Specific Determination of Plasmatic Thrombin Activity

Abstract

Thrombin is the key enzyme of coagulation. Its activity can be determined via fibrinogen øfibrin conversion or via cleavage of a chromogenic substrate. The latter method is easier than the first one, but in plasma it is hampered due to unspecific cleavage of the chromogenic substrate by thrombin-like enzymes of hemostasis, especially those of the contact phase. The concentration of the thrombin substrate (HD-CHG-Ala-Arg-pNA) was optimized, using final substrate concentrations of 0 to 5 mM, a final arginine concentration of 1.13 M, and samples of 10 mIU/mL purified thrombin in 7% human albumin or pooled normal citrated plasma without and with EDTA. Twenty microliters pooled normal citrated plasma (frozen/thawed) or factor II–deficient plasma (lyophilized) were incubated with 10 µL 0% to 0.5% Thromborel S® (100% = 162 ng/mL tissue factor [TF]) in 6% BSA or with 10 µL 0% (physiol. NaCl) to 50% Pathromtin SL®and with 20 µL 25 mM CaCl2. After 0 to 22 minutes (37°C), 20 µL 1.7 M arginine, pH 8.7 were added. Fifteen microliters 0.9 mM HD-CHGAla-Arg-pNA in 2.3 M arginine, pH 8.6, were added and the increase in absorbance (δA) at 405 nm was determined. Thrombin activity was standardized against the 3A measured for 1 IU/mL thrombin in 7% human albumin (8.8 mA/min RT). The optimal final chromogenic substrate concentration to detect thrombin in this assay system is less than 0.6 mM. Higher substrate concentrations in a plasma milieu result in unspecific cleavage of the substrate. Using final concentrations of chromogenic substrate less than 0.4 mM (the approximate Km- value for thrombin) and final concentrations of arginine greater than 800 mM, in factor II–depleted plasma, when activated either by TF or by the contact phase, there is no significant thrombin generation. The circulating thrombin activity measured in EDTA plasma of 39 healthy donors is 100 ± 20% of norm (mean value ± 1 SD; 100% = 5.5 mIU/mL thrombin). This chromogenic assay detects thrombin activity independent of clotting seconds or fibrin mediated turbidity increases. This technique allows to standardize the thrombin activity generated in any biologic system in international thrombin units.