Functional Characterization of Antithrombin Mutations by Monitoring of Thrombin Inhibition Kinetics

Figures

• 

  Figure 1. Inactivation kinetics of thrombin in representative human plasma samples. Purified thrombin (FIIa) was added to citrate-anticoagulated plasma obtained from a healthy individual (human thrombin, blue circles) or a patient with hereditary antithrombin (AT) deficiency (human thrombin, red filled circles; bovine thrombin, red unfilled circles) to achieve a final concentration of 2 ng/mL. At the indicated time points, thrombin inactivation was stopped by the addition of argatroban at a final concentration of 200 µmol/L and the residual thrombin activation measured using the thrombin-oligonucleotide-based enzyme capture assay.

• 

  Figure 2. Inactivation rates of thrombin in healthy individuals and antithrombin (AT)-deficient patients. The half-life of exogenously added thrombin (2 ng/mL final concentration) was determined in healthy controls (n = 26) and patients with AT mutations (n = 36, human thrombin; n = 19, bovine thrombin). The boxes show quartiles and the median of the data, the whiskers extend up to 1.5 times of the interquartile range from the box, and circles show outlying data values. X indicates the mean.

• 

  Figure 3. Dependence of inhibition kinetics of human thrombin (FIIa) on antithrombin (AT) plasma levels. (A) The half-life of exogenously added human thrombin (2 ng/mL final concentration) was determined in citrated plasma obtained from patients with AT mutations (red circles, n = 36)) and in normal pooled plasma (NPP) mixed with AT-deficient plasma to achieve the indicated AT levels (blue circles). Data points in blue represent means of three independent determinations on three different days, error bars represent the standard deviation. The intersected lines parallel to the x-axis indicate the 95th and 5th percentiles of half-life times measured in healthy controls (n = 26). Functional AT levels were determined by a thrombin-based assay. (B) Functional AT levels were determined by an activated factor X (FXa)-based assay. Data points filled white indicate plasma from patients under therapy with FXa inhibitors. (C) AT antigen levels were determined by an immunoassay. (D) Correlation of the ratio of AT activity determined by a FIIa-based assay and AT antigen concentration with the plasma half-life of exogenously added human thrombin. r indicates the Pearson correlation coefficient.

• 

  Figure 4. Dependence of inhibition kinetics of bovine thrombin (FIIa) on antithrombin (AT) plasma levels. (A) The half-life of exogenously added bovine thrombin (2 ng/mL final concentration) was determined in citrated plasma obtained from patients with AT mutations (red circles, n = 19) and in normal pooled plasma (NPP) mixed with AT-deficient plasma to achieve the indicated AT levels (blue circles). Data points in blue represent means of three independent determinations on three different days, error bars represent the standard deviation. The intersected lines parallel to the x-axis indicate the 95th and 5th percentiles of half-life times measured in healthy controls (n = 26). Functional AT levels were determined by a thrombin-based assay. (B) Functional AT levels were determined by an activated factor X (FXa)-based assay. Data points filled white indicate plasma from patients under therapy with FXa inhibitors. (C) AT antigen levels were determined by an immunoassay. (D) Correlation of the ratio of AT activity determined by a FIIa-based assay and AT antigen concentration with the plasma half-life of exogenously added bovine thrombin. r indicates the Pearson correlation coefficient.