The effect of sialic acid content in glycans on thrombolytic activity of tissue plasminogen activators

2020 ◽  
pp. 10-17
Author(s):  
D. V. Chaschinova ◽  
N. V. Stratonova ◽  
D. A. Kudlay
Keyword(s):  
Sialic Acid ◽  
Plasminogen Activators ◽  
Fibrin Clot ◽  
Sialic Acids ◽  
Target Range ◽  
Clot Lysis ◽  
Batch Mode ◽  
Sialic Acid Content ◽  
Thrombolytic Activity ◽  
Tissue Plasminogen

Purpose: to investigate the dependence of the thrombolytic activity of tissue plasminogen activators (tPA) alteplase and tenecteplase on the degree of sialylation of the oligosaccharide component of molecules. Materials and methods: tPA specimens with an average degree of sialylation were obtained by cultivating CHO clones-producers in the fed-batch mode; desialylated forms of tPA were obtained by processing with neuraminidase, and hypersialylated forms of tPA were obtained by cultivating clones-producers in a medium with the addition of butyrate. The content of sialic acids was determined by resorcinol method, and the activity of tPA was determined by fibrin clot lysis. Results: the dependence of tenecteplase activity on the content of sialic acids in the molecule is demonstrated. The activity of tenecteplase falls below the limits of the target range when the content of sialic acids is more than 5 residues per tenecteplase molecule. No such relation was found for alteplase. Conclusion: the content of sialic acid residues affects the biological activity of tenecteplase; the activity of alteplase does not depend on the degree of sialylation of the molecule.

In Vitro Studies on the Fibrinolytic, Thrombolytic and Fibrinogenolytic Properties of a Tissue Plasminogen Activator from Guinea Pig Keratocytes

1985 ◽  
Vol 53 (02) ◽  
pp. 200-203 ◽  
Author(s):  
A Electricwala ◽  
R J Ling ◽  
P M Sutton ◽  
B Griffiths ◽  
P A Riley ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Human Plasma ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Fibrin Clot ◽  
Clot Lysis ◽  
Thrombolytic Activity ◽  
Tissue Plasminogen ◽  
In Vitro Systems

SummaryThe fibrinolytic and thrombolytic properties of a tissue plasminogen activator (tPA) purified from the conditioned medium of an established guinea pig keratocyte (GPK) cell line were investigated in in vitro systems and compared with urokinase. Using the fibrin clot lysis assay, GPK activator appears to be similar to human melanoma tPA and not to human urokinase. GPK activator also caused negligible fibrinogen breakdown, when incubated with human plasma at 37° C over 23 hr. Urokinase on the other hand caused significant fibrinogenolysis, under similar conditions. Comparison of the lysis of plasma clots by GPK activator and human urokinase have shown that GPK activator was a much more effective fibrinolytic agent than urokinase, especially at lower concentrations (<50 IU/ml). Studies on the thrombolytic effect of GPK activator on the lysis of aged and cross-linked whole human blood clots and plasma clots hanging in artificially circulating human plasma suggest that GPK activator can lyse both these types of clots equally well. The lysis is dose dependent, attaining complete lysis within 3–6 hr with the concentration of GPK activator in the range of 1–5 μg/ml plasma. It is concluded that GPK activator has a higher fibrinolytic and thrombolytic activity and lower fibrinogenolytic activity than urokinase.

scholarly journals Erythrocyte Sialic Acid Content during Aging in Humans: Correlation with Markers of Oxidative Stress

2012 ◽  
Vol 32 (3) ◽  
pp. 179-186 ◽  
Author(s):  
Mohammad Murtaza Mehdi ◽  
Prabhakar Singh ◽  
Syed Ibrahim Rizvi
Keyword(s):  
Oxidative Stress ◽  
Sialic Acid ◽  
Erythrocyte Membrane ◽  
Acid Content ◽  
Lipid Hydroperoxide ◽  
Plasma Lipid ◽  
Sialic Acids ◽  
Sialic Acid Content ◽  
Total Antioxidant ◽  
The Relationship

Sialic acids are substituted neuraminic acid derivatives which are typically found at the outermost end of glycan chains on the membrane in all cell types. The role of erythrocyte membrane sialic acids during aging has been established however the relationship between sialic acid and oxidative stress is not fully understood. The present work was undertaken to analyze the relationship between erythrocyte membrane sialic acid with its plasma level, membrane and plasma lipid hydroperoxide levels and plasma total antioxidant capacity. Results show that sialic acid content decreases significantly (P< 0.001) in RBC membrane (r= −0.901) and increases in plasma (r= 0.860) as a function of age in humans. Lipid peroxidation measured in the form of hydroperoxides increases significantly (P< 0.001) in plasma (r= 0.830) and RBC membranes (r= 0.875) with age in humans. The Trolox Equivalent Total Antioxidant Capacity (TETAC) of plasma was found to be significantly decreased (P< 0.001,r= −0.844). We observe significant correlations between decrease of erythrocyte membrane sialic acid and plasma lipid hydroperoxide and TETAC. Based on the observed correlations, we hypothesize that increase in oxidative stress during aging may influence the sialic acid decomposition from membrane thereby altering the membrane configuration affecting many enzymatic and transporter activities. Considering the importance of plasma sialic acid as a diagnostic parameter, it is important to establish age-dependent reference.

scholarly journals Differential Sialic Acid Content in Adult and Neonatal Fibrinogen Mediate Differences in Clot Polymerization

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 21-22
Author(s):  
Kimberly A Nellenbach ◽  
Nina A Guzzetta ◽  
Ashley C Brown
Keyword(s):  
Sialic Acid ◽  
Acid Concentration ◽  
Acid Content ◽  
Blood Clot ◽  
Enzymatic Digestion ◽  
Fibrin Clot ◽  
Blood Products ◽  
Sialic Acid Content ◽  
Fibrin Polymerization ◽  
Fibrin Clots

Recent studies have identified several major qualitative and quantitative differences in important hemostatic proteins between adult and neonatal humans, including the primary coagulation protein fibrinogen.Despite these differences, neonates with post-operative bleeding from procedures requiring cardiopulmonary bypass (CPB) or extracorporeal membrane oxygenation (ECMO) are treated with transfusions of adult blood products, namely adult fibrinogen. The effectiveness of such transfusions is inconsistent in neonates and often results in a deficient fibrin matrix structure which may not be sufficient for mitigating bleeding. Our recent studies have also identified differences at the bulk clot level between purified neonatal and adult fibrin clots, including major structural and functional distinctions, which could contribute to these outcomes. Notably, adult fibrinogen degrades slower than neonatal clots, therefore transfusion of adult blood products to neonatal patients could contribute to thrombotic complications. Given the inconsistent results and potential complications from the transfusion of adult blood products to neonatal patients, there is a critical need to better understand the mechanistic differences in hemostatic processes between adults and neonates. Neonates possess a molecular variant of fibrinogen known as fetal fibrinogen. Increased sialic acid concentration compared to adults has been identified in many neonatal glycoproteins across physiological systems, including fetal fibrinogen. Studies have shown that fibrin clot properties are influenced by fibrin polymerization mechanisms and post translational modifications (e.g. sialic acid). For example, an increased sialic acid content in the dysfibrinogenemia associated with liver disease has been associated with an altered fibrin clot structure. Additionally, recent work from our group has found that the increased sialic acid in neonatal fibrin networks results in significantly greater fibroblast attachment than adult networks. Therefore, we hypothesized that differences in neonatal and adult fibrin clot properties are due to mechanistic differences in fibrin polymerization between neonates and adults owing to altered sialic acid concentrations. The activation of fibrinogen and conversion into fibrin by the proteolytic enzyme thrombin is essential for the formation of a stable blood clot and the cessation of bleeding. Thrombin converts soluble fibrinogen to insoluble fibrin via cleavage of fibrinopeptides A and B, exposing fibrin knobs A and B. Fibrin protofibrils are then formed from the noncovalent binding of fibrin knobs to complementary fibrin holes a and b on adjacent proteins. In adults, fibrin A:a knob:hole interactions are critical for polymerization. However, these mechanisms have not been explored in neonates. Therefore, we characterized the influence of sialic acid on the knob:hole interactions in neonatal fibrin polymerization. We first investigated the influence of sialic acid concentration on neonatal fibrin polymerization by removing sialic acid via enzymatic digestion and performing structural and functional analysis on desialylated fibrinogen. Desialylated adult and neonatal fibrinogen had roughly equivalent structure, polymerization kinetics, and clottability results. These results indicate that differential sialylation may at least partially explain functional differences been adult and neonatal clots. Additionally, we investigated the role that sialylation plays on neonatal fibrin polymerization dynamics by comparing neonatal or adult fibrin clots formed with snake venom thrombin-like enzymes (svTLE) that preferentially cleave either A or B fibrinogen fibrinopeptides or thrombin. Structural, mechanical, fibrinolytic, and polymerization assays were conducted. Quantitative release of fibrinopeptides was determined via ELISA. Results indicate neonatal fibrin polymerization mechanisms are more dependent on B:b knob:hole interactions than adult fibrin. Results from this study provide insight into the mechanism of the neonatal clotting process and are a critical contribution for the development of neonatal-specific treatments for bleeding and thrombosis. Disclosures Brown: Selsym Biotech, Inc.: Other: Founder and CEO.

scholarly journals Differential sialic acid content in adult and neonatal fibrinogen mediates differences in clot polymerization dynamics

2021 ◽  
Author(s):  
Kimberly Nellenbach ◽  
Alexander Kyu ◽  
Nina Guzzetta ◽  
Ashley Brown
Keyword(s):  
Sialic Acid ◽  
Acid Content ◽  
Fibrin Clot ◽  
Fibrinopeptide A ◽  
Sialic Acid Content ◽  
Fibrin Polymerization ◽  
Fibrin Networks ◽  
Molecular Variant ◽  
Mixed Efficacy ◽  
And Function

Neonates possess a molecular variant of fibrinogen, known as fetal fibrinogen, characterized by increased sialic acid, a greater negative charge, and decreased activity compared to adults. Despite these differences, adult fibrinogen is used for treatment of bleeding in neonates, with mixed efficacy. In order to determine safe and efficacious bleeding protocols for neonates, more information on neonatal fibrin clot formation and the influence of sialic acid on these processes is needed. Here, we examine the influence of sialic acid on neonatal fibrin polymerization. We hypothesized that the increased sialic acid content of neonatal fibrinogen promotes fibrin B:b knob hole interactions and consequently influences the structure and function of the neonatal fibrin matrix. We explored this hypothesis through analysis of structural properties and knob:hole polymerization dynamics of normal and desialylated neonatal fibrin networks and compare to those formed with adult fibrinogen. We then characterized normal neonatal fibrin knob:hole interactions by forming neonatal and adult clots with either thrombin or snake-venom thrombin like enzymes (SVTLEs) that preferentially cleave fibrinopeptide A or B. We determined that sialic acid content of neonatal fibrinogen is a key determinant of resulting clot properties. Experiments analyzing knob:hole dynamics indicated typical neonatal fibrin clots are formed with the release of more fibrinopeptide B and less fibrinopeptide A than adults. After the removal of sialic acid, fibrinopeptide release was roughly equivalent between adults and neonates indicating the influence of sialic acid on fibrin neonatal fibrin polymerization mechanisms. These results could inform future studies developing neonatal specific treatments of bleeding.

scholarly journals Pharmacokinetic and thrombolytic properties of chimeric plasminogen activators consisting of a single-chain Fv fragment of a fibrin- specific antibody fused to single-chain urokinase

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 696-703
Author(s):  
P Holvoet ◽  
Y Laroche ◽  
JM Stassen ◽  
HR Lijnen ◽  
B Van Hoef ◽  
...  
Keyword(s):  
Steady State ◽  
Fold Increase ◽  
Plasminogen Activators ◽  
Clot Lysis ◽  
Chimeric Antibody ◽  
Maximal Rate ◽  
Single Chain ◽  
Thrombolytic Activity ◽  
Single Chain Fv ◽  
Single Chain Fv Fragment

The pharmacokinetic and thrombolytic properties were determined of two recombinant single-chain chimeric plasminogen activators (PA) consisting of u-PA-33k, a low-molecular weight derivative of single- chain urokinase-type PA (scu-PA) comprising amino acids Ala132 through Leu411, and of either a single-chain variable region fragment (Fv) derived from the fibrin fragment D-dimer-specific monoclonal antibody MA-15C5 (K12G0S32) or of the deglycosylated single-chain Fv fragment obtained by substitution of Asn88 with Glu (K12G2S32). Following bolus injection in hamsters, clearances of recombinant scu-PA (rscu-PA) and of K12G0S32 were similar. In contrast, clearance of K12G2S32 was fourfold slower than that of rscu-PA. The thrombolytic potency (percent lysis per u-PA administered in milligrams per kilogram body weight) and specific thrombolytic activity (percent lysis per microgram per milliliter steady-state plasma u-PA antigen level) of these compounds were studied in hamsters with an experimental pulmonary embolus consisting of a human plasma clot injected via the jugular vein. The doses of K12G0S32 and K12G2S32 required to obtain maximal rate of clot lysis were sixfold and 11-fold lower than that of rscu-PA. The steady- state u-PA-related plasma antigen levels of K12G0S32 and K12G2S32 required to obtain maximal rate of clot lysis were 10-fold and fourfold lower than that of rscu-PA. Thus, targeting of K12G0S32 to the clot surface by means of its glycosylated Fv fragment results in a 10-fold increase of its specific thrombolytic activity and sixfold increase of its thrombolytic potency as compared with those of rscu-PA. Targeting of K12G2S32 to the clot surface by means of its deglycosylated Fv fragment results in only a twofold increase of its thrombolytic activity. However, its fourfold slower clearance, combined with its twofold higher specific thrombolytic activity, results in an 11-fold increase of its thrombolytic potency over that of rscu-PA. These findings indicate that the thrombolytic potency of chimeric antibody- targeted PA may be increased by increasing the specific thrombolytic activity, reducing the clearance, or both.

scholarly journals Physico-Chemical and Biological Properties of Biosimilar and Reference Tissue Plasminogen Activator Products

2019 ◽  
Vol 19 (1) ◽  
pp. 39-49
Author(s):  
V. D. Gusarova ◽  
M. S. Pantyushenko ◽  
V. M. Simonov ◽  
R. R. Shukurov ◽  
R. A. Khamitov ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Specific Activity ◽  
Reference Product ◽  
Fibrin Clot ◽  
Biological Properties ◽  
Clot Lysis ◽  
Physico Chemical ◽  
Tissue Plasminogen

Recombinant tissue plasminogen activator (international nonproprietary name — alteplase) which was developed by «GENERIUM» (Russia) and received a marketing authorisation in Russia is completely analogous to Actilyse® which is used to treat medical conditions accompanied by thrombosis, such as acute myocardial infarction, pulmonary embolism, and ischemic stroke. The aim of the study was to carry out a comprehensive comparison of physico-chemical and biological properties of Revelyse® and the reference product Actilyse® in order to assess their biosimilarity. Materials and Methods: comparative peptide mapping and determination of comparability of chromatographic profiles of tryptic hydrolysates was performed using RP-HPLC and massspectrometry; the molecular weight distribution was determined by mass-spectrometry and polyacrylamide gel electrophoresis (Laemmli method). The purity and homogeneity of products as well as the content of related impurities (oligomers and fragments) were determined using gel filtration; N-glycosylation profile was analysed by hydrophilic HPLC, total sialic acid was quantified by the Svennerholm resorcinol method. Protein binding to fibrin and human fibrinogen was assessed by surface plasmon resonance, and the specific activity was compared by fibrin clot lysis. Results: the research demonstrated a complete overlap of the products’ peptide maps, which indicates the identity of аlteplase amino acid sequences in the two medicines being compared. The authors of the study also determined the molecular weight and the content of the intact single-stranded form of the protein, and quantified post-translational modifications, the content of sialic acids and neutral sugars. The analysis of the N-glycosylation profile revealed insignificant differences in the percentage of multiantenna complex glycans. The specificity of alteplase was evaluated by analysing the formation of protein complexes with natural alteplase ligands – fibrin and plasminogen activator inhibitor-1, but no significant differences were found. The comparison of specific activation of plasminogen fibrinolytic activity was performed based on the results of the assay analysing the fibrin clot lysis rate, and it demonstrated comparability of Revelyse® and Actilyse®. Conclusions: comparative experimental studies have shown no differences in the structure, charge distribution heterogeneity, impurities content, and specific activity of alteplase as a component of Revelyse® and the reference product Actilyse®, which leads to the conclusion that they are similar in terms of physicochemical and biological properties.

Fibrin Clot Lysis by Thrombolytic Agents Is Impaired in Newborns due to a Low Plasminogen Concentration

1992 ◽  
Vol 68 (03) ◽  
pp. 325-330 ◽  
Author(s):  
M Andrew ◽  
L Brooker ◽  
M Leaker ◽  
B Paes ◽  
J Weitz
Keyword(s):  
Thrombolytic Agent ◽  
Fibrin Clot ◽  
Clot Lysis ◽  
Therapeutic Effectiveness ◽  
Thrombolytic Agents ◽  
Cord Plasma ◽  
Tissue Plasminogen ◽  
Adult Plasma ◽  
Low Levels ◽  
Fibrin Clots

SummaryAlthough thrombolytic drugs have been extensively used in adults, there is sparse information on their effectiveness in newborns whose fibrinolytic system differs significantly from adults. The purpose of this study was to determine if low plasma levels of plasminogen in cord plasma limited the therapeutic effectiveness of thrombolytic agents. Urokinase (UK), streptokinase (SK) and tissue plasminogen activator (TPA) were compared for their ability to lyse washed 125I-labelled adult or cord fibrin clots suspended in cord or adult plasma. 125I-labelled fibrin clots were prepared by recalcifying cord or adult plasma spiked with labelled fibrinogen and then placed into cord or adult plasma which contained either saline or differing amounts of a specific thrombolytic agent. After a 60 min incubation, the remaining 125I-fibrin in clots released 125I-fibrin fragments, and concentrations of fibrinogen, α2-antiplasmin, and plasminogen in the bathing plasma were measured and compared to starting values. Cord fibrin clots were more resistant than adult fibrin clots to all thrombolytic drugs tested (p <0.001). On average, the cord system retained 27% more 125I-fibrin in clots, and released 32% less 125I-fibrin fragments into plasma. Fibrinogenolysis was also decreased in cord plasmas compared to adult plasmas. The degree of fibrinolysis and fibrinogenolysis in cord plasma increased to adult values when plasminogen concentrations were increased in the bathing plasma. Thus, cord fibrin clots have an impaired response to thrombolytic agents secondary to low levels of plasminogen. We speculate that the clinical response of newborns to thrombolytic agents is also impaired and will not be enhanced by increasing doses of thrombolytic agents but may be enhanced by increasing the concentration of plasminogen.

Removal of O-acetylated sialic acids from rat colonic epithelial glycoproteins by cell-free extracts of rat faeces

1983 ◽  
Vol 61 (8) ◽  
pp. 868-874 ◽  
Author(s):  
H. Poon ◽  
P. E. Reid ◽  
C. W. Ramey ◽  
W. L. Dunn ◽  
M. G. Clay
Keyword(s):  
Sialic Acid ◽  
Acid Content ◽  
Minimal Medium ◽  
Wistar Rat ◽  
Sialic Acids ◽  
Side Chain ◽  
Sialic Acid Content ◽  
Vibrio Cholera ◽  
Sterile Cell

Sterile, cell-free, extracts of freshly defaecated Wistar rat faeces in a pH 7.0 "minimal medium" contain neuraminidase(s), capable of removing sialic acids both with and without side-chain substituents from bovine submandibular mucin and rat colonic epithelial glycoproteins, and an esterase which removes O-acetyl substituents from the side chain of sialic acid residues. Studies of the removal of sialic acids from bovine submandibular mucin and rat colonic epithelial glycoproteins indicated that (i) the faecal enzymes removed a greater proportion of the sialic acid of both the de-O-acetylated and native glycoproteins than was removed with Vibrio cholera neuraminidase, (ii) sialic acids were removed more rapidly from de-O-acetylated glycoproteins, and (iii) the resistance to removal of sialic acids was apparently dependent at least in part upon the O-acetyl sialic acid content of the substrate.

Sialoglycoproteins and sialic acids ofPlasmodium knowlesischizont-infected erythrocytes and normal rhesus monkey erythrocytes

Parasitology ◽  
1986 ◽  
Vol 92 (3) ◽  
pp. 527-543 ◽  
Author(s):  
R. J. Howard ◽  
G. Reuter ◽  
J. W. Barnwell ◽  
R. Schauer
Keyword(s):  
Sialic Acid ◽  
Rhesus Monkey ◽  
Acid Content ◽  
Sialic Acids ◽  
Galactose Oxidase ◽  
Parasite Development ◽  
Sialic Acid Content ◽  
Acetylneuraminic Acid ◽  
Total Sialic Acid ◽  
Significant Difference

SUMMARYThe effects of malaria infection on RBC sialic acids and sialoglycoproteins were studied with asexual blood-stage infections ofPlasmodium knowlesiin rhesus monkeys. Glycoprotein radio-isotope labelling methods were used to compare the sialoglycoproteins of normal RBC andP. knowlesischizont-infected RBC (SI-RBC). Tritiation of glycoproteins from SI-RBC with the standard sialidase + galactose oxidase/NaB3H4method or standard periodate/NaB3H4method was significantly decreased when compared to normal RBC. However, tritium uptake into glycoproteins was normal when SI-RBC were treated with 5-fold higher concentrations of both enzymes in the first labelling method, or with a 5-fold increase in the molar ratio of periodate to sialic acid in the second method. The mobility of tritiated host cell glycoproteins on SDS–polyacrylamide gels was identical for SI-RBC and normal RBC. New bands, possibly glycoproteins, of 230, 160, 90, 52, and 30 kDa were detected after labelling SI-RBC by the modified periodate/NaB3H4method. Sialic acid analysis of normal rhesus monkey RBC (62μg/1010RBC) revealed that 46% of the total sialic acid wasN-glycolylneuraminic acid, 33% wasN-acetyl-9-O-acetylneuraminic acid, and the remainderN-acetylneuraminic acid. SI-RBC collected either directly from infected monkeys or afterin vitroculture of ring-infected RBC in horse serum, had increased total sialic acid (126 or 115μg/1010RBC, respectively). The sialic acid content of infected RBC must increase during parasite development since RBC infected with ring-stageP. knowlesihad the same content as normal RBC. There was no significant difference in the ratio of the three sialic acids of SI-RBC and normal RBC. In contrast, the uninfected RBC from infected blood of different monkeys showed marked variation in sialic acid composition and generally had a lower sialic acid content than normal RBC.

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