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

Proteolytic reactions in the coagulation cascade are regulated by multiple plasma inhibitors.^1,2 These inhibitors often target >1 coagulation proteinases.^1,2 Thrombin is regulated by multiple plasma inhibitors.^3-5 Among these inhibitors are the serine proteinase inhibitors antithrombin (AT), heparin cofactor II (HCII), and α₁-proteinase inhibitor (A1PI).^3,6-10 Thrombin can also be inhibited by the broad-spectrum non–serine proteinase inhibitor α₂-macroglobulin (A2M).^4,5,11-13 A2M acts through a unique mechanism. Its bait region has cleavage sites for a very wide range of proteinases. Cleavage of the bait region triggers a dramatic structural change that traps the proteinase within the A2M molecule¹⁴ (supplemental Figure 1).

To understand the role of a particular thrombin inhibitor, it is necessary to know both the intrinsic activity of that inhibitor toward thrombin and the plasma concentration of the inhibitor. The activity is expressed as the rate at which the inhibitor can react with thrombin (second-order rate constant).¹⁵ An inhibitor may have weak inhibitory ability but may nonetheless play an important role because of a high plasma concentration.

A novel strategy for treating hemophilia by rebalancing hemostasis has recently completed phase 3 clinical trials.^16,17 This strategy involves reducing the levels of AT with a target of 15% to 35% of the normal plasma levels.^18,19 In this report, we have explored how the overall rate of thrombin inhibition is changed as AT levels are lowered in the presence of other inhibitors. We have generated experimental data on thrombin inhibition as AT is decreased from normal levels to 0 in the presence of plasma levels of HCII, A1PI, and A2M. To analyze these data, we used computational models of inhibition.

Thrombin, AT, and HCII were purchased from Prolytix (Essex Junction, VT). A1PI was purchased from MyBioSource (San Diego, CA). A2M was purified according to the method of Imber and Pizzo.²⁰ Chromogenic substrate Tos-Gly-Pro-Arg-pNA was purchased from Pentapharm (Aesch, Switzerland). Substrate cleavage was monitored on a Molecular Devices (San Jose, CA) ThermoMax. For all studies, the buffer was 20 mM HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid; pH 7.4), 150 mM NaCl, 5 mM Ca²⁺, and 0.02% weight-to-weight ratio polyethylene glycol (Sigma-Aldrich, Saint Louis, MO).

The second-order rate constant was calculated by dividing the measured rate of inhibition by the concentration of the inhibitor.

Thrombin was incubated either with AT alone or AT with plasma concentrations of the other inhibitors (HCII, A1PI, and A2M). At timed intervals, chromogenic substrate for thrombin was added to give a final concentration of 500 μM. Substrate cleavage was monitored at an absorbance of 405 nm in 12-second intervals for 10 minutes. At each time point, the rate of substrate cleavage was expressed relative to the rate of substrate cleavage in the absence of inhibitors.

Uncertainty in the data fits was derived from the covariance of optimized fits to provide the 95% confidence interval and ranges assuming a normal distribution of experimental errors.

Substrate cleavage was modeled as the concentration of free thrombin + $IIa-A2M∗Vtime=∞Vtime=0$ (from equation 2).

Thrombin generation was modeled using a modification of the hockin.py module taken from Owen et al²⁴ (https://github.com/mjowen/MathematicalModelsOfCoagulation-AreWeThereYet/).

To model hemophilia, the factor VIII concentration was set to 0. Concentrations and second-order rate constants for HCII, A1PI, and A2M were added to mirror the existing equations for AT inhibition of thrombin and meizothrombin (supplemental Code 3). Integrated thrombin was calculated using the scipy module for the composite trapezoidal rule. Code was run in numerical python using the program Jupyter as a wrapper (supplemental Code 4).²⁵ 

Published values for the second-order rate constants for thrombin inhibition differ by as much as 10-fold.^3,6,7,11,26 Thus, we measured the second-order rate constants of the proteins used in this study (Figure 1; Table 1; supplemental Table 1; supplemental Code 1). Note that thrombin complexed with A2M retains the ability to cleave the small molecule chromogenic substrate used in our assays, although at a reduced rate compared with uncomplexed thrombin (supplemental Code 1). This results in a plateau in the A2M inhibition curve at ∼56% residual substrate cleavage (Figure 1, lower right panel).

To investigate the interplay of the thrombin inhibitors, inhibition of 4 nM thrombin was studied with either (1) varied AT levels (left side of Figure 2) or (2) varied AT levels in the presence of plasma levels of A2M, HCII, and A1PI (right side of Figure 2). Figure 2A shows the time course of inhibition monitored by substrate cleavage. Thrombin inhibition was modeled using equation 3 and the reactions shown in reaction scheme 1 (supplemental Code 2 showing ODE model). The concentrations of modeled free thrombin and modeled thrombin-A2M complex were used to model substrate cleavage (Figure 2B). The modeled curves show a close match to the experimental data. Note that substrate cleavage is not expected to reach 0 because thrombin in complex with A2M retains its ability to cleave the chromogenic substrate (Figure 1; supplemental Code 1). However, thrombin trapped within A2M is unable to interact with its natural (protein) substrates and is functionally inactive in coagulation.²⁷ Figure 2C models the decrease in free thrombin over time. In the presence of other inhibitors, the time required to inhibit half the thrombin increases from ∼15 seconds at plasma levels of AT to slightly >60 seconds in the complete absence of AT.

The typical plasma concentration of prothrombin is ∼100 μg/mL (1400 nM).^28,29 If converted completely to thrombin, the concentration of thrombin would exceed the concentration of AT when AT is reduced below ∼40%. The ability of these 4 major thrombin inhibitors was studied in the extreme case in which all available prothrombin was converted to thrombin. Experimental data measured the inhibition of 1400 nM thrombin over time with varied AT levels added to plasma levels of A2M, HCII, and A1PI (Figure 3A). Thrombin inhibition was modeled using equation 3 and the reactions shown in reaction scheme 1 (supplemental Code 2). The concentrations of modeled free thrombin and modeled thrombin-A2M complex were used to model substrate cleavage (Figure 3B). The contribution of IIa-A2M was removed to give the modeled free thrombin shown in Figure 3C. The amount of thrombin modeled to be in complex with each inhibitor is shown in Figure 3D. As expected, a decrease in AT results in an increase in the fraction of thrombin inhibited by A2M, HCII, and A1PI instead. With the other inhibitors present, all of the thrombin is eventually inhibited even when the molar concentration of thrombin exceeds the molar concentration of AT.

The effect of changes in the rate of thrombin inhibition was studied in a model of thrombin generation. Recently, Owen et al published an examination of a number of current mathematical models of thrombin generation.²⁴ Owen et al²⁴ included python code that implemented the ODE that described the models. The venerable model of Hockin et al was used to study the role of other inhibitors in thrombin generation in hemophilia A.³⁰ To the existing Hockin model, equations for the inhibition of all thrombin species by HCII, A1PI, and A2M were added using the rate constants and plasma concentrations shown in Figure 1 (code provided in the supplemental Code 3 and supplemental Figures 2 and 3). As expected, there was little thrombin generation in the model of hemophilia A (Figure 4A).^31-34 Decreasing AT gave increasing thrombin peak (Figure 4A) and an increasing area under the thrombin curve (Figure 4B).

This study examines the effects of 4 thrombin inhibitors. The concentrations for each inhibitor are consistent with the plasma concentrations. Antithrombin is varied from its plasma concentration, through the range used for therapeutic reduction in rebalancing therapy in hemophilia, to 0.¹⁸ There are several limitations to this study. The reactions studied are in the absence of proteoglycans such as heparan sulfate and dermatan sulfate, which are found in vivo on endothelium. These proteoglycans accelerate thrombin inhibition by AT and HCII.^35,36 This study also does not consider “inhibition” of thrombin bound to fibrin. Such thrombin sequestration into a fibrin clot has a procoagulant function that is considered to be important for long-term stabilization and maintenance of clots.³⁷ These studies only consider inhibition of thrombin after it has been formed. This work does not address AT as an inhibitor of the reactions that promote thrombin generation.

As AT levels are decreased, the rate of thrombin inhibition is also decreased. This result is consistent with previous data from patients with low AT levels because of liver disease, which showed that the rate of thrombin inhibition was decreased as AT levels decreased.³⁸ Kremers et al examined thrombin inhibition in AT deficient plasma reconstituted at 40% to 230% of their measured average normal plasma concentration (1.94 μM).⁴ They also observed that decreasing AT gave decreasing rates of thrombin inhibition.

At normal levels of all of the thrombin inhibitors, AT is the dominant inhibitor in plasma and accounts for three-fourths of the thrombin inhibition. When AT is reduced to 30% levels, AT and A2M each account for just under half of the thrombin inhibition with HCII and A1PI accounting for the remaining inhibition. At 15% AT, A2M accounts for almost twice as much of the thrombin inhibition as does AT. This result is consistent with data from multiple patient populations in which a trend toward reduced thrombin-AT complexes was associated with a trend toward increased thrombin-A2M complexes (appendix of de Laat-Kremers et al⁵). Even in the complete absence of AT, all of the thrombin was inhibited. This is expected even when all prothrombin is converted to thrombin (1.4 μM). At reduced AT, the concentration of thrombin might exceed the concentration of AT; however, the concentration of thrombin cannot exceed the combined concentrations of HCII (1.2 μM), A2M (2.4 μM), and A1PI (32 μM).

This decrease in the rate of inhibition has dramatic effects on modeled thrombin generation. A decreased rate of inhibition cannot overcome the longer lag phase seen in hemophilia, but peak thrombin increases. The effect of this reduced rate of thrombin inhibition and increased thrombin peak can be seen in the integrated area under the thrombin curve, a measure of how much thrombin is generated and how long that thrombin is available to clot fibrinogen and bind to the fibrin clot. This value is similar to the endogenous thrombin potential (ETP) in a calibrated automated thrombogram (CAT) assay.³⁹ The integrated area increases as the levels of AT are decreased. Tsuchida et al examined thrombin generation in reconstituted AT deficient (but otherwise normal) plasma.³⁹ They showed that peak thrombin increased less than twofold as AT decreased to 0 and the cumulative thrombin production increased fivefold.³⁹ The increase in cumulative thrombin generation in Figure 4B is greater because the baseline level of thrombin is so low.

There are practical consequences to this shift in the pattern of thrombin inhibition. Thrombin generation assays are being used to monitor the effects of reduced AT in patients with hemophilia.⁴⁰ Because the complex of thrombin and A2M can still cleave a fluorescent substrate, assays such as the CAT that monitor cleavage of substrate will have increased fluorescent signal due to thrombin-A2M. In CAT assays, this increased signal is accounted for by algorithms that remove the effect of thrombin-A2M.^41,42 However, in cases of very low AT the increased substrate cleavage can give a high background signal leading to inaccurate estimates of thrombin levels.^39,42,43 In some settings all of the substrate is consumed, further complicating the data analysis, particularly calculation of the ETP.^42,44 

In addition to AT, HCII, A2M, and A1PI can contribute to thrombin inhibition. In plasma with normal levels of AT, AT is the dominant inhibitor with A2M, HCII, and A1PI making smaller contributions to thrombin inhibition. However, at reduced AT levels, these other thrombin inhibitors become the dominant inhibitors and provide a mechanism to maintain regulation of thrombin activity. These findings may be relevant for therapies that reduce AT levels to rebalance hemostasis in people with hemophilia A or B, with or without inhibitors.

Future studies will be done with plasma to directly test the hypothesis that formation of thrombin complexes with HCII and A1PI are increased as AT is reduced. A2M complex formation will also be assessed. Upstream effects of AT reduction on the rate and extent of prothrombin activation on platelets will be probed to assess the role of AT inhibition of factor Xa in the context of the physiologic surface and all of the plasma factor Xa inhibitors.

The authors thank Jen-Yea Chang for his advice.

Funding for this study was provided by a research grant from Sanofi to The University of North Carolina.

Contribution: A.M. performed the experiments, analyzed the data, and wrote the manuscript; D.M.M. designed and supervised the study, analyzed the data, and wrote the manuscript; and M.H. designed and supervised the study and edited the manuscript.

Conflict-of-interest disclosure: M.H. reports research funding from Takeda and served on advisory boards for Sanofi. The remaining authors declare no competing financial interests.

Correspondence: Dougald M. Monroe, UNC Blood Research Center, 8202B Mary Ellen Jones Building, 116 Manning Dr, Chapel Hill, NC 27599-7035; email: dmonroe@med.unc.edu.