Fluorescent Activity-Based Probe To Image and Inhibit Factor XIa Activity in Human Plasma.

Anticoagulation therapy is a mainstay of the treatment of thrombotic disorders; however, conventional anticoagulants trade antithrombotic benefits for bleeding risk. Factor (f) XI deficiency, known as hemophilia C, rarely causes spontaneous bleeding, suggesting that fXI plays a limited role in hemostasis. In contrast, individuals with congenital fXI deficiency display a reduced incidence of ischemic stroke and venous thromboembolism, indicating that fXI plays a role in thrombosis. For these reasons, there is intense interest in pursuing fXI/factor XIa (fXIa) as targets for achieving antithrombotic benefit with reduced bleeding risk. To obtain selective inhibitors of fXIa, we employed libraries of natural and unnatural amino acids to profile fXIa substrate preferences. We developed chemical tools for investigating fXIa activity, such as substrates, inhibitors, and activity-based probes (ABPs). Finally, we demonstrated that our ABP selectively labels fXIa in the human plasma, making this tool suitable for further studies on the role of fXIa in biological samples.

Parallel imaging of coagulation pathway proteases activated protein C, thrombin, and factor Xa in human plasma.

Activated protein C (APC), thrombin, and factor (f) Xa are vitamin K-dependent serine proteases that are key factors in blood coagulation. Moreover, they play important roles in inflammation, apoptosis, fibrosis, angiogenesis, and viral infections. Abnormal activity of these coagulation factors has been related to multiple conditions, such as bleeding and thrombosis, Alzheimer's disease, sepsis, multiple sclerosis, and COVID-19. The individual activities of APC, thrombin, and fXa in coagulation and in various diseases are difficult to establish since these proteases are related and have similar substrate preferences. Therefore, the development of selective chemical tools that enable imaging and discrimination between coagulation factors in biological samples may provide better insight into their roles in various conditions and potentially aid in the establishment of novel diagnostic tests. In our study, we used a large collection of unnatural amino acids, and this enabled us to extensively explore the binding pockets of the enzymes' active sites. Based on the specificity profiles obtained, we designed highly selective substrates, inhibitors, and fluorescent activity-based probes (ABPs) that were used for fast, direct, and simultaneous detection of APC, thrombin, and fXa in human plasma.

Design and characterization of an APC-specific serpin for the treatment of hemophilia.

Hemophilia is a bleeding disorder caused by deficiency in factors VIII or IX, the two components of the intrinsic Xase complex. Treatment with replacement factor can lead to the development of inhibitory antibodies, requiring the use of bypassing agents such as factor VIIa and factor concentrates. An alternative approach to bypass the Xase complex is to inhibit endogenous anticoagulant activities. Activated protein C (APC) breaks down the complex that produces thrombin by proteolytically inactivating factor Va. Defects in this mechanism (eg, factor V Leiden) are associated with thrombosis but result in less severe bleeding when co-inherited with hemophilia. Selective inhibition of APC might therefore be effective for the treatment of hemophilia. The endogenous inhibitors of APC are members of the serpin family: protein C inhibitor (PCI) and α1-antitrypsin (α1AT); however, both exhibit poor reactivity and selectivity for APC. We mutated residues in and around the scissile P1-P1' bond in PCI and α1AT, resulting in serpins with the desired specificity profile. The lead candidate was shown to promote thrombin generation in vitro and to restore fibrin and platelet deposition in an intravital laser injury model in hemophilia B mice. The power of targeting APC was further demonstrated by the complete normalization of bleeding after a severe tail clip injury in these mice. These results demonstrate that the protein C anticoagulant system can be successfully targeted by engineered serpins and that administration of such agents is effective at restoring hemostasis in vivo.

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.

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.

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.

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.

Molecular basis of thrombin recognition by protein C inhibitor revealed by the 1.6-A 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 A resolution. Thrombin interacts with PCI in an unusual fashion that depends on the length of PCI's reactive center loop (RCL) to align the heparin-binding sites of the two proteins. The principal exosite contact is engendered by movement of thrombin's 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.

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 beta-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.

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 Ibalpha, 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.

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.

Crystal structure of wild-type human thrombin in the Na+-free state.

Regulation of thrombin activity is critical for haemostasis and the prevention of thrombosis. Thrombin has several procoagulant substrates, including fibrinogen and platelet receptors, and essential cofactors for stimulating its own formation. However, thrombin is also capable of serving an anticoagulant function by activating protein C. The specificity of thrombin is primarily regulated by binding to the cofactor TM (thrombomodulin), but co-ordination of Na+ can also affect thrombin activity. The Na+-free form is often referred to as 'slow' because of reduced rates of cleavage of procoagulant substrates, but the slow form is still capable of rapid activation of protein C in the presence of TM. The molecular basis of the slow proteolytic activity of thrombin has remained elusive, in spite of two decades of solution studies and many published crystallographic structures. In the present paper, we report the first structure of wild-type unliganded human thrombin grown in the absence of co-ordinating Na+. The Na+-binding site is observed in a highly ordered position 6 A (1 A=0.1 nm) removed from that seen in the Na+-bound state. The movement of the Na+ loop results in non-catalytic hydrogen-bonding in the active site and blocking of the S1 and S2 substrate-binding pockets. Similar, if more dramatic, changes were observed in a previous structure of the constitutively slow thrombin variant E217K. The slow behaviour of thrombin in solutions devoid of Na+ can now be understood in terms of an equilibrium between an inert species, represented by the crystal structure described in the present paper, and an active form, where the addition of Na+ populates the active state.

The crystal structure of activated protein C-inactivated bovine factor Va: Implications for cofactor function.

In vertebrate hemostasis, factor Va serves as the cofactor in the prothrombinase complex that results in a 300,000-fold increase in the rate of thrombin generation compared with factor Xa alone. Structurally, little is known about the mechanism by which factor Va alters catalysis within this complex. Here, we report a crystal structure of protein C inactivated factor Va (A1.A3-C1-C2) that depicts a previously uncharacterized domain arrangement. This orientation has implications for binding to membranes essential for function. A high-affinity calcium-binding site and a copper-binding site have both been identified. Surprisingly, neither shows a direct involvement in chain association. This structure represents the largest physiologically relevant fragment of factor Va solved to date and provides a new scaffold for the future generation of models of coagulation cofactors.

The oxalate effect on release of iron from human serum transferrin explained.

A unique feature of the mechanism of iron binding to the transferrin (TF) family is the synergistic relationship between metal binding and anion binding. Little or no iron will bind to the protein without concomitant binding of an anion, physiologically identified as carbonate. Substitution of oxalate for carbonate produces no significant changes in polypeptide folding or domain orientation in the N-lobe of human serum TF (hTF) as revealed by our 1.2A structure. The oxalate is able to bind to the iron in a symmetric bidentate fashion, which, combined with the low pK(a) of the oxalate anion, makes iron displacement more difficult as documented by both iron release kinetic and equilibrium data. Characterization of an N-lobe in which the arginine at position 124 is mutated to alanine reveals that the stabilizing effect of oxalate is even greater in this mutant and nearly cancels the destabilizing effect of the mutation. Importantly, incorporation of oxalate as the synergistic anion appears to completely inhibit removal of iron from recombinant full-length hTF by HeLa S(3) cells, strongly indicating that oxalate also replaces carbonate in the C-lobe to form a stable complex. Kinetic studies confirm this claim. The combination of structural and functional data provides a coherent delineation of the effect of oxalate binding on hTF and rationalizes the results of many previous studies. In the context of iron uptake by cells, substitution of carbonate by oxalate effectively locks the iron into each lobe of hTF, thereby interfering with normal iron metabolism.

The position of arginine 124 controls the rate of iron release from the N-lobe of human serum transferrin. A structural study.

Human serum transferrin (hTF) is a bilobal iron-binding and transport protein that carries iron in the blood stream for delivery to cells by a pH-dependent mechanism. Two iron atoms are held tightly in two deep clefts by coordination to four amino acid residues in each cleft (two tyrosines, a histidine, and an aspartic acid) and two oxygen atoms from the "synergistic" carbonate anion. Other residues in the binding pocket, not directly coordinated to iron, also play critical roles in iron uptake and release through hydrogen bonding to the liganding residues. The original crystal structures of the iron-loaded N-lobe of hTF (pH 5.75 and 6.2) revealed that the synergistic carbonate is stabilized by interaction with Arg-124 and that both the arginine and the carbonate adopt two conformations (MacGillivray, R. T. A., Moore, S. A., Chen, J., Anderson, B. F., Baker, H., Luo, Y. G., Bewley, M., Smith, C. A., Murphy, M. E., Wang, Y., Mason, A. B., Woodworth, R. C., Brayer, G. D., and Baker, E. N. (1998) Biochemistry 37, 7919-7928). In the present study, we show that the two conformations are also found for a structure at pH 7.7, indicating that this finding was not strictly a function of pH. We also provide structures for two single point mutants (Y45E and L66W) designed to force Arg-124 to adopt each of the previously observed conformations. The structures of each mutant show that this goal was accomplished, and functional studies confirm the hypothesis that access to the synergistic anion dictates the rate of iron release. These studies highlight the importance of the arginine/carbonate movement in the mechanism of iron release in the N-lobe of hTF. Access to the carbonate via a water channel allows entry of protons and anions, enabling the attack on the iron.