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Staphylokinase (Sak), a small 15 kDa globular protein that is secreted by certain strains of Staphylococcus aureus, shows a potent fibrin-selective thrombolytic activity. Earlier work has shown that Sak could potentially become a low-cost alternative to currently used thrombolytic agents, such as tissue plasminogen activator (tPA). In attempts to improve its potential for clinical applications, numerous modifications of Sak have already been investigated. Here, we have characterized a novel Sak modification, cyclized Sak (cyc-Sak), which was prepared through split-intein mediated protein backbone cyclization. We have characterized the structure, stability and the activity of cyc-Sak using biophysical techniques, limited proteolysis studies and plasminogen (PG)-activation assays. Our results show that cyc-Sak possesses an identical structure, enhanced stability, resistance to proteolysis by exoproteases and improved PG-activation properties compared to its linear counterpart. It can be over-expressed with high yield in the cytoplasm of Escherichia coli and is easily purified in a two-step process. The intein-mediated cyclization occurs spontaneously in vivo during protein expression and does not necessitate further modification steps after purification of the protein. Furthermore, covalent Sak cyclization could be readily combined with other Sak modifications previously proposed, to generate an effective thrombolytic agent with lower immunogenicity and improved stability and activity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

To effectively solve the problem of significant loss of transplanted cells caused by thrombosis during cell transplantation, this study simulates the human fibrinolytic system and combines metabolic oligosaccharide engineering with strain-promoted azide-alkyne cycloaddition (SPAAC) click chemistry to construct a cell surface with fibrinolytic activity. First, a copolymer (POL) of oligoethylene glycol methacrylate (OEGMA) and 6-amino-2-(2-methylamido)hexanoic acid (Lys) was synthesized by reversible addition-fragmentation chain transfer (RAFT) copolymerization, and the dibenzocyclooctyne (DBCO) functional group was introduced into the side chain of the copolymer through an active ester reaction, resulting in a functionalized copolymer DBCO-PEG4-POL with ε-lysine ligands. Then, azide functional groups were introduced onto the surface of HeLa model cells through metabolic oligosaccharide engineering, and DBCO-PEG4-POL was further specifically modified onto the surface of HeLa cells via the SPAAC "click" reaction. In vitro investigations revealed that compared with unmodified HeLa cells, modified cells not only resist the adsorption of nonspecific proteins such as fibrinogen and human serum albumin but also selectively bind to plasminogen in plasma while maintaining good cell viability and proliferative activity. More importantly, upon the activation of adsorbed plasminogen into plasmin, the modified cells exhibited remarkable fibrinolytic activity and were capable of promptly dissolving the primary thrombus formed on their surfaces. This research not only provides a novel approach for constructing transplantable cells with fibrinolytic activity but also offers a new perspective for effectively addressing the significant loss of transplanted cells caused by thrombosis.

A new family of antifibrinolytic drugs has been recently discovered, combining a triazole moiety, an oxadiazolone, and a terminal amine. Two of the molecules of this family have shown activity that is greater than or similar to that of tranexamic acid (TXA), the current antifibrinolytic gold standard, which has been associated with several side effects and whose use is limited in patients with renal impairment. The aim of this work was to thoroughly examine the mechanism of action of the two ideal candidates of the 1,2,3-triazole family and compare them with TXA, to identify an antifibrinolytic alternative active at lower dosages. Specifically, the antifibrinolytic activity of the two compounds ( 1 and 5 ) and TXA was assessed in fibrinolytic isolated systems and in whole blood. Results revealed that despite having an activity pathway comparable to that of TXA, both compounds showed greater activity in blood. These differences could be attributed to a more stable ligand-target binding to the pocket of plasminogen for compounds 1 and 5 , as suggested by molecular dynamic simulations. This work presents further evidence of the antifibrinolytic activity of the two best candidates of the 1,2,3-triazole family and paves the way for incorporating these molecules as new antifibrinolytic therapies.

The potent angiogenesis inhibitor known as human plasminogen Kringle 5 has shown promise in the treatment of vascular disorders and malignancies. The study aimed to investigate the recognition and interaction between Kringle 5 and the A2M domain of human complement component C5 using bio-specific methodologies and molecular dynamics (MD) simulation. Initially, the specific interaction between Kringle 5 and A2M was confirmed and characterized through Ligand Blot and ELISA, yielding the dissociation constant (K d ) of 1.70 × 10 -7  mol/L. Then, Kringle 5 showcased a dose-dependent inhibition of the production of C5a in lung cancer A549 cells, consequently impeding their proliferation and migration. Following the utilization of frontal affinity chromatography (FAC), it was revealed that there exists a singular binding site with the binding constant (K a ) of 3.79 × 10 5  L/mol. Following the implementation of homology modeling and MD optimization, the detailed results indicate that only a specific segment of the N-terminal structure of the A2M molecule engages in interaction with Kringle 5 throughout the binding process and the principal driving forces encompass electrostatic force, hydrogen bonding, and van der Waals force. In conclusion, the A2M domain of human complement C5 emerges as a plausible binding target for Kringle 5 in vivo., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Lipoprotein(a) (Lp(a)), an independent, causal cardiovascular risk factor, is a lipoprotein particle that is formed by the interaction of a low-density lipoprotein (LDL) particle and apolipoprotein(a) (apo(a)) 1,2 . Apo(a) first binds to lysine residues of apolipoprotein B-100 (apoB-100) on LDL through the Kringle IV (K IV ) 7 and 8 domains, before a disulfide bond forms between apo(a) and apoB-100 to create Lp(a) (refs. 3-7 ). Here we show that the first step of Lp(a) formation can be inhibited through small-molecule interactions with apo(a) K IV 7-8. We identify compounds that bind to apo(a) K IV 7-8, and, through chemical optimization and further application of multivalency, we create compounds with subnanomolar potency that inhibit the formation of Lp(a). Oral doses of prototype compounds and a potent, multivalent disruptor, LY3473329 (muvalaplin), reduced the levels of Lp(a) in transgenic mice and in cynomolgus monkeys. Although multivalent molecules bind to the Kringle domains of rat plasminogen and reduce plasmin activity, species-selective differences in plasminogen sequences suggest that inhibitor molecules will reduce the levels of Lp(a), but not those of plasminogen, in humans. These data support the clinical development of LY3473329-which is already in phase 2 studies-as a potent and specific orally administered agent for reducing the levels of Lp(a)., (© 2024. The Author(s).)

The zymogen protease Plasminogen (Plg) and its active form plasmin (Plm) carry out important functions in the blood clot disintegration (breakdown of fibrin fibers) process. Inhibition of plasmin effectively reduces fibrinolysis to circumvent heavy bleeding. Currently, available Plm inhibitor tranexamic acid (TXA) used for treating severe hemorrhages is associated with an increased incidence of seizures which in turn were traced to gamma-aminobutyric acid antagonistic activity (GABAa) in addition to having multiple side effects. Fibrinolysis can be suppressed by targeting the three important protein domains: the kringle-2 domain of tissue plasminogen activator, the kringle-1 domain of plasminogen, and the serine protease domain of plasminogen. In the present study, one million molecules were screened from the ZINC database. These ligands were docked to their respective protein targets using Autodock Vina, Schrödinger Glide, and ParDOCK/BAPPL+. Thereafter, the drug-likeness properties of the ligands were evaluated using Discovery Studio 3.5. Subsequently, we subjected the protein-ligand complexes to molecular dynamics simulation of 200 ns in GROMACS. The identified ligands P76(ZINC09970930), C97(ZINC14888376), and U97(ZINC11839443) for each protein target are found to impart higher stability and greater compactness to the protein-ligand complexes. Principal component analysis (PCA) implicates, that the identified ligands occupy smaller phase space, form stable clusters, and provide greater rigidity to the protein-ligand complexes. Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) analysis reveals that P76, C97, and U97 exhibit better binding free energy (ΔG) when compared to that of the standard ligands. Thus, our findings can be useful for the development of promising anti-fibrinolytic agents.Communicated by Ramaswamy H. Sarma.

Aim: Streptokinase has poor selectivity and provokes the immune response. In this study, we used in silico studies to design a fusion protein to achieve targeted delivery to the thrombus. Materials & methods: Streptokinase was analyzed computationally for mapping. The fusion protein modeling and quality assessment were carried out on several servers. The enzymatic activity and the stability of the fusion protein and its complex with plasminogen were assessed through molecular docking analysis and molecular dynamics simulation respectively. Results: Physicochemical properties analysis, protein quality assessments, protein-protein docking and molecular dynamics simulations predicted that the designed fusion protein is functionally active. Conclusion: Our results showed that this fusion protein might be a prospective candidate as a novel thrombolytic agent with better selectivity.

Background: High levels of Lp(a) (lipoprotein(a)) are associated with multiple forms of cardiovascular disease. Lp(a) consists of an apoB 100 -containing particle attached to the plasminogen homologue apo(a). The pathways for Lp(a) clearance are not well understood. We previously discovered that the plasminogen receptor PlgRKT (plasminogen receptor with a C-terminal lysine) promoted Lp(a) uptake in liver cells. Here, we aimed to further define the role of PlgRKT and to investigate the role of 2 other plasminogen receptors, annexin A2 and S100A10 (S100 calcium-binding protein A10) in the endocytosis of Lp(a)., Methods: Human hepatocellular carcinoma (HepG2) cells and haploid human fibroblast-like (HAP1) cells were used for overexpression and knockout of plasminogen receptors. The uptake of Lp(a), LDL (low-density lipoprotein), apo(a), and endocytic cargos was visualized and quantified by confocal microscopy and Western blotting., Results: The uptake of both Lp(a) and apo(a), but not LDL, was significantly increased in HepG2 and HAP1 cells overexpressing PlgRKT, annexin A2, or S100A10. Conversely, Lp(a) and apo(a), but not LDL, uptake was significantly reduced in HAP1 cells in which PlgRKT and S100A10 were knocked out. Surface binding studies in HepG2 cells showed that overexpression of PlgRKT, but not annexin A2 or S100A10, increased Lp(a) and apo(a) plasma membrane binding. Annexin A2 and S100A10, on the other hand, appeared to regulate macropinocytosis with both proteins significantly increasing the uptake of the macropinocytosis marker dextran when overexpressed in HepG2 and HAP1 cells and knockout of S100A10 significantly reducing dextran uptake. Bringing these observations together, we tested the effect of a PI3K (phosphoinositide-3-kinase) inhibitor, known to inhibit macropinocytosis, on Lp(a) uptake. Results showed a concentration-dependent reduction confirming that Lp(a) uptake was indeed mediated by macropinocytosis., Conclusions: These findings uncover a novel pathway for Lp(a) endocytosis involving multiple plasminogen receptors that enhance surface binding and stimulate macropinocytosis of Lp(a). Although the findings were produced in cell culture models that have limitations, they could have clinical relevance since drugs that inhibit macropinocytosis are in clinical use, that is, the PI3K inhibitors for cancer therapy and some antidepressant compounds., Competing Interests: Disclosures None.

Plasminogen Kringle 5 is one of the most potent cytokines identified to inhibit the proliferation and migration of vascular endothelial cells. Herein, six aptamer candidates that specifically bind to Kringle 5 were generated by the systematic evolution of ligands by exponential enrichment (SELEX). After 10 rounds of screening against Kringle 5, a highly enriched ssDNA pool was sequenced and the representative aptamers were subjected to binding assays to evaluate their affinity and specificity. The preferred aptamer KG-4, which demonstrated a low dissociation constant (K d ) of ∼ 432 nM and excellent selectivity for Kringle 5. A conserved "motif" of eight bases located at the stem-loop intersection, common to the aptamer, was further confirmed as the recognition element for binding with Kringle 5. The bulge formed by the motif and depression on the lysine binding site of Kringle 5 were both located at the binding interface, and the "induced fit" between their structures played a central role in the recognition process. Kringle 5 interacts KG-4 primarily through enthalpy-driven van der Waals forces and hydrogen bond. The key nucleotides A34 and C35 at motif on KG-4 and the positively charged amino acids in the loop 1 and loop 4 regions on Kringle 5 play a major role in the interaction. Furthermore, KG-4 dose-dependently reduced the proliferation inhibition of vascular endothelial cells by Kringle 5 and had a blocking effect on the function of Kringle 5 in inhibiting migration and promoting apoptosis of vascular endothelial cells in vitro. This study put a new light on protein-aptamer binding mechanism and may provide insight into the treatment of ischemic diseases by target depletion of Kringle 5., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Liujiao Bian reports financial support was provided by National Natural Science Foundation of China., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Staphylokinase (SAK), a potent fibrin-specific plasminogen activator secreted by Staphylococcus aureus, carries a pair of lysine at the carboxy-terminus that play a key role in plasminogen activation. The underlaying mechanism by which C-terminal lysins of SAK modulate its function remains unknown. This study has been undertaken to unravel role of C-terminal lysins of SAK in plasminogen activation. While deletion of C-terminal lysins (Lys135, Lys136) drastically impaired plasminogen activation by SAK, addition of lysins enhanced its catalytic activity 2-2.5-fold. Circular dichroism analysis revealed that C-terminally modified mutants of SAK carry significant changes in their beta sheets and secondary structure. Structure models and RING (residue interaction network generation) studies indicated that the deletion of lysins has conferred extensive topological alterations in SAK, disrupting vital interactions at the interface of SAK.plasmin complex, thereby leading significant impairment in its functional activity. In contrast, addition of lysins at the C-terminus enhanced its conformational flexibility, creating a stronger coupling at the interface of SAK.plasmin complex and making it more efficient for plasminogen activation. Taken together, these studies provided new insights on the role of C-terminal lysins in establishment of precise intermolecular interactions of SAK with the plasmin for the optimal function of activator complex., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Streptococcus pyogenes (GAS) is a human bacterial pathogen that generates various mild to severe diseases. Worldwide, there are approximately 700 million cases of GAS infections per year. In some strains of GAS, the surface-resident M-protein, plasminogen-binding group A streptococcal M-protein (PAM), binds directly to human host plasminogen (hPg), where it is activated to plasmin through a mechanism involving a Pg/bacterial streptokinase (SK) complex as well as endogenous activators. Binding to Pg and its activation are dictated by selected sequences within the human host Pg protein, making it difficult to generate animal models to study this pathogen., Objectives: To develop a murine model for studying GAS infection by minimally modifying mouse Pg to enhance the affinity to bacterial PAM and sensitivity to GAS-derived SK., Methods: We used a targeting vector that contained a mouse albumin-promoter and mouse/human hybrid plasminogen cDNA targeted to the Rosa26 locus. Characterization of the mouse strain consisted of both gross and histological techniques and determination of the effects of the modified Pg protein through surface plasmon resonance measurements, Pg activation analyses, and mouse survival post-GAS infection., Results: We generated a mouse line expressing a chimeric Pg protein consisting of 2 amino acid substitutions in the heavy chain of Pg and a complete replacement of the mouse Pg light chain with the human Pg light chain., Conclusion: This protein demonstrated an enhanced affinity for bacterial PAM and sensitivity to activation by the Pg-SK complex, making the murine host susceptible to the pathogenic effects of GAS., Competing Interests: Declaration of competing interests All authors declare no conflict of interest., (Copyright © 2023 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Cellular plasminogen (Pg) receptors (PgRs) are utilized to recruit Pg; stimulate its activation to the serine protease, plasmin (Pm); and sterically protect the surface Pm from inactivation by host inhibitors. One such PgR is the moonlighting enzyme, enolase, some of which leaves the cytoplasm and resides at the cell surface to potentially function as a PgR. Since microbes employ conscription of host Pg by PgRs as one virulence mechanism, we explored the structural basis of the ability of Streptococcus pyogenes enolase (Sen) to function in this manner. Employing single-particle cryo-electron microscopy (cryo-EM), recombinant Sen from S. pyogenes was modeled at 2.6 Å as a stable symmetrical doughnut-shaped homooctamer with point group 422 (D4) symmetry, with a monomeric subunit molecular weight of ∼49 kDa. Binding sites for hPg were reported in other studies to include an internal K 252,255 and the COOH-terminal K 434,435 residues of Sen. However, in native Sen, the latter are buried within the minor interfaces of the octamer and do not function as a Pg-binding epitope. Whereas Sen and hPg do not interact in solution, when Sen is bound to a surface, hPg interacts with Sen independently of K 252,255,434,435 . PgRs devoid of COOH-terminal lysine utilize lysine isosteres comprising a basic residue, " i ", and an anionic residue at " i + 3" around one turn of an α-helix. We highlight a number of surface-exposed potential hPg-binding lysine isosteres and further conclude that while the octameric structure of Sen is critical for hPg binding, disruption of this octamer without dissociation exposes hPg-binding epitopes.

Tetranectin-plasminogen interaction plays a defining role in extracellular matrix degradation, enabling tumor cell invasion and metastasis. This interaction occurs via the carbohydrate recognition domain (CRD) and Kringle 4 domain of tetranectin and plasminogen, respectively, leading to activation of the plasminogen-cascade that triggers the proteolytic processes. Thus targeting this interaction represents an important strategy to suppress tumor cell migration and invasion. In this direction, we attempted to target the CRD of tetranectin to inhibit its interaction with the Kringle-4 domain of plasminogen using natural bioactive compounds. A cheminformatics pipeline for drug designing and screening was utilized to obtain lead compound(s) that exhibit conformationally and energetically viable CRD binding. Out of 206 compounds screened, diosgenin and scytonemin displayed the most favorable interactions with CRD. Short-term molecular dynamics simulations of 20 ns were employed to further study the conformational stability of both compounds with tetranectin CRD which reflected at the increased stability of diosgenin in the CRD binding pocket compared to scytonemin. Finally, an extended molecular dynamic simulation of 100 ns affirmed the robust and stable interaction of diosgenin with CRD. Furthermore, diosgenin was observed to exert a pronounced anti-proliferative effect on high tetranectin-expressing MDA-MB-231 breast cancer cells. The inhibitory effect of diosgenin on the tetranectin-plasminogen interaction was corroborated by the reduced migration and invasiveness of MDA-MB-231 cells under diosgenin treatment. Overall the study presents an alternate and safer approach to impede breast cancer metastasis and delineates the novel anti-metastatic activity of diosgenin.Communicated by Ramaswamy H. Sarma.

Background: Streptokinase, one of the most widely used thrombolytic medicines, is a favorable protein for site-specific PEGylation as it lacks any cysteine residues in its amino acid sequence; however, any changes in the protein's structure should be carefully planned to avoid undesired changes in its function., Objectives: This study aimed to design and produce novel di/tri-cysteine variants of streptokinase from previously developed cysteine analogues, Arg45, Glu263, and Arg319, as candidates for multiple site-specific PEGylation., Methods: Using bioinformatics tools and site-directed mutagenesis, we incorporated concurrent mutations at Arg45, Glu263, and Arg319 (carried out in our previous study) to create di/tri-cysteine variants of streptokinase proteins (SK 45-319cys , SK 263-319cys , and SK 45-263-319cys ) and evaluated their kinetic activity parameters by a colorimetric method, using H-D-Val-Leu-Lys-pNA.2HCl (S2251) as substrate., Results: Based on the kinetic results, SK 263-319cys with 44% enzyme efficiency increment compared to wild-type SK was the superior protein in terms of activity; as well, SK45-319cys and SK45-263-319cys showed 17 and 22% activity enhancement, respectively. Docking of the mutant streptokinase proteins with μ-plasmin demonstrated that changes in intermolecular interactions caused by amino acid substitution could be the reason for activity difference., Conclusion: The novel mutant proteins created in this study exhibit remarkable biological activity and may be uniquely suitable for simultaneous PEGylation on two/three domains. As well, PEGylated derivates of these variants might prove to be more proficient proteins, compared to the singlecysteine analogs of streptokinase; because of their more surface coverage and increased molecular weight., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

In this study, we examined for the first time the effect of the HOCl/OCl - - and H 2 O 2 -induced oxidation of Glu-plasminogen on damage to its primary structure and the biological activity of plasmin. The consolidated results obtained with the aid of MS/MS, electrophoresis, and colourimetry, demonstrated that none of the oxidised amino acid residues found in the proenzyme treated with 25 μM HOCl/OCl - or 100 μM H 2 O 2 were functionally significant for plasminogen. However, the treatment of plasminogen with increasing concentrations of HOCl/OCl - from 25 μM to 100 μM or H 2 O 2 from 100 μM to 300 μM promoted a partial loss in the activity of oxidised plasmin. Several methionine residues (Met57, Met182, Met385, Met404, Met585, and Met788) localized in different protein domains have been shown to serve as ROS traps, thus providing an efficient defense mechanism against oxidative stress. Oxidised Trp235, Trp417, Trp427, Trp761, and Tyr672 are most likely responsible for the reduced biological activity of Glu-plasminogen subjected to strong oxidation. The results of the present study, along with those of previous studies, indicate that the structure of Glu-plasminogen is adapted to oxidation to withstand oxidative stress induced by ROS., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The direct binding of human plasminogen (hPg), via its kringle-2 domain (K2 hPg ), to streptococcal M-protein (PAM), largely contributes to the pathogenesis of Pattern D Group A Streptococcus pyogenes (GAS). However, the mechanism of complex formation is unknown. In a system consisting of a Class II PAM from Pattern D GAS isolate NS88.2 (PAM NS88.2 ), with one K2 hPg binding a-repeat in its A-domain, we employed biophysical techniques to analyze the mechanism of the K2 hPg /PAM NS88.2 interaction. We show that apo-PAM NS88.2 is a coiled-coil homodimer (M.Wt. ~80 kDa) at 4°C-25°C, and is monomeric (M.Wt. ~40 kDa) at 37°C, demonstrating a temperature-dependent dissociation of PAM NS88.2 over a narrow temperature range. PAM NS88.2 displayed a single tight binding site for K2 hPg at 4°C, which progressively increased at 25°C through 37°C. We isolated the K2 hPg /PAM NS88.2 complexes at 4°C, 25°C, and 37°C and found molecular weights of ~50 kDa at each temperature, corresponding to a 1:1 (m:m) K2 hPg /PAM NS88.2  monomer complex. hPg activation experiments by streptokinase demonstrated that the hPg/PAM NS88.2  monomer complexes are fully functional. The data show that PAM dimers dissociate into functional monomers at physiological temperatures or when presented with the active hPg module (K2 hPg ) showing that PAM is a functional monomer at 37°C., (© 2021 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Plasminogen is a zymogenic form of plasmin, an enzyme that plays a fundamental role in the dissolution of fibrin clots as well as in many other physiological processes. For the first time, by the method of gas chromatography-mass spectrometry, post-translational modifications in the primary structure of plasminogen treated with physiologically relevant amounts of hydrogen peroxide were identified. It was found that methionine and tryptophan residues located in different structural regions of plasminogen served as targets of the oxidant. Plasminogen oxidation caused a dose-dependent effect in decreasing the fibrinogenolytic activity of plasmin evidenced by the formation of fibrinogen degradation products. The possible antioxidant role of methionines in the oxidative modification of plasminogen is discussed., (© 2021. Pleiades Publishing, Ltd.)

Tetraselmis subcordiformis , a unicellular marine green alga, is used widely in aquaculture as an initial feeding for fish, bivalve mollusks, penaeid shrimp larvae, and rotifers because of its rich content of amino acids and fatty acids. A stable nuclear transformation system using the herbicide phosphinothricin (PPT) as a selective reagent was established previously. In this research, the recombinant expression in T. subcordiformis was investigated by particle bombardment with the rt-PA gene that encodes the recombinant human tissue-type plasminogen activator (Reteplase), which is a thrombolytic agent for acute myocardial infarction treatment. Transgenic algal strains were selected by their resistance to PPT, and expression of rt-PA was validated by PCR, Southern blotting, and Western blotting, and bioactivity of rt-PA was confirmed by the fibrin agarose plate assay for bioactivity. The results showed that rt-PA was integrated into the genome of T. subcordiformis , and the expression product was bioactive, indicating proper post-transcriptional modification of rt-PA in T. subcordiformis . This report contributes to efforts that take advantage of marine microalgae as cell factories to prepare recombinant drugs and in establishing a characteristic pathway of oral administration in aquaculture.

Plasminogen is an abundant plasma protein that exists in various zymogenic forms. Plasmin, the proteolytically active form of plasminogen, is known for its essential role in fibrinolysis. To date, therapeutic targeting of the fibrinolytic system has been for 2 purposes: to promote plasmin generation for thromboembolic conditions or to stop plasmin to reduce bleeding. However, plasmin and plasminogen serve other important functions, some of which are unrelated to fibrin removal. Indeed, for >40 years, the antifibrinolytic agent tranexamic acid has been administered for its serendipitously discovered skin-whitening properties. Plasmin also plays an important role in the removal of misfolded/aggregated proteins and can trigger other enzymatic cascades, including complement. In addition, plasminogen, via binding to one of its dozen cell surface receptors, can modulate cell behavior and further influence immune and inflammatory processes. Plasminogen administration itself has been reported to improve thrombolysis and to accelerate wound repair. Although many of these more recent findings have been derived from in vitro or animal studies, the use of antifibrinolytic agents to reduce bleeding in humans has revealed additional clinically relevant consequences, particularly in relation to reducing infection risk that is independent of its hemostatic effects. The finding that many viruses harness the host plasminogen to aid infectivity has suggested that antifibrinolytic agents may have antiviral benefits. Here, we review the broadening role of the plasminogen-activating system in physiology and pathophysiology and how manipulation of this system may be harnessed for benefits unrelated to its conventional application in thrombosis and hemostasis., (© 2021 by The American Society of Hematology.)

Fibrinolysis is an important process in hemostasis responsible for dissolving the clot during wound healing. Plasmin is a central enzyme in this process via its capacity to cleave fibrin. The kinetics of plasmin generation (PG) and inhibition during fibrinolysis have been poorly understood until the recent development of assays to quantify these metrics. The assessment of plasmin kinetics allows for the identification of fibrinolytic dysfunction and better understanding of the relationships between abnormal fibrin dissolution and disease pathogenesis. Additionally, direct measurement of the inhibition of PG by antifibrinolytic medications, such as tranexamic acid, can be a useful tool to assess the risks and effectiveness of antifibrinolytic therapy in hemorrhagic diseases. This review provides an overview of available PG assays to directly measure the kinetics of plasmin formation and inhibition in human and mouse plasmas and focuses on their applications in defining the role of plasmin in diseases, including angioedema, hemophilia, rare bleeding disorders, COVID-19, or diet-induced obesity. Moreover, this review introduces the PG assay as a promising clinical and research method to monitor antifibrinolytic medications and screen for genetic or acquired fibrinolytic disorders.

Borrelia miyamotoi, a relapsing fever spirochete transmitted by Ixodid ticks causes B. miyamotoi disease (BMD). To evade the human host´s immune response, relapsing fever borreliae, including B. miyamotoi, produce distinct variable major proteins. Here, we investigated Vsp1, Vlp15/16, and Vlp18 all of which are currently being evaluated as antigens for the serodiagnosis of BMD. Comparative analyses identified Vlp15/16 but not Vsp1 and Vlp18 as a plasminogen-interacting protein of B. miyamotoi. Furthermore, Vlp15/16 bound plasminogen in a dose-dependent fashion with high affinity. Binding of plasminogen to Vlp15/16 was significantly inhibited by the lysine analog tranexamic acid suggesting that the protein-protein interaction is mediated by lysine residues. By contrast, ionic strength did not have an effect on binding of plasminogen to Vlp15/16. Of relevance, plasminogen bound to the borrelial protein cleaved the chromogenic substrate S-2251 upon conversion by urokinase-type plasminogen activator (uPa), demonstrating it retained its physiological activity. Interestingly, further analyses revealed a complement inhibitory activity of Vlp15/16 and Vlp18 on the alternative pathway by a Factor H-independent mechanism. More importantly, both borrelial proteins protect serum sensitive Borrelia garinii cells from complement-mediated lysis suggesting multiple roles of these two variable major proteins in immune evasion of B. miyamotoi.

Virulent strains of Streptococcus pyogenes (gram-positive group A Streptococcus pyogenes [GAS]) recruit host single-chain human plasminogen (hPg) to the cell surface-where in the case of Pattern D strains of GAS, hPg binds directly to the cells through a surface receptor, plasminogen-binding group A streptococcal M-protein (PAM). The coinherited Pattern D GAS-secreted streptokinase (SK2b) then accelerates cleavage of hPg at the R 561 -V 562 peptide bond, resulting in the disulfide-linked two-chain protease, human plasmin (hPm). hPm localizes on the bacterial surface, assisting bacterial dissemination via proteolysis of host defense proteins. Studies using isolated domains from PAM and hPg revealed that the A-domain of PAM binds to the hPg kringle-2 module (K2 hPg ), but how this relates to the function of the full-length proteins is unclear. Herein, we use intact proteins to show that the lysine-binding site of K2 hPg is a major determinant of the activation-resistant T-conformation of hPg. The binding of PAM to the lysine-binding site of K2 hPg relaxes the conformation of hPg, leading to a greatly enhanced activation rate of hPg by SK2b. Domain swapping between hPg and mouse Pg emphasizes the importance of the Pg latent heavy chain (residues 1-561) in PAM binding and shows that while SK2b binds to both hPg and mouse Pg, the activation properties of streptokinase are strictly attributed to the serine protease domain (residues 562-791) of hPg. Overall, these data show that native hPg is locked in an activation-resistant conformation that is relaxed upon its direct binding to PAM, allowing hPm to form and provide GAS cells with a proteolytic surface., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is an evolutionarily conserved essential enzyme in the glycolytic pathway. GAPDH is also involved in a wide spectrum of non-catalytic cellular 'moonlighting' functions. Bacterial surface-associated GAPDHs engage in many host interactions that aid in colonization, pathogenesis, and virulence. We have structurally and functionally characterized the recombinant GAPDH of the obligate intracellular bacteria Chlamydia trachomatis, the leading cause of sexually transmitted bacterial and ocular infections. Contrary to earlier speculations, recent data confirm the presence of glucose-catabolizing enzymes including GAPDH in both stages of the biphasic life cycle of the bacterium. The high-resolution crystal structure described here provides a close-up view of the enzyme's active site and surface topology and reveals two chemically modified cysteine residues. Moreover, we show for the first time that purified C. trachomatis GAPDH binds to human plasminogen and plasmin. Based on the versatility of GAPDH's functions, data presented here emphasize the need for investigating the Chlamydiae GAPDH's involvement in biological functions beyond energy metabolism., (© 2020 The Protein Society.)

The emergence of SARS-CoV-2 has prompted a worldwide health emergency. There is an urgent need for therapeutics, both through the repurposing of approved drugs and the development of new treatments. In addition to the viral drug targets, a number of human drug targets have been suggested. In theory, targeting human proteins should provide an advantage over targeting viral proteins in terms of drug resistance, which is commonly a problem in treating RNA viruses. This paper focuses on the human protein TMPRSS2, which supports coronavirus life cycles by cleaving viral spike proteins. The three-dimensional structure of TMPRSS2 is not known and so we have generated models of the TMPRSS2 in the apo state as well as in complex with a peptide substrate and putative inhibitors to aid future work. Importantly, many related human proteases have 80% or higher identity with TMPRSS2 in the S1-S1' subsites, with plasminogen and urokinase-type plasminogen activator (uPA) having 95% identity. We highlight 376 approved, investigational or experimental drugs targeting S1A serine proteases that may also inhibit TMPRSS2. Whilst the presence of a relatively uncommon lysine residue in the S2/S3 subsites means that some serine protease inhibitors will not inhibit TMPRSS2, this residue is likely to provide a handle for selective targeting in a focused drug discovery project. We discuss how experimental drugs targeting related serine proteases might be repurposed as TMPRSS2 inhibitors to treat coronaviruses., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Significant amounts of enolase-a cytosolic enzyme involved in the glycolysis pathway-are exposed on the cell surface of Candida yeast. It has been hypothesized that this exposed enolase form contributes to infection-related phenomena such as fungal adhesion to human tissues, and the activation of fibrinolysis and extracellular matrix degradation. The aim of the present study was to characterize, in structural terms, the protein-protein interactions underlying these moonlighting functions of enolase. The tight binding of human vitronectin, fibronectin and plasminogen by purified C. albicans and C. tropicalis enolases was quantitatively analyzed by surface plasmon resonance measurements, and the dissociation constants of the formed complexes were determined to be in the 10 -7 -10 -8 M range. In contrast, the binding of human proteins by the S. cerevisiae enzyme was much weaker. The chemical cross-linking method was used to map the sites on enolase molecules that come into direct contact with human proteins. An internal motif 235 DKAGYKGKVGIAMDVASSEFYKDGK 259 in C. albicans enolase was suggested to contribute to the binding of all three human proteins tested. Models for these interactions were developed and revealed the sites on the enolase molecule that bind human proteins, extensively overlap for these ligands, and are well-separated from the catalytic activity center.

One of the main culprits of Alzheimer's disease (AD) is the formation of toxic amyloid-β (Aβ) peptide polymers and the aggregation of Aβ to form plaques in the brain. We have developed techniques to purify the catalytic domain of plasmin, micro-plasmin (µPlm), which can be used for an Aβ-clearance based AD therapy. However, in serum, µPlm is irreversibly inhibited by its principal inhibitor α2-antiplasmin (α2-AP). In this study, we engineered and selected mutant forms of µPlm that are both catalytically active and insensitive to α2-AP inhibition. We identified surface residues of μPlm that might interact and bind α2-AP, and used an alanine-scanning mutagenesis method to select residues having higher activity but lower α2-AP inhibition. Then we employed saturation mutagenesis for further optimize both properties. Modeled complex structure of µPlm/α2-AP shows that F587 is a critical contact residue, which can be used as a starting position for further investigation.

To understand the role of substrate plasminogen kringles in its differential catalytic processing by the streptokinase - human plasmin (SK-HPN) activator enzyme, Fluorescence Resonance Energy Transfer (FRET) model was generated between the donor labeled activator enzyme and the acceptor labeled substrate plasminogen (for both kringle rich Lys plasminogen - LysPG, and kringle less microplasminogen - µPG as substrates). Different steps of plasminogen to plasmin catalysis i.e. substrate plasminogen docking to scissile peptide bond cleavage, chemical transformation into proteolytically active product, and the decoupling of the nascent product from the SK-HPN activator enzyme were segregated selectively using (1) FRET signal as a proximity sensor to score the interactions between the substrate and the activator during the cycle of catalysis, (2) active site titration studies and (3) kinetics of peptide bond cleavage in the substrate. Remarkably, active site titration studies and the kinetics of peptide bond cleavage have shown that post docking chemical transformation of the substrate into the product is independent of kringles adjacent to the catalytic domain (CD). Stopped-flow based rapid mixing experiments for kringle rich and kringle less substrate plasminogen derivatives under substrate saturating and single cycle turnover conditions have shown that the presence of kringle domains adjacent to the CD in the macromolecular substrate contributes by selectively speeding up the final step, namely the product release/expulsion step of catalysis by the streptokinase-plasmin(ogen) activator enzyme., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Hyperfibrinolytic situations can lead to life-threatening bleeding, especially during cardiac surgery. The approved antifibrinolytic agents such as tranexamic acid, ε-aminocaproic acid, 4-aminomethylbenzoic acid, and aprotinin were developed in the 1960s without the structural insight of their respective targets. Crystal structures of the main antifibrinolytic targets, the lysine binding sites on plasminogen's kringle domains, and plasmin's serine protease domain greatly contributed to the structure-based drug design of novel inhibitor classes. Two series of ligands targeting the lysine binding sites have been recently described, which are more potent than the most-widely used antifibrinolytic agent, tranexamic acid. Furthermore, four types of promising active site inhibitors of plasmin have been developed: tranexamic acid conjugates targeting the S1 pocket and primed sites, substrate-analogue linear homopiperidylalanine-containing 4-amidinobenzylamide derivatives, macrocyclic inhibitors addressing nonprimed binding regions, and bicyclic 14-mer SFTI-1 analogues blocking both, primed and nonprimed binding sites of plasmin. Furthermore, several allosteric plasmin inhibitors based on heparin mimetics have been developed.

Neisseria meningitidis is a human-restricted bacterium that can invade the bloodstream and cross the blood-brain barrier resulting in life-threatening sepsis and meningitis. Meningococci express a cytoplasmic peroxiredoxin-glutaredoxin (Prx5-Grx) hybrid protein that has also been identified on the bacterial surface. Here, recombinant Prx5-Grx was confirmed as a plasminogen (Plg)-binding protein, in an interaction which could be inhibited by the lysine analogue ε-aminocapronic acid. rPrx5-Grx derivatives bearing a substituted C-terminal lysine residue (rPrx5-Grx K244A ), but not the active site cysteine residue (rPrx5-Grx C185A ) or the sub-terminal rPrx5-Grx K230A lysine residue, exhibited significantly reduced Plg-binding. The absence of Prx5-Grx did not significantly reduce the ability of whole meningococcal cells to bind Plg, but under hydrogen peroxide-mediated oxidative stress, the N. meningitidis Δpxn5-grx mutant survived significantly better than the wild-type or complemented strains. Significantly, using human whole blood as a model of meningococcal bacteremia, it was found that the N. meningitidis Δpxn5-grx mutant had a survival defect compared with the parental or complemented strain, confirming an important role for Prx5-Grx in meningococcal pathogenesis., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Bartonella infection is distributed worldwide with animal and public health. Recent studies have shown that host cells infection by Bartonella has a series of different infection stages, beginning with encounter and adherence to the cells. In this study, we expressed and purified recombinant Bartonella henselae (B. henselae) α-enolase. And we found that B. henselae α-enolase is highly conserved in Bartonella species. The interacting protein partners of B. henselae α-enolase were showed by String-11. The interactions between B. henselae α-enolase and human plasminogen were subsequently confirmed by ELISA, pull down, T7 phage display and molecular docking assays. And the plasminogen-binding sites of B. henselae α-enolase are predicted at 247 FYKNGSYFY 255 . These findings will help elucidate and improve the understanding of the molecular mechanisms of Bartonella infection., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Plasminogen, the precursor of plasmin, is a serine protease that plays a fundamental role in the intravascular thrombolysis. For the first time, by using high-resolution mass spectrometry, data on the oxidative modifications of the plasminogen molecule under induced oxidation were obtained. The FTIR data show that, under oxidation on the protein, its secondary structure also undergoes the rearrangements. The high tolerance of plasminogen to oxidation can be due to both the closed conformation and the ability of some Met residues to serve as ROS trap.

Streptokinase (SK) is a plasminogen activator which converts inactive plasminogen (Pg) to active plasmin (Pm), which cleaves fibrin clots. SK secreted by groups A, C, and G Streptococcus (SKA/SKC/SKG) is composed of three domains: SKα, SKβ and SKγ. Previous domain-swapping studies between SK1/SK2b-cluster variants revealed that SKβ plays a major role in the activation of human Pg. Here, we carried out domain-swapping between skcg-SK/SK2-cluster variants to determine the involvement of SKβ in several SK functionalities, including specific/proteolytic activity kinetics, fibrinogen-bound Pg activation and α 2 -antiplasmin resistance. Our results indicate that SKβ has a minor to determining role in these diverse functionalities for skcg-SK and SK2b variants, which might potentially be accompanied by few critical residues acting as hot spots. Our findings enhance our understanding of the roles of SKβ and hot spots in different functional characteristics of SK clusters and may aid in the engineering of fibrin-specific variants of SK for breaking down blood clots with potentially higher efficacy and safety., (© 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

M-proteins (M-Prts) are major virulence determinants of Group A Streptococcus pyogenes (GAS) that are covalently anchored to the cell wall at their conserved COOH-termini while the NH 2 -terminal regions extend through the capsule into extracellular space. Functional M-Prts are also secreted and/or released from GAS cells where they exist as helical coiled-coil dimers in solution. Certain GAS strains (Pattern D) uniquely express an M-protein (plasminogen-binding group A streptococcal M-protein; PAM) that directly interacts with human plasminogen (hPg), a process strongly implicated in the virulence of these strains. M-Prt expressed by the emm gene is employed to serotype over 250 known strains of GAS, ∼20 of which are hitherto found to express PAMs. We have developed a modular structural model of the PAM dimer that describes the roles of different domains of this protein in various functions. While the helical COOH-terminal domains of PAM are essential for dimerization in solution, regions of its NH 2 -terminal domains also exhibit a weak potential to dimerize. We find that temperature controls the open (unwound) or closed (wound) states of the functional NH 2 -terminal domains of PAM. As temperature increases, α-helices are dramatically reduced, which concomitantly destabilizes the helical coiled-coil PAM dimers. PAMs with two a-repeats within the variable NH 2 -terminal A-domain (class I/III) bind to hPg tightly, but natural PAM isolates with a single a-repeat in this domain (class II) display dramatic changes in hPg binding with temperature. We conclude that coexistence of two a-repeats in PAM is critical to achieve optimal binding to hPg, especially in its monomeric form, at the biologically relevant temperature., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The grass carp reovirus (GCRV) has been shown to cause lethal infections in the grass carp Ctenopharyngodon idella (C. idella). In order to investigate the immune response to GCRV infection, the full-length cDNA sequences of coagulation factor VIII (CiFVIII) and plasminogen (CiPLG) from C. idella were cloned and their involvement in the immune response was studied. The immunity factor levels in C. idella with different GCRV resistances were also analyzed. The full-length 2478 bp cDNA of CiFVIII contained an open reading frame of 1965 bp and encoded a putative polypeptide of 654 amino acid residues. The full-length 2907 bp cDNA of CiPLG contained an open reading frame of 2133 bp and encoded a putative polypeptide of 710 amino acid residues. CiFVIII was closely clustered with that of Clupea harengus. CiPLG was first clustered with those of Cyprinus carpio and Danio rerio. CiFVIII transcripts were most abundant in the liver and least in the skin. The highest expression level of CiPLG was observed in liver and the lowest in muscle. Expression levels of CiFVIII in gill, head kidney and spleen, and expression levels of CiPLG in gill, intestine and liver all reached the maximum at 72 h post GCRV infection. In spleen, expression levels of CiFVIII and CiPLG were significantly positively correlated. The activities of T-AOC, LSZ and IgM in R♂ were significantly higher than those in O♂. Likewise, T-AOC and LSZ activities in F1 were significantly higher than f1 individuals (P < 0.01). These results indicated that CiFVIII and CiPLG may play important roles in the immune response to GCRV infection. In addition, antioxidant ability and serum immune factor activity may confer a different viral resistance to C. idella., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Mycoplasma bovis is an extremely small cell wall-deficient pathogenic bacterium in the genus Mycoplasma that causes serious economic losses to the cattle industry worldwide. Fructose-1,6-bisphosphate aldolase (FBA), a key enzyme in the glycolytic pathway, is a multifunctional protein in several pathogenic bacterial species, but its role in M. bovis remains unknown. Herein, the FBA gene of the M. bovis was amplified by PCR, and subcloned into the prokaryotic expression vector pET28a (+) to generate the pET28a-FBA plasmid for recombinant expression in Escherichia coli Transetta. Expression of the 34 kDa recombinant rMbFBA protein was confirmed by electrophoresis, and enzymatic activity assays based on conversion of NADH to NAD+ revealed Km and Vmax values of 48 μM and 43.8 μmoL/L/min, respectively. Rabbit anti-rMbFBA and anti-M. bovis serum were generated by inoculation with rMbFBA and M. bovis, and antigenicity and immunofluorescence assay demonstrated that FBA is an immunogenic protein expressed on the cell membrane in M. bovis cells. Enzyme-linked immunosorbent assays revealed equal distribution of FBA in the cell membrane and cytoplasm. Complement-dependent mycoplasmacidal assays showed that rabbit anti-rMbFBA serum killed 44.1% of M. bovis cells in the presence of complement. Binding and ELISA assays demonstrated that rMbFBA binds native bovine plasminogen and in a dose-dependent manner. Fluorescent microscopy revealed that pre-treatment with antibodies against rMbFBA decreased the adhesion of M. bovis to embryonic bovine lung (EBL) cells. Furthermore, adherence inhibition assays revealed 34.4% inhibition of M. bovis infection of EBL cells following treatment with rabbit anti-rMbFBA serum, suggesting rMbFBA participates in bacterial adhesion to EBL cells., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Anti-angiogenesis therapy is an established therapeutic strategy for cancer. The endogenous angiogenic inhibitor angiostatin contains the first 3-4 kringle domains of plasminogen and inhibits both angiogenesis and vascular permeability. We present here a 10-residue peptide, Angio-3, derived from plasminogen kringle 3, which retains the functions of angiostatin in inhibiting both angiogenesis and vascular permeability. NMR studies indicate that Angio-3 holds a solution structure similar to the corresponding region of kringle 3. Mechanistically, Angio-3 inhibited both VEGF- and bFGF-induced angiogenesis by inhibiting EC proliferation and migration while inducing apoptosis. Inhibition of VEGF-induced vascular permeability results from its ability to impede VEGF-induced dissociation of adherens junction and tight junction proteins as well as the formation of actin stress fibers. When administered intravenously, Angio-3 inhibited subcutaneous breast cancer and melanoma growth by suppressing both tumor angiogenesis and intra-tumor vascular permeability. Hence, Angio-3 is a novel dual inhibitor of angiogenesis and vascular permeability. It is valuable as a lead peptide that can be further developed as therapeutics for diseases involving excessive angiogenesis and/or vascular permeability.

Nanoparticles used in cellular applications encounter free serum proteins that adsorb onto the surface of the nanoparticle, forming a protein corona. This protein layer controls the interaction of nanoparticles with cells. For nanomedicine applications, it is important to consider how intravenous injection and the subsequent shear flow will affect the protein corona. Our goal was to determine if shear flow changed the composition of the protein corona and if these changes affected cellular binding. Colorimetric assays of protein concentration and gel electrophoresis demonstrate that polystyrene nanoparticles subjected to flow have a greater concentration of serum proteins adsorbed on the surface, especially plasminogen. Plasminogen, in the absence of nanoparticles, undergoes changes in structure in response to flow, characterized by fluorescence and circular dichroism spectroscopy. The protein-nanoparticle complexes formed from fetal bovine serum after flow had decreased cellular binding, as measured with flow cytometry. In addition to the relevance for nanomedicine, these results also highlight the technical challenges of protein corona studies. The composition of the protein corona was highly dependent on the initial mixing step: rocking, vortexing, or flow. Overall, these results reaffirm the importance of the protein corona in nanoparticle-cell interactions and point toward the challenges of predicting corona composition based on nanoparticle properties., (Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

In this study, we have isolated and characterized a fibrinolytic enzyme from the GRAS (Generally Recognized as Safe) fungus, Neurospora sitophila. The enzyme was purified by fractional ammonium sulfate precipitation, hydrophobic interaction, ion exchange and gel filtration chromatography to 45.2 fold with a specific activity of 415.6U/mg protein. The native molecular mass of the enzyme was 49kDa, while the denatured molecular mass was 30kDa and 17.5kDa, indicating that the enzyme was a hetero-dimer. It was optimally active at 50°C and pH 7.4 and stable at human physiological temperature and pH. It was found to be a chymotrypsin-like serine protease which cleaved the synthetic chromogenic substrate, N-Succinyl-Ala-Ala-Pro-Phe-pNA for which the apparent K m and V max values were 0.24mM and 4.17×10 -5 mM/s, respectively. The enzyme hydrolyzed all the chains of fibrinogen by cleaving α chain first, followed by β chain and then γ chain. Moreover, the enzyme possessed dual function of direct fibrinolysis as well as plasminogen activation. Due to its attractive biochemical and fibrinolytic properties and being from a GRAS fungus, the fibrinolytic enzyme has application as a safe and efficient thrombolytic drug., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Hereditary angioedema (HAE) is a genetically heterogeneous disease that is characterized by recurrent skin swelling, abdominal pain attacks, and potentially life-threatening upper airway obstruction. The two classic types, HAE types I and II, are both caused by mutations in the complement C1 inhibitor (SERPING1) gene resulting either in a quantitative or a qualitative deficiency of C1 inhibitor. In so-called HAE type III, in contrast, patients show normal C1 inhibitor measurements in plasma ('HAE with normal C1 inhibitor'). As previously shown by us, one subgroup of 'HAE with normal C1 inhibitor' is caused by mutations of the coagulation factor XII (F12) gene. For the present study, following the exclusion of numerous candidate genes, we screened eight unrelated index patients representing eight 'HAE families with normal C1 inhibitor and no F12 mutation' for mutations in the plasminogen (PLG) gene. A rare non-conservative missense mutation was newly identified in exon 9 of the PLG gene. This mutation (c.1100A > G), encountered in three out of eight patients, predicts a lysine-to-glutamic acid substitution in position 311 of the mature protein (p.Lys311Glu). Using isoelectric focusing of plasma samples followed by an immunoblotting procedure we demonstrated that the presence of the mutation is associated with a dysplasminogenemia, namely the presence of an aberrant plasminogen protein. The predicted structural and functional impact of the mutation, its absence in 139 control individuals, and its co-segregation with the phenotype in three large families provide strong support that it causes disease. Extending a previously proposed gene-based alphabetic nomenclature for the various HAE types one may use the term 'HAE type C' for the HAE entity described here., (Copyright © 2017. Published by Elsevier Inc.)

The coagulation system does not only offer protection against bleeding, but also aids in our defense against invading microorganisms. The hemostatic system and innate immunity are strongly entangled, which explains why so many infections are complicated by either bleeding or thrombosis. Staphylococcus aureus (S. aureus), currently the most deadly infectious agent in the developed world, causes devastating intravascular infections such as sepsis and infective endocarditis. During these infections S. aureus comes in close contact with the host hemostatic system and proves to be a master in manipulating coagulation. The coagulases of S. aureus directly induce coagulation by activating prothrombin. S. aureus also manipulates fibrinolysis by triggering plasminogen activation via staphylokinase. Furthermore, S. aureus binds and activates platelets and interacts with key coagulation proteins such as fibrin(ogen), fibronectin and von Willebrand factor. By manipulating the coagulation system S. aureus gains a significant advantage over the host defense mechanisms. Studying the interplay between S. aureus and the hemostatic system can therefore lead to new innovative therapies for battling S. aureus infections., (© 2017 International Society on Thrombosis and Haemostasis.)

Surface localized microbial enolases' binding with human plasminogen has been increasingly proven to have an important role in initial infection cycle of several human pathogens. Likewise, surface localized Mycobacterium tuberculosis (Mtb) enolase also binds to human plasminogen, and this interaction may entail crucial consequences for granuloma stability. The current study is the first attempt to explore the plasminogen interacting residues of enolase from Mtb. Beginning with the structural modeling of Mtb enolase, the binding pose of Mtb enolase and human plasminogen was predicted using protein-protein docking simulations. The binding pose revealed the interface region with interacting residues and molecular interactions. Next, the interacting residues were refined and ranked by using various criteria. Finally, the selected interacting residues were tested experimentally for their involvement in plasminogen binding. The two consecutive lysine residues, Lys-193 and Lys-194, turned out to be active residues for plasminogen binding. These residues when substituted for alanine along with the most active residue Lys-429, that is, the triple mutant (K193A + K194A + K429A) Mtb enolase, exhibited 40% reduction in plasminogen binding. It is worth noting that Mtb enolase lost nearly half of the plasminogen binding activity with only three simultaneous substitutions, without any significant secondary structure perturbation. Further, the sequence comparison between Mtb and human enolase isoforms suggests the possibility of selective targeting of Mtb enolase to obstruct binding of human plasminogen., (© 2017 Wiley Periodicals, Inc.)

Thrombotic cardiovascular disease, including acute myocardial infarction, ischemic stroke, and venous thromboembolic disease, is the leading cause of morbidity and mortality worldwide. While reperfusion therapy with thrombolytic agents reduces mortality from acute myocardial infarction and disability from stroke, thrombolysis is generally less effective than mechanical reperfusion and is associated with fatal intracerebral hemorrhage in up to 2-5% of patients. To address these limitations, we propose the tobacco mosaic virus (TMV)-based platform technology for targeted delivery of thrombolytic therapies. TMV is a plant virus-based nanoparticle with a high aspect ratio shape measuring 300 × 18 nm. These soft matter nanorods have favorable flow and margination properties allowing the targeting of the diseased vessel wall. We have previously shown that TMV homes to thrombi in a photochemical mouse model of arterial thrombosis. Here we report the synthesis of TMV conjugates loaded with streptokinase (STK). Various TMV-STK formulations were produced through bioconjugation of STK to TMV via intervening PEG linkers. TMV-STK was characterized using SDS-PAGE and Western blot, transmission electron microscopy, cryo-electron microscopy, and cryo-electron tomography. We investigated the thrombolytic activity of TMV-STK in vitro using static phantom clots, and in a physiologically relevant hydrodynamic model of shear-induced thrombosis. Our findings demonstrate that conjugation of STK to the TMV surface does not compromise the activity of STK. Moreover, the nanoparticle conjugate significantly enhances thrombolysis under flow conditions, which can likely be attributed to TMV's shape-mediated flow properties resulting in enhanced thrombus accumulation and dissolution. Together, these data suggest TMV to be a promising platform for the delivery of thrombolytics to enhance clot localization and potentially minimize bleeding risk.

The binding of human plasminogen (hPg) to the surface of the human pathogen group A Streptococcus pyogenes (GAS) and subsequent hPg activation to the protease plasmin generate a proteolytic surface that GAS employs to circumvent host innate immunity. Direct high-affinity binding of hPg/plasmin to pattern D GAS is fully recapitulated by the hPg kringle 2 domain (K2 hPg ) and a short internal peptide region (a1a2) of a specific subtype of bacterial surface M protein, present in all GAS pattern D strains. To better understand the nature of this binding, critical to the virulence of many GAS skin-tropic strains, we used high-resolution NMR to define the interaction of recombinant K2 hPg with recombinant a1a2 (VKK38) of the M protein from GAS isolate NS455. We found a 2:1 (m/m) binding stoichiometry of K2 hPg /VKK38, with the lysine-binding sites of two K2 hPg domains anchored to two regions of monomeric VKK38. The K2 hPg /VKK38 binding altered the VKK38 secondary structure from a helical apo-peptide with a flexible center to an end-to-end K2 hPg -bound α-helix. The K2 hPg residues occupied opposite faces of this helix, an arrangement that minimized steric clashing of K2 hPg We conclude that VKK38 provides two conformational lysine isosteres that each interact with the lysine-binding sites in K2 hPg Further, the adoption of an α-helix by VKK38 upon binding to K2 hPg sterically optimizes the side chains of VKK38 for maximal binding to K2 hPg and minimizes steric overlap between the K2 hPg domains. The mechanism for hPg/M protein binding uncovered here may facilitate targeting of GAS virulence factors for disease management., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

The misfolding of proteins and their accumulation in extracellular tissue compartments as insoluble amyloid or amorphous protein aggregates are a hallmark feature of many debilitating protein deposition diseases such as Alzheimer's disease, prion diseases, and type II diabetes. The plasminogen activation system is best known as an extracellular fibrinolytic system but was previously reported to also be capable of degrading amyloid fibrils. Here we show that amorphous protein aggregates interact with tissue-type plasminogen activator and plasminogen, via an exposed lysine-dependent mechanism, to efficiently generate plasmin. The insoluble aggregate-bound plasmin is shielded from inhibition by α 2 -antiplasmin and degrades amorphous protein aggregates to release smaller, soluble but relatively hydrophobic fragments of protein (plasmin-generated protein fragments (PGPFs)) that are cytotoxic. In vitro , both endothelial and microglial cells bound and internalized PGPFs before trafficking them to lysosomes. Clusterin and α 2 -macroglobulin bound to PGPFs to significantly ameliorate their toxicity. On the basis of these findings, we hypothesize that, as part of the in vivo extracellular proteostasis system, the plasminogen activation system may work synergistically with extracellular chaperones to safely clear large and otherwise pathological protein aggregates from the body., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Retinal fibrosis, including epiretinal membrane (ERM) and proliferative vitreoretinopathy (PVR), is an ocular disease that can lead to blindness. An efficacious therapy remains an unmet medical challenge. In this study, we intended to explore the inhibitory effects of the nanoparticle-mediated delivery of plasminogen kringle 5 (K5-NPs) on laser-induced ERM and to identify the potential anti-fibrosis targets of K5-NPs. A rat model of laser-induced ERM was used. Control-NPs or K5-NPs were intravitreally injected. ERM formation was observed in vivo. The expression of fibrosis-associated factors including TGF-β, CTGF, and α-SMA was detected by qRT-PCR, Western blot, or immunohistochemistry. Our results showed that K5-NPs effectively inhibit laser-induced ERM formation. The expression of α-SMA, the marker of myofibroblast, apparently decreased in immunostained ERMs in the K5-NPs group. The expression of CTGF, a pro-fibrotic factor, in our ERM rat model was significantly downregulated by K5-NPs. Meanwhile, the expression of TGF-β, the upstream regulator of CTGF, was found to be suppressed by K5-NPs as well. The anti-fibrosis effect of K5-NPs might be achieved by interfering with TGF-β expression, thus abrogating the TGF-β-induced upregulation of CTGF and α-SMA. Our study has an important clinical relevance, demonstrating that K5-NPs might offer an additional efficacious clinical option for treating fibrosis-related diseases.

Dimeric M-proteins (M-Prt) in group A Streptococcus pyogenes (GAS) are surface-expressed virulence factors implicated in processes that contribute to the pathogenicity of infection. Sequence analyses of various GAS M-Prts have shown that they contain a highly conserved sortase A-dependent cell wall-anchored C terminus, whereas the surface-exposed N terminus is highly variable, a feature used for identification and serotyping of various GAS strains. This variability also allows for strain-specific responses that suppress host defenses. Previous studies have indeed identified the N-terminal M-Prt B-domain as the site interacting with antiphagocytotic human-host fibrinogen (hFg). Herein, we show that hFg strongly interacts with M-Prts containing highly variable B-domains. We further demonstrate that specific GAS clinical isolates display high affinity for the D-domain of hFg, and this interaction allowed for subsequent surface binding of human-host plasminogen (hPg) to the E-domain of hFg. This GAS surface-bound hPg is then activated by GAS-secreted streptokinase, leading to the generation of an invasive proteolytic bacterial surface. Our results underscore the importance of the human fibrinolytic system in host-pathogen interactions in invasive GAS infections., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

A fibrinolytic enzyme was produced by the medicinal mushroom, Cordyceps militaris using submerged fermentation. The enzyme was purified from culture supernatant by hydrophobic interaction, ion exchange and gel filtration chromatographies. It was purified by 36 fold, with a specific activity of 1,467.4U/mg protein and the final yield was 5.8%. The molecular weight of the enzyme as determined by SDS-PAGE and gel filtration was 28kDa and 24.5kDa, respectively, and its isoelectric point (pI) was 9.0±0.2. It was found to be a glycoprotein with carbohydrate content of 1.67% (w/v). The enzyme was optimally active at 37°C and pH 7.2. The enzyme activity was strongly inhibited by soybean trypsin inhibitor (SBTI) and aprotinin which indicated it to be a serine protease, while other inhibitors like N-α-tosyl-l-phenylalanine chloromethyl ketone (TPCK), phenyl methane sulfonyl fluoride (PMSF), pepstatin and metal chelator EDTA did not inhibit its activity. Amino acid sequences of the purified enzyme were determined partially by Q-TOF2 and they were IEDFPYQVDLR; ANCGGTVISEK; YVLTAGHCAEGYTGLNIR; TNYASVTPITADMICAGFPEGK; KDSCSGDSGGPLVTGGK; VVGIVSFGTGCAR; ANKPGVYSSVASAEIR. Sequences of the seven peptides completely matched with those of a trypsin-like serine protease from Cordyceps militaris CM01 (accession no. EGX95217.1). The purified enzyme degraded α chains of fibrinogen first and then β and γ chains and also activated plasminogen into plasmin. It can act as an anticoagulant and prevent clot formation by degrading fibrinogen. Based on these studies, the purified enzyme has great potential to be developed as a natural agent for prevention and treatment of thrombolytic diseases., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Essentials Plg-R KT is a novel integral membrane plasminogen receptor. The functions of Plg-R KT in vivo are not known. Plg-R KT is a key player in macrophage recruitment in the inflammatory response in vivo. Plg-R KT deficiency is not compatible with survival of the species., Summary: Background Plg-R KT is a novel integral membrane plasminogen receptor that binds plasminogen via a C-terminal lysine exposed on the cell surface and promotes plasminogen activation on the cell surface by both tissue plasminogen activator and urokinase plasminogen activator. Objectives To evaluate the role of Plg-R KT in vivo we generated Plg-R KT -/- mice using a homologous recombination technique. Methods We characterized the effect of Plg-R KT deletion on reproduction, viability, health and spontaneous thrombosis and inflammation. Results Plg-R KT -/- mice were viable and fertile. Survival of Plg-R KT -/- mice and Plg-R KT +/+ littermates was not significantly different. However, quite strikingly, all pups of Plg-R KT -/- females died within 2 days of birth, consistent with a lactation defect in Plg-R KT -/- mothers. Additionally, there was a significant effect of Plg-R KT deficiency on the growth rates of female, but not male, mice. In experimental peritonitis studies, Plg-R KT -/- mice exhibited a marked defect in macrophage recruitment. As a contributing mechanism, the capacity of Plg-R KT -/- macrophages for plasminogen binding was markedly decreased. Conclusions These studies demonstrate that Plg-R KT is required for plasminogen binding and macrophage migration in vivo. In addition, Plg-R KT deficiency is not compatible with survival of the species, due to the death of all offspring of Plg-R KT -/- females. This new mouse model will be important for future studies aimed at delineating the role of cell surface plasminogen activation in challenge and disease models in vivo., Competing Interests: Authors state no conflicts of interests., (© 2016 International Society on Thrombosis and Haemostasis.)

Although plasminogen is a key protein in fibrinolysis and several mutations in the plasminogen gene (PLG) have been identified that result in plasminogen deficiency, there are conflicting reports to associate it with the risk of thrombosis. Our aim was to unravel the genetic architecture of PLG in families with plasminogen deficiency and its relationship with spontaneous thrombotic events in these families. A total of 13 individuals from 4 families were recruited. Their genetic risk profile of thromboembolism was characterized using the Thrombo inCode kit. Only one family presented genetic risk of thromboembolism (homozygous carrier of F12 rs1801020 and F13A1 rs5985). The whole PLG was tested using Next Generation Sequencing (NGS) and 5 putative pathogenic mutations were found (after in silico predictions) and associated with plasminogen deficiency. Although we can not find genetic risk factors of thrombosis in 3 of 4 families, even the mutations associated with plasminogen deficiency do not cosegregated with thrombosis, we can not exclude plasminogen deficiency as a susceptibility risk factor for thrombosis, since thrombosis is a multifactorial and complex disease where unknown genetic risk factors, in addition to plasminogen deficiency, within these families may explain the thrombotic tendency.

Kringle 5, the fifth fragment of plasminogen, is known to be important for inhibiting the proliferation and migration of vascular endothelial cell (VEC), while not having any effects on normal endothelial cells. Therefore, it may be a potential tumor therapy candidate. However, the ligand of the Kringle 5 in VEC has not yet been identified. In this study, the possible ligand of Kringle 5 in vitro was screened and validated using Ph.D.-7 phage display peptide library with molecular docking, along with surface plasma resonance (SPR). After four rounds of panning, the specific clones of Kringle 5 were confirmed using enzyme-linked immunosorbent assay (ELISA). The gene sequence analysis showed that they expressed the common amino sequence IGNSNTL. Then, using a NCBI BLAST, 103 matching sequences were found. Following the molecular docking evaluation and considering the acting function and pathway of the plasminogen Kringle 5 in the human body, the most promising candidate was determined to be voltage-dependent anion channel-1 (VDAC-1), which was able to bind to Kringle 5 at -822.65 J·mol-1 of the binding energy at the residues of Lys12, Thr19, Ser57, Thr188, Arg139, Asn214, Ser240 and Lys274. A strong dose-dependent interaction occurred between the VDAC-1 and Kringle 5 (binding constant 2.43 × 103 L·mol-1) in SPR observation. Therefore, this study proposed that VDAC-1 was a potential ligand of plasminogen Kringle 5, and also demonstrated that the screening and validation of protein ligand using phage display peptide library with the molecular docking, along with SPR, was a practicable application., Competing Interests: The authors have declared that no competing interests exist.