Ty E. Adams

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.

Thrombin-Cofactor Interactions Structural Insights Into Regulatory Mechanisms

Precise modulation of thrombin activity throughout the hemostatic response is essential for efficient cessation of bleeding while preventing inappropriate clot growth or dissemination which causes thrombosis. Regulating thrombin activity is made difficult by its ability to diffuse from the surface on which it was generated and its ability to cleave at least 12 substrates. To overcome this challenge, thrombin recognition of substrates is largely controlled by cofactors that act by localizing thrombin to various surfaces, blocking substrate binding to critical exosites, engendering new exosites for substrate recognition and by allosterically modulating the properties of the active site of thrombin. Thrombin cofactors can be classified as either pro- or anticoagulants, depending on how substrate preference is altered. The procoagulant cofactors include glycoprotein Ibα, fibrin, and Na+, and the anticoagulants are heparin and thrombomodulin. Over the last few years, crystal structures have been reported for all of the thrombin-cofactor complexes. The purpose of this article is to summarize the features of these structures and to discuss the mechanisms and physiological relevance of cofactor binding in thrombin regulation.

Molecular basis of thrombin recognition by protein C inhibitor revealed by the 1.6-Å structure of the heparin-bridged complex

Protein C inhibitor (PCI) is a serpin with many roles in biology, including a dual role as pro- and anticoagulant in blood. The protease specificity and local function of PCI depend on its interaction with cofactors such as heparin-like glycosaminoglycans (GAGs) and thrombomodulin (TM). Both cofactors significantly increase the rate of thrombin inhibition, but GAGs serve to promote the anticoagulant activity of PCI, and TM promotes its procoagulant function. To gain insight into how PCI recognition of thrombin is aided by these cofactors, we determined a crystallographic structure of the Michaelis complex of PCI, thrombin, and heparin to 1.6 Å resolution. Thrombin interacts with PCI in an unusual fashion that depends on the length of PCIs reactive center loop (RCL) to align the heparin-binding sites of the two proteins. The principal exosite contact is engendered by movement of thrombins 60-loop in response to the unique P2 Phe of PCI. This mechanism of communication between the active site of thrombin and its recognition exosite is previously uncharacterized and may relate to other thrombin substrate–cofactor interactions. The cofactor activity of heparin thus depends on the formation of a heparin-bridged Michaelis complex and substrate-induced exosite contacts. We also investigated the cofactor effect of TM, establishing that TM bridges PCI to thrombin through additional direct interactions. A model of the PCI–thrombin–TM complex was built and evaluated by mutagenesis and suggests distinct binding sites for heparin and TM on PCI. These data significantly improve our understanding of the cofactor-dependent roles of PCI in hemostasis.

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.

Structure of Native Protein C Inhibitor Provides Insight into Its Multiple Functions

Protein C inhibitor (PCI) is a multifunctional serpin with wide ranging protease inhibitory functions, unique cofactor binding activities, and potential non-inhibitory functions akin to the hormone-transporting serpins. To gain insight into the molecular mechanisms utilized by PCI we developed a robust expression system in Escherichia coli and solved the crystal structure of PCI in its native state. The five monomers obtained from our two crystal forms provide an NMR-like ensemble revealing regions of inherent flexibility. The reactive center loop (RCL) of PCI is long and highly flexible with no evidence of hinge region incorporation into β-sheet A, as seen for other heparin-binding serpins. We adapted an extrinsic fluorescence method for determining dissociation constants for heparin and find that the N-terminal tail of PCI and residues adjacent to helix H are not involved in heparin binding. The minimal heparin length capable of tight binding to PCI was determined to be chains of eight monosaccharide units. A large hydrophobic pocket occupied by hydrophobic crystal contacts was found in an analogous position to the hormone-binding site in thyroxine-binding globulin. In conclusion, the data presented here provide important insights into the mechanisms by which PCI exercises its multiple inhibitory and non-inhibitory functions.

Predicting the pharmacology of thrombin inhibitors.

Summary.  Thrombotic disorders can lead to uncontrolled thrombin generation and clot formation within the circulatory system leading to vascular thrombosis. Direct inhibitors of thrombin have been developed and tested in clinical trials for the treatment of a variety of these thrombotic disorders. The bleeding complications observed during these trials have raised questions about their clinical use. The development of a computer‐based model of coagulation using the kinetic rates of individual reactions and concentrations of the constituents involved in each reaction within blood has made it possible to study coagulation pathologies in silico. We present an extension of our initial model of coagulation to include several specific thrombin inhibitors. Using this model we have studied the effect of a variety of inhibitors on thrombin generation and compared these results with the clinically observed data. The data suggest that numerical models will be useful in predicting the effectiveness of inhibitors of coagulation.

Molecular basis of thrombomodulin activation of slow thrombin

Summary.  Background: Coagulation is a highly regulated process where the ability to prevent blood loss after injury is balanced against the maintenance of blood fluidity. Thrombin is at the center of this balancing act. It is the critical enzyme for producing and stabilizing a clot, but when complexed with thrombomodulin (TM) it is converted to a powerful anticoagulant. Another cofactor that may play a role in determining thrombin function is the monovalent cation Na+. Its apparent affinity suggests that half of the thrombin generated is in a Na+‐free ‘slow’ state and half is in a Na+‐coordinated ‘fast’ state. While slow thrombin is a poor procoagulant enzyme, when complexed to TM it is an effective anticoagulant. Methods: To better understand this molecular transformation we solved a 2.4 Å structure of thrombin complexed with EGF domains 4–6 of TM in the absence of Na+ and other cofactors or inhibitors. Results: We find that TM binds as previously observed, and that the thrombin component resembles structures of the fast form. The Na+ binding loop is observed in a conformation identical to the Na+‐bound form, with conserved water molecules compensating for the missing ion. Using the fluorescent probe p‐aminobenzamidine we show that activation of slow thrombin by TM principally involves the opening of the primary specificity pocket. Conclusions: These data show that TM binding alters the conformation of thrombin in a similar manner as Na+ coordination, resulting in an ordering of the Na+ binding loop and an opening of the adjacent S1 pocket. We conclude that other, more subtle subsite changes are unlikely to influence thrombin specificity toward macromolecular substrates.

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.

Effect of O-glycosylation and tyrosine sulfation of leech-derived peptides on binding and inhibitory activity against thrombin

Synthesis of sulfated and unsulfated (glyco)peptide fragments of Hirudin P6 (a potent anticoagulant from the leech Hirudinaria manillensis) is described. The effect of O-glycosylation and tyrosine sulfation on thrombin binding and peptidolytic activity was investigated, together with the inhibition of fibrinogen cleavage.

Structural transitions during prothrombin activation: On the importance of fragment 2.

Prothrombin is activated to thrombin by the prothrombinase complex through sequential cleavage at two distinct sites. This occurs at sites of vascular injury in a highly regulated cascade of serine protease and cofactor activation, where activated platelets provide a suitable surface for protease/cofactor/substrate assembly. The precise structural and conformational changes undergone during the transition from prothrombin to thrombin have been studied for decades, and several structures of prothrombin fragments along the activation pathway have been solved. Here we present a new structure analyzed in context of other recent structures and biochemical studies. What emerges is an unexpected mechanism that involves a change in the mode of binding of the F2 domain (fragment 2) on the catalytic domain after cleavage at Arg320, and a subsequent reorientation of the linker between the F2 and catalytic domain to present the Arg271 site for cleavage.