Rebalancing Hemostasis in Hemophilia: Interplay between Antithrombin and Ancillary Thrombin Inhibitors

Introduction: Antithrombin (AT) is the dominant thrombin inhibitor under normal conditions. Hemophilia is a defect in thrombin generation (TG). One novel therapeutic approach for rebalancing hemostasis in hemophilia is to reduce the levels of AT to compensate for the reduced level of TG. Kinetic studies suggest that other inhibitors, including heparin cofactor II (HCII), alpha-1-protease inhibitor (a1PI), and alpha-2-macroglobulin (a2M) might assume greater importance when AT is low. We hypothesize that HCII, a1PI, and a2M contribute to the hemostatic balance in hemophilia when AT levels are reduced.

Methods: We studied the effects of AT, HCII, a1PI, and a2M on TG in models of hemophilia. TG assays used lipid or 200,000 platelets/µL as the procoagulant surface and tissue factor as the initiator. Plasma assays used AT deficient plasma with a polyclonal anti-factor VIII antibody to model hemophilia A with inhibitors (HA plasma). Synthetic plasma contained normal plasma concentrations of factors II, V, VII, IX, X, XI, protein S and TFPI. AT concentration was varied in the presence or absence of plasma levels of HCII, a1PI, and a2M. ELISA assays were developed that recognized thrombin in complex with the Serpin inhibitors a1PI and HCII. Thrombin complexes with a2M could not be assessed by ELISA, since a2M does not uniformly form a covalent complex with thrombin. a2M inhibits thrombin by trapping it within the a2M protein, and blocking access of macromolecular substrates to its active site. Thrombin-a2M complexes were distinguished from free thrombin by measuring residual thrombin activity after addition of heparin and AT to inhibit all free thrombin. Prothrombin conversion to thrombin on platelets in a synthetic hemophilia A plasma was measured by gel electrophoresis and Western blotting with an anti-prothrombin antibody.

Results: TG assays based on a fluorogenic thrombin substrate measure the concentration of free (uninhibited) thrombin as a function of time. In hemophilia, at 100% AT, TG is reduced relative to a non-hemophilic state. As AT levels are decreased, TG is increased. However, TG assays cannot be used to study very low levels of AT, because all of the fluorescent substrate is consumed.

To assess the role of a1PI and HCII at very low AT levels, covalent complexes with thrombin were measured by ELISA in HA plasma. Decreasing AT increased the amount of thrombin complexed with a1PI or HCII. However, those complexes still only accounted for a small fraction of total thrombin inhibition, suggesting that a2M might play a dominant role in thrombin inhibition. a2M inhibits thrombin by forming a "caged" complex that blocks macromolecular substrates from being cleaved by the entrapped thrombin. Levels of these complexes were assessed by measuring cleavage of a small molecular weight substrate. As AT was decreased from 100% to 15%, a2M became a more dominant inhibitor of thrombin, with a2M-IIa complexes increasing from <20% to >50% of the thrombin generated.

Thrombin is formed by activation of prothrombin. At 100% AT, prothrombin activation was minimal suggesting that AT not only inhibits formed thrombin but also reduces thrombin formation. Decreasing AT gave increasing levels of prothrombin conversion. At reduced levels of AT, addition of a2M reduced prothrombin conversion.

Conclusions: In normal plasma, AT is the dominant regulator of hemostasis. AT inhibits thrombin and, surprisingly, regulates conversion of prothrombin to thrombin. In hemophilia, normal levels of AT results in thrombin activity too low to provide hemostasis. Reducing AT in a cell-based model of hemophilia rebalances hemostasis so that more prothrombin is converted to thrombin, and the thrombin is inhibited more slowly. Auxiliary thrombin inhibitors A2M, HCII, and A1PI maintain effective thrombin inhibition with A2M becoming a dominant thrombin inhibitor. One limitation of these in vitro studies is that they do not reflect any contribution of heparin like molecules from vessel walls or other sources. In particular, HCII activity is accelerated by heparin-like molecules, and so may have a greater role in vivo than in our studies. Even with this limitation, our data suggest that secondary thrombin inhibitors may serve as ‘back-up’ to prevent deleterious consequences of lowering AT levels, and may be relevant for therapies that lower AT to rebalance hemostasis by increasing TG in people with hemophilia A or B, with or without inhibitors.