constant (TARC). For dilutions of normal plasma log (b) appears to be linearly related to log (plasma concentration) and to log (PT) where PT is the standard one stage clotting time. For plasmas deficient in extrinsic clotting factors, b was linearly related to log (factor concentrations). The chromogenic assay was most sensitive to changes in prothrombin; least sensitive to changes in factor VII; and unaffected by changes in concentration of factors VIII and IX. The standard PT was least sensitive to prothrombin and more sensitive to factors X and VII. For oral anticoagulated plasmas log b and log PT were linearly correlated with a regression slope which was more negative than that from normal plasma. From theses results it was concluded that TARC is a chromogenic assay sensitive to all factors in the extrinsic pathway; that TARC might be used as both a screening assay for extrinsic deficiencies and a monitoring assay for anticoagulants; and that TARC may be more sensitive than the standard PT to the defects from oral anticoagulation.