Thrombin-activable Factor X Re-establishes an Intrinsic Amplification in Tenase-deficient Plasmas

Abstract

Classical hemophilia results from a defect of the intrinsic tenase complex, the main factor X (FX) activator. Binding of factor VIIa to tissue factor triggers coagulation, but little amplification of thrombin production occurs. Handling of hemophilia by injection of the deficient or missing (thus foreign) factor often causes immunological complications. Several strategies have been designed to bypass intrinsic tenase complex, but none induce true auto-amplification of thrombin production. In an attempt to re-establish a cyclic amplification of prothrombin activation in the absence of tenase, we prepared a chimera of FX having fibrinopeptide A for the activation domain (FX FpA ). We reasoned that cascade initiation would produce traces of thrombin that would activate FX FpA (contrary to its normal homologue). Given that the activation domain of FX is released upon activation, thrombin cleavage would produce authentic FXa that would produce more thrombin, which in turn would activate more chimeras. Blood clotting results from a cascade of zymogen activation that culminates with a large production of thrombin (1-4). Injury initiates the process by uncovering tissue factor (TF), 3 which captures circulating activated factor VII (FVIIa) to form a complex that activates factors IX (FIX) and X (FX). Traces of activated FX (FXa) are sufficient to produce a small amount of thrombin empowering the cascade to inflate. The two main amplification complexes within this cascade are intrinsic tenase, composed of activated factor VIII (FVIIIa) associated with FIXa, and prothrombinase, composed of activated factor V (FVa) associated with FXa. The former activates FX into FXa, the latter, prothrombin into thrombin. Hemophilias A and B are coagulopathies resulting from a dysfunction of the intrinsic tenase complex, following a defect of FVIII or FIX, respectively (5). As a consequence, there is an inadequate production of FXa and subsequently of thrombin. Despite normal triggering, little amplification of thrombin production occurs. To date, replacement therapy is the only treatment that re-establishes an intrinsic auto-amplification of thrombin production (6). Therapy simply consists of administering the deficient FVIII or FIX. The main drawback of this approach resides in the potential antigenicity of the injected molecule, because it is often seen as foreign by the recipient. Neutralizing allo-antibodies makes replacement therapy gradually ineffective. Three approaches that bypass the intrinsic tenase complex without re-establishing auto-amplification of thrombin formation have been used or envisaged. In the first, injection of a mixture of the vitamin K-dependent factors (FVII, FIX, FX, and prothrombin) effectively bypasses tenase. Treatment however induces rare but serious side effects, including anaphylactic shock and thrombotic incidents. A mixture of recombinant prothrombin and FXa is currently under consideration to improve this approach (7). An efficient alternative relies on supraphysiologic injection of FVIIa (8 -9). Un...

Zymogen-like factor Xa variants restore thrombin generation and effectively bypass the intrinsic pathway in vitro

“…wt-FXa, FXa I16L , or FXa V17A were incubated in HA plasma for various periods of time, and residual clotting activity was monitored ( Figure 5 and Table 2). Consistent with previous reports, 19,44 wt-FXa is rapidly inhibited in plasma with a half-life of approximately 1 minute. In contrast, the half-lives of FXa I16L and FXa V17A were markedly prolonged and estimated to be approximately 1 hour.…”

“…Several groups have initiated this process and include a full range of strategies such as high-activity FVIIa variants, 46 FXIIa implants, 47 reversibly acylated FXa, 48 and thrombin-activable FX. 44 Previous work in the 1980s using a canine hemophilia model examined the possibility of using FXa or FXa in combination with anionic phospholipids as a hemostatic agent to treat hemophilia. 22 However, these attempts ultimately proved unsuccessful because of FXa's very short half-life and its potential to induce disseminated intravascular coagulation.…”

Engineered Factor VII, Factor IX, and Factor X Variants for Hemophilia Gene Therapy

“…The FX hybrid protein can be directly activated by thrombin cleavage and in turn activate prothrombin to thrombin. This shortcut re-establishes a shortened intrinsic amplification loop of thrombin formation in absence of the Xase complex [66]. Since an effective production of fibrin is accompanied by rapidly release of FpA from fibrinogen, the investigators reasoned that chimera FX FpA may display equally high susceptibility to activation by thrombin as its natural substrate fibrinogen.…”

“…During the last years, two major strategies based on either FXa or FX variants were envisaged for bypassing of FVIII-and FIX-deficiencies in hemophiliacs. In the first approach FXa variants with zymogen-like properties are generated which display a higher stability and extended half-life in comparison to wild-type FXa [65] whereas the second approach relies on the generation of zymogenic FX chimera proteins which can be activated by thrombin [66].…”

“…To generate a thrombin-activable zymogenic FX chimera Louvain-Quintard et al replaced the activation domain in FX by the corresponding sequence of fibrinogen referred to as fibrinopeptide A (FpA) [66]. The FX hybrid protein can be directly activated by thrombin cleavage and in turn activate prothrombin to thrombin.…”