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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.)

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.)

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.)

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.)

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.)

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.)

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.)

Unlabelled: Essentials Physiologic variations in blood plasminogen (Pg) levels may affect ischemic stroke outcomes. We tested Pg effects in a model with translational relevance to human thromboembolic stroke. A dose-response exists between Pg levels and brain injury, fibrinolysis, barrier breakdown. Higher Pg levels reduce microvascular thrombosis and improve outcomes in ischemic stroke., Summary: Background and Objectives Plasminogen appears to affect brain inflammation, cell movement, fibrinolysis, neuronal excitotoxicity, and cell death. However, brain tissue and circulating blood plasminogen may have different roles, and there is wide individual variation in blood plasminogen levels. The aim of this study was to determine the integrated effect of blood plasminogen levels on ischemic brain injury. Methods We examined thromboembolic stroke in mice with varying, experimentally determined, blood plasminogen levels. Ischemic brain injury, blood-brain barrier breakdown, matrix metalloproteinase-9 expression and microvascular thrombosis were determined. Results Within the range of normal variation, plasminogen levels were strongly associated with ischemic brain injury; higher blood plasminogen levels had dose-related, protective effects. Higher plasminogen levels were associated with increased dissolution of the middle cerebral artery thrombus. Higher plasminogen levels decreased blood-brain barrier breakdown, matrix metalloproteinase-9 expression and microvascular thrombosis in the ischemic brain. In plasminogen-deficient mice, selective restoration of blood plasminogen levels reversed the harmful effects of plasminogen deficiency on ischemic brain injury. Specific inhibition of thrombin also reversed the effect of plasminogen deficiency on ischemic injury by decreasing microvascular thrombosis, blood-brain barrier breakdown, and matrix metalloproteinase-9 expression. Conclusions Variation in blood plasminogen levels, within the range seen in normal individuals, had marked effects on experimental ischemic brain injury. Higher plasminogen levels protected against ischemic brain injury, and decreased blood-brain barrier breakdown, matrix metalloproteinase-9 expression, and microvascular thrombosis. The protective effects of blood plasminogen appear to be mediated largely through a decrease in microvascular thrombosis in the ischemic territory., (© 2016 International Society on Thrombosis and Haemostasis.)

Unlabelled: Essentials Factor Xa (FXa) acquires cleavage-mediated tissue plasminogen activator (tPA) cofactor activity. Recombinant (r) tPA is the predominant thrombolytic drug, but it may cause systemic side effects. Chemically modified, non-enzymatic FXa was produced (Xai-K), which rapidly lysed thrombi in mice. Unlike rtPA, Xai-K had no systemic fibrinolysis activation markers, indicating improved safety., Summary: Background Enzymatic thrombolysis carries the risk of hemorrhage and re-occlusion must be evaded by co-administration with an anticoagulant. Toward further improving these shortcomings, we report a novel dual-functioning molecule, Xai-K, which is both a non-enzymatic thrombolytic agent and an anticoagulant. Xai-K is based on clotting factor Xa, whose sequential plasmin-mediated fragments, FXaβ and Xa33/13, accelerate the principal thrombolytic agent, tissue plasminogen activator (tPA), but only when localized to anionic phospholipid. Methods The effect of Xai-K on fibrinolysis was measured in vitro by turbidity, thromboelastography and chromogenic assays, and measured in a murine model of occlusive carotid thrombosis by Doppler ultrasound. The anticoagulant properties of Xai-K were evaluated by normal plasma clotting assays, and in murine liver laceration and tail amputation hemostatic models. Results Xa33/13, which participates in fibrinolysis of purified fibrin, was rapidly inhibited in plasma. Cleavage was blocked at FXaβ by modifying residues at the active site. The resultant Xai-K (1 nm) enhanced plasma clot dissolution by ~7-fold in vitro and was dependent on tPA. Xai-K alone (2.0 μg g(-1) body weight) achieved therapeutic patency in mice. The minimum primary dose of the tPA variant, Tenecteplase (TNK; 17 μg g(-1) ), could be reduced by > 30-fold to restore blood flow with adjunctive Xai-K (0.5 μg g(-1) ). TNK-induced systemic markers of fibrinolysis were not detected with Xai-K (2.0 μg g(-1) ). Xai-K had anticoagulant activity that was somewhat attenuated compared with a previously reported analogue. Conclusion These results suggest that Xai-K may ameliorate the safety profile of therapeutic thrombolysis, either as a primary or tPA/TNK-adjunctive agent., (© 2016 International Society on Thrombosis and Haemostasis.)

Elevated plasma concentrations of lipoprotein (a) [Lp(a)] have been determined to be a causal risk factor for coronary heart disease, and may similarly play a role in other atherothrombotic disorders. Lp(a) consists of a lipoprotein moiety indistinguishable from LDL, as well as the plasminogen-related glycoprotein, apo(a). Therefore, the pathogenic role for Lp(a) has traditionally been considered to reflect a dual function of its similarity to LDL, causing atherosclerosis, and its similarity to plasminogen, causing thrombosis through inhibition of fibrinolysis. This postulate remains highly speculative, however, because it has been difficult to separate the prothrombotic/antifibrinolytic functions of Lp(a) from its proatherosclerotic functions. This review surveys the current landscape surrounding these issues: the biochemical basis for procoagulant and antifibrinolytic effects of Lp(a) is summarized and the evidence addressing the role of Lp(a) in both arterial and venous thrombosis is discussed. While elevated Lp(a) appears to be primarily predisposing to thrombotic events in the arterial tree, the fact that most of these are precipitated by underlying atherosclerosis continues to confound our understanding of the true pathogenic roles of Lp(a) and, therefore, the most appropriate therapeutic target through which to mitigate the harmful effects of this lipoprotein., (Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.)

The aim of the present study was the investigation of peroxynitrite (ONOO(-)) effects on fibrinolysis in vitro and in silico. The exposure of human plasminogen to ONOO(-) (10-1000μM) resulted in a decrease of t-PA-induced amidolytic activity of plasmin; the inhibitory effect was associated with the increasing level of 3-nitrotyrosine in plasminogen/plasmin molecule. Furthermore, ONOO(-) displayed both the ability to impair the t-PA-induced activation of plasminogen to plasmin, and to reduce the rate of fibrin lysis by plasmin. The susceptibility of plasminogen in blood plasma to nitrative action of ONOO(-) was revealed by the immunoprecipitation technique. To confirm the hypothesis that 3-nitrotyrosine generation is crucial for the impairment of plasmin activity, (-)-epicatechin, a polyphenolic antioxidant that selectively prevents tyrosine nitration, was used both for in vitro experiments as well as for in silico studies on ONOO(-), ONOOH and (-)-epicatechin binding and plasminogen nitration. (-)-Epicatechin effectively protected plasminogen against ONOO(-)-induced inactivation and significantly reduced the level of 3-nitrotyrosine. The obtained results revealed tyrosine nitration as the most likely mechanism of the inhibitory effect of ONOO(-) on plasmin(ogen) functions. The possible role of tyrosine modifications was additionally confirmed by bioinformatics calculations with indication of nitration susceptible tyrosine residues., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The potent fibrinolytic enzyme, plasmin has numerous clinical applications for recannulizing vessels obstructed by thrombus. Despite its diminutive size, 91 kDa, success in the recombinant expression of this serine protease has been limited. For this reason, a truncated non-glycosylated plasmin variant was developed capable of being expressed and purified from E. coli. This mutated plasmin, known as δ-plasmin, eliminates four of the five kringle domains present on native plasmin, retaining only kringle 1 fused directly to the unmodified catalytic domain of plasmin. This study demonstrates that δ-plasmin exhibits similar kinetic characteristics to full length plasmin despite its heavily mutated form; KM = 268.78 ± 19.12, 324.90 ± 8.43 μM and Kcat = 770.48 ± 41.73, 778.21 ± 1.51 1/min for plasmin and δ-plasmin, respectively. A comparative analysis was also carried out to investigate the inhibitory effects of a range of benzamidine based small molecule inhibitors: benzamidine, p-aminobenzamidine, 4-carboxybenzamidine, 4-aminomethyl benzamidine, and pentamidine. All of the small molecule inhibitors, with the exception of unmodified benzamidine, demonstrated comparable competitive inhibition constants (Ki) for both plasmin and δ-plasmin ranging from Ki < 4 μM for pentamidine to Ki > 1000 μM in the case of aminomethyl benzamidine. This result further supports that δ-plasmin retains much of the same functionality as native plasmin despite its greatly reduced size and complexity. This study serves the purpose of demonstrating the tunable inhibition of plasmin and δ-plasmin with potential applications for the improved clinical delivery of δ-plasmin to treat various thrombi., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Most proteins in nature are chemically modified after they are made to control how, when, and where they function. The 3 core features of proteins are posttranslationally modified: amino acid side chains can be modified, peptide bonds can be cleaved or isomerized, and disulfide bonds can be cleaved. Cleavage of peptide bonds is a major mechanism of protein control in the circulation, as exemplified by activation of the blood coagulation and complement zymogens. Cleavage of disulfide bonds is emerging as another important mechanism of protein control in the circulation. Recent advances in our understanding of control of soluble blood proteins and blood cell receptors by functional disulfide bonds is discussed as is how these bonds are being identified and studied.

PfbA is a surface adhesin and invasin of Streptococcus pneumoniae that binds to human fibronectin and plasminogen of the host extracellular matrix. It is a virulence factor for its pathogenesis. The crystal structure of recombinant PfbA150-607 from S. pneumoniae strain R6, was determined using multiwavelength anomalous dispersion (MAD) method and refined to 1.90Å resolution. The structure of rPfbA150-607 revealed that residues Thr150 to Lys570 form a rigid parallel beta helix, followed by a short disordered region (571-607) that consists of beta hairpins. The structural organization of the beta helix resembles that of polysaccharide-modifying enzymes. The structural and sequence features essential for fibronectin-binding observed in the well characterized fibronectin-binding proteins such as FnBPA of Staphylococcus aureus, SfbI of Streptococcus pyogenes and BBK32 of Borrelia burgdorferi has been found in rPfbA150-607. Based on this, it is predicted that the disordered region following the beta helix could be the fibronectin-binding region in PfbA. PfbA150-607 contains relatively high number of surface exposed lysines and these residues are probably involved in binding plasmin(ogen) as observed in other plasminogen-binding proteins., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Cluster 2b streptokinase (SK2b), secreted by invasive skin-trophic strains of Streptococcus pyogenes (GAS), is a human plasminogen (hPg) activator that optimally functions when human plasma hPg is bound, via its kringle-2 domain, to cognizant bacterial cells through the a1a2 domain of the major cellular hPg receptor, Plasminogen-binding group A streptococcal M-like protein (PAM). Another class of streptokinases (SK1), secreted primarily by GAS strains that possess affinity for pharyngeal infections, does not require PAM-bound hPg for optimal activity. We find herein that replacement of the central β-domain of SK2b with the same module from SK1 reduces the dependency of SK2b on PAM, and the converse is true when the β-domain of SK1 is replaced with this same region of SK2b. These data suggest that simple evolutionary shuttling of protein domains in GAS can be employed by GAS to rapidly generate strains that differ in tissue tropism and invasive capability and allow the bacteria to survive different challenges by the host., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Plasminogen (PLG) is the zymogen of plasmin, the major enzyme that degrades fibrin clots. In addition to its binding and activation on fibrin clots, PLG also specifically interacts with cell surfaces where it is more efficiently activated by PLG activators, compared with the reaction in solution. This results in association of the broad-spectrum proteolytic activity of plasmin with cell surfaces that functions to promote cell migration. Here, we review emerging data establishing a role for PLG, plasminogen receptors and the newly discovered plasminogen receptor, Plg-RKT, in macrophage recruitment in the inflammatory response, and we address mechanisms by which the interplay between PLG and its receptors regulates inflammation., (© 2014 Elsevier Inc. All rights reserved.)

Background: The activity of alpha-2-antiplasmin (α2AP), the main fibrinolytic inhibitor, is modified by N- and C-terminal proteolytic cleavages. C-terminal cleavage converts plasminogen-binding α2AP (PB-α2AP) into a non-plasminogen-binding derivative. N-terminal cleavage by antiplasmin-cleaving enzyme (APCE), a soluble, circulating derivative of fibroblast activation protein (FAP), turns native Met-α2AP into Asn-α2AP, which is more quickly crosslinked into fibrin., Objectives: We developed two novel enzyme-linked immunosorbent assays (ELISAs) to determine the N-terminal variation of α2AP to test the hypothesis that liver cirrhosis, characterized by increased expression of FAP/APCE, results in increased N-terminal cleavage of α2AP., Patients/methods: α2AP and FAP/APCE antigen levels were measured in the plasma samples of 75 patients with cirrhosis with different severities and 30 healthy control individuals. The percentage of N-terminal cleavage of α2AP was calculated., Results: Compared with levels (median [interquartile range]) in control individuals, total PB-α2AP levels and Met-PB-α2AP levels were reduced in cirrhosis patients (27.3 [21.4-41.3] μg mL(-1) vs. 56.2 [49.6-62.8] μg mL(-1) , P < 0.001, and 2.7 [1.7-5.5] μg mL(-1) vs. 12.1 [11.0-15.3] μg mL(-1) , P < 0.001, respectively). Interestingly, the percentage of N-terminal cleavage was increased in the patients (87.8 [85.0-91.6]% vs. 77.2 [72.2-79.8]% in controls, P < 0.001), as well as the plasma FAP/APCE levels (166 [60-550] ng mL(-1) in patients vs. 107 [67-157] ng mL(-1) in controls, P < 0.001). Additionally, all variables significantly correlated with the severity of disease., Conclusions: Using our novel ELISAs we found increased N-terminal cleavage of α2AP in liver cirrhosis patients, which correlated with the severity of disease and is likely to have reflected the increased FAP/APCE levels in these patients., (© 2013 International Society on Thrombosis and Haemostasis.)

Human tissue kallikreins (KLKs) are a group of serine proteases found in many tissues and biological fluids and are differentially expressed in several specific pathologies. Here, we present evidences of the ability of these enzymes to activate plasminogen. Kallikreins 3 and 5 were able to induce plasmin activity after hydrolyzing plasminogen, and we also verified that plasminogen activation was potentiated in the presence of glycosaminoglycans compared with plasminogen activation by tPA. This finding can shed new light on the plasminogen/plasmin system and its involvement in tumor metastasis, in which kallikreins appear to be upregulated., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Binding of tissue-type plasminogen (Pgn) activator (t-PA) and Pgn to fibrin regulates plasmin generation, but there is no consistent, quantitative understanding of the individual contribution of t-PA finger and kringle 2 domains to the regulation of fibrinolysis. Kringle domains bind to lysines in fibrin, and this interaction can be studied by competition with lysine analogs and removal of C-terminal lysines by carboxypeptidase B (CPB)., Methods: High-throughput, precise clot lysis assays incorporating the lysine analog tranexamic acid (TA) or CPB and genetically engineered variants of t-PA were performed. In particular, wild-type (WT) t-PA (F-G-K1-K2-P) and a domain-switched variant K1K1t-PA (F-G-K1-K1-P) that lacks kringle 2 but retains normal t-PA structure were compared to probe the importance of fibrin lysine binding by t-PA kringle 2., Results: WT t-PA showed higher rates of fibrinolysis than K1K1t-PA, but the inhibitory effects of TA or CPB were very similar for WT t-PA and the variant t-PA (< 10% difference). Urokinase plasminogen activator (u-PA)-catalyzed fibrinolysis was also inhibited by TA, even though Pgn activation could be stimulated. Fibrin treated with factor XIIIa (FXIIIa) generates crosslinked degradation products, but these did not affect the results obtained with WT t-PA and K1K1t-PA., Conclusions: t-PA kringle 2 has a minor role in the initial interaction of t-PA and fibrin, but stimulation of fibrinolysis by C-terminal lysines (or inhibition by carboxypeptidases or TA) operates through Pgn and plasmin binding, not through t-PA. This is also true when fibrin is crosslinked by treatment with FXIIIa., (© 2012 International Society on Thrombosis and Haemostasis.)

Background: Plasminogen is the zymogen form of plasmin and the precursor of angiostatin. It has been implicated in a variety of disease states, including thrombosis, bleeding and cancers. The native plasminogen, known as Glu-plasminogen, contains seven domains comprising the N-terminal peptide domain (NTP), five kringle domains (K1-K5) and the C-terminal serine protease domain (SP). Previous studies have established that the lysine binding site (LBS) of the conserved kringle domains plays a crucial role in mediating the regulation of plasminogen function. However, details of the related conformational mechanism are unknown., Objectives: We aim to understand in more detail the conformational mechanism of plasminogen activation involving the kringles., Methods: We crystallized the native plasminogen under physiologically relevant conditions and determined the structure at 3.5 Å resolution. We performed structural analyses and related these to the literature data to gain critical understanding of the plasminogen activation., Results and Conclusions: The structure reveals the precise architecture of the quaternary complex. It shows that the Glu-plasminogen renders its compact form as an activation-resistant conformation for the proteolytic activation. The LBSs of all kringles, except K1, are engaged in intra-molecular interactions while only K1-LBS is readily available for ligand binding or receptor anchorage. The structure also provides insights into the interactions between plasminogen and α2-antiplasmin, the primary physiological inhibitor of plasmin. Furthermore, the data presented explain why a conformational transition to the open form is necessary for plasminogen activation as well as angiostatin generation, and provide a rationale for the functional hierarchy of the different kringles., (© 2012 International Society on Thrombosis and Haemostasis.)

We previously demonstrated that streptokinase (SK) can be used to generate active site-labeled fluorescent analogs of plasminogen (Pg) by virtue of its nonproteolytic activation of the zymogen. The method is versatile and allows stoichiometric and active site-specific incorporation of any one of many molecular probes. The limitation of the labeling approach is that it is both time-consuming and low yield. Here we demonstrate an improved method for the preparation of labeled Pg analogs by the use of an engineered SK mutant fusion protein with both COOH- and NH(2)-terminal His(6) tags. The NH(2)-terminal tag is followed by a tobacco etch virus proteinase cleavage site to ensure that the SK Ile(1) residue, essential for conformational activation of Pg, is preserved. The SK COOH-terminal Lys(414) residue and residues Arg253-Leu260 in the SK β-domain were deleted to prevent cleavage by plasmin (Pm) and to disable Pg substrate binding to the SK·Pg(∗)/Pm catalytic complexes, respectively. Near elimination of Pm generation with the SKΔ(R253-L260)ΔK414-His(6) mutant increased the yield of labeled Pg 2.6-fold and reduced the time required more than 2-fold. The versatility of the labeling method was extended to the application of Pg labeled with a near-infrared probe to quantitate Pg receptors on immune cells by flow cytometry., (Copyright © 2011 Elsevier Inc. All rights reserved.)

When Glu-plasminogen binds to cells, its activation to plasmin is markedly enhanced compared with the reaction in solution, suggesting that Glu-plasminogen on cell surfaces adopts a conformation distinct from that in solution. However, direct evidence for such conformational changes has not been obtained. Therefore, we developed anti-plasminogen mAbs to test the hypothesis that Glu-plasminogen undergoes conformational changes on its interaction with cells. Six anti-plasminogen mAbs (recognizing 3 distinct epitopes) that preferentially recognized receptor-induced binding sites (RIBS) in Glu-plasminogen were obtained. The mAbs also preferentially recognized Glu-plasminogen bound to the C-terminal peptide of the plasminogen receptor, Plg-R(KT), and to fibrin, plasmin-treated fibrinogen, and Matrigel. We used trypsin proteolysis, immunoaffinity chromatography, and tandem mass spectrometry and identified Glu-plasminogen sequences containing epitopes recognized by the anti-plasminogen-RIBS mAbs: a linear epitope within a domain linking kringles 1 and 2; a nonlinear epitope contained within the kringle 5 domain and the latent protease domain; and a nonlinear epitope contained within the N-terminal peptide of Glu-plasminogen and the latent protease domain. Our results identify neoepitopes latent in soluble Glu-plasminogen that become available when Glu-plasminogen binds to cells and demonstrate that binding of Glu-plasminogen to cells induces a conformational change in Glu-plasminogen distinct from that of Lys-Pg.

Clotting and thrombosis remain the most serious problems in the development of blood contacting devices such as heart valves, vascular stents, grafts and catheters. No material exists that does not provoke these phenomena and coagulation appears to be inevitable when a foreign (i.e. non-endothelial) surface is in contact with blood. As an alternative to a surface that prevents coagulation, the concept of a clot-lysing or fibrinolytic surface is attractive. By designing the surface effectively to mimic the fibrinolytic system in the vasculature, it may be possible for clots to be lysed (effectively dissolved) as they form. In this review we elaborate on this concept and discuss ways in which such a surface could be realized. Developments in this area to the present time are reviewed, and some perspectives for future research are presented., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: A synthetic nonadecapeptide (SP; GPYLMVNVTGVDGKGNELL) previously enhanced the activation of plasminogen by the SAK/plasmin complex., Objectives: To identify the binding site for SP on plasminogen and elucidate the effects of SP on plasminogen activation by the tissue-type plasminogen activator (t-PA)., Methods: The effects of SP on plasminogen activation were estimated using a chromogenic substrate and from the cleavage of plasmin on SDS-PAGE under reduced conditions. The binding to SP of various peptides derived from the amino acid sequence of plasminogen was analyzed with an IAsys biosensor. The SP-mediated structural change to plasminogen was analyzed by circular dichroism (CD) spectroscopy. The thrombolytic effects of SP were examined using a mouse model of thrombosis., Results: SP enhanced the activation of plasminogen by t-PA. The catalytic efficiency (k(cat)/K(m)) of Glu-plasminogen activation by t-PA was 11.4-fold higher in the presence than absence of SP. The binding of SP to plasminogen was greatly inhibited by a synthetic peptide, FEKDKYILQGVTSWGLG, located close to the C-terminal of the plasminogen B region. Near-ultraviolet CD spectra of the complex between SP and Glu-plasminogen significantly differed from those of Glu-plasminogen. When SP was administered in a mouse model of thrombosis, early recanalization was observed in a dose-dependent manner. However, SP did not cause recanalization in t-PA gene-deficient mice., Conclusions: SP bound to the B region and promoted the activation of plasminogen by t-PA, and then induced effective thrombolysis., (© 2011 International Society on Thrombosis and Haemostasis.)

We have developed a potentially fibrinolytic surface in which a bioinert polymer is used as a spacer to immobilize lysine such that the ε-amino group is free to capture plasminogen when in contact with blood. Adsorbed plasminogen can be activated to plasmin and potentially dissolve nascent clots formed on the surface. In previous work lysine was immobilized through a poly(ethylene glycol) (PEG) spacer; however, the graft density of PEG was limited and the resulting adsorbed quantity of plasminogen was insufficient to dissolve clots efficiently. The aim of the present work was to optimize the surface using graft-polymerized poly(2-hydroxyethyl methacrylate) (poly(HEMA)) as a spacer to increase the grafting density of lysine. Such a poly(HEMA)-lysine modified polyurethane (PU) surface is expected to have increased plasminogen binding capacity and clot lysing efficiency compared with PEG-lysine modified PU. A lysine density of 2.81 nmol cm(-2) was measured on the PU-poly(HEMA)-Lys surface vs. 0.76 nmol cm(-2) on a comparable PU-PEG-Lys surface reported previously. The poly(HEMA)-lysine-modified surface was shown to reduce non-specific (fibrinogen) adsorption while binding plasminogen from plasma with high affinity. With increased plasminogen binding capacity these surfaces showed more rapid clot lysis (20 min) in a standard in vitro assay than the corresponding PEG-lysine system (40 min). The data suggest that poly(HEMA) is superior to PEG when used as a spacer in the immobilization of bioactive molecules at high density. This method of modification may also provide a generic approach for preparing bioactive PU surfaces of high activity and low non-specific adsorption of proteins., (Copyright © 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

A simple methodology for the identification of hemostatic proteins that are subjected to posttranslational tyrosine sulfation was developed. The procedure involves sequence analysis of members of the three hemostatic pathways using the Sulfinator prediction algorithm, followed by [(35)S]sulfate labeling of cultured HepG2 human hepatoma cells, immunoprecipitation of targeted [(35)S]sulfate-labeled hemostatic proteins, and tyrosine O-[(35)S]sulfate analysis of immunoprecipitated proteins. Three new tyrosine-sulfated hemostatic proteins-protein S, prekallikrein, and plasminogen-were identified. Such a target-specific approach will allow investigation of tyrosine-sulfated proteins of other biochemical/physiological pathways/processes and contribute to a better understanding of the functional role of posttranslational tyrosine sulfation.

The characteristics of a streptococcal plasminogen activator (PA) displaying specificity for ruminant plasminogen (Plg) were defined using molecular approaches. The 16-kDa secreted protein PadA was found to be prevalent in Streptococcus dysgalactiae subspecies dysgalactiae isolated from cases of bovine mastitis and septic arthritis in lambs. PadA was able to activate bovine, ovine and caprine Plg, but not human Plg. Amino acid sequence analysis identified a limited level of homology to other streptococcal PAs, including streptokinase; however, PadA was found to align well with and match in size the staphylococcal PA, staphylokinase. Recombinant PadA was used to investigate interaction with bovine Plg, leading to formation of an activator complex that was capable of recruiting and converting further substrate Plg into plasmin. Individual non-overlapping peptides of PadA or bovine microplasminogen were found to block the interaction between PadA and bovine Plg, preventing the formation of the activation complex. Homology modelling based upon structures of staphylokinase complexed with human microplasminogen supported these findings by placing critical residues in close proximity to the plasmin component of the activation complex.

Streptococcus mutans is the etiologic agent of dental caries and is a causative agent of infective endocarditis. While the mechanisms by which S. mutans cells colonize heart tissue is not clear, it is thought that bacterial binding to extracellular matrix and blood components is crucial in the development of endocarditis. Previously, we have demonstrated that S. mutans cells have the capacity to bind and activate plasminogen to plasmin. Here we report the first cloning and characterization of an alpha-enolase of S. mutans that binds plasminogen. The functional identity of the purified recombinant alpha-enolase protein was confirmed by its ability to catalyze the conversion of 2-phosphoglycerate to phosphoenolpyruvate. The protein exhibited a Km of 9.5 mM and a Vmax of 31.0 mM/min/mg. The alpha-enolase protein was localized in the cytoplasmic, cell wall and extracellular fractions of S. mutans. Binding studies using an immunoblot analysis revealed that human plasminogen binds to the enolase enzyme of S. mutans. These findings identify S. mutans alpha-enolase as a binding molecule used by this oral pathogen to interact with the blood component, plasminogen. Further studies of this interaction may be critical to understand the pathogenesis of endocarditis caused by S. mutans.

Plasminogen (plg) deficiency has been classified as (i) hypoplasminogenemia or 'true' type I plg deficiency, and (ii) dysplasminogenemia, also called type II plg deficiency. Both forms, severe hypoplasminogenemia and dysplasminogenemia, are not causally linked to venous thrombosis. Dysplasminogenemia does not lead to a specific clinical manifestation and probably represents only a polymorphic variation in the general population, mainly in Asian countries. Severe hypoplasminogenemia is associated with compromised extracellular fibrin clearance during wound healing, leading to pseudomembraneous (ligneous) lesions on affected mucous membranes (eye, middle ear, mouth, pharynx, duodenum, upper and lower respiratory tract and female genital tract). Ligneous conjunctivitis is by far the most common clinical manifestation. More than 12% of patients with severe hypoplasminogenemia exhibit congenital occlusive hydrocephalus. In milder cases of ligneous conjunctivitis, topical application of plg-containing eye drops, fresh frozen plasma, heparin, corticosteroids or certain immunosuppressive agents (such as azathioprine) may be more or less effective. Oral treatment with sex hormones was successful in two female patients with ligneous conjunctivitis. In severe cases with possibly life-threatening multi-organ involvement, true therapeutic options are not available at present. The plg-knockout mouse is a useful tool to study the many different properties of plg in a variety of settings, such as wound healing, tissue repair and tissue remodeling, virulence and invasiveness of certain bacteria in the human host, tumor growth and dissemination, as well as arteriosclerosis.

alpha(2)-Antiplasmin (alpha(2)-AP) is the fast serpin inhibitor of plasmin and appears to limit the success of treatment for thrombosis. We examined the mechanisms through which monoclonal antibodies (mAbs) against alpha(2)-AP amplify fibrinolysis. The mAbs RWR, 49 and 77 interfered with the ability of alpha(2)-AP to inhibit plasmin, microplasmin and trypsin. In solution, mAbs 49 and 77 bound to alpha(2)-AP with 5-fold to 10-fold higher relative affinity than mAb-RWR, while mAb-RWR bound with greater avidity to immobilized or denatured alpha(2)-AP. Binding studies with chimeric alpha(2)-APs revealed that none of the mAbs bound to sites in alpha(2)-AP that form putative contacts with plasmin, namely the carboxy terminal lysines of alpha(2)-AP, or the reactive center loop in the serpin domain of alpha(2)-AP. Rather, mAb-RWR recognized an epitope in the amino-terminus of alpha(2)-AP (L(13)GNQEPGGQTALKSPPGVCS(32)) near the site at which alpha(2)-AP cross-links to fibrin. mAbs 49 and 77 bound to another conformational epitope in the serpin domain of alpha(2)-AP. mAbs 49 and 77 markedly increased the stoichiometry of plasmin inhibition by alpha(2)-AP (from 1.1 +/- 0.1 to 51 +/- 4 and 67 +/- 7) indicating that they convert alpha(2)-AP from an inhibitor to a substrate of plasmin. This was confirmed by sodium dodecylsulfate polyacrylamide gel electrophoresis analysis showing cleavage of alpha(2)-AP by plasmin in the presence of these mAbs. In summary, these mAbs appear to act at sites distinct from known alpha(2)-AP-plasmin contacts to enhance fibrinolysis by converting alpha(2)-AP from an inhibitor to a plasmin substrate.

Inhibition of endothelial cell proliferation and angiogenesis is emerging as an important strategy in cancer therapeutics. Kringle 5 (K5) of human plasminogen is a potent angiogenesis inhibitor. Previous studies have shown K5 exposure promotes caspase activity and apoptosis in endothelial cells. Here we report that K5 treatment evokes an autophagic response in endothelial cells that is specific and initiated even in the absence of nutritional stress. Endothelial cells exposed to K5 up-regulated Beclin 1 levels within a few hours. Furthermore, progressively increasing amounts of antiapoptotic Bcl-2 were found to be complexed with Beclin 1, although total levels of Bcl-2 remained unchanged. Prolonged exposure to K5 ultimately led to apoptosis via mitochondrial membrane depolarization and caspase activation in endothelial cells. Knocking down Beclin 1 levels by RNA interference decreased K5 induced autophagy but accelerated K5-induced apoptosis. These studies suggest that interfering with the autophagic survival response can potentiate the antiangiogenic effects of Kringle 5 in endothelial cells.

Allosteric disulfide bonds control protein function by mediating conformational change when they undergo reduction or oxidation. The known allosteric disulfide bonds are characterized by a particular bond geometry, the -RHStaple. A number of thrombosis and thrombolysis proteins contain one or more disulfide bonds of this type. Tissue factor (TF) was the first hemostasis protein shown to be controlled by an allosteric disulfide bond, the Cys186-Cys209 bond in the membrane-proximal fibronectin type III domain. TF exists in three forms on the cell surface: a cryptic form that is inert, a coagulant form that rapidly binds factor VIIa to initiate coagulation, and a signaling form that binds FVIIa and cleaves protease-activated receptor 2, which functions in inflammation, tumor progression and angiogenesis. Reduction and oxidation of the Cys186-Cys209 disulfide bond is central to the transition between the three forms of TF. The redox state of the bond appears to be controlled by protein disulfide isomerase and NO. Plasmin(ogen), vitronectin, glycoprotein 1balpha, integrin beta(3) and thrombomodulin also contain -RHStaple disulfides, and there is circumstantial evidence that the function of these proteins may involve cleavage/formation of these disulfide bonds.

Severe type I plasminogen (PLG) deficiency has been causally linked to a rare chronic inflammatory disease of the mucous membranes that may be life threatening. Here we report clinical manifestations, PLG plasma levels, and molecular genetic status of the PLG gene of 50 patients. The most common clinical manifestations among these patients were ligneous conjunctivitis (80%) and ligneous gingivitis (34%), followed by less common manifestations such as ligneous vaginitis (8%), and involvement of the respiratory tract (16%), the ears (14%), or the gastrointestinal tract (2%). Four patients showed congenital occlusive hydrocephalus, 2 with Dandy-Walker malformation of cerebellum. Venous thrombosis was not observed. In all patients, plasma PLG levels were markedly reduced. In 38 patients, distinct mutations in the PLG gene were identified. The most common genetic alteration was a K19E mutation found in 34% of patients. Transient in vitro expression of PLG mutants R134K, delK212, R216H, P285T, P285A, T319&#95;N320insN, and R776H in transfected COS-7 cells revealed significantly impaired secretion and increased degradation of PLG. These results demonstrate impaired secretion of mutant PLG proteins as a common molecular pathomechanism in type I PLG deficiency.

Plasminogen (Pl), a circulating protease synthesized in the liver, is also present in several tissues. In the thyroid gland a Pl-like protease was found in the apical lumen where it is involved, through its proteolytic activity, in luminal degradation of thyroglobulin (Tg). Here, we showed for the first time that the Pl-like protease apically secreted by epithelial thyroid cells is sulfated, both on tyrosine residue(s) and on oligosaccharide side chains. The Pl molecule is composed of a large N-terminal moiety made of five distinct Kringle domains (K1-K5) separated by small peptidic fragments, and of a C-terminal domain with serine protease activity. Using a software tool able to predict tyrosine sulfation sites in protein sequences we localized the potential tyrosine sulfation sites of Pl. Then, we became aware that, whatever the species considered, at least three of the four potential tyrosine sulfation sites of Pl were located on Kringle sites, and more precisely, for K1, on the highly conserved binding domain of K1. We determined with the same software tool which potential sulfation sites were the most likely to be really sulfated. We hypothesize that the sulfation of these sites modulates the binding properties of Pl.

Objective: While coronary artery disease (CAD) is associated with disturbances of the plasma fibrinolytic system, the nature of these disturbances is not fully defined. Fibrinolysis is regulated by plasmin, whose production is mediated by plasminogen activator conversion of plasminogen (Plg) to plasmin. The cascade is modulated by feedback loops that include Plg activator inhibitor 1 (PAI-1). Molecular interactions with Plg kringle domains play an important role in regulating plasmin production and its modulation of fibrinolysis. We hypothesized that interactions of tissue plasminogen activator (tPA) with Plg kringle domains regulates plasmin levels in patients with stable CAD., Methods: Plasma was collected from patients (n = 33) with an angiographically significant CAD and controls (n = 18) with angiographically established normal or minimally diseased arteries. Plasmin activity, tPA activity, and plasma levels of Plg, PAI-1, uPA, and tPA were determined., Results: CAD patients had 1.7-fold greater plasmin activity (P = 0.02) that correlated with 1.5-fold higher tPA activity when compared to controls. Epitope mapping of Plg domains showed Plg differences in epitope exposure between the two groups. Plasma from CAD patients had 50% less (P < 0.001) detectable kringle 4 and 48% less (P = 0.007) detectable kringles 1-3., Conclusions: Based on detectable differences in Plg, we conclude that in patients with stable CAD, Plg complexed with tPA exists in a conformation that enables increased tPA activity and Plg conversion to plasmin.

Background: BB-10153 is an engineered variant of human plasminogen, modified to be activated to plasmin by thrombin. Thrombin-activatable plasminogen was designed as a novel thrombolytic agent which would persist in the blood as a prodrug and be activated to plasmin only at fresh or forming thrombi by the thrombin that is tightly localized there. We previously described the construction of several thrombin-activatable plasminogens and their in vitro clot lysis activity., Objectives and Methods: The current study was an examination of the thrombolytic properties of BB-10153 in vivo; comparison was made with tissue-type plasminogen activator (t-PA) in a femoral artery copper coil thrombosis model in the anesthetized dog and rabbit. Heparin was not coadministered so that the fundamental activity of the agents could be compared., Results: BB-10153, administered as an intravenous bolus of 5 mg kg(-1) in the dog and 10 mg kg(-1) in the rabbit, produced a comparable incidence of reperfusion to 3 mg kg(-1) t-PA. Reocclusion at these doses occurred in 4/4 dogs and 5/7 rabbits treated with t-PA and in 2/6 dogs and 0/10 rabbits treated with BB-10153. There was no reocclusion in three dogs dosed with 10 mg kg(-1) BB-10153. BB-10153 did not affect plasma alpha2-antiplasmin levels or the bleeding time, whereas 3 mg kg(-1) t-PA caused marked depletion of alpha2-antiplasmin and fibrinogen and increased the bleeding time. The plasma half-life of BB-10153 was 3-4 h., Conclusions: The long half-life and thrombus-selective thrombolytic activity of BB-10153 might allow it to overcome the bleeding and reocclusion shortfalls in the performance of current thrombolytics.

An international collaborative study was organized to calibrate a replacement for the current (2nd) International Standard (IS) for Streptokinase, stocks of which are almost exhausted. Two candidate preparations were assayed against the 2nd IS in a study involving 16 laboratories in 12 countries: preparation 88/824 (coded B), and preparation 00/464 (C and D, coded duplicates). Laboratories could use two methods provided, either a fibrin clot lysis assay or a solution chromogenic method, or an in-house method. Laboratories were encouraged to perform more than one method if possible. With the exception of one laboratory which gave outlying results for preparation 00/464, there was good agreement within and between laboratories and no significant differences between potencies using the different methods employed. This study demonstrates that a solution chromogenic assay is an acceptable format for potency determination of the streptokinase preparations in this study and fibrin is not necessary. It has now been agreed that a solution chromogenic plasminogen activation assay replace the current euglobulin reference method for streptokinase activity determination in the European Pharmacopoeia. Study participants, SSC of the International Society on Thrombosis and Haemostasis and the Expert Committee on Biological Standardization (ECBS) at the World Health Organization approved preparation 00/464 (C,D in the study) as the 3rd IS for Streptokinase with a potency of 1030 IU per ampoule.

The principle of affinity chromatography was used for preparation of thromboresistant bilayer coatings. The outer biospecific layer containing epsilon-aminocaproic acid residues (from 2.2 up to 5.5 nmol/cm2) was synthesized using a copolymer of maleic anhydride with N-vinylpyrrolidone and L-lysine dihydrochloride or N-epsilon-tert-BOC-L-lysine. This surface can selectively adsorb plasminogen (fibrinolytic zymogen) from blood. The biospecific layer (from 2.0 up to 3.6 microg/cm2) was applied for covering chitosan (native or modified) or albumin interlayer. Such bilayer coatings (BCs) were stable and represented the insoluble polyelectrolyte complexes. BCs were proposed for bilayer modification of synthetic vascular grafts, polyethylene, and other materials contacting with blood. This technique allowed us to significantly reduce thrombogenic properties of polyethylene surfaces.

Earthworm fibrinolytic enzyme II (EFE-II) from Eisenia fetida has a broad hydrolytic specificity for peptide bonds. Our experiments show that EFE-II can hydrolyze the specific chromogenic substrates of thrombin (Chromozym TH), trypsin (Chromozym TRY) and elastase (Chromozym ELA). The Michaelis-Menten constant (K(m)) for Chromozym ELA (approximately 245 microM) is much higher than those for the thrombin (approximately 90 microM) and trypsin (approximately 60 microM) substrates. On the other hand, EFE-II is inhibited most strongly by soybean trypsin inhibitor (SBTI), and weakly inhibited by elastinal, suggesting that EFE-II has a trypsin-like activity. Degradation of plasminogen (PLg) and fibrinogen by EFE-II was investigated after EFE-II had been immobilized onto 1,1'-carboryl-diimidazole (CDI)-activated Sepharose CL-6B. The immobilized EFE-II has 55-60% activity of the native enzyme with a higher thermal and pH resistance. EFE-II cleaves PLg at four hydrolytic sites: Lys(77)-Arg(78), Arg(342)-Met(343), Ala(444)-Ala(445) and Arg(557)-Ile(558). The site Arg(557)-Ile(558) is also recognized and cleaved by tissue plasminogen activator (t-PA) and urokinase (UK), producing active plasmin. Cleaving Ala(444)-Ala(445) released mini-plasmin with secondary activity to hydrolyze fibrin. Immobilized EFE-II degrades not only the Aalpha chain of fibrinogen in the C-terminal region (like human neutrophil elastase, HNE), but also in the N-terminal region at the Val(21)-Glu(22) site.

Low-density lipoprotein (LDL) oxidation is stimulated by copper. Addition of a recombinant form of apolipoprotein(a) (apo(a); the distinguishing protein component of lipoprotein(a)) containing 17 plasminogen kringle IV-like domains (17K r-apo(a)) protects LDL against oxidation by copper. Protection is specific to apo(a) and is not achieved by plasminogen or serum albumin. When Cu(2+) is added to 17K r-apo(a), its intrinsic fluorescence is quenched in a concentration-dependent and saturable manner. Quenching is unchanged whether performed aerobically or anaerobically and is reversible by ethylenediaminetetraacetate, suggesting that it is due to equilibrium binding of Cu(2+) and not to oxidative destruction of tryptophan residues. The fluorescence change exhibits a sigmoid dependence on copper concentration, and time courses of quenching are complex. At copper concentrations below 10 microM there is little quenching, whereas above 10 microM quenching proceeds immediately as a double-exponential decay. The affinity and kinetics of copper binding to 17K r-apo(a) are diminished in the presence of the lysine analogue epsilon -aminocaproic acid. We propose that copper binding to the kringle domains of 17K is mediated by a His-X-His sequence that is located about 5A from the closest tryptophan residue of the lysine binding pocket. Copper binding may account for the natural resistance to copper-mediated oxidation of lipoprotein(a) relative to LDL that has been previously reported and for the protection afforded by apo(a) from copper-mediated oxidation of LDL that we describe in the present study.

Angiostatin, an inhibitor of angiogenesis, contains 3 to 4 kringle domains that are derived from proteolytic cleavage of plasminogen. The antiangiogenic effects of angiostatin occur, in part, from its inhibition of endothelial cell surface adenosine triphosphate synthase, integrin functions, and pericellular proteolysis. Angiostatin has structural similarities to hepatocyte growth factor (HGF; "scatter factor"), a promoter of angiogenesis, that induces proliferation and migration of both endothelial and smooth muscle cells via its cell surface receptor, c-met. We hypothesized that angiostatin might block HGF-induced signaling in endothelial and smooth muscle cells. Angiostatin inhibited HGF-induced phosphorylation of c-met, Akt, and ERK1/2. Angiostatin also significantly inhibited proliferation of human umbilical vein endothelial cells (HUVECs) induced by HGF. In contrast, angiostatin did not inhibit vascular endothelial growth factor (VEGF)-or basic fibroblast growth factor (bFGF)-induced signaling events or HUVEC proliferation. Angiostatin bound to immobilized truncated c-met produced by A431 cells and could be immunoprecipitated as a complex with soluble c-met. HGF inhibited the binding of (125)I-angiostatin to HUVECs. Soluble c-met, produced by several tumor cell lines, could inhibit the antiangiogenic effect of angiostatin. The disruption of HGF/c-met signaling is a novel mechanism for the antiangiogenic effect of angiostatin.

We determined the pattern of cross-reactivity of a panel of anti-streptokinase (SK) monoclonal antibodies (mAbs) with SK variants in order to map the antigenic and functional epitope of SK. Comparison of the pattern of cross-reactivity of the anti-SK mAb A4.3 with SK variants and sequence alignments of SK variants and native (n) SK suggested that mutation of Ser 138 to Lys results in loss of binding of mAb A4.3 to SK variants. However, this mutation does not affect formation of activator complex by these proteins. The epitope specificity of the mAb A4.3 was further confirmed by mutating Ser 138 to Lys in n SK. Monoclonal Ab A4.3 did not bind to mutant SK (Ser138Lys). Activator activity of mutant SK (Ser138Lys) was indistinguishable from that of n SK and recombinant n SK. Since addition of A4.3 mAb to an equimolar mixture of SK and human plasminogen inhibits activator complex formation, the sequences spanning position 138 are likely important for formation of streptokinase-plasminogen activator complex or processing of the plasminogen substrate.

To select sequences complementary to their binding sites, two anti-streptokinase (SK) monoclonal antibodies (mAbs), A4.5 and A5.5, were used in biopanning of 15-mer and hexamer phage-displayed peptide libraries, respectively. mAb A4.5 inhibits the catalytic activity of streptokinase-plasminogen activator complex (SKPAC), the binding of plasminogen to SK and the binding of human anti-SK polyclonal Abs to SK. All clones selected from the 15-mer peptide library by mAb A4.5 had identical nucleotide and amino acid sequences, RSVYRCSPFVGCWFG. An 11-mer peptide (peptide A4.5, YRCSPFVGCWF) derived from this sequence inhibited the binding of mAb A4.5 and human anti-SK polyclonal Abs to SK as well as the catalytic activity of both SKPAC and plasmin. The binding of the second mAb (mAb A5.5) to SK is lost upon interaction of SK with plasminogen, suggesting that sequences selected by this mAb are likely associated with the C-terminal cleavage site of SK. Biopanning of a hexamer peptide library with mAb A5.5 selected the sequence RYLQDY that is homologous to residues 324-328, adjacent to one possible C-terminal cleavage site in SK. A 10-mer synthetic peptide (LDFRDLYDPR) corresponding to residues 321-330 in SK specifically inhibited the binding of mAb A5.5 to SK. The selection and characterization of these two peptides enhances our understanding of SK structure, maps an antigenic epitope, and identifies a peptide inhibitor of plasminogen activation.