New anticoagulant protein from medicinal leech.

The saliva of the medicinal leech contains various anticoagulants. Some of them, such as hirudin, are well known. However, it is reasonable to believe that not all anticoagulant proteins from medicinal leech saliva have been identified. We previously performed a comprehensive study of the transcriptome, genome, and proteome of leech salivary gland cells, which led to the discovery of several previously unknown hypothetical proteins that may have anticoagulant properties. Subsequently, we obtained a series of recombinant proteins and investigated their impact on coagulation in in vitro assays. We identified a previously undescribed protein that exhibited a high ability to suppress coagulation. The His-tagged recombinant protein was expressed in Escherichia coli and purified using metal chelate chromatography. To determine its activity, commonly used coagulation methods were used: activated partial thromboplastin time, prothrombin time, and thrombin inhibition clotting assay. Clotting and chromogenic assays for factor Xa inhibition were performed to evaluate anti-Xa activity. We used recombinant hirudin as a control anticoagulant protein in all experiments. The new protein showed significantly greater inhibition of coagulation than hirudin at the same molar concentrations in the activated partial thrombin time assay. However, hirudin demonstrated better results in the direct thrombin inhibition test, although the tested protein also exhibited the ability to inhibit thrombin. The chromogenic analysis of factor Xa inhibition revealed no activity, whereas the clotting test for factor Xa showed the opposite result. Thus, a new powerful anticoagulant protein has been discovered in the medicinal leech. This protein is homologous to antistatin, with 28 % identical amino acid residues. The recombinant protein was expressed in E. coli. This protein is capable of directly inhibiting thrombin, and based on indirect evidence, other proteases of the blood coagulation cascade have been identified.

Tyrosine phosphorylation of recombinant hirudin increases affinity to thrombin and antithrombotic activity.

Thrombin is one of the key enzymes of the blood coagulation system and a promising target for the development of anticoagulants. One of the most specific natural thrombin inhibitors is hirudin, contained in the salivary glands of medicinal leeches. The medicinal use of recombinant hirudin is limited because of the lack of sulfation on Tyr63, resulting in a 10-fold decrease in activity compared to native (sulfated) hirudin. In the present work, a set of hirudin derivatives was tested for affinity to thrombin: phospho-Tyr63, Tyr63(carboxymethyl)Phe, and Tyr63Glu mutants, which mimic Tyr63 sulfation and Gln65Glu mutant and lysine-succinylated hirudin, which enhance the overall negative charge of hirudin, as well as sulfo-hirudin and desulfo-hirudin as references. Using steered molecular dynamics simulations with subsequent umbrella sampling, phospho-hirudin was shown to exhibit the highest affinity to thrombin among all hirudin analogs, including native sulfo-hirudin; succinylated hirudin was also prospective. Phospho-hirudin exhibited the highest antithrombotic activity in in vitro assay in human plasma. Taking into account the modern methods for obtaining phospho-hirudin and succinylated hirudin, they are prospective as anticoagulants in clinical practice.

Effect of Sulfotyrosine and Negatively Charged Amino Acid of Leech-Derived Peptides on Binding and Inhibitory Activity Against Thrombin.

Hirudins, natural sulfo(glyco)proteins, are clinical anticoagulants that directly inhibit thrombin, a key coagulation factor. Their potent thrombin inhibition primarily results from antagonistic interactions with both the catalytic and non-catalytic sites of thrombin. Hirudins often feature sulfate moieties on Tyr residues in their anionic C-terminus region, enabling strong interactions with thrombin exosite-I and effectively blocking its engagement with fibrinogen. Although sulfotyrosines have been identified in various hirudin variants, the precise relationship between sulfotyrosine and the number of negatively charged amino acids within the anionic-rich C-terminus peptide domain for the binding of thrombin has remained elusive. By using Fmoc-SPPS, hirudin dodecapeptides homologous to the C-terminus of hirudin variants from various leech species were successfully synthesized, and the effect of sulfotyrosine and the number of negatively charged amino acids on hirudin-thrombin interactions was investigated. Our findings did not reveal any synergistic effect between an increasing number of sulfotyrosines or negatively charged amino acids and their inhibitory activity on thrombin or fibrinolysis in the assays, despite a higher binding level toward thrombin in the sulfated dodecapeptide Hnip_Hirudin was observed in SPR analysis.

Revisiting the Asian Buffalo Leech (Hirudinaria manillensis) Genome: Focus on Antithrombotic Genes and Their Corresponding Proteins.

Leeches are well-known annelids due to their obligate blood-feeding habits. Some leech species secrete various biologically active substances which have important medical and pharmaceutical value in antithrombotic treatments. In this study, we provided a high-quality genome of the Asian buffalo leech (Hirudinaria manillensis), based on which we performed a systematic identification of potential antithrombotic genes and their corresponding proteins. Combining automatic and manual prediction, we identified 21 antithrombotic gene families including fourteen coagulation inhibitors, three platelet aggregation inhibitors, three fibrinolysis enhancers, and one tissue penetration enhancer. A total of 72 antithrombotic genes, including two pseudogenes, were identified, including most of their corresponding proteins forming three or more disulfide bonds. Three protein families (LDTI, antistasin, and granulin) had internal tandem repeats containing 6, 10, and 12 conserved cysteines, respectively. We also measured the anticoagulant activities of the five identified hirudins (hirudin_Hman1 ~ hirudin_Hman5). The results showed that three (hirudin_Hman1, hirudin_Hman2, and hirudin_Hman5), but not the remaining two, exhibited anticoagulant activities. Our study provides the most comprehensive collection of antithrombotic biomacromolecules from a leech to date. These results will greatly facilitate the research and application of leech derivatives for medical and pharmaceutical purposes in the treatment of thrombotic diseases.

Exploiting Chemical Protein Synthesis to Study the Role of Tyrosine Sulfation on Anticoagulants from Hematophagous Organisms.

Tyrosine sulfation is a post-translational modification (PTM) that modulates function by mediating key protein-protein interactions. One of the early proteins shown to possess this PTM was hirudin, produced in the salivary glands of the medicinal leech Hirudo medicinalis, whereby tyrosine sulfation led to a ∼10-fold improvement in α-thrombin inhibitory activity. Outside of this pioneering discovery, the involvement of tyrosine sulfation in modulating the activity of salivary proteins from other hematophagous organisms was unknown. We hypothesized that the intrinsic instability of the tyrosine sulfate functionality, particularly under the acidic conditions used to isolate and analyze peptides and proteins, has led to poor detection during the isolation and/or expression of these molecules.Herein, we summarize our efforts to interrogate the functional role of tyrosine sulfation in the thrombin inhibitory and anticoagulant activity of salivary peptides and proteins from a range of different blood feeding organisms, including leeches, ticks, mosquitoes, and flies. Specifically, we have harnessed synthetic chemistry to efficiently generate homogeneously sulfated peptides and proteins for detailed structure-function studies both in vitro and in vivo.Our studies began with the leech protein hirudin P6 (from Hirudinaria manillensis), which is both sulfated on tyrosine and O-glycosylated at a nearby threonine residue. Synthetically, this was achieved through solid-phase peptide synthesis (SPPS) with a late-stage on-resin sulfation, followed by native chemical ligation and a folding step to generate six differentially modified variants of hirudin P6 to assess the functional interplay between O-glycosylation and tyrosine sulfation. A one-pot, kinetically controlled ligation of three peptide fragments was used to assemble homogeneously sulfoforms of madanin-1 and chimadanin from the tick Haemaphysalis longicornis. Dual tyrosine sulfation at two distinct sites was shown to increase the thrombin inhibitory activity by up to 3 orders of magnitude through a novel interaction with exosite II of thrombin. The diselenide-selenoester ligation developed by our lab provided us with a means to rapidly assemble a library of different sulfated tick anticoagulant proteins: the andersonins, hyalomins, madanin-like proteins, and hemeathrins, thus enabling the generation of key structure-activity data on this family of proteins. We have also confirmed the presence of tyrosine sulfation in the anticoagulant proteins of Anopheles mosquitoes (anophelins) and the Tsetse fly (TTI) via insect expression and mass spectrometric analysis. These molecules were subsequently synthesized and assessed for thrombin inhibitory and anticoagulant activity. Activity was significantly improved by the addition of tyrosine sulfate modifications and led to molecules with potent antithrombotic activity in an in vivo murine thrombosis model.The Account concludes with our most recent work on the design of trivalent hybrids that tandemly occupy the active site and both exosites (I and II) of α-thrombin, with a TTI-anophelin hybrid (Ki = 20 fM against α-thrombin) being one of the most potent protease inhibitors and anticoagulants ever generated. Taken together, this Account highlights the importance of the tyrosine sulfate post-translational modification within salivary proteins from blood feeding organisms for enhancing anticoagulant activity. This work lays the foundation for exploiting native or engineered variants as therapeutic leads for thrombotic disorders in the future.

Targeting endothelial coagulation signaling ameliorates liver obstructive cholestasis and dysfunctional angiogenesis.

Cholestatic fibrogenesis is a pathobiological process in which cumulative injury to the bile ducts coincides with progressive liver fibrosis. The pathobiologic mechanisms underlying fibrogenesis and disease progression remain poorly understood. Currently, there is no effective treatment for liver fibrosis. In this study, we discovered that components of the coagulation cascade were associated with the advanced progression of obstructive cholestasis, and anticoagulant therapy could improve liver cholestasis-induced fibrosis. In a mouse model of common bile duct ligation (BDL), which mimics cholestatic liver injury, RNA sequencing analysis revealed an increased expression of coagulation factors in endothelial cells. Pharmacological targeting of the coagulation signaling by hirudin, an anticoagulatory antagonist of thrombin, ameliorated obstructive cholestasis, and attenuated liver fibrosis symptoms. Hirudin attenuated fibrosis-associated angiogenesis, endothelial-to-mesenchymal transition (EndMT), and tissue hypoxia and reduced liver inflammation after BDL. Furthermore, hirudin suppressed YAP (Yes-associated protein) signaling and its downstream effectors in vascular endothelial cells, which are considered with profibrotic characteristics. In conclusion, we demonstrated that pharmacological targeting of coagulation signaling by hirudin has the potential to alleviate liver obstructive cholestasis and fibrosis.

Intracerebral hirudin injection alleviates cognitive impairment and oxidative stress and promotes hippocampal neurogenesis in rats subjected to cerebral ischemia.

Cerebral ischemia starts with cerebral blood flow interruption that causes severely limited oxygen and glucose supply, eliciting a cascade of pathological events, such as excitotoxicity, oxidative stress, calcium dysregulation, and inflammatory response, which could ultimately result in neuronal death. Hirudin has beneficial effects in ischemic stroke and possesses antioxidant and anti-inflammatory properties. Therefore, we investigated the biological functions of hirudin and its related mechanisms in cerebral ischemia. The ischemia-like conditions were induced by transient middle cerebral artery occlusion (MCAO). To investigate hirudin roles, intracerebroventricular injection of 10 U hirudin was given to the rats. Cognitive and motor functions were examined by beam walking and Morris water maze tests. 2,3,5-triphenyl tetrazolium chloride-stained brain sections were used to measure infarct volume. Oxidative stress was determined by assessment of oxidative stress markers. The proliferated cells were labeled by BrdU and Nestin double staining. Western blotting was performed to measure protein levels. Hirudin administration improved cognitive and motor deficits post-ischemia. Hirudin reduced brain infarction and neurological damage in MCAO-subjected rats. Hirudin alleviated oxidative stress and enhanced neurogenesis in ischemic rats. Hirudin facilitated the promotion of phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and serine-threonine kinase. In sum, hirudin alleviates cognitive deficits by attenuating oxidative stress and promoting hippocampal neurogenesis through the regulation of ERK1/2 and serine-threonine kinase in MCAO-subjected rats.

Inferring Pathways of Oxidative Folding from Prefolding Free Energy Landscapes of Disulfide-Rich Toxins.

Short, cysteine-rich peptides can exist in stable or metastable structural ensembles due to the number of possible patterns of formation of their disulfide bonds. One interesting subset of this peptide group is the conotoxins, which are produced by aquatic snails in the family Conidae. The µ conotoxins, which are antagonists and blockers of the voltage-gated sodium channel, exist in a folding spectrum: on one end of the spectrum are more hirudin-like folders, which form disulfide bonds and then reshuffle them, leading to an ensemble of kinetically trapped isomers, and on the other end are more BPTI-like folders, which form the native disulfide bonds one by one in a particular order, leading to a preponderance of conformations existing in a single stable state. In this Article, we employ the composite diffusion map approach to study the unified free energy surface of prefolding µ-conotoxin equilibrium. We identify the two most important nonlinear collective modes of the unified folding landscape and demonstrate that in the absence of their disulfides, the conotoxins can be thought of as largely disordered polymers. A small increase in the number of hydrophobic residues in the protein shifts the free energy landscape toward hydrophobically collapsed coil conformations responsible for cysteine proximity in hirudin-like folders, compared to semiextended coil conformations with more distal cysteines in BPTI-like folders. Overall, this work sheds important light on the folding processes and free energy landscapes of cysteine-rich peptides and demonstrates the extent to which sequence and length contribute to these landscapes.

Hirudin ameliorates diabetic nephropathy by inhibiting Gsdmd-mediated pyroptosis.

Our group previously reported that hirudin ameliorated diabetic nephropathy (DN) in streptozotocin (STZ)-injected rats, but the mechanism remained largely unknown. Therefore, we further explored its possible mechanism. We subcutaneously injected 5 U hirudin into STZ-induced WT mice or Gasdermin D (Gsdmd)-/- (KO) mice daily for 12 weeks, respectively, and evaluated their kidney injury. Next, glomerular endothelial cells (GECs), renal tubular epithelial cells (RTECs), and bone-marrow-derived macrophages (BMDMs) were isolated from WT mice and treated with hirudin in the presence of high glucose/lipopolysaccharides and ATP to measure the release of interleukin-18 and interleukin-1ß. Kidney injury induced by STZ injection was significantly ameliorated by hirudin through inhibiting Gsdmd-mediated pyroptosis in the mice, not Caspase 1-mediated apoptosis. Meanwhile, hirudin also suppressed pyroptosis in primary GECs, RTECs, and BMDMs in vitro. Moreover, the deletion of Gsdmd reduced pyroptosis and kidney injury both in vivo and in vitro. We also found that hirudin regulated the expression of Gsdmd by inhibiting interferon regulatory factor 2 (Irf2). Hirudin ameliorated Gsdmd-mediated pyroptosis by inhibiting irf2, leading to the improvement of kidney injury. Therefore, hirudin might serve as a potential therapeutic strategy to treat DN.

Coagulation Factor XIIIa and Activated Protein C Activate Platelets via GPVI and PAR1.

Platelet and coagulation activation are highly reciprocal processes driven by multi-molecular interactions. Activated platelets secrete several coagulation factors and expose phosphatidylserine, which supports the activation of coagulation factor proteins. On the other hand, the coagulation cascade generates known ligands for platelet receptors, such as thrombin and fibrin. Coagulation factor (F)Xa, (F)XIIIa and activated protein C (APC) can also bind to platelets, but the functional consequences are unclear. Here, we investigated the effects of the activated (anti)coagulation factors on platelets, other than thrombin. Multicolor flow cytometry and aggregation experiments revealed that the 'supernatant of (hirudin-treated) coagulated plasma' (SCP) enhanced CRP-XL-induced platelet responses, i.e., integrin αIIbß3 activation, P-selectin exposure and aggregate formation. We demonstrated that FXIIIa in combination with APC enhanced platelet activation in solution, and separately immobilized FXIIIa and APC resulted in platelet spreading. Platelet activation by FXIIIa was inhibited by molecular blockade of glycoprotein VI (GPVI) or Syk kinase. In contrast, platelet spreading on immobilized APC was inhibited by PAR1 blockade. Immobilized, but not soluble, FXIIIa and APC also enhanced in vitro adhesion and aggregation under flow. In conclusion, in coagulation, factors other than thrombin or fibrin can induce platelet activation via GPVI and PAR receptors.

Make it double: identification and characterization of a Tandem-Hirudin from the Asian medicinal leech Hirudinaria manillensis.

Haematophagous leeches express a broad variety of secretory proteins in their salivary glands, among them are hirudins and hirudin-like factors. Here, we describe the identification, molecular and initial functional characterization of Tandem-Hirudin (TH), a novel salivary gland derived factor identified in the Asian medicinal leech, Hirudinaria manillensis. In contrast to the typical structure of hirudins, TH comprises two globular domains arranged in a tandem-like orientation and lacks the elongated C-terminal tail. Similar structures of thrombin inhibitors have so far been identified only in kissing bugs and ticks. Expression of TH was performed in both cell-based and cell-free bacterial systems. A subsequent functional characterization revealed no evidence for a thrombin-inhibitory potency of TH.

Exploring the mechanism of hirudin in the treatment of diabetic kidney disease using network pharmacology combined with molecular docking verification.

OBJECTIVE: To explore the mechanism of hirudin in the treatment of diabetic kidney disease (DKD). METHOD: Cytoscape software was used to analyze the network between hirudin targets and active components in the treatment of DKD. The biological function and mechanism of effective targets of hirudin for DKD treatment were analyzed by the Database for Annotation, Visualization and Integrated Discovery (DAVID) database. Molecular docking technology was used to simulate the docking of key targets, and the DKD rat model was used to verify the first 4 key targets with high "Hydrogen number" among the top 10 targets verified by molecular docking. RESULTS: Total of 12334 DKD targets were screened in GeneCards, OMIM and other databases, Hirudin and DKD had 247 common target genes, and the protein interaction network got 2115 edges. The DAVID database was used for the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis, confirming that hirudin in treatment of DKD involves multiple signaling pathways such as the forkhead box O signaling pathway, the phosphatidylinositol 3-kinase-protein kinase B signaling pathway, the vascular endothelial-derived growth factor signaling pathway and other signaling pathways. The top ten key targets of hirudin in treatment of DKD were verified by molecular docking. Animal experiments showed that hirudin could decrease the expression of caspase-3 in renal tissue of DKD rats, and increase the expression of RAC-alpha serine/threonine-protein kinase, Catalase, and Heat shock protein HSP 90-alpha in renal tissue of DKD rats. CONCLUSION: This study preliminarily reveals that hirudin treats DKD through multiple targets and pathways, and molecular docking and animal experiments indicates the feasibility of this study. Hirudin may be directly or indirectly involved in the regulation of cell metabolism, oxidative stress and other mechanisms in the treatment of DKD, which will lay the foundation for future molecular biological experiments of hirudin in the treatment of DKD.

Tyrosine-O-sulfation is a widespread affinity enhancer among thrombin interactors.

Tyrosine-O-sulfation is a common post-translational modification (PTM) of proteins following the cellular secretory pathway. First described in human fibrinogen, tyrosine-O-sulfation has long been associated with the modulation of protein-protein interactions in several physiological processes. A number of relevant interactions for hemostasis are largely dictated by this PTM, many of which involving the serine proteinase thrombin (FIIa), a central player in the blood-clotting cascade. Tyrosine sulfation is not limited to endogenous FIIa ligands and has also been found in hirudin, a well-known and potent thrombin inhibitor from the medicinal leech, Hirudo medicinalis. The discovery of hirudin led to successful clinical application of analogs of leech-inspired molecules, but also unveiled several other natural thrombin-directed anticoagulant molecules, many of which undergo tyrosine-O-sulfation. The presence of this PTM has been shown to enhance the anticoagulant properties of these peptides from a range of blood-feeding organisms, including ticks, mosquitos and flies. Interestingly, some of these molecules display mechanisms of action that mimic those of thrombin's bona fide substrates.

Hirudin protects against isoproternol-induced myocardial infraction by alleviating oxidative via an Nrf2 dependent manner.

Oxidative stress plays a critical role in the progression of myocardial injury. Increasing evidence suggests that hiruidin can treat patients with cardio-injury. However, the mechanism of hirudin against myocardial infraction remains unknown. In the present study, we evaluated the potential role and mechanism of hirudin on both isoproterenol (ISO)-induced myocardial infraction (MI) in rats and Hypoxia-Reoxygenation model in H9C2 cells. Compared with the model group, hirudin apparently decreased the levels of myocardial Creatine Kinase Isoenzyme-MB (CK-MB), lactate dehydrogenase (LDH), and alleviated myocardial histopathological changes induced by ISO injection. The underlying mechanisms were revealed by the following observations: Hirudin exerted its cardioprotective effect via restoring super oxide dismutase (SOD), attenuating reactive oxygen species (ROS) and malondialdehyde (MDA). It induced the activation of Nuclear factor erythroid 2-related factor 2 (Nrf2) signal pathway through disrupting Keap1-Nrf2 complex, thus Nrf2 translocated from cytoplasm to nucleus to regulate Nrf2-dependent gene (HO-1, SOD) expressions. Furthermore, it should be noted that hirudin restored mitochondrial membrane potential in addition to cytochrome C-related apoptosis.

Draft genome of the European medicinal leech Hirudo medicinalis (Annelida, Clitellata, Hirudiniformes) with emphasis on anticoagulants.

The European medicinal leech has been used for medicinal purposes for millennia, and continues to be used today in modern hospital settings. Its utility is granted by the extremely potent anticoagulation factors that the leech secretes into the incision wound during feeding and, although a handful of studies have targeted certain anticoagulants, the full range of anticoagulation factors expressed by this species remains unknown. Here, we present the first draft genome of the European medicinal leech, Hirudo medicinalis, and estimate that we have sequenced between 79-94% of the full genome. Leveraging these data, we searched for anticoagulation factors across the genome of H. medicinalis. Following orthology determination through a series of BLAST searches, as well as phylogenetic analyses, we estimate that fully 15 different known anticoagulation factors are utilized by the species, and that 17 other proteins that have been linked to antihemostasis are also present in the genome. We underscore the utility of the draft genome for comparative studies of leeches and discuss our results in an evolutionary context.

The hirudin-like factors HLF3 and HLF4-hidden hirudins of European medicinal leeches.

The hirudin-like factors 3 (HLF3) and 4 (HLF4) belong to a new class of leech-derived factors and are present in specimens of the three European medicinal leeches, Hirudo medicinalis, Hirudo verbana, and Hirudo orientalis, respectively. Here we describe the functional analysis of natural and synthetic variants of HLF3 and HLF4. Whereas the natural variants display only very low or no detectable anti-coagulatory activities, modifications within the N-termini in combination with an exchange of the central globular domain have the potency to greatly enhance the inhibitory effects of respective HLF3 and HLF4 variants on blood coagulation. Our results support previous observations on the crucial importance of all parts (both the N- and C-termini as well as the central globular domains) of hirudin and HLF molecules for thrombin inhibition.

Hirudin Reduces the Expression of Markers of the Extracellular Matrix in Renal Tubular Epithelial Cells in a Rat Model of Diabetic Kidney Disease Through the Hypoxia-Inducible Factor-1α (HIF-1α)/Vascular Endothelial Growth Factor (VEGF) Signaling Pathway.

BACKGROUND This study aimed to investigate the effects of hirudin on the production of extracellular matrix (ECM) factors by renal tubular epithelial cells in a rat model of diabetic kidney disease (DKD) and HK-2 human renal tubule epithelial cells. MATERIAL AND METHODS Sprague-Dawley rats were divided into the normal control group (n=10), the normal control+hirudin group (n=10), the DKD model group (n=12) and the DKD+hirudin group (n=12). At the end of the study, renal histopathology was undertaken, and the expression of type IV collagen, fibronectin, hypoxia-inducible factor-1alpha (HIF-1alpha), and vascular endothelial growth factor (VEGF) were evaluated using immunohistochemistry, Western blot, and quantitative real-time polymerase chain reaction (qRT-PCR). HK-2 cells were cultured in glucose and treated with hirudin. Protein and mRNA expression of fibronectin, type IV collagen, HIF-1alpha, and VEGF were evaluated following knockdown or overexpression of HIF-1alpha. RESULTS Hirudin significantly improved renal function in the rat model of DKD (P<0.01), and significantly down-regulated the expression of fibronectin, type IV collagen, HIF-1alpha, and VEGF proteins (P<0.05). The expression of ECM associated proteins was increased in HK-2 cells treated with high glucose and reduced in the high glucose+shRNA HIF-1alpha group (P<0.05). Compared with the control group, the expression of ECM associated proteins was increased in the HIF-1alpha over-expressed group, and decreased following treatment with hirudin (P<0.05). CONCLUSIONS Hirudin reduced the expression of markers of ECM by inhibiting the HIF-1alpha/VEGF signaling pathway in DKD renal tubular epithelial cells.

Hirudin or hirudin-like factor - that is the question: insights from the analyses of natural and synthetic HLF variants.

The hirudin-like factor 1 (HLF1) of Hirudo medicinalis belongs to a new class of leech-derived factors. In previous investigations, HLF1 did not exhibit anticoagulatory activities. Here, we describe the analysis of natural and synthetic variants of HLF1 and HLF-Hyb, a yet uncharacterized member of the HLF family. Modifications within the N terminus of HLF1 have a strong impact on its activity. Some variants of HLF1 exhibit thrombin-inhibiting activity comparable to hirudins, whereas others have reduced or no activity. The analyses of HLF-Hyb variants revealed a strong impact of the central globular domain on activity. Our results indicate a comparable mode of action of hirudins and thrombin-inhibiting HLF variants. Finally, we propose and discuss criteria for classifying hirudins and HLFs.

Transcriptomic Analysis of the Influence of Methanol Assimilation on the Gene Expression in the Recombinant Pichia pastoris Producing Hirudin Variant 3.

Hirudin and its variants, as strong inhibitors against thrombin, are present in the saliva of leeches and are recognized as potent anticoagulants. However, their yield is far from the clinical requirement up to now. In this study, the production of hirudin variant 3 (HV3) was successfully realized by cultivating the recombinant Pichia pastoris GS115/pPIC9K-hv3 under the regulation of the promoter of AOX1 encoding alcohol oxidase (AOX). The antithrombin activity in the fermentation broth reached the maximum value of 5000 ATU/mL. To explore an effective strategy for improving HV3 production in the future, we investigated the influence of methanol assimilation on the general gene expression in this recombinant by transcriptomic study. The results showed that methanol was partially oxidized into CO2, and the rest was converted into glycerone-P which subsequently entered into central carbon metabolism, energy metabolism, and amino acid biosynthesis. However, the later metabolic processes were almost all down-regulated. Therefore, we propose that the up-regulated central carbon metabolism, energy, and amino acid metabolism should be beneficial for methanol assimilation, which would accordingly improve the production of HV3.

Hirudins of the Asian medicinal leech, Hirudinaria manillensis: same same, but different.

Blood coagulation in vertebrates is a complex mechanism that involves the precisely coordinated and regulated action of a cascade of factors in order to prevent excessive blood loss upon wounding. Any blood sucking ectoparasite, however, has to circumvent this mechanism to ensure the uptake of an adequate blood meal. Inhibitors of blood coagulation in the saliva are hence widespread among these animals. Thrombin as a key factor of blood coagulation is a prominent target of such inhibitors, and hirudin is probably the best known among the thrombin inhibitors. Hirudin was originally described in the genus Hirudo, but occurs in other leech genera like Hirudinaria and Macrobdella as well. Besides several isoforms of hirudin, a new class of putative leech saliva components, the hirudin-like factors (HLFs), was identified in both genera Hirudo and Hirudinaria. Here, we describe the expression, purification, and functional characterization of three HLFs (HLF5, 6, and 8, respectively) and two additional hirudins (HM3 and HM4) of Hirudinaria manillensis. While HLF6 lacked any inhibitory activity on thrombin, HLF5 as well as HLF8 clearly exhibited anticoagulatory properties. The inhibitory activity of HLF5 and HLF8, however, was much lower compared with both HM3 and HM4 of Hirudinaria manillensis as well as the hirudin variants 1 (HV1) and 2 (HV2) of Hirudo medicinalis. Neither an inhibition of trypsin nor a platelet aggregation was caused by HLF8. Our data indicates the presence of two classes (rather than isoforms) of hirudins in Hirudinaria manillensis with markedly different inhibitory activity on human thrombin.