The kinetics and interplay of thrombin inhibition by 4 plasma proteinase inhibitors

Abstract

A novel therapeutic approach for restoring hemostasis in hemophilia is to reduce antithrombin (AT) to rebalance reduced thrombin generation. In plasma, multiple inhibitors including AT, heparin cofactor II (HCII), α₂-macroglobulin (A2M), and α₁-proteinase inhibitor (A1PI) play a role in thrombin inhibition. The goal was to study the kinetics of thrombin inhibition and the roles of various inhibitors across a broad range of AT levels. Thrombin inhibition was measured at varied concentrations of AT with and without A2M, HCII, and A1PI. Reducing AT to 0 from plasma levels in the presence HCII, A2M, and A1PI, results in slower thrombin inhibition with the time required to inhibit half the thrombin increasing approximately fourfold. Computational models of thrombin inhibition and thrombin generation in hemophilia were constructed and used to analyze thrombin inhibition and the relative contribution of each inhibitor. In a model of thrombin generation, decreased thrombin inhibition resulted in increased peak thrombin and increased area under the thrombin curve. Even at high concentrations of thrombin, all of the thrombin was inhibited with the relative contribution of other inhibitors increasing as AT was decreased. These studies show that in a system without heparin-like glycosaminoglycans, AT is the dominant inhibitor of thrombin, followed by A2M, HCII, and, finally, A1PI. As AT levels decrease, thrombin inhibition is slower, resulting in higher levels of thrombin in a computational model of thrombin generation. Ultimately, the other inhibitors compensate for AT to maintain a level of thrombin regulation.

Graphical abstract

Figure 1.

Calculation of second-order rate constants. Thrombin (10 nM) and inhibitor (AT, 0.3 μM; A1PI, 135 μM; HCII, 1.2 μM; and A2M, 2.4 μM) were incubated. At time points, residual thrombin was measured by addition of a chromogenic thrombin substrate. Note that each plot has a separate time course. The rate of thrombin inhibition was calculated from an exponential decay curve fit to the data (equation 1). As expected, the A2M/IIa complex retains some ability to cleave substrate (equation 2). The shaded area indicates the 95% confidence interval of the fit to the data. The rate measured from each curve fit was divided by the concentration of inhibitor to give the second-order rate constant shown in Table 1. Also shown in Table 1 is the plasma concentration of each inhibitor.

Figure 2.

Thrombin inhibition without or with other plasma inhibitors. (A) Thrombin (4 nM) was incubated either with the indicated concentration of AT (expressed as a percentage of the plasma concentration of 3500 nM) or with the indicated concentration of AT plus plasma concentrations of A1PI, HCII, and A2M. At time points residual thrombin was measured by addition of a thrombin substrate. The rate of thrombin inhibition was calculated from a decay curve fit to the data. As expected, the thrombin-A2M complex retains some ability to cleave substrate. The shaded area indicates the 95% confidence interval of the fit to the data. (B) Substrate cleavage during thrombin inhibition was modeled as a series of ordinary differential equations (equation 3) using the rate constants determined in Figure 1. The model showed good correlation with the experimental data. (C) The model of substrate cleavage includes the contribution of thrombin bound to A2M. Because the IIa-A2M complex is functionally inactive in coagulation, the contribution of IIa-A2M was removed to give a model of residual free thrombin as a function of time (ie, thrombin not bound to an inhibitor). As expected, in the model with only AT, there was no effect of IIa-A2M.

Figure 3.

Thrombin inhibition without or with other plasma inhibitors. (A) Thrombin (1400 nM) was incubated with the indicated concentration of AT (expressed as a percentage of the plasma concentration) plus plasma concentrations of A1PI, HCII, and A2M. At time points, residual thrombin was measured by diluting the sample into a thrombin substrate. As expected, the thrombin-A2M complex retains some ability to cleave substrate. (B) Substrate cleavage was modeled from the thrombin and thrombin-A2M concentrations determined by a series of ordinary differential equations using the rate constants determined in Figure 1 (see supplemental Code 2). (C) The model of substrate cleavage was converted to a model of residual free thrombin (ie, thrombin not bound to an inhibitor). (D) Modeled concentrations of each inhibitor complex at 10 minutes was plotted for each AT concentration.

Figure 4.

Model of thrombin generation in hemophilia. (A) Thrombin generation was modeled using the parameters of hockin.py modified to include rate constants and concentrations for inhibitors taken from Table 1. Plasma concentrations of factor VIII with 100% AT are indicated with “normal.” The shaded area encompasses the modeled range of thrombin generation values when the inhibitors are collectively set to the extremes of their reference ranges (AT, 86%-128%; HC, 70%-130%; A2M, 70%-155%; and A1PI, 53%-133%³⁴). In hemophilia A, the factor VIII concentration was set to 0. HC, A2M, and A1PI were set to the values shown in Table 1. AT was set to the indicated value. Modeled free thrombin is shown as a function of time. (B) The integrated thrombin was calculated from the free thrombin shown in panel A.