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Reteplase (recombinant plasminogen activator, rPA) is a mutant non-glycosylated tissue-type plasminogen activator (tPA) containing 355 amino acids with longer half-life and promising thrombolytic activity than its original counterpart, full length tPA. In this study, we aimed to produce and optimize the purification process of recombinant tissue-type plasminogen activator (tPA) known as Reteplase (rPA). Reteplase cDNA synthesized from total mRNA isolated from human placenta was PCR amplified, cloned into a pET-28a(+) E. coli expression vector and expressed in Rosetta-gami 2 E. coli (Novagen Ⓡ ) host. rPA was expressed as an inclusion body in E. coli and its biological activity was achieved after single step solubilization, purification and refolding. We exploited the strategy of Slow Refolding using Gradual Dialysis (SRGD) in which a refolding buffer containing glutathione oxidized (1 mM GSSG) and glutathione reduced (3 mM GSH) and pH 9.0 was used. Using the SRGD method, we were able to successfully obtain the protein in its active form. We obtained 4.26 mg of active refolded protein from a 50 mL culture that was scaled up in a bioreactor. The purity and homogeneity of rPA was evaluated by SDS-PAGE, Western blotting and mass spectrometry. Circular dichroism spectroscopy was conducted to evaluate the refolding and stability of the refolded rPA in comparison to reference standard rPA. The thrombolytic potential of rPA was assessed by fibrin plate assay and In Vitro clot lysis assay. The presented protocol offers a viable approach for enhancing both the yield and refolding efficiency of reteplase, potentially resulting in an increase in yield., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Disseminated intravascular coagulation (DIC) is a pathologic state that follows systemic injury and other diseases. Often a complication of sepsis or trauma, DIC causes coagulopathy associated with paradoxical thrombosis and hemorrhage. DIC upregulates the thrombotic pathways while simultaneously downregulating the fibrinolytic pathways that cause excessive fibrin deposition, microcirculatory thrombosis, multiorgan dysfunction, and consumptive coagulopathy with excessive bleeding. Given these opposing disease phenotypes, DIC management is challenging and includes treating the underlying disease and managing the coagulopathy. Currently, no therapies are approved for DIC. We have developed clot-targeted therapeutics that inhibit clot polymerization and activate clot fibrinolysis to manage DIC. We hypothesize that delivering both an anticoagulant and a fibrinolytic agent directly to clots will inhibit active clot polymerization while also breaking up pre-existing clots; therefore, reversing consumptive coagulopathy and restoring hemostatic balance. To test this hypothesis, we single- and dual-loaded fibrin-specific nanogels (FSNs) with antithrombinIII (ATIII) and/or tissue plasminogen activator (tPA) and evaluated their clot preventing and clot lysing abilities in vitro and in a rodent model of DIC. In vivo , single-loaded ATIII-FSNs decreased fibrin deposits in DIC organs and reduced blood loss when DIC rodents were injured. We also observed that the addition of tPA in dual-loaded ATIII-tPA-FSNs intensified the antithrombotic and fibrinolytic mechanisms, which proved advantageous for clot lysis and restoring platelet counts. However, the addition of tPA may have hindered wound healing capabilities when an injury was introduced. Our data supports the benefits of delivering both anticoagulants and fibrinolytic agents directly to clots to reduce the fibrin load and restore hemostatic balance in DIC.

Thrombosis is a leading global cause of death, in part due to the low efficacy of thrombolytic therapy. Here, we describe a method for precise delivery and accurate dosing of tissue plasminogen activator (tPA) using an intelligent DNA nanodevice. We use DNA origami to integrate DNA nanosheets with predesigned tPA binding sites and thrombin-responsive DNA fasteners. The fastener is an interlocking DNA triplex structure that acts as a thrombin recognizer, threshold controller and opening switch. When loaded with tPA and intravenously administrated in vivo, these DNA nanodevices rapidly target the site of thrombosis, track the circulating microemboli and expose the active tPA only when the concentration of thrombin exceeds a threshold. We demonstrate their improved therapeutic efficacy in ischaemic stroke and pulmonary embolism models, supporting the potential of these nanodevices to provide accurate tPA dosing for the treatment of different thromboses., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

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.

Recombinant tissue-type plasminogen activator (rtPA, or Alteplase) is the first approved thrombolytic drug for acute ischemic stroke, but suffers from a short half-life and poor resistance to plasminogen activator inhibitor (PAI-1), limiting its clinical use. The development of novel thrombolytic agents with improved benefit/risk balance has always been of great significance. In this study, we identified a mutant of serine protease domain of tPA (named ΔtPA A146V ) capable of escaping the inhibition by endogenous PAI-1 with 66-fold increased resistance compared to the wild type tPA. Based on this mutant, we generated a triple fusion ΔtPA (TriF-ΔtPA) containing albumin and fibrin binding peptide(FBP). The fusion with albumin effectively prolonged the plasma half-life of ΔtPA in mice to 144 min, which is much longer than ΔtPA and did not affect its thrombolytic activity. Furthermore, FBP rendered fibrin specificity of the fusion protein, giving a dissociation constant of ∼ 25 ± 0.9 μM. In a novel murine carotid embolism-induced stroke (CES) model, i.v. administration of TriF-ΔtPA promoted vascular recanalization, reduced infarct volume, and mitigated neurobehavioral deficits more significantly compared to ΔtPA-HSA or Alteplase, showing little bleeding risk. Together, this long-acting PAI-1-resistant thrombolytic agent holds great potential for clinical applications., 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 © 2023. Published by Elsevier B.V.)

Fibrin is the fibrous protein network that comprises blood clots; it is uniquely capable of bearing very large tensile strains (up to 200%) due to multiscale force accommodation mechanisms. Fibrin is also a biochemical scaffold for numerous enzymes and blood factors. The biomechanics and biochemistry of fibrin have been independently studied. However, comparatively little is known about how fibrin biomechanics and biochemistry are coupled: how does fibrin deformation influence its biochemistry? In this study, we show that mechanically induced protein structural changes in fibrin affect fibrin biochemistry. We find that tensile deformation of fibrin leads to molecular structural transitions of α-helices to β-sheets, which reduced binding of tissue plasminogen activator (tPA), an enzyme that initiates fibrin lysis. Moreover, binding of tPA and Thioflavin T, a commonly used β-sheet marker, were mutually exclusive, further demonstrating the mechano-chemical control of fibrin biochemistry. Finally, we demonstrate that structural changes in fibrin suppressed the biological activity of platelets on mechanically strained fibrin due to reduced αIIbβ3 integrin binding. Our work shows that mechanical strain regulates fibrin molecular structure and biological activity in an elegant mechano-chemical feedback loop, which possibly extends to other fibrous biopolymers.

Branch Retinal Vein Occlusion (BRVO) is the second chronic branch retinal vascular disease that causes abnormal vision loss after acute branch retinal disease in type 2 diabetes. There is no scientific conclusion about its specific pathogenic mechanism at present. Most clinical scholars generally support the theory that the partial human anatomical structure and various systemic risk psychological factors cause insufficient oxygen supply and hemostasis in the local branch retinal arteries. The research results of this article aim to reconstruct a non-nanocell-targeted thrombolytic drug delivery system without modification of rtPA without polyethylene glycol-methyl polycaprolactone and to re-evaluate its thrombus targeting and dissolution. The effect and safety of thrombus provide a new strategy for realizing combined treatment of thrombus. It is a study on the targeting of rtPA-NP to thrombus and its thrombolytic properties. HPLC method was used to detect the binding of fibrin clot prepared in vitro with coumarin-6 labeled NP and rtPA-NP; immunofluorescence technique was used to observe the location of nanomedicine and fibrin clot in branch retinal vein occlusion model Condition. The rtPA-NP drug delivery system constructed in this study not only retains the activity of rtPA and good thrombus targeting but also significantly prolongs its half-life and simplifies the way of administration. The therapeutic efficiency of rtPA-NP thrombus targeted administration on branch retinal vein occlusion reached 85.64%. The successful construction of the rtPA-NP thrombus targeted drug delivery system provides a new way for thrombosis treatment and lays the foundation for the future combination of anticoagulants and vascular protection drugs to achieve the combined treatment of thrombosis and the development of safe and efficient thrombolytic drugs.

Background: Details of the molecular interaction between tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor type-1 (PAI-1) remain unknown., Methods and Results: Three distinct forms of high-molecular-weight complexes are demonstrated. Two of the forms were detected by mass spectrometry. The high molecular mass detected by MALDI-TOF MS (matrix-assisted laser desorption ionization-time of flight mass spectrometry) was 107,029 Da, which corresponds to the sum of molecular masses of the intact tPA (65,320 Da) and the intact PAI-1 (42,416 Da). The lower molecular mass was 104,367 Da and is proposed to lack the C-terminal bait peptide of PAI-1 (calculated mass: 3,804 Da), which was detected as a 3,808 Da fragment. When the complex was analyzed by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), only a single band was observed. However, after treatment by SDS and Triton X-100, two distinct forms of the complex with different mobilities were shown by SDS-PAGE. The higher molecular weight band demonstrated specific tPA activity on fibrin autography, whereas the lower molecular weight band did not. Peptide sequence analysis of these two bands, however, unexpectedly revealed the existence of the C-terminal cleavage peptide in both bands and its amount was less in the upper band. In the upper band, the sequences corresponding to the regions at the interface between two molecules in its Michaelis intermediate were diminished. Thus, these two bands corresponded to distinct nonacyl-enzyme complexes, wherein only the upper band liberated free tPA under the conditions employed., Conclusion: These data suggest that under physiological conditions a fraction of the tPA-PAI-1 population exists as nonacylated-enzyme inhibitor complex., Competing Interests: None declared., (Thieme. All rights reserved.)

Thrombolytic (clot-busting) therapies with plasminogen activators (PAs) are first-line treatments against acute thrombosis and ischemic stroke. However, limitations such as narrow therapeutic windows, low success rates, and bleeding complications hinder their clinical use. Drug-loaded polyphenol-based nanoparticles (NPs) could address these shortfalls by delivering a more targeted and safer thrombolysis, coupled with advantages such as improved biocompatibility and higher stability in vivo. Herein, a template-mediated polyphenol-based supramolecular assembly strategy is used to prepare nanocarriers of thrombolytic drugs. A thrombin-dependent drug release mechanism is integrated using tannic acid (TA) to cross-link urokinase-type PA (uPA) and a thrombin-cleavable peptide on a sacrificial mesoporous silica template via noncovalent interactions. Following drug loading and template removal, the resulting NPs retain active uPA and demonstrate enhanced plasminogen activation in the presence of thrombin (1.14-fold; p < 0.05). Additionally, they display lower association with macrophage (RAW 264.7) and monocytic (THP-1) cell lines (43 and 7% reduction, respectively), reduced hepatic accumulation, and delayed blood clearance in vivo (90% clearance at 60 min vs 5 min) compared with the template-containing NPs. Our thrombin-responsive, polyphenol-based NPs represent a promising platform for advanced drug delivery applications, with potential to improve thrombolytic therapies.

Structural engineering of the recombinant thrombolytic drug, Reteplase, and its cost-effective production are important goals in the pharmaceutical industry. In this study, a single-point mutant of the protein was rationally designed and evaluated in terms of physicochemical characteristics, enzymatic activity, as well as large-scale production settings. An accurate homology model of Reteplase was used as the input to appropriate tools to identify the aggregation-prone sites, while considering the structural stability. Selected variants underwent extensive molecular dynamic simulations (total 540 ns) to assess their solvation profile and their thermal stability. The Reteplase-fibrin interaction was investigated by docking. The best variant was expressed in E. coli, and Box-Behnken design was used through response surface methodology to optimize its expression conditions. M72R mutant demonstrated appropriate stability, enhanced enzymatic activity (p < 0.05), and strengthened binding to fibrin, compared to the wild type. The optimal conditions for the variant's production in a bioreactor was shown to be 37 ºC, induction with 0.5 mM IPTG, for 2 h of incubation. Under these conditions, the final amount of the produced enzyme was increased by about 23 mg/L compared to the wild type, with an increase in the enzymatic activity by about 2 IU/mL. This study thus offered a new Reteplase variant with nearly all favorable properties, except solubility. The impact of temperature and incubation time on its large-scale production were underlined as well., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Thrombolytic and fibrinolytic therapies are effective treatments to dissolve blood clots in stroke therapy. Thrombolytic drugs activate plasminogen to its cleaved form plasmin, a proteolytic enzyme that breaks the crosslinks between fibrin molecules. The FDA-approved human tissue plasminogen activator Reteplase (rPA) is a non-glycosylated protein produced in E. coli. rPA is a deletion mutant of the wild-type Alteplase that benefits from an extended plasma half-life, reduced fibrin specificity and the ability to better penetrate into blood clots. Different methods have been proposed to improve the production of rPA. Here we show for the first time the transient expression in Nicotiana benthamiana of rPA fused to the immunoglobulin fragment crystallizable (Fc) domain on an IgG1, a strategy commonly used to improve the stability of therapeutic proteins. Despite our success on the expression and purification of dimeric rPA-Fc fusions, protein instability results in high amounts of Fc-derived degradation products. We hypothesize that the "Y"- shape of dimeric Fc fusions cause steric hindrance between protein domains and leads to physical instability. Indeed, mutations of critical residues in the Fc dimerization interface allowed the expression of fully stable rPA monomeric Fc-fusions. The ability of rPA-Fc to convert plasminogen into plasmin was demonstrated by plasminogen zymography and clot lysis assay shows that rPA-Fc is able to dissolve blood clots ex vivo. Finally, we addressed concerns with the plant-specific glycosylation by modulating rPA-Fc glycosylation towards serum-like structures including α2,6-sialylated and α1,6-core fucosylated N-glycans completely devoid of plant core fucose and xylose residues., Competing Interests: The authors have declared that no competing interests exist.

Tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) are serine proteases and major activators of fibrinolysis in mammalian systems. Because fibrinolysis is an essential component of the response to tissue injury, diverse cells, including cells that participate in the response to injury, have evolved receptor systems to detect tPA and uPA and initiate appropriate cell-signaling responses. Formation of functional receptor systems for the plasminogen activators requires assembly of diverse plasma membrane proteins, including but not limited to: the urokinase receptor (uPAR); integrins; N -formyl peptide receptor-2 (FPR2), receptor tyrosine kinases (RTKs), the N -methyl-d-aspartate receptor (NMDA-R), and low-density lipoprotein receptor-related protein-1 (LRP1). The cell-signaling responses elicited by tPA and uPA impact diverse aspects of cell physiology. This review describes rapidly evolving knowledge regarding the structure and function of plasminogen activator receptor assemblies. How these receptor assemblies regulate innate immunity and inflammation is then considered.

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.

Plasmin is the key enzyme in fibrinolysis. Upon interaction with plasminogen activators, the zymogen plasminogen is converted to active plasmin. Some studies indicate plasminogen activation is regulated by cation-independent mannose 6-phosphate receptor (CI-MPR), a protein that facilitates lysosomal enzyme trafficking and insulin-like growth factor 2 downregulation. Plasminogen regulation may be accomplished by CI-MPR binding to plasminogen or urokinase plasminogen activator receptor. We asked whether other members of the plasminogen activation system, such as tissue plasminogen activator (tPA), also interact with CI-MPR. Because tPA is a glycoprotein with three N-linked glycosylation sites, we hypothesized that tPA contains mannose 6-phosphate (M6P) and binds CI-MPR in a M6P-dependent manner. Using surface plasmon resonance, we found that two sources of tPA bound the extracellular region of human and bovine CI-MPR with low-mid nanomolar affinities. Binding was partially inhibited with phosphatase treatment or M6P. Subsequent studies revealed that the five N-terminal domains of CI-MPR were sufficient for tPA binding, and this interaction was also partially mediated by M6P. The three glycosylation sites of tPA were analyzed by mass spectrometry, and glycoforms containing M6P and M6P-N-acetylglucosamine were identified at position N448 of tPA. In summary, we found that tPA contains M6P and is a CI-MPR ligand.

Nature utilizes endothelium as a blood interface that perfectly controls hemostasis, preventing the uncontrolled formation of thrombi. The management of positive and negative feedback that finely tunes thrombosis and fibrinolysis is essential for human life, especially for patients who undergo extracorporeal circulation (ECC) after a severe respiratory or cardiac failure. The exposure of blood to a surface different from healthy endothelium inevitably initiates coagulation, drastically increasing the mortality rate by thromboembolic complications. In the present study, an ultrathin antifouling fibrinolytic coating capable of disintegrating thrombi in a self-regulated manner is reported. The coating system is composed of a polymer brush layer that can prevent any unspecific interaction with blood. The brushes are functionalized with a tissue plasminogen activator (tPA) to establish localized fibrinolysis that solely and exclusively is active when it is required. This interactive switching between the dormant and active state is realized through an amplification mechanism that increases (positive feedback) or restores (negative feedback) the activity of tPA depending on whether a thrombus is detected and captured or not. Thus, only a low surface density of tPA is necessary to lyse real thrombi. Our work demonstrates the first report of a coating that self-regulates its fibrinolytic activity depending on the conditions of blood.

Background: Pulmonary surfactant dysfunction is an important pathological factor in acute respiratory distress syndrome (ARDS) and pulmonary fibrosis (PF)., Objective: In this study, the characteristics of recombinant mature surfactant protein B (SP-B) and reteplase (rPA) fusion protein maintaining good pulmonary surface activity and rPA fibrinolytic activity in acute lung injury cell model were studied., Methods: We studied the characteristics of SP-B fusion expression, cloned rPA gene and N-terminal rPA/C-terminal SP-B co-expression gene, and constructed them into eukaryotic expression vector pEZ-M03 to obtain recombinant plasmids pEZ-rPA and pEZ-rPA/SP-B. The recombinant plasmids was transfected into Chinese hamster ovary (CHO) K1 cells and the expression products were analyzed by Western Blot. Lipopolysaccharide (LPS) was used to induce CCL149 (an alveolar epithelial cell line) cell injury model. Fluorescence staining of rPA and rPA/SP-B was carried out with the enhanced green fluorescent protein (eGFP) that comes with pEZ-M03; the cell Raman spectroscopy technique was used to analyze the interaction between rPA/SP-B fusion protein and the phospholipid structure of cell membrane in CCL149 cells. The enzyme activity of rPA in the fusion protein was determined by fibrin-agarose plate method., Results: The rPA/SP-B fusion protein was successfully expressed. In the CCL149 cell model of acute lung injury (ALI), the green fluorescence of rPA/SP-B is mainly distributed on the CCL149 cell membrane. The rPA/SP-B fusion protein can reduce the disorder of phospholipid molecules and reduce cell membrane damage. The enzyme activity of rPA/SP-B fusion protein was 3.42, and the fusion protein still had good enzyme activity., Conclusion: The recombinant eukaryotic plasmid pEZ-rPA/SP-B is constructed and can be expressed in the eukaryotic system. Studies have shown that rPA/SP-B fusion protein maintains good SP-B lung surface activity and rPA enzyme activity in acute lung injury cell model., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Fibrinolytic enzymes have been considered promising for treatment and protection of healthy circulation due its ability to dissolve the fibrin in blood clots. Extractive fermentation is a not explored and efficient downstream process which segregates the desired product simultaneously in a fermentation process fast and economically. Extraction of fibrinolytic enzymes by Bacillus stearothermophilus DPUA 1729 employing conventional aqueous two-phase systems (ATPS) and extractive fermentation with ATPS was evaluated. The results of both systems were compared using a factorial design with PEG molar mass, PEG and salt concentrations as independent variables and extraction parameters as a response. In all conditions evaluated it was observed a similar partitioning of fibrinolytic enzymes through the phases, both in conventional ATPS and extractive fermentation. Salt concentration and interaction among PEG and salt concentration influenced in the partition coefficient. The fibrinolytic activity was determined by hydrolysis of fibrin in plate using the extract of one condition from extractive fermentation. The zone degradation presented a diameter of 7.03 ± 0.94 mm. In conclusion, there was no significant difference among the results obtained using conventional ATPS and extractive fermentation, however, the second one presents more advantages and can integrate production and extraction in one single step, reducing the costs.

Reteplase is a deleted variant of human tissue plasminogen activator with a complex structure containing nine disulfide bonds. Reteplase is expressed as inclusion bodies in Escherichia coli and needs the additional step of refolding for activation. In this study an experimental design was performed to find the optimal refolding condition for reteplase. The influence of 14 chemical additives was assessed by one factor at a time method and then Taguchi design followed by response surface methodology was employed to find compounds with most significant effects on reteplase refolding and their optimum concentration. We found that 0.13 M histidine, 1.64 M methionine, 0.33 M cysteine, and 0.34 M arginine in addition to the GSH/GSSG is the optimal condition for refolding of reteplase. We also investigated the refolding yield for inclusion bodies obtained from different E. coli strains and found that BL21 (DE3) has the best recovery yield in comparison to Rosetta-gami and Shuffle T7., (Copyright © 2020. Published by Elsevier B.V.)

Rubisco, the key enzyme of CO 2 fixation in photosynthesis, is prone to inactivation by inhibitory sugar phosphates. Inhibited Rubisco undergoes conformational repair by the hexameric AAA+ chaperone Rubisco activase (Rca) in a process that is not well understood. Here, we performed a structural and mechanistic analysis of cyanobacterial Rca, a close homolog of plant Rca. In the Rca:Rubisco complex, Rca is positioned over the Rubisco catalytic site under repair and pulls the N-terminal tail of the large Rubisco subunit (RbcL) into the hexamer pore. Simultaneous displacement of the C terminus of the adjacent RbcL opens the catalytic site for inhibitor release. An alternative interaction of Rca with Rubisco is mediated by C-terminal domains that resemble the small Rubisco subunit. These domains, together with the N-terminal AAA+ hexamer, ensure that Rca is packaged with Rubisco into carboxysomes. The cyanobacterial Rca is a dual-purpose protein with functions in Rubisco repair and carboxysome organization., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

New-era soft microrobots for biomedical applications need to mimic the essential structures and collective functions of creatures from nature. Biocompatible interfaces, intelligent functionalities, and precise locomotion control in a collective manner are the key parameters to design soft microrobots for the complex bio-environment. In this work, a biomimetic magnetic microrobot (BMM) inspired by magnetotactic bacteria (MTB) with speedy motion response and accurate positioning is developed for targeted thrombolysis. Similar to the magnetosome structure in MTB, the BMM is composed of aligned iron oxide nanoparticle (MNP) chains embedded in a non-swelling microgel shell. Linear chains in BMMs are achieved due to the interparticle dipolar interactions of MNPs under a static magnetic field. Simulation results show that, the degree and speed of assembly is proportional to the field strength. The BMM achieves the maximum speed of 161.7 µm s -1 and accurate positioning control under a rotating magnetic field with less than 4% deviation. Importantly, the locomotion analyses of BMMs demonstrate the frequency-dependent synchronization under 8 Hz and asynchronization at higher frequencies due to the increased drag torque. The BMMs can deliver and release thrombolytic drugs via magneto-collective control, which is promising for ultra-minimal invasive thrombolysis., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The activation of the nuclear factor-κB (NF-κB) pathway plays a central role in the initiation and progression of inflammation, which contributes to the pathogenesis and progression of various human diseases including kidney, brain, and other diseases. Tissue plasminogen activator (tPA), a serine protease regulating homeostasis of blood coagulation, fibrinolysis, and matrix degradation, has been shown to act as a cytokine to trigger profound receptor-mediated intracellular events, modulate the NF-κB pathway, and mediate organ dysfunction and injury. In this review, we focus on the current understanding of NF-κB and tPA signaling in the development and progression of kidney disease. Their roles in the nervous and cardiovascular system are also briefly discussed.

Rapid cut-off of blood supply in diseases involving thrombosis is a major cause of morbidity and mortality worldwide. However, the current thrombolysis strategies offer limited results due to the therapeutics' short half-lives, low targeting ability, and unexpected bleeding complications. Inspired by the innate roles of platelets in hemostasis and pathological thrombus, platelet membrane-camouflaged polymeric nanoparticles (nanoplatelets) are developed for targeting delivery of the thrombolytic drug, recombinant tissue plasminogen activator (rt-PA), to local thrombus sites. The tailor-designed nanoplatelets efficiently accumulate at the thrombi in pulmonary embolism and mesenteric arterial thrombosis model mice, eliciting a significantly enhanced thrombolysis activity compared to free rt-PA. In addition, the nanoplatelets exhibit improved therapeutic efficacy over free rt-PA in an ischemic stroke model. Analysis of in vivo coagulation indicators suggests the nanoplatelets might possess a low risk of bleeding complications. The hybrid biomimetic nanoplatelets described offer a promising solution to improve the efficacy and reduce the bleeding risk of thrombolytic therapy in a broad spectrum of thrombosis diseases., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The goal of stem cell therapy for spinal cord injury (SCI) is to restore motor function without exacerbating pain. Induced pluripotent stem cells (iPSC) may be administered by autologous transplantation, avoiding immunologic challenges. Identifying strategies to optimize iPSC-derived neural progenitor cells (hiNPC) for cell transplantation is an important objective. Herein, we report a method that takes advantage of the growth factor-like and anti-inflammatory activities of the fibrinolysis protease, tissue plasminogen activator tPA, without effects on hemostasis. We demonstrate that conditioning hiNPC with enzymatically-inactive tissue-type plasminogen activator (EI-tPA), prior to grafting into a T3 lesion site in a clinically relevant severe SCI model, significantly improves motor outcomes. EI-tPA-primed hiNPC grafted into lesion sites survived, differentiated, acquired markers of motor neuron maturation, and extended βIII-tubulin-positive axons several spinal segments below the lesion. Importantly, only SCI rats that received EI-tPA primed hiNPC demonstrated significantly improved motor function, without exacerbating pain. When hiNPC were treated with EI-tPA in culture, NMDA-R-dependent cell signaling was initiated, expression of genes associated with stemness (Nestin, Sox2) was regulated, and thrombin-induced cell death was prevented. EI-tPA emerges as a novel agent capable of improving the efficacy of stem cell therapy in SCI.

The vascular transport of molecules, cells, and nanoconstructs is a fundamental biophysical process impacting tissue regeneration, delivery of nutrients and therapeutic agents, and the response of the immune system to external pathogens. This process is often studied in single-channel microfluidic devices lacking the complex tridimensional organization of vascular networks. Here, soft lithography is employed to replicate the vein system of a Hedera elix leaf on a polydimethilsiloxane (PDMS) template. The replica is then sealed and connected to an external pumping system to realize an authentically complex microvascular network. This satisfies energy minimization criteria by Murray's law and comprises a network of channels ranging in size from capillaries (∼50 μm) to large arterioles and venules (∼400 μm). Micro-PIV (micro-particle image velocimetry) analysis is employed to characterize flow conditions in terms of streamlines, fluid velocity, and flow rates. To demonstrate the ability to reproduce physiologically relevant transport processes, two different applications are demonstrated: vascular deposition of tumor cells and lysis of blood clots. To this end, conditions are identified to culture cells within the microvasculature and realize a confluent endothelial monolayer. Then, the vascular deposition of circulating breast (MDA-MB 231) cancer cells is documented throughout the network under physiologically relevant flow conditions. Firm cell adhesion mostly occurs in channels with low mean blood velocity. As a second application, blood clots are formed within the chip by mixing whole blood with a thrombin solution. After demonstrating the blood clot stability, tissue plasminogen activator (tPA) and tPA-carrying nanoconstructs (tPA-DPNs) are employed as thrombolytics. In agreement with previous data, clot dissolution is equally induced by tPA and tPA-DPNs. The proposed leaf-inspired chip can be efficiently used to study a variety of vascular transport processes in complex microvascular networks, where geometry and flow conditions can be modulated and monitored throughout the experimental campaign.

Ischemic stroke caused by a thrombus clog and ischemia is one of the most lethal and disabling cerebrovascular diseases. A sequentially targeted delivery system is highly desired to deliver thrombolytics and neuroprotectant to the site of the thrombus and ischemic penumbra, respectively, to pursue a maximized combinational effect. Inspired by the vital roles that platelets play in thrombus formation, herein, we develop a bioengineered "nanoplatelet" (tP-NP-rtPA/ZL006e) for sequentially site-specific delivery of recombinant tissue plasminogen activator (rtPA) and neuroprotectant (ZL006e) for ischemic stroke treatment. The tP-NP-rtPA/ZL006e consists of a ZL006e-loaded dextran derivative polymeric nanoparticle core and platelet membrane shell conjugated with thrombin-cleavable Tat-peptide-coupled rtPA. Mediated by the cloak of the platelet membrane, tP-NP-rtPA/ZL006e targets the thrombus site and rtPA is triggered to release by the upregulated thrombin. Subsequently, the in situ exposed Tat peptide enhanced penetration of the "nanoplatelet" across the blood-brain barrier into ischemic brain for ZL006e site-specific delivery. From the in vitro and in vivo evaluation, tP-NP-rtPA/ZL006e is demonstrated to significantly enhance the anti-ischemic stroke efficacy in the rat model  with middle cerebral artery occlusion, showing a 63 and 72% decrease in ischemic area and reactive oxygen species level compared to that with free drug combination, respectively.

Objective: We recently investigated the pathways of immunoreactive bradykinin (iBK) formation in fresh blood of normal volunteers and of patients with hereditary angioedema due to C1-esterase inhibitor deficiency (HAE-1/-2). Herein, we adapted the techniques to small volumes (200 μl) of previously frozen citrated plasma and further analyzed the mechanisms of iBK formation with additional biotechnological inhibitors., Results: Measurable iBK formation was observed under stimulation with tissue kallikrein (KLK-1, 10 nM), the particulate material Kontact-APTT (concentration reduced to 2% v/v) or recombinant tissue plasminogen activator (tPA, 169 nM), with little background in unstimulated plasma incubated for up to 2 h. Plasma samples from HAE-1/-2 patients responded earlier to tPA than those from controls, as previously reported with whole blood. Lanadelumab inhibited iBK formation induced by Kontact-APTT and tPA. A highly specific plasmin inhibitor, DX-1000, abolished tPA-induced iBK formation in plasma but had no effect against Kontact-APTT, confirming the role of fibrinolysis in tPA-induced kinin formation. The anti-lanadelumab neutralizing antibody M293-D02 reversed the inhibitory effects of lanadelumab. Frozen plasma is a suitable material for measuring iBK formation kinetics, with possible applications such as investigating the effect of rare disease states on the kallikrein-kinin system and monitoring the effect of HAE prophylactic treatments.

Multidomain biotherapeutic proteins present additional behavioral and analytical challenges for the optimization of their kinetic stability by formulation. Tissue-type plasminogen activator (tPA) comprises six protein domains that exhibit a complex and pH-dependent thermal unfolding profile, due to partially independent domain unfolding. Here we have used tPA as a model for evaluating the relationships between various thermal unfolding and aggregation parameters in multidomain proteins. We show that changes in the thermal unfolding profile of tPA were parametrized by the overall thermal midpoint transition temperature, T m , and the Van't Hoff entropy for unfolding, Δ S vh , which is a measure of unfolding cooperativity. The kinetics of degradation at 45 °C, leading to aggregation, were measured as rates of monomer and activity loss. These two rates were found to be coincident at all pH. Aggregation accelerated at pH 4 due to the early unfolding of the serine protease N-terminal domain (SP-N), whereas at pH 5-8, the fraction unfolded at 45 °C ( f 45 ) was <1%, resulting in a baseline rate of aggregation from the native ensemble. We used a Design of Experiments (DoE) approach to evaluate how formulation excipients impact and control the thermal unfolding profile for tPA and found that the relative stability of each of the tPA domains was dependent on the formulation. Therefore, the optimization of formulations for complex multidomain proteins such as tPA may need to be multiobjective, with careful selection of the desired attributes that improve stability. As aggregation rates (ln v) correlated well to T m ( R 2 = 0.77) and Δ S vh ( R 2 = 0.71) but not T agg ( R 2 = 0.01), we analyzed how formulation excipients and pH would be able to optimize T m and Δ S vh . Formulation excipient behaviors were found to group according to their combined impact on T m and Δ S vh . The effects of each excipient were often selectively stabilizing or destabilizing to specific tPA domains and changed the stability of particular domains relative to the others. The types of mechanism by which this could occur might involve specific interactions with the protein surface, or otherwise effects that are mediated via the solvent as a result of the different surface hydrophobicities and polarities of each domain.

Urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) are two serine proteases that contribute to initiating fibrinolysis by activating plasminogen. uPA is also an important tumour-associated protease due to its role in extracellular matrix remodelling. Overexpression of uPA has been identified in several different cancers and uPA inhibition has been reported as a promising therapeutic strategy. Although several peptide-based uPA inhibitors have been developed, the extent to which uPA tolerates different tetrapeptide sequences that span the P1-P4 positions remains to be thoroughly explored. In this study, we screened a sequence-defined peptide aldehyde library against uPA and tPA. Preferred sequences from the library screen yielded potent inhibitors for uPA, led by Ac-GTAR-H (K i =18 nm), but not for tPA. Additionally, synthetic peptide substrates corresponding to preferred inhibitor sequences were cleaved with high catalytic efficiency by uPA but not by tPA. These findings provide new insights into the binding specificity of uPA and tPA and the relative activity of tetrapeptide inhibitors and substrates against these enzymes., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Essentials Activated clotting factor X (FXa) acquires fibrinolytic cofactor function after cleavage by plasmin. FXa-mediated plasma fibrinolysis is enabled by active site modification blocking a second cleavage. FXa-directed oral anticoagulants (DOACs) alter FXa cleavage by plasmin. DOACs enhance FX-dependent fibrinolysis and plasmin generation by tissue plasminogen activator., Background: When bound to an anionic phospholipid-containing membrane, activated clotting factor X (FXa) is sequentially cleaved by plasmin from the intact form, FXaα, to FXaβ and then to Xa33/13. Tissue-type plasminogen activator (t-PA) produces plasmin and is the initiator of fibrinolysis. Both FXaβ and Xa33/13 enhance t-PA-mediated plasminogen activation. Although stable in experiments using purified proteins, Xa33/13 rapidly loses t-PA cofactor function in plasma. Bypassing this inhibition, covalent modification of the FXaα active site prevents Xa33/13 formation by plasmin, and the persistent FXaβ enhances plasma fibrinolysis. As the direct oral anticoagulants (DOACs) rivaroxaban and apixaban bind to the FXa active site, we hypothesized that they similarly modulate FXa fibrinolytic function., Methods: DOAC effects on fibrinolysis and the t-PA cofactor function of FXa were studied in patient plasma, normal pooled plasma and purified protein experiments by the use of light scattering, chromogenic assays, and immunoblots., Results: The plasma of patients taking rivaroxaban showed enhanced fibrinolysis correlating with FXaβ. In normal pooled plasma, the addition of rivaroxaban or apixaban also shortened fibrinolysis times. This was related to the cleavage product, FXaβ, which increased plasmin production by t-PA. It was confirmed that these results were not caused by DOACs affecting activated FXIII-mediated fibrin crosslinking, clot ultrastructure and thrombin-activatable fibrinolysis inhibitor activation in plasma., Conclusion: The current study suggests a previously unknown effect of DOACs on FXa in addition to their well-documented anticoagulant role. By enabling the t-PA cofactor function of FXaβ in plasma, DOACs also enhance fibrinolysis. This effect may broaden their therapeutic indications., (© 2018 International Society on Thrombosis and Haemostasis.)

Tissue plasminogen activator (tPA) is the only FDA approved thrombolytic drug for acute ischemic stroke but concerns regarding its limitations remain. Here, we developed a new strategy by incorporating tPA into porous magnetic iron oxide (Fe 3 O 4 )-microrods (tPA-MRs) for targeted thrombolytic therapy in ischemic stroke induced by distal middle cerebral artery occlusion. We showed that intra-arterial injection of tPA-MRs could target the cerebral blood clot in vivo under the guidance of an external magnet, where tPA was subsequently released at the site of embolism. When applied with an external rotating magnetic field, rotating tPA-MRs significantly improved not only the mass transport of the tPA-clot reaction, but also mechanically disrupted the clot network, which thus increased clot interaction and penetration of tPA. Importantly, intravenously injected MRs could be discharged from the kidney, and the function of liver and kidney were not damaged at different durations after administration of tPA-MRs. Our data suggest that tPA-MRs overcome the limitations of thrombolytic therapy with tPA alone, which may be not only just for the treatment of ischemic stroke but also have majorly impact on other thrombotic diseases.

Introduction: Rural and critical access hospitals rely on the "drip and ship" practice using helicopter emergency medical services (HEMS). But those helicopter flights are an unusual environment with physical factors such as vibration and accelerations that could potentially affect the stability, and pharmacological properties of IV rtPA, an issue that has not been previously addressed., Materials and Methods: This was a prospective cohort study of consecutive acute ischemic stroke patients receiving IV rtPA through a Comprehensive Stroke Center from November 2015 to February 2017 to measure the effects of HEMS on the integrity and activity of rtPA by collecting residual medication left in the vial., Clinical Trial Registration: http://www.clinicaltrials.gov. NCT02752256 RESULTS: A total of 33 patients and rtPA samples were included; 18 patients who presented directly to the Comprehensive Stroke Center emergency department and 15 patients who received rtPA during air ambulance transfer. The median rtPA antigen concentration in the residual medication vial was 3.04 mg/mL (IQR: 1.24-3.87) in the HEMS group and 1.91 mg/mL (IQR: 1.33-2.60) in the controls (P = .168). There were no significant differences in rtPA activity or specific activity between the HEMS and control groups and there was no association between total HEMS flight time on overall rtPA specific activity., Conclusions: In summary, this study provides supportive evidence of the lack of a detrimental effect of the HEMS physical environment on the integrity of rtPA, therefore endorsing current drip and ship practices without infusion adjustments., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Human tissue-type plasminogen activator is one of the most important therapeutic proteins involved in the breakdown of blood clots following the stroke. A mutation was found at position 1541 bp (G514E) and the mutated form was cloned into the binary vector pTRAc-ERH. In silico analysis showed that this mutation might have no significant effect on the active site of the tissue plasminogen activator enzyme. Accordingly, zymography assay confirmed the serine protease activity of the mutated form and its derivatives. The expression of the mutated form was verified with/without co-agroinjection of the P19 gene silencing suppressor in both Nicotiana tabacum and N. benthamiana. The ELISA results showed that the concentration of the mutated form in the absence of P19 was 0.65% and 0.74% of total soluble protein versus 0.141% and 1.36% in the presence of P19 in N. benthamiana and N. tabacum, respectively. In N. tabacum, co-agroinjection of P19 had the synergistic effect and increased the mutated tissue plasminogen activator production two-fold higher. However, in N. benthamiana, the presence of P19 had the adverse effect of five-fold reduction in the concentration. Moreover, results showed that the activity of the mutated form and its derivatives was more than that of the purified commercial tissue plasminogen activator.

The aim of the present study was to investigate the effect of three routine drug excipients, as osmolytes, in three different concentrations, on structure, thermal stability and the activity of single-chain (sc-) tenecteplase. To see the influence of trehalose, mannitol, and sucrose on the structure, stability and function of sc-tenecteplase, thermal stability, fluorescence, circular dichroism (CD) and enzyme kinetic measurements and molecular docking studies were carried out. To measure the effect of osmolytes on stability of sc-tenecteplase, thermodynamic parameters (transition temperature (T m ), standard enthalpy change (ΔH°), standard entropy change (ΔS°) and ΔG°, the standard Gibbs free energy change, were determined from heat-induced transition curves of the protein in absence and presence of each osmolyte. It was observed that all three osmolytes acted as an enhancer for the sc-tenecteplase stability, with varying efficacies and efficiencies. The results of the kinetic study showed that the activity of sc-tenecteplase is increased in the presence of osmolytes. The near-UV and far-UV CD studies showed transfer of Trp, Phe and Tyr residues to a more flexible environment in the presence of osmolytes. The sc-tenecteplase fluorescence quenching suggested the more polar location of Trp residues. Molecular docking studies revealed that (i) Gibbs free energy of interaction between the osmolyte and sc-tenecteplase is negative, and (ii) hydrogen bond and hydrophobic interactions dominate within the interaction sites., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Ischemic stroke occurs when a blood clot obstructs or narrows the arteries that supply blood to the brain. Currently, tissue plasminogen activator (tPA), a thrombolytic agent, is the only United States Food and Drug Administration (FDA)-approved pharmacologic treatment for ischemic stroke. Despite its effective usage, the major limitation of tPA that stems from its short half-life in plasma (≈5 min) is the potential for increased risk of hemorrhagic complications. To circumvent these limitations, herein, the first proof-of-principle demonstration of a theranostic nanoconstruct system derived from erythrocytes doped with the FDA-approved near-infrared (NIR) imaging agent, indocyanine green, and surface-functionalized with tPA is reported. Using a clot model, the dual functionality of these nanoconstructs in NIR fluorescence imaging and clot lysis is demonstrated. These biomimetic theranostic nanoconstructs may ultimately be effective in imaging and treatment of blood clots involved in ischemic stroke., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Therapeutic approaches that enhance thrombolysis by combining recombinant tissue plasminogen activator (rtPA), ultrasound, and/or microbubbles (MBs) are known as sonothrombolysis techniques. To date, sonothrombolysis approaches have primarily utilized commercially available MB formulations (or derivatives thereof) with diameters in the range 1-4 µm and circulation lifetimes between 5 and 15 min. The present study evaluated the in vitro sonothrombolysis efficacy of large diameter MBs (d MB  ≥ 10 µm) with much shorter lifetimes that were produced on demand and in close proximity to the blood clot using a flow-focusing microfluidic device. MBs with a N 2 gas core and a non-crosslinked bovine serum albumin shell were produced with diameters between 10 and 20 µm at rates between 50 and 950 × 10 3 per second. Use of these large MBs resulted in approximately 4.0-8.8 fold increases in thrombolysis rates compared to a clinical rtPA dose and approximately 2.1-4.2 fold increases in thrombolysis rates compared to sonothrombolysis techniques using conventional MBs. The results of this study indicate that the large diameter microbubbles with transient stability are capable of significantly enhanced in vitro sonothrombolysis rates when delivered directly to the clot immediately following production by a flow focusing microfluidic device placed essentially in situ adjacent to the clot.

Down syndrome (DS), also known as "trisomy 21", is a genetic disorder caused by the presence of all or part of a third copy of chromosome 21. Silencing these extra genes is beyond existing technology and seems to be impractical. A number of pharmacologic options have been proposed to change the quality of life and lifespan of individuals with DS. It was reported that treatment with epigallocatechin gallate (EGCG) improves cognitive performance in animal models and in humans, suggesting that EGCG may alleviate symptoms of DS. Traditionally, EGCG has been associated with the ability to reduce dual specificity tyrosine phosphorylation regulated kinase 1A activity, which is overexpressed in trisomy 21. Based on the data available in the literature, we propose an additional way in which EGCG might affect trisomy 21-namely by modifying the proteolytic activity of the enzymes involved. It is known that, in Down syndrome, the nerve growth factor (NGF) metabolic pathway is altered: first by downregulating tissue plasminogen activator (tPA) that activates plasminogen to plasmin, an enzyme converting proNGF to mature NGF; secondly, overexpression of metalloproteinase 9 (MMP-9) further degrades NGF, lowering the amount of mature NGF. EGCG inhibits MMP-9, thus protecting NGF. Urokinase (uPA) and tPA are activators of plasminogen, and uPA is inhibited by EGCG, but regardless of their structural similarity tPA is not inhibited. In this review, we describe mechanisms of proteolytic enzymes (MMP-9 and plasminogen activation system), their role in Down syndrome, their inhibition by EGCG, possible degradation of this polyphenol and the ability of EGCG and its degradation products to cross the blood-brain barrier. We conclude that known data accumulated so far provide promising evidence of MMP-9 inhibition by EGCG in the brain, which could slow down the abnormal degradation of NGF., Competing Interests: The authors declare no conflict of interest.

Acute ischaemic stroke, myocardial infarction and pulmonary embolism are the main causes of mortality and morbidity worldwide. Thrombolysis by intravenous injection of recombinant tissue plasminogen activator (rtPA) remains the most common non-interventional treatment to recanalize occluded vessels. However, this procedure is limited by significant drawbacks, including high doses and bleeding complications. Recent studies showed that fucoidan targets the intraluminal thrombus in vivo. We have developed a chimaera covalently linking fucoidan, able to target platelets within the thrombus, to dilysine, able to non-covalently bind rtPA. We hypothesize that this construct should vectorize rtPA to the thrombus, thus increasing its fibrinolytic efficacy and avoiding its deleterious effects. In vitro, rtPA mixed to dilysine fucoidan (DLF) shows a greater fibrinolytic effect than rtPA alone, both on platelet-rich thrombus and in whole blood. In vivo, occluded mesenteric vessels, carotid artery and vena cava were more efficiently recanalized by DLF complexed to rtPA than by rtPA alone. This study thus provides evidence that DLF may be a promising therapeutic tool to fight against acute thrombosis by enhancing rtPA fibrinolytic efficiency., Competing Interests: Disclosures: The chimer was patented (no. 14715449.6–1453, 2015, Aminofucoidan) as a vector for fibrinolysis in thrombotic disease., (Schattauer GmbH Stuttgart.)

How AAA+ chaperones conformationally remodel specific target proteins in an ATP-dependent manner is not well understood. Here, we investigated the mechanism of the AAA+ protein Rubisco activase (Rca) in metabolic repair of the photosynthetic enzyme Rubisco, a complex of eight large (RbcL) and eight small (RbcS) subunits containing eight catalytic sites. Rubisco is prone to inhibition by tight-binding sugar phosphates, whose removal is catalyzed by Rca. We engineered a stable Rca hexamer ring and analyzed its functional interaction with Rubisco. Hydrogen/deuterium exchange and chemical crosslinking showed that Rca structurally destabilizes elements of the Rubisco active site with remarkable selectivity. Cryo-electron microscopy revealed that Rca docks onto Rubisco over one active site at a time, positioning the C-terminal strand of RbcL, which stabilizes the catalytic center, for access to the Rca hexamer pore. The pulling force of Rca is fine-tuned to avoid global destabilization and allow for precise enzyme repair., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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.)

Thrombi that occlude blood vessels can be resolved with fibrinolytic agents that degrade fibrin, the polymer that forms between and around platelets to provide mechanical stability. Fibrinolysis rates however are often constrained by transport-limited delivery to and penetration of fibrinolytics into the thrombus. Here, these limitations are overcome with colloidal microwheel (µwheel) assemblies functionalized with the fibrinolytic tissue-type plasminogen activator (tPA) that assemble, rotate, translate, and eventually disassemble via applied magnetic fields. These microwheels lead to rapid fibrinolysis by delivering a high local concentration of tPA to induce surface lysis and, by taking advantage of corkscrew motion, mechanically penetrating into fibrin gels and platelet-rich thrombi to initiate bulk degradation. Fibrinolysis of plasma-derived fibrin gels by tPA-microwheels is fivefold faster than with 1 µg mL -1 tPA. µWheels following corkscrew trajectories can also penetrate through 100 µm sized platelet-rich thrombi formed in a microfluidic model of hemostasis in ≈5 min. This unique combination of surface and bulk dissolution mechanisms with mechanical action yields a targeted fibrinolysis strategy that could be significantly faster than approaches relying on diffusion alone, making it well-suited for occlusions in small or penetrating vessels not accessible to catheter-based removal., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A single-step fabrication method, coaxial electrohydrodynamic atomization (CEHDA), was developed to synthesize drug-loaded microbubbles (MBs) for combination treatment of ischemic stroke. The bioactivity of therapeutic agent (tPA, tissue plasminogen activator) after preparation was evaluated, showing that CEHDA could be very promising method for producing MBs with therapeutic functions. The bubble performance and tPA release profiles were also examined by exposing the bubbles to 2MHz ultrasound of various intensities. The results showed that the mean diameter of tPA-loaded MBs was found to fluctuate about its original diameter when exposed to ultrasound and higher intensity ultrasound was more effective in triggering the burst of CEHDA MBs. High ultrasound-triggered bubble disintegration effectiveness in a short period (first 5min) fits well with the requirement of short ultrasound exposure time for human brain. Moreover, a numerical model was also applied to investigate the stability of the fabricated MBs in the bloodstream. It was found that MB dissolution time increased with initial radius, decreased with initial surface tension and increased with initial shell resistance but it was barely affected by the average excessive bloodstream pressure., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Objective: To explore the feasibility and safety of using tissue-type plasminogen activator (t-PA) to prevent graft restenosis after coronary artery bypass grafting (CABG)., Methods: In this prospective observational study, 37 patients underwent CABG between June 2009 and May 2013. These patients were grouped according to the anti-coagulation strategy after surgery: t-PA (n = 12) and conventional treatments (n = 25). In the t-PA group, the patients received acetylsalicylic acid (ASA) and clopidogrel plus intravenous infusion of t-PA (0.25 mg/kg/day) starting at 24 h after surgery and that lasted for 3 days. In the conventional group, the patients received only ASA and clopidogrel. 64-row spiral computed tomographic coronary angiography was performed at 1 week, 1, and 3 months after surgery to evaluate the patency of the graft vessel., Results: The mean stenosis severity of the saphenous vein grafts was lower in the t-PA group compared with the conventional group at 3 months after surgery (p < 0.05), but there was no significant difference at 1 week and 1 month (p > 0.05). The patency rate of the grafts was not significantly different between the two groups at 1 week, 1, and 3 months after surgery (p > 0.05)., Conclusion: Early application of t-PA after CABG was feasible and safe, and might help prevent early restenosis of SV grafts. Additional clinical randomized trials are necessary to address this issue.

The recombinant protein TK1-2, which consists of two kringle domains of tissue-type plasminogen activator (t-PA), inhibits angiogenesis and tumor growth. ɪn this study, we examined the anti-angiogenic activities of peptides derived from kringle 2 domain of t-PA to identify the functional core sequence. Seven peptides were constructed from the kringle 2 sequence, based on the structure and characteristics of amino acid residues, and were analyzed for their inhibitory effects on endothelial cells (ECs). Among them, TP-7 (derived from a β-sheet motif) potently inhibited proliferation, tube formation, and migration of ECs in a dose-dependent manner, whereas truncation of 3-9 amino acid residues from either N or C terminus of TP-7 abrogated its inhibitory effects on ECs. TP-7 also potently inhibited angiogenesis in a Matrigel plug assay in vivo. Moreover, TP-7 dose-dependently suppressed corneal neovascularization induced by an acute chemical burn in a rat model. At the molecular level, TP-7 inhibited VEGF- or bFGF-induced phosphorylation of FAK and ERK1/2 and drastically disrupted VEGF- or bFGF-induced formation of stress fibers and focal adhesion complexes. In addition, TP-7 markedly suppressed attachment and spreading of ECs on a collagen type I or fibronectin matrix. Adhesion of ECs to immobilized TP-7 increased dose-dependently, which was disrupted strongly by pretreatment with soluble TP-7 and slightly by an integrin α2β1-blocking antibody. These results suggest that TP-7 is a potent anti-angiogenic peptide in part affecting the integrin α2β1-dependent pathway and that it can be used for treatment of corneal neovascularization by targeting VEGF and non-VEGF pathways. J. Cell. Biochem. 118: 1132-1143, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Background: Functionalized nanoparticles (NPs) are one promising tool for detecting specific molecular targets and combine molecular biology and nanotechnology aiming at modern imaging. We aimed at ligand-directed delivery with a suitable target-biomarker to detect early pancreatic ductal adenocarcinoma (PDAC). Promising targets are galectins (Gal), due to their strong expression in and on PDAC-cells and occurrence at early stages in cancer precursor lesions, but not in adjacent normal tissues., Results: Molecular probes (10-29 AA long peptides) derived from human tissue plasminogen activator (t-PA) were selected as binding partners to galectins. Affinity constants between the synthesized t-PA peptides and Gal were determined by microscale thermophoresis. The 29 AA-long t-PA-peptide-1 with a lactose-functionalized serine revealed the strongest binding properties to Gal-1 which was 25-fold higher in comparison with the native t-PA protein and showed additional strong binding to Gal-3 and Gal-4, both also over-expressed in PDAC. t-PA-peptide-1 was selected as vector moiety and linked covalently onto the surface of biodegradable iron oxide nanoparticles (NPs). In particular, CAN-doped maghemite NPs (CAN-Mag), promising as contrast agent for magnetic resonance imaging (MRI), were selected as magnetic core and coated with different biocompatible polymers, such as chitosan (CAN-Mag-Chitosan NPs) or polylactic co glycolic acid (PLGA) obtaining polymeric nanoparticles (CAN-Mag@PNPs), already approved for drug delivery applications. The binding efficacy of t-PA-vectorized NPs determined by exposure to different pancreatic cell lines was up to 90%, as assessed by flow cytometry. The in vivo targeting and imaging efficacy of the vectorized NPs were evaluated by applying murine pancreatic tumor models and assessed by 1.5 T magnetic resonance imaging (MRI). The t-PA-vectorized NPs as well as the protease-activated NPs with outer shell decoration (CAN-Mag@PNPs-PEG-REGAcp-PEG/tPA-pep1 Lac ) showed clearly detectable drop of subcutaneous and orthotopic tumor staining-intensity indicating a considerable uptake of the injected NPs. Post mortem NP deposition in tumors and organs was confirmed by Fe staining of histopathology tissue sections., Conclusions: The targeted NPs indicate a fast and enhanced deposition of NPs in the murine tumor models. The CAN-Mag@PNPs-PEG-REGAcp-PEG/tPA-pep1 Lac interlocking steps strategy of NPs delivery and deposition in pancreatic tumor is promising.

Intraperitoneal adhesion occurs frequently after pelvic and abdominal surgery, which plays an enormous burden on patients. Various drugs and barrier agents were studied and used to prevent adhesions, but few of them were satisfactory. A thermosensitive hydrogel was developed in our previous work and was effective in preventing adhesions. But in our preliminary experiment, it showed limited activity in a more rigorous rat repeated-injury adhesion model, which was much closer to clinical. In this study, tissue-type plasminogen activator (tPA) loaded thermosensitive hydrogel (tPA-hydrogel) was prepared, which combined barrier functions with sustained release of antiadhesion drug. The obtained tPA-hydrogel was injectable and degraded in vivo gradually in four weeks. Both hematoxylin and eosin and Masson trichrome staining confirmed that the tPA-hydrogel exhibited excellent antiadhesion effects on repeated-injury adhesion. Scanning electron microscopy was used to observe the injured abdominal wall, and cecum remesothelialized after treated with tPA-hydrogel for 14 d. In addition, the PAI-1 and tPA levels were measured by enzyme-linked immunosorbent assay. Results showed the PAI-1 concentrations in peritoneal lavage fluids of tPA-hydrogel treated rats were lower than that of other groups, leading to decreased fibrin formation, while there were no significant differences observed in tPA blood levels at any point in time (P > 0.05). This study demonstrated that the effectiveness of thermosensitive hydrogel in preventing adhesions could be enhanced by delivering antiadhesion drugs, and the tPA-hydrogel might be a promising system for clinical application.

Objective: To determine whether the ratio single chain (sc)/(sc + 2 chain [tc]) recombinant tissue plasminogen activator (rtPA) influences outcomes in patients with cerebral ischemia., Methods: We prospectively included consecutive patients treated with IV rtPA for cerebral ischemia in 13 stroke centers and determined the sc/(sc + tc) ratio in the treatment administered to each patient. We evaluated the outcome with the modified Rankin Scale (mRS) at 3 months (prespecified analysis) and occurrence of epileptic seizures (post hoc analysis). We registered Outcome of Patients Treated by IV Rt-PA for Cerebral Ischaemia According to the Ratio Sc-tPA/Tc-tPA (OPHELIE) under ClinicalTrials.gov identifier no. NCT01614080., Results: We recruited 1,004 patients (515 men, median age 75 years, median onset-to-needle time 170 minutes, median NIH Stroke Scale score 10). We found no statistical association between sc/(sc + tc) ratios and handicap (mRS > 1), dependency (mRS > 2), or death at 3 months. Patients with symptomatic intracerebral hemorrhages had lower ratios (median 69% vs 72%, adjusted p = 0.003). The sc/(sc + tc) rtPA ratio did not differ between patients with and without seizures, but patients with early seizures were more likely to have received a sc/(sc + tc) rtPA ratio >80.5% (odds ratio 3.61; 95% confidence interval 1.26-10.34)., Conclusions: The sc/(sc + tc) rtPA ratio does not influence outcomes in patients with cerebral ischemia. The capacity of rtPA to modulate NMDA receptor signaling might be associated with early seizures, but we observed this effect only in patients with a ratio of sc/(sc + tc) rtPA >80.5% in a post hoc analysis., (© 2016 American Academy of Neurology.)

Streptokinase is a valuable fibrinolytic agent used to cope with myocardial infarction and brain stroke. Despite its high efficiency in dissolving blood clots, streptokinase (SK) has no specificity in binding fibrin, causing some problems such as internal bleedings following its administration. To make streptokinase fibrin specific and limit the fibrinolytic process to the clot location, we engineered a chimeric streptokinase by fusing the fibrin binding Kringle 2 domain of tissue plasminogen activator (TPA) to the streptokinase N-terminal end. The chimeric SK construct (KSK) with inserted Kringle 2 domain was cloned into pET28a expression vector. The expression of recombinant protein was carried out in Escherichia coli origami (DE3) and confirmed by SDS-PAGE and Western blotting analyses. We used the chromogenic substrate S-2251 method to assess the specific activities of the chimeric and control wild-type proteins. Then, the two proteins were added in amounts with equal activity to fibrin clots of identical size. Finally, the supernatant above the fibrin clots was collected and subjected to the chromogenic assay to analyze the specificity of the chimeric protein. The specific activities of the chimeric and wild-type proteins were found to be 0.06 U/mg and 0.07 U/mg, respectively. Because of the binding of the chimeric protein to fibrin, the mean specific activity was significantly lower in the KSK supernatant (0.01) compared with the control (approximately 0.06) (p < 0.05). Our in vitro results indicate that the chimeric streptokinase protein has strong fibrin-specific activity compared to the wild-type protein. However, further in vivo studies are needed to evaluate its potential fibrinolytic effects., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Tissue engineered heart valve is a promising alternative to current heart valve surgery, for its capability of growth, repair, and remodeling. However, extensive development is needed to ensure tissue compatibility, durability and antithrombotic potential. This study aims to investigate the biological effects of multi-signal composite material of polyethyl glycol-cross-linked decellularized valve on adhesion and proliferation of endothelial progenitor cells. Group A to E was decellularized valve leaflets, composite material of polyethyl glycol-cross-linked decellularized valves leaflets, vascular endothelial growth factor-composite materials, Arg-Gly-Asp peptide-composite materials and multi-signal modified materials of polyethyl glycol-cross-linked decellularized valve leaflets, respectively. The endothelial progenitor cells were seeded for each group, cell adhesion and proliferation were detected and neo-endothelium antithrombotic function of the multi-signal composite materials was evaluated. At 2, 4, and 8 h after the seeding, the cell numbers and 3H-TdR incorporation in group D were the highest. At 2, 4, and 8 days after the seeding, the cell numbers and 3H-TdR incorporation were significantly higher in groups C, D, and E compared with groups A and B (P < 0.05) and cell numbers and the expression of t-PA and eons in the neo-endothelium were quite similar to those in the human umbilical vein endothelial cells at 2, 4, and 8 days after the seeding. The Arg-Gly-Asp- peptides (a sequential peptide composed of arginine (Arg), glycine (Gly) and aspartic acid (Asp)) and VEGF-conjugated onto the composite material of PEG-crosslinked decellularized valve leaflets synergistically promoted the adhesion and proliferation of endothelial progenitor cells on the composite material, which may help in tissue engineering of heart valves.