Molecular basis of factor IXa recognition by heparin-activated antithrombin revealed by a 1.7-Å structure of the ternary complex

Abstract

Factor (f) IXa is a critical enzyme for the formation of stable blood clots, and its deficiency results in hemophilia. The enzyme functions at the confluence of the intrinsic and extrinsic pathways by binding to fVIIIa and rapidly generating fXa. In spite of its importance, little is known about how fIXa recognizes its cofactor, its substrate, or its only known inhibitor, antithrombin (AT). However, it is clear that fIXa requires extensive exosite interactions to present substrates for efficient cleavage. Here we describe the 1.7-Å crystal structure of fIXa in its recognition (Michaelis) complex with heparin-activated AT. It represents the highest resolution structure of both proteins and allows us to address several outstanding issues. The structure reveals why the heparin-induced conformational change in AT is required to permit simultaneous active-site and exosite interactions with fIXa and the nature of these interactions. The reactive center loop of AT has evolved to specifically inhibit fIXa, with a P2 Gly so as not to clash with Tyr99 on fIXa, a P4 Ile to fit snugly into the S4 pocket, and a C-terminal extension to exploit a unique wall-like feature of the active-site cleft. Arg150 is at the center of the exosite interface, interacting with AT residues on β-sheet C. A surprising crystal contact is observed between the heparin pentasaccharide and fIXa, revealing a plausible mode of binding that would allow longer heparin chains to bridge the complex.hemophilia | hemostasis | pentasaccharide | protease | thrombosis B lood coagulation (hemostasis) is traditionally described as two separate cascades of proteolytic activation events, the so-called intrinsic and extrinsic pathways (1, 2) (for a recent review, see ref.3). The extrinsic pathway is initiated by tissue damage that exposes tissue factor (TF) and subendothelial matrix proteins to the blood. Platelets adhere to collagen, and circulating factor (f) VIIa binds to and is activated by TF. The fVIIa-TF complex (extrinsic Xase) activates fX, and fXa in the presence of fVa produces thrombin. Just enough thrombin is generated at this stage for the formation of an initial clot through platelet activation; however, unless much more thrombin is produced in short order, the clot will not persist and bleeding will result. The second stage of hemostasis utilizes components of the intrinsic pathway, factors VIIIa and IXa, and deficiency of these critical proteins is the cause of hemophilia A and B, respectively. The fIXa-fVIIIa complex is known as intrinsic Xase and forms on the surface of activated platelets to efficiently produce fXa, resulting in a burst of thrombin formation.Regulatory proteins guard against overgrowth or dissemination of the clot, and all proteases generated in the cascade must eventually be inhibited. Factors VIIa and Xa are inhibited during the initiation phase by tissue factor pathway inhibitor (4), a triple Kunitz-domain canonical inhibitor, but the principal inhibitor of the coagulation proteases is the aptly named member of the serpin fam...

The Allosteric Mechanism of Activation of Antithrombin as an Inhibitor of Factor IXa and Factor Xa

“…(ii) How has a likely successful attempt to induce helix D extension failed to result in RCL hinge expulsion and large increase in heparin affinity (true for both hD(131-136) and hD(132-136))? Although one can only speculate at this point as to how this decoupling might have occurred and why these two variants are not identical to H5-bound WT, it is noteworthy that, although the side chain of Lys-133 points into solution in heparin-free antithrombin (32), it points inward to make a salt bridge with Glu-414 in all H5-bound antithrombin complexes examined in which the RCL hinge has been expelled, including antithrombin-H5 (32), antithrombin-H5-anhydrothrombin (33), antithrombin-H5-S195A factor Xa (30), and antithrombin-H5-S195A factor IXa (31). In these complexes the lysine side chain is further flanked by another acidic residue (Asp-278).…”

Saturation Mutagenesis of the Antithrombin Reactive Center Loop P14 Residue Supports a Three-step Mechanism of Heparin Allosteric Activation Involving Intermediate and Fully Activated States

“…Notably, the RCL P14 residuevated state. Mutagenesis studies (8,9) and x-ray structures of heparin pentasaccharide-activated antithrombin complexes with catalytically disabled factor Xa (10) and factor IXa (11) have shown that allosteric activation is mediated by a common exosite outside the RCL whose engagement by RCL-bound factors Xa and IXa augments the protease interaction with the serpin and thereby enhances reactivity. Recent mutagenesis studies have shown that the exosite is accessible to RCL-bound proteases in the native structure despite RCL insertion into sheet A and contributes to the low antithrombin reactivity with these proteases in the native state (12).…”

Heparan Sulfate Proteoglycans Mediate Factor XIIa Binding to the Cell Surface

“…Similarly, the number of basic amino acids on the surface of activated protein C provides a binding site for heparin and HS (27). Moreover, structural studies on factor IX or factor X indicate that these proteins interact with heparin via positively charged surfaces homologous to the heparin binding site described in thrombin (28,29). Interestingly, the study of FXII architecture reveals the presence of an electropositive path on the protein surface formed by the fibronectin type II domain and the epidermal growth factorlike region on the protein N terminus (30).…”