Disseminated intravascular coagulation (DIC), an acute syndrome of systemic thrombus formation in microvasculatures throughout the body, can be induced by severe infections, e.g. sepsis. Anticoagulants are clinically used to alleviate the intensities of DIC. However, anticoagulants only reduce the thrombus formation but have negligible effects on the inflammatory conditions. We previously reported embelin, a natural product, as an inhibitor of plasminogen activator inhibitor-1 (PAI-1), suggesting the potent antithrombotic property. In this study, we used three thrombotic mice models to confirm the antithrombotic property of embelin. By combining the anti-inflammatory and the antithrombotic properties, we proposed embelin as a potent therapeutic agent for sepsis-induced DIC, which involves both inflammation and thrombosis. In a lipopolysaccharides-induced septic mice model, embelin not only significantly ameliorated the inflammation levels, but also effectively reduced the pulmonary hemorrhages and the micro-thrombi formations in lung. In contrast, low-molecular-weight-heparin, an anticoagulant, only moderately ameliorated the pulmonary hemorrhages and thrombotic obstructions, but had non-measurable effect on the inflammatory conditions. In addition, embelin alleviated the dysregulation of the global coagulation in septic mice, but did not affect the global coagulation in normal mice. Our current study demonstrates the antithrombotic property of embelin and the potency of the treatment or prevention of syndromes combining inflammation and thrombosis, e.g. sepsis-induced DIC.

BackgroundThe plasminogen (PLG) activation system is composed by a series of serine proteases, inhibitors and several binding proteins, which together control the temporal and spatial generation of the active serine protease plasmin. As this proteolytic system plays a central role in human physiology and pathophysiology it has been extensively studied in mammals. The serine proteases of this system are believed to originate from an ancestral gene by gene duplications followed by domain gains and deletions. However, the identification of ancestral forms in primitive chordates supporting these theories remains elusive. In addition, evolutionary studies of the non-proteolytic members of this system are scarce.ResultsOur phylogenetic analyses place lamprey PLG at the root of the vertebrate PLG-group, while lamprey PLG-related growth factors represent the ancestral forms of the jawed-vertebrate orthologues. Furthermore, we find that the earliest putative orthologue of the PLG activator group is the hyaluronan binding protein 2 (HABP2) gene found in lampreys. The prime plasminogen activators (tissue- and urokinase-type plasminogen activator, tPA and uPA) first occur in cartilaginous fish and phylogenetic analyses confirm that all orthologues identified compose monophyletic groups to their mammalian counterparts. Cartilaginous fishes exhibit the most ancient vitronectin of all vertebrates, while plasminogen activator inhibitor 1 (PAI-1) appears for the first time in cartilaginous fishes and is conserved in the rest of jawed vertebrate clades. PAI-2 appears for the first time in the common ancestor of reptiles and mammals, and represents the latest appearing plasminogen activator inhibitor. Finally, we noted that the urokinase-type plasminogen activator receptor (uPAR)and three-LU domain containing genes in generaloccurred later in evolution and was first detectable after coelacanths.ConclusionsThis study identifies several primitive orthologues of the mammalian plasminogen activation system. These ancestral forms provide clues to the origin and diversification of this enzyme system. Further, the discovery of several membershitherto unknown in mammalsprovide new perspectives on the evolution of this important enzyme system.

Urokinase-type plasminogen activator (uPA) is a diagnostic marker for breast and prostate cancers recommended by American Society for Clinical Oncology and German Breast Cancer Society. Inhibition of uPA was proposed as an efficient strategy for cancer treatments. In this study, we report peptide-based uPA inhibitors with high potency and specificity comparable to monoclonal antibodies. We revealed the binding and inhibitory mechanisms by combining crystallography, molecular dynamic simulation, and other biophysical and biochemical approaches. Besides, we showed that our peptides efficiently inhibited the invasion of cancer cells via intervening with the processes of the degradation of extracellular matrices. Furthermore, our peptides significantly suppressed the tumor growth and the cancer metastases in tumor-bearing mice. This study demonstrates that these uPA peptides are highly potent anticancer agents and reveals the mechanistic insights of these uPA inhibitors, which can be useful for developing other serine protease inhibitors.

Fibrosis is a prevalent pathological condition arising from the chronic activation of fibroblasts. This activation results from the extensive intercellular crosstalk mediated by both soluble factors and direct cell-cell connections. Prominent among these are the interactions of fibroblasts with immune cells, in which the fibroblast-mast cell connection, although acknowledged, is relatively unexplored. We have used a Tg mouse model of skin fibrosis, based on expression of the transcription factor Snail in the epidermis, to probe the mechanisms regulating mast cell activity and the contribution of these cells to this pathology. We have discovered that Snail-expressing keratinocytes secrete plasminogen activator inhibitor type 1 (PAI1), which functions as a chemotactic factor to increase mast cell infiltration into the skin. Moreover, we have determined that PAI1 upregulates intercellular adhesion molecule type 1 (ICAM1) expression on dermal fibroblasts, rendering them competent to bind to mast cells. This heterotypic cell-cell adhesion, also observed in the skin fibrotic disorder scleroderma, culminates in the reciprocal activation of both mast cells and fibroblasts, leading to the cascade of events that promote fibrogenesis. Thus, we have identified roles for PAI1 in the multifactorial program of fibrogenesis that expand its functional repertoire beyond its canonical role in plasmin-dependent processes.

Some peptide sequences can behave as either substrates or inhibitors of serine proteases. Working with a cyclic peptidic inhibitor of the serine protease urokinase-type plasminogen activator (uPA), we have now demonstrated a new mechanism for an inhibitor-to-substrate switch. The peptide, CSWRGLENHAAC (upain-2), is a competitive inhibitor of human uPA, but is also slowly converted to a substrate in which the bond between Arg4 and Gly5 (the P1-P1' bond) is cleaved. Substituting the P2 residue Trp3 to an Ala or substituting the P1 Arg4 residue with 4-guanidino-phenylalanine strongly increased the substrate cleavage rate. We studied the structural basis for the inhibitor-to-substrate switch by determining the crystal structures of the various peptide variants in complex with the catalytic domain of uPA. While the slowly cleaved peptides bound clearly in inhibitory mode, with the oxyanion hole blocked by the side chain of the P3' residue Glu7, peptides behaving essentially as substrates with a much accelerated rate of cleavage was observed to be bound to the enzyme in substrate mode. Our analysis reveals that the inhibitor-to-substrate switch was associated with a 7 Å translocation of the P2 residue, and we conclude that the inhibitor-to-substrate switch of upain-2 is a result of a major conformational change in the enzyme-bound state of the peptide. This conclusion is in contrast to findings with so-called standard mechanism inhibitors in which the inhibitor-to-substrate switch is linked to minor conformational changes in the backbone of the inhibitory peptide stretch.

The catalytic activity of trypsin-like serine proteases is in many cases regulated by conformational changes initiated by binding of physiological modulators to exosites located distantly from the active site. A trypsin-like serine protease of particular interest is urokinase-type plasminogen activator (uPA), which is involved in extracellular tissue remodeling processes. Herein, we used hydrogen/deuterium exchange mass spectrometry (HDXMS) to study regulation of activity in the catalytic domain of the murine version of uPA (muPA) by two muPA specific monoclonal antibodies. Using a truncated muPA variant (muPA(16-243)), containing the catalytic domain only, we show that the two monoclonal antibodies, despite binding to an overlapping epitope in the 37s and 70s loops of muPA(16-243), stabilize distinct muPA(16-243) conformations. Whereas the inhibitory antibody, mU1 was found to increase the conformational flexibility of muPA(16-243), the stimulatory antibody, mU3, decreased muPA(16-243) conformational flexibility. Furthermore, the HDXMS data unveil the existence of a pathway connecting the 70s loop to the active site region. Using alanine scanning mutagenesis, we further identify the 70s loop as an important exosite for the activation of the physiological uPA substrate plasminogen. Thus, the data presented here reveal important information about dynamics in uPA by demonstrating how various ligands can modulate uPA activity by mediating long-range conformational changes. Moreover, the results provide a possible mechanism of plasminogen activation.

Serine proteases play important roles in numerous physiological and pathophysiological processes. Moreover, serine proteases arc classical subjects for studies of catalytic and inhibitory mechanisms of enzymes. Here, we determined the crystal structures of a serine protease, murine plasma kallikrein (mPK), and its complex with a peptidic inhibitor. Although mPK in the complex adopts a canonical protease structure, the apo-mPK exhibits a previously unobserved structural feature: the entrance of the intact Si pocket is blocked by Clu217. In addition, molecular dynamics simulations and functional assays support the flexibility of Glu217 and suggest that this flexibility play s a role in regulating the activity of serine proteases.

Summary Plasminogen activator inhibitor type 1 (PAI-1) is a central regulator of fibrinolysis and tissue remodelling. PAI-1 belongs to the serpin super-family and unlike other inhibitory serpins undergoes a spontaneous inactivation process under physiological conditions, termed latency transition. During latency transition the solvent exposed reactive centre loop is inserted into the central β–sheet A of the molecule, and is no longer accessible to reaction with the protease. More than three decades of research on mammalian PAI-1 has not been able to clarify the evolutionary advantage and physiological relevance of latency transition. In order to study the origin of PAI-1 latency transition, we produced PAI-1 from Spiny dogfish shark (Squalus acanthias) and African lungfish (Protopterus sp.), which represent central species in the evolution of vertebrates. Although human PAI-1 and the non-mammalian PAI-1 variants share only approximately 50 % sequence identity, our results showed that all tested PAI–1 variants undergo latency transition with a similar rate. Since the functional stability of PAI–1 can be greatly increased by substitution of few amino acid residues, we conclude that the ability to undergo latency transition must have been a specific selection criterion for the evolution of PAI-1. It appears that all PAI-1 molecules must harbour latency transition to fulfil their physiological function, stressing the importance to further pursue a complete understanding of this molecular phenomenon with possible implication to pharmacological intervention. Our results provide the next step in understanding how the complete role of this important protease inhibitor evolved along with the fibrinolytic system. Supplementary Material to this article is available online at www.thrombosis-online.com.

Plasma kallikrein (PK), a serine protease in the trypsin clan (SA), plays critical roles in many physiological and pathological pathways. Regulating the abnormal activity of PK has been successfully used in the clinical therapy of hereditary angioedema. In this study, the serine protease domain of murine plasma kallikrein (mPK) was expressed in the pichia pastoris system. The recombinant protein was a glycosylated active enzyme after purification by the cation exchange and size-exclusion chromatography, and was crystallized at the precipitant condition of 25% PEG 3350, 0.1 M Tris-HCl pH 8.5 and 0.1 M NaCl. The crystal structure of mPK was determined at 2.6 angstrom. This is the first published crystal structure of mPK, showing some distinctive features at S2' and S3' pockets when compared to its human analogue (human plasma kallikrein, hPK). In addition, mPK show unique structural features in the non-conservative 67-72 and 76-81 loops when compared to other serine proteases. These results provide insights for the design of potent and selective PK inhibitors.

Although trypsin-like serine proteases have flexible surface-exposed loops and are known to adopt higher and lower activity conformations, structural determinants for the different conformations have remained largely obscure. The trypsin-like serine protease, urokinase-type plasminogen activator (uPA), is central in tissue remodeling processes and also strongly implicated in tumor metastasis. We solved five X-ray crystal structures of murine uPA (muPA) in the absence and presence of allosteric molecules and/or substrate-like molecules. The structure of unbound muPA revealed an unsuspected non-chymotrypsin-like protease conformation in which two β-strands in the core of the protease domain undergoes a major antiparallel-to-parallel conformational transition. We next isolated two anti-muPA nanobodies; an active-site binding nanobody and an allosteric nanobody. Crystal structures of the muPA:nanobody complexes and hydrogen-deuterium exchange mass spectrometry revealed molecular insights about molecular factors controlling the antiparallel-to-parallel equilibrium in muPA. Together with muPA activity assays, the data provide valuable insights into regulatory mechanisms and conformational flexibility of uPA and trypsin-like serine proteases in general.

This review summarizes our studies in the development of small cyclic peptides for specifically modulating enzyme activity. Serine proteases share highly similar active sites but perform diverse physiological and pathological functions. From a phage-display peptide library, we isolated two mono-cyclic peptides, upain-1 (CSWRGLENHRMC) and mupain-1 (CPAYSRYLDC), which inhibit the activity of human and murine urokinase-type plasminogen activators (huPA and muPA) with Ki values in the micromolar or sub-micromolar range, respectively. The following affinity maturations significantly enhanced the potencies of the two peptides, 10-fold and >250-fold for upain-1 and mupain-1, respectively. The most potent muPA inhibitor has a potency (Ki = 2 nM) and specificity comparable to mono-clonal antibodies. Furthermore, we also found an unusual feature of mupain-1 that its inhibitory potency can be enhanced by increasing the flexibility, which challenges the traditional viewpoint that higher rigidity leading to higher affinity. Moreover, by changing a few key residues, we converted mupain-1 from a uPA inhibitor to inhibitors of other serine proteases, including plasma kallikrein (PK) and coagulation factor XIa (fXIa). PK and fXIa inhibitors showed Ki values in the low nanomolar range and high specificity. Our studies demonstrate the versatility of small cyclic peptides to engineer inhibitory potency against serine proteases and to provide a new strategy for generating peptide inhibitors of serine proteases.

The spiny dogfish shark (Squalus acanthias) is one of the most commonly used cartilaginous fishes in biological research, especially in the fields of nitrogen metabolism, ion transporters and osmoregulation. Nonetheless, transcriptomic data for this organism is scarce. In the present study, a multi-tissue RNA-seq experiment and de novo transcriptome assembly was performed in four different spiny dogfish tissues (brain, liver, kidney and ovary), providing an annotated sequence resource. The characterization of the transcriptome greatly increases the scarce sequence information for shark species. Reads were assembled with the Trinity de novo assembler both within each tissue and across all tissues combined resulting in 362,690 transcripts in the combined assembly which represent 289,515 Trinity genes. BUSCO analysis determined a level of 87% completeness for the combined transcriptome. In total, 123,110 proteins were predicted of which 78,679 and 83,164 had significant hits against the SwissProt and Uniref90 protein databases, respectively. Additionally, 61,215 proteins aligned to known protein domains, 7,208 carried a signal peptide and 15,971 possessed at least one transmembrane region. Based on the annotation, 81,582 transcripts were assigned to gene ontology terms and 42,078 belong to known clusters of orthologous groups (eggNOG). To demonstrate the value of our molecular resource, we show that the improved transcriptome data enhances the current possibilities of osmoregulation research in spiny dogfish by utilizing the novel gene and protein annotations to investigate a set of genes involved in urea synthesis and urea, ammonia and water transport, all of them crucial in osmoregulation. We describe the presence of different gene copies and isoforms of key enzymes involved in this process, including arginases and transporters of urea and ammonia, for which sequence information is currently absent in the databases for this model species. The transcriptome assemblies and the derived annotations generated in this study will support the ongoing research for this particular animal model and provides a new molecular tool to assist biological research in cartilaginous fishes.

Uncontrolled bleeding is a major cause of mortality. Lysine analogues are routinely used in the management of bleeding, but several studies indicate a risk of serious detrimental effects upon their administration. In this study, we report a bivalent conjugate "3218" of two RNA aptamers selected for binding to the serine protease tissue-type plasminogen activator (tPA), the principal initiator of fibrinolysis in mammals. The constituent monomeric aptamers, K32v2 and K18v2, were previously demonstrated to weakly inhibit fibrinolysis. We now show that K32v2 and K18v2 recognize distinct binding sites, presumably in the A- and B-chain of tPA, respectively. Both aptamers bind tPA with low nanomolar affinity and inhibit tPA-mediated activities in a way that is consistent with the proposed localization of their binding sites. The 3218 conjugate possesses the inhibitory activities of both K32v2 and K18v2 and additionally exhibits increased inhibitory efficiency relative to the monomeric aptamers. The 3218 conjugate proved an efficient inhibitor of fibrinolysis and may find application in the management of bleeding as a substitute for, or in combination with, currently used lysine analogues.

Thrombosis is a leading cause of death worldwide [1]. Recombinant tissue-type plasminogen activator (tPA) is the FDA-approved thrombolytic drug for ischemic strokes, myocardial infarction and pulmonary embolism. tPA is a multi-domain serine protease of the trypsin-family [2] and catalyses the critical step in fibrinolysis [3], converting the zymogen plasminogen to the active serine protease plasmin, which degrades the fibrin network of thrombi and blood clots. tPA is rapidly inactivated by endogenous plasminogen activators inhibitor-1 (PAI-1) [4] (Fig. 1). Engineering on tPA to reduce its inhibition by PAI-1 without compromising its thrombolytic effect is a continuous effort [5]. Tenecteplase (TNK-tPA) is a newer generation of tPA variant showing slower inhibition by PAI-1 [6]. Extensive studies to understand the molecular interactions between tPA and PAI-1 have been carried out [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], however, the precise details at atomic resolution remain unknown. We report the crystal structure of tPA·PAI-1 complex here. The methods required to achieve these data include: (1) recombinant expression and purification of a PAI-1 variant (14-1B) containing four mutations (N150H, K154T, Q319L, and M354I), and a tPA serine protease domain (tPA-SPD) variant with three mutations (C122A, N173Q, and S195A, in the chymotrypsin numbering) [19]; (2) formation of a tPA-SPD·PAI-1 Michaëlis complex in vitro [19]; and (3) solving the three-dimensional structure for this complex by X-ray crystallography [deposited in the PDB database as 5BRR]. The data explain the specificity of PAI-1 for tPA and uPA [19], [20], and provide structural basis to design newer generation of PAI-1-resistant tPA variants as thrombolytic agents [19].

The membrane-associated matrix metalloproteinase-14, MT1-MMP, has been implicated in pericellular proteolysis with an important role in cellular invasion of collagenous tissues. It is substantially upregulated in various cancers and rheumatoid arthritis, and has been considered as a potential therapeutic target. Here, we report the identification of antibody fragments to MT1-MMP that potently and specifically inhibit its cell surface functions. Lead antibody clones displayed inhibitory activity towards pro-MMP-2 activation, collagen-film degradation and gelatin-film degradation, and were shown to bind to the MT1-MMP catalytic domain outside the active site cleft, inhibiting binding to triple helical collagen. Affinity maturation using CDR3 randomization created a second generation of antibody fragments with dissociation constants down to 0.11 nM, corresponding to an improved affinity of 332-fold with the ability to interfere with cell-surface MT1-MMP functions, displaying IC50 values down to 5 nM. Importantly, the new inhibitors were able to inhibit collagen invasion by tumor-cells in vitro and in vivo primary tumor growth and metastasis of MDAMB-231 cells in a mouse orthotopic xenograft model. Herein is the first demonstration that an inhibitory antibody targeting sites outside the catalytic cleft of MT1-MMP can effectively abrogate its in vivo activity during tumorigenesis and metastasis.

RNA molecules with high affinity to specific proteins can be isolated from libraries of up to 1016 different RNA sequences by systematic evolution of ligands by exponential enrichment (SELEX). These so-called protein-binding RNA aptamers are often interesting, e.g., as modulators of protein function for therapeutic use, for probing the conformations of proteins, for studies of basic aspects of nucleic acid-protein interactions, etc. Studies on the interactions between RNA aptamers and proteins display a number of expected and unexpected features, including the chemical nature of the interacting RNA-protein surfaces, the conformation of protein-bound aptamer versus free aptamer, the conformation of aptamer-bound protein versus free protein, and the effects of aptamers on protein function. Here, we review current insights into the details of RNA aptamer-protein interactions. WIREs RNA 2016, 7:744-757. doi: 10.1002/wrna.1360 For further resources related to this article, please visit the WIREs website.

Most serpins are fast and specific inhibitors of extracellular serine proteases controlling biological processes such as blood coagulation, fibrinolysis, tissue remodeling, and inflammation. The inhibitory activity of serpins is based on a conserved metastable structure and their conversion to a more stable state during reaction with the target protease. However, the metastable state also makes serpins vulnerable to mutations, resulting in disease caused by inactive and misfolded monomeric or polymeric forms ("serpinopathy''). Misfolding can occur either intracellularly (type-I serpinopathies) or extracellularly (type-II serpinopathies). We have isolated a 20-fluoropyrimidine-modified RNA aptamer, which inhibits a mutation-induced inactivating misfolding of the serpin alpha(1)-antichymotrypsin. It is the first agent able to stabilize a type-II mutation of a serpin without interfering with the inhibitory mechanism, thereby presenting a solution for the long-standing challenge of preventing pathogenic misfolding without compromising the inhibitory function.

A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30-40% of the nanobody remainedintactandinhibitoryafterprolongedincubationwiththe protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors.

Components of the plasminogen activation system including urokinase (uPA), its inhibitor (PAI-1) and its cell surface receptor (uPAR) have been implicated in a wide variety of biological processes related to tissue homoeostasis. Firstly, the binding of uPA to uPAR favours extracellular proteolysis by enhancing cell surface plasminogen activation. Secondly, it promotes cell adhesion and signalling through binding of the provisional matrix protein vitronectin. We now report that uPA and plasmin induces a potent negative feedback on cell adhesion through specific cleavage of the RGD motif in vitronectin. Cleavage of vitronectin by uPA displays a remarkable receptor dependence and requires concomitant binding of both uPA and vitronectin to uPAR Moreover, we show that PAI-1 counteracts the negative feedback and behaves as a proteolysis-triggered stabilizer of uPAR-mediated cell adhesion to vitronectin. These findings identify a novel and highly specific function for the plasminogen activation system in the regulation of cell adhesion to vitronectin. The cleavage of vitronectin by uPA and plasmin results in the release of N-terminal vitronectin fragments that can be detected in vivo, underscoring the potential physiological relevance of the process.

Fibrinolysis is a process responsible for the dissolution of formed thrombi to re-establish blood flow after thrombus formation. Plasminogen activator inhibitor-1 (PAI-1) inhibits urokinase-type and tissue-type plasminogen activator (uPA and tPA) and is the major negative regulator of fibrinolysis. Inhibition of PAI-1 activity prevents thrombosis and accelerates fibrinolysis. However, a specific antagonist of PAI-1 is currently unavailable for therapeutic use. We screened a panel of uPA variants with mutations at and near the active site to maximize their binding to PAI-1 and identified a potent PAI-1 antagonist, PAItrap. PAItrap is the serine protease domain of urokinase containing active-site mutation (S195A) and four additional mutations (G37bR-R217L-C122A-N145Q). PAItrap inhibits human recombinant PAI-1 with high potency (Kd = 0.15 nM) and high specificity. In vitro using human plasma, PAItrap showed significant thrombolytic activity by inhibiting endogenous PAI-1. In addition, PAItrap inhibits both human and murine PAI-1, allowing the evaluation in murine models. In vivo, using a laser-induced thrombosis mouse model in which thrombus formation and fibrinolysis are monitored by intravital microscopy, PAItrap reduced fibrin generation and inhibited platelet accumulation following vascular injury. Therefore, this work demonstrates the feasibility to generate PAI-1 inhibitors using inactivated urokinase.