Daniel J. D. Johnson

Structure of the antithrombin-thrombin-heparin ternary complex reveals the antithrombotic mechanism of heparin.

The maintenance of normal blood flow depends completely on the inhibition of thrombin by antithrombin, a member of the serpin family. Antithrombin circulates at a high concentration, but only becomes capable of efficient thrombin inhibition on interaction with heparin or related glycosaminoglycans. The anticoagulant properties of therapeutic heparin are mediated by its interaction with antithrombin, although the structural basis for this interaction is unclear. Here we present the crystal structure at a resolution of 2.5 Å of the ternary complex between antithrombin, thrombin and a heparin mimetic (SR123781). The structure reveals a template mechanism with antithrombin and thrombin bound to the same heparin chain. A notably close contact interface, comprised of extensive active site and exosite interactions, explains, in molecular detail, the basis of the antithrombotic properties of therapeutic heparin.

Crystal structure of a stable dimer reveals the molecular basis of serpin polymerization

Repeating intermolecular protein association by means of β-sheet expansion is the mechanism underlying a multitude of diseases including Alzheimer’s, Huntington’s and Parkinson’s and the prion encephalopathies. A family of proteins, known as the serpins, also forms large stable multimers by ordered β-sheet linkages leading to intracellular accretion and disease. These ‘serpinopathies’ include early-onset dementia caused by mutations in neuroserpin, liver cirrhosis and emphysema caused by mutations in α1-antitrypsin (α1AT), and thrombosis caused by mutations in antithrombin. Serpin structure and function are quite well understood, and the family has therefore become a model system for understanding the β-sheet expansion disorders collectively known as the conformational diseases. To develop strategies to prevent and reverse these disorders, it is necessary to determine the structural basis of the intermolecular linkage and of the pathogenic monomeric state. Here we report the crystallographic structure of a stable serpin dimer which reveals a domain swap of more than 50 residues, including two long antiparallel β-strands inserting in the centre of the principal β-sheet of the neighbouring monomer. This structure explains the extreme stability of serpin polymers, the molecular basis of their rapid propagation, and provides critical new insights into the structural changes which initiate irreversible β-sheet expansion.

Antithrombin–S195A factor Xa-heparin structure reveals the allosteric mechanism of antithrombin activation

Regulation of blood coagulation is critical for maintaining blood flow, while preventing excessive bleeding or thrombosis. One of the principal regulatory mechanisms involves heparin activation of the serpin antithrombin (AT). Inhibition of several coagulation proteases is accelerated by up to 10 000‐fold by heparin, either through bridging AT and the protease or by inducing allosteric changes in the properties of AT. The anticoagulant effect of short heparin chains, including the minimal AT‐specific pentasaccharide, is mediated exclusively through the allosteric activation of AT towards efficient inhibition of coagulation factors (f) IXa and Xa. Here we present the crystallographic structure of the recognition (Michaelis) complex between heparin‐activated AT and S195A fXa, revealing the extensive exosite contacts that confer specificity. The heparin‐induced conformational change in AT is required to allow simultaneous contacts within the active site and two distinct exosites of fXa (36‐loop and the autolysis loop). This structure explains the molecular basis of protease recognition by AT, and the mechanism of action of the important therapeutic low‐molecular‐weight heparins.

NMR resonance assignments of thrombin reveal the conformational and dynamic effects of ligation

The serine protease thrombin is generated from its zymogen prothrombin at the end of the coagulation cascade. Thrombin functions as the effector enzyme of blood clotting by cleaving several procoagulant targets, but also plays a key role in attenuating the hemostatic response by activating protein C. These activities all depend on the engagement of exosites on thrombin, either through direct interaction with a substrate, as with fibrinogen, or by binding to cofactors such as thrombomodulin. How thrombin specificity is controlled is of central importance to understanding normal hemostasis and how dysregulation causes bleeding or thrombosis. The binding of ligands to thrombin via exosite I and the coordination of Na+ have been associated with changes in thrombin conformation and activity. This phenomenon has become known as thrombin allostery, although direct evidence of conformational change, identification of the regions involved, and the functional consequences remain unclear. Here we investigate the conformational and dynamic effects of thrombin ligation at the active site, exosite I and the Na+-binding site in solution, using modern multidimensional NMR techniques. We obtained full resonance assignments for thrombin in seven differently liganded states, including fully unliganded apo thrombin, and have created a detailed map of residues that change environment, conformation, or dynamic state in response to each relevant single or multiple ligation event. These studies reveal that apo thrombin exists in a highly dynamic zymogen-like state, and relies on ligation to achieve a fully active conformation. Conformational plasticity confers upon thrombin the ability to be at once selective and promiscuous.

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

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.

Crystal Structure of Monomeric Native Antithrombin Reveals a Novel Reactive Center Loop Conformation

The poor inhibitory activity of circulating antithrombin (AT) is critical to the formation of blood clots at sites of vascular damage. AT becomes an efficient inhibitor of the coagulation proteases only after binding to a specific heparin pentasaccharide, which alters the conformation of the reactive center loop (RCL). The molecular basis of this activation event lies at the heart of the regulation of hemostasis and accounts for the anticoagulant properties of the low molecular weight heparins. Although several structures of AT have been solved, the conformation of the RCL in native AT remains unknown because of the obligate crystal contact between the RCL of native AT and its latent counterpart. Here we report the crystallographic structure of a variant of AT in its monomeric native state. The RCL shifted ∼20Å, and a salt bridge was observed between the P1 residue (Arg-393) and Glu-237. This contact explains the effect of mutations at the P1 position on the affinity of AT for heparin and also the properties of AT-Truro (E237K). The relevance of the observed conformation was verified through mutagenesis studies and by solving structures of the same variant in different crystal forms. We conclude that the poor inhibitory activity of the circulating form of AT is partially conferred by intramolecular contacts that restrain the RCL, orient the P1 residue away from attacking proteases, and additionally block the exosite utilized in protease recognition.

Factor Xa and thrombin, but not factor VIIa, elicit specific cellular responses in dermal fibroblasts

Summary.  Coagulation factors (F)VIIa, FXa and thrombin are implicated in cellular responses in vascular, mesenchymal and inflammatory cells. Fibroblasts are the most abundant cells in connective tissue, and damage to blood vessels places coagulation factors in contact with these and other cell types. Objectives: To investigate cellular responses of primary dermal fibroblasts to FVIIa, FXa and thrombin by following changes in expression of candidate proteins: monocyte chemotactic protein‐1 (MCP‐1), interleukin‐8 (IL‐8), interleukin‐6 (IL‐6) and vascular endothelial growth factor (VEGF), and to determine the expression of receptors implicated in signaling by these coagulation factors. Methods: Steady‐state mRNA levels were quantified by RNase protection assay, and protein secretion by ELISA. PAR gene expression was assessed by ribonuclease protection assay and conventional and quantitative reverse‐transcription–polymerase chain reaction. Results: FVIIa did not induce the candidate genes. In contrast, FXa and thrombin induced MCP‐1 mRNA and protein secretion strongly, IL‐8 moderately, and IL‐6 weakly. Neither FXa nor thrombin induced VEGF mRNA or protein secretion, although FXa induced VEGF protein secretion in lung fibroblasts. Comparison of the presence of candidate receptors in the two fibroblast subtypes demonstrated higher levels of PAR‐1 and PAR‐3 in lung fibroblasts relative to their dermal counterparts and the additional expression of PAR‐2. Conclusions: FXa and thrombin induce expression of MCP‐1, IL‐8 and IL‐6, and distribution and expression of PARs on dermal fibroblasts is reduced relative to their lung counterparts. Tissue origin may influence the cellular response of fibroblasts to coagulation proteases.

A Tyr346 -> Cys substitution in the interdomain acidic region a1 of factor VIII in an individual with factor VIII : C assay discrepancy

Summary. The interdomain acidic region a1 is a unique structural feature of coagulation factor VIII (FVIII) and may mediate the proteolytic activation of FVIII and the inactivation of FVIIIa. We report an individual with a Tyr346→Cys substitution within region a1, who presented with a one‐stage FVIII activity (FVIII:C) of 0·34 iu/ml (normal range 0·5–2·0) but normal two‐stage FVIII:C and FVIII antigen values. In a factor Xa (FXa)‐generation assay for FVIII in which the activation time with thrombin was varied, the variant plasma showed normal FVIII:C at both short and long activation times. However, at intermediate activation times the FXa generation of the variant plasma was less than that of normal pooled plasma. In a modified one‐stage FVIII:C assay in which partially purified FVIII was activated with thrombin at low concentrations, the variant FVIII showed less activation than wild‐type FVIII, although this defect corrected with increasing concentrations of thrombin. When partially purified variant FVIII was activated with a large molar excess of thrombin, the subsequent rate of decay of FVIII:C was greater for variant FVIII. The complex defects in activation and inactivation displayed by FVIII Tyr346→Cys support the hypothesis that the a1 sequence is a key regulator of FVIII activity.

Crystal structure of the prothrombinase complex from the venom of Pseudonaja textilis

The prothrombinase complex, composed of the protease factor (f)Xa and cofactor fVa, efficiently converts prothrombin to thrombin by specific sequential cleavage at 2 sites. How the complex assembles and its mechanism of prothrombin processing are of central importance to human health and disease, because insufficient thrombin generation is the root cause of hemophilia, and excessive thrombin production results in thrombosis. Efforts to determine the crystal structure of the prothrombinase complex have been thwarted by the dependence of complex formation on phospholipid membrane association. Pseutarin C is an intrinsically stable prothrombinase complex preassembled in the venom gland of the Australian Eastern Brown Snake (Pseudonaja textilis). Here we report the crystal structures of the fX-fV complex and of activated fXa from P textilis venom and the derived model of active pseutarin C. Structural analysis supports a single substrate binding channel on fVa, to which prothrombin and the intermediate meizothrombin bind in 2 different orientations, providing insight into the architecture and mechanism of the prothrombinase complex-the molecular engine of blood coagulation.

Thrombin Inhibition by Serpins Disrupts Exosite II

Thrombin uses three principal sites, the active site, exosite I, and exosite II, for recognition of its many cofactors and substrates. It is synthesized in the zymogen form, prothrombin, and its activation at the end of the blood coagulation cascade results in the formation of the active site and exosite I and the exposure of exosite II. The physiological inhibitors of thrombin are all serpins, whose mechanism involves significant conformational change in both serpin and protease. It has been shown that the formation of the thrombin-serpin final complex disorders the active site and exosite I of thrombin, but exosite II is thought to remain functional. It has also been hypothesized that thrombin contains a receptor-binding site that is exposed upon final complex formation. The position of this cryptic site may depend on the regions of thrombin unfolded by serpin complexation. Here we investigate the conformation of thrombin in its final complex with serpins and find that in addition to exosite I, exosite II is also disordered, as reflected by a loss of affinity for the γ′-peptide of fibrinogen and for heparin and by susceptibility to limited proteolysis. This disordering of exosite II occurs for all tested natural thrombin-inhibiting serpins. Our data suggest a novel framework for understanding serpin function, especially with respect to thrombin inhibition, where serpins functionally “rezymogenize” proteases to ensure complete loss of activity and cofactor binding.