Preparation of PEG-grafted chitosan/streptokinase nanoparticles to improve biological half-life and reduce immunogenicity of the enzyme.

Streptokinase, as a thrombolytic drug, is widely used in treatment of cardiovascular disorders and deep vein thrombosis. Streptokinase is immunogenic due to its prokaryotic source, having short biological half-life (i.e. 15 to 30 min) that is not enough for an efficient therapy. In this study, nanoparticles (NPs) of chitosan/streptokinase and polyethylene glycol (PEG)-grafted chitosan/streptokinase were prepared by polyelectrolyte complex method. Particle size of chitosan and PEG-grafted chitosan NPs were 154 ± 42 and 211 ± 47 nm, respectively. Results showed that using PEG in preparation of nanoparticles leads to ~24% decrease in encapsulation efficiency. Encapsulation of streptokinase in the NPs also resulted in a slight reduction in enzymatic activity. However, in vivo findings indicated that response of the immune system was delayed for 20 days and blood circulation time of the enzyme increased up to 120 min by using PEG. Biological half-life of the drug also increased up to twice in PEG-grafted chitosan. In conclusion, PEG-grafted chitosan NPs could be an alternative for delivery of streptokinase to reduce its clinical limitations.

Immunoglobulin Attenuates Streptokinase-Mediated Virulence in Streptococcus dysgalactiae Subspecies equisimilis Necrotizing Fasciitis.

Background: Necrotizing fasciitis (NF) retains a very high mortality rate despite prompt and adequate antibiotic treatment and surgical debridement. Necrotizing fasciitis has recently been associated with Streptococcus dysgalactiae subspecies equisimilis (SDSE). Methods: We investigated the causes of a very severe clinical manifestation of SDSE-NF by assessing both host and pathogen factors. Results: We found a lack of streptokinase-function blocking antibodies in the patient resulting in increased streptokinase-mediated fibrinolysis and bacterial spread. At the same time, the clinical SDSE isolate produced very high levels of streptokinase. Exogenous immunoglobulin Gs (ex-IgGs) efficiently blocked streptokinase-mediated fibrinolysis in vitro, indicating a protective role against the action of streptokinase. In vivo, SDSE infection severity was also attenuated by ex-IgGs in a NF mouse model. Conclusions: These findings illustrate for the first time that the lack of specific antibodies against streptococcal virulence factors, such as streptokinase, may contribute to NF disease severity. This can be counteracted by ex-IgGs.

PEGylation of Truncated Streptokinase Leads to Formulation of a Useful Drug with Ameliorated Attributes.

Streptokinase (SK) remains a favored thrombolytic agent in the developing world as compared to the nearly 10-fold more expensive human tissue-plasminogen activator (tPA) for the dissolution of pathological fibrin clots in myocardial infarction. However, unlike the latter, SK induces systemic activation of plasmin which results in a greater risk of hemorrhage. Being of bacterial origin, it elicits generation of unwanted antibody and has a relatively short half-life in vivo that needs to be addressed to make it more efficacious clinically. In order to address these lacunae, in the present study we have incorporated cysteine residues specifically at the N- and C-termini of partially truncated SK and these were then PEGylated successfully. Some of the obtained derivatives displayed enhanced plasmin resistance, longer half-life (upto several hours), improved fibrin clot-specificity and reduced immune-reactivity as compared to the native SK (nSK). This paves the way for devising next-generation SK-based thrombolytic agent/s that besides being fibrin clot-specific are endowed with an improved efficacy by virtue of an extended in vivo half-life.

Antistreptokinase antibodies and outcome of fibrinolytic therapy with streptokinase for left-sided prosthetic valve thrombosis.

BACKGROUND: Left-sided prosthetic valve thrombosis (PVT) is a serious complication of valve replacement. In developing countries, fibrinolysis with streptokinase (SK) is often used as the first line of treatment. Anti-streptokinase (anti-SK) antibodies are widely prevalent in the general population, but their effect on the efficacy and outcome of fibrinolysis with SK in patients with PVT is not known. METHODS: Patients with rheumatic heart disease and prosthetic valve replacement presenting with a first episode of left-sided PVT were enrolled. All patients underwent fibrinolysis with SK. An indirect enzyme-linked immunosorbent assay was used to detect anti-SK antibodies before fibrinolysis. Relationship of these antibodies to the outcome of fibrinolysis was evaluated. RESULTS: Forty-four patients treated for left-sided PVT were included. Thrombosis affected 33 mitral and 11 aortic prosthetic valves. On fibrinolysis with SK, 32 (73%) patients achieved complete success, whereas it was unsuccessful in the remaining 12 patients. There were 3 bleeding events, 1 stroke, and 3 deaths. Mean anti-SK antibody levels were not significantly different between patients who had complete success and those who did not (8.81 ± 2.43 vs 7.67 ± 1.26 Au/mL; P = .13) and did not correlate with the outcome after adjustment with other variables. Patients in New York Heart Association class III or IV had a greater chance of failed fibrinolytic therapy, even after adjustment for other prognostic variables (odds ratio 9.0; 95% CI 1.29-63.02; P = .027). CONCLUSION: Anti-SK antibody titers are not associated with success of fibrinolytic therapy using SK in patients with left-sided PVT.

Plasmin(ogen) acquisition by group A Streptococcus protects against C3b-mediated neutrophil killing.

The globally significant human pathogen group A Streptococcus (GAS) sequesters the host protease plasmin to the cell surface during invasive disease initiation. Recent evidence has shown that localized plasmin activity prevents opsonization of several bacterial species by key components of the innate immune system in vitro. Here we demonstrate that plasmin at the GAS cell surface resulted in degradation of complement factor C3b, and that plasminogen acquisition is associated with a decrease in C3b opsonization and neutrophil-mediated killing in vitro. Furthermore, the ability to acquire cell surface plasmin(ogen) correlates directly with a decrease in C3b opsonization, neutrophil phagocytosis, and increased bacterial survival in a humanized plasminogen mouse model of infection. These findings demonstrate that localized plasmin(ogen) plays an important role in facilitating GAS escape from the host innate immune response and increases bacterial virulence in the early stages of infection.

Comparative study of the reactivity of natural and mutated streptokinase with total antistreptokinase antibodies in human sera.

Streptokinase is widely used as an anticoagulant drug for the treatment of heart attacks. Because of antibody production against injected drug in individuals consuming streptokinase and causing allergic reactions, streptokinase treatment effects become neutral. Recombinant mutant type of streptokinase was prepared by removing of 42 amino acids from the C terminal region. ELISA plates were coated by natural and mutant streptokinase as antigen. Ninety-six normal serum samples as well as 27 streptokinase consumer serum samples (patients with acute myocardial infraction) were analyzed. The results showed that serum antibodies against natural streptokinase were three times more than those against the mutated streptokinase. In case of preserving thrombolytic activity, mutated streptokinase can be used as an alternative of the natural form.

Can anti-streptokinase antibody predict myocardial infarction outcomes after streptokinase treatment?

BACKGROUND: The aim of this study was finding the association between anti- Streptokinase (SK) levels based on previous streptococcus infection and the clinical outcome of acute myocardial infarction (AMI) among Iranian patients after SK treatment. METHODS: In this prospective study, 31 consecutive patients presented to the emergency room of a referral university hospital within six hours of the onset of symptoms of AMI were recruited over a 3-year period (2007-2010). Blood samples for the analysis of the effect of neutralizing antibodies to SK assays were obtained immediately on arrival at the hospital. In-hospital and out-hospital clinical outcome defined as including return of typical chest pain after 48 hours, appearance of complex arrhythmia after 24 hours, maximum CPK serum concentration during first three days of admission, Left Venticular Ejection Fraction (EF) on the last day of admission, surgical interventions (CABG, PTCA), re-MI and re-admission due to cardiac problems during the one-year follow-up. RESULTS: Overall, 31 patients (7 female, 24 male with the mean age of 56.83 +/- 2.21 years) were included in this study. The recurrence of typical ischemic chest pain 48 hours after AMI, appearance of complex arrhythmia during the admission to CCU and 24 hours after AMI, maximum CPK serum concentration during the first three days of admission, and left EF on the last day of admission were not significantly different between the two compared groups (p > 0.05). CONCLUSION: According to this study, previous exposure to streptococcal infections may not reduce the efficacy of a single dose of SK and it does not seem necessary that its titer be measured before SK administration.

A comparative study on the activity and antigenicity of truncated and full-length forms of streptokinase.

Application of streptokinase (SK) as a common and cost-effective thrombolytic drug is limited by its antigenicity and undesired hemorrhagic effects. Prior structural/functional and epitope-mapping studies on SK suggested that removal of 59 N-terminal residues led to its fibrin dependency and identified SK antigenic regions, respectively. Following in silico analyses two truncated SK proteins were designed and compared for their fibrin specificity and antigenicity with the full-length SK. Computer-based modeling was used to predict the effect of vector (pET41a)-born protein tags on the conformation of SK fragments. SK60-386, SK143-386 and full-length SK (1-414) were separately cloned, expressed in BL21 E. coli cells and confirmed by Western-blotting. Functional activity of the purified proteins was evaluated with chromogenic and clot lysis assays and their antigenicity was tested by ELISA assay using rabbit anti-streptokinase antibody. As expected, chromogenic bioassay showed a major activity decline for SK60-386 and SK143-386 (83 and 91 percent, respectively), compared to SK1-414. However, in clot lysis assay, which is a fibrin-dependent pharmacopoeia-approved test, SK60-386 and SK143-386 were respectively 35 and 31 percent more active though lysed the clots slower than full-length SK. Antigenic analysis also indicated significant decrease in ELISA signals obtained for truncated proteins compared to SK1-414 (45 and 28 percent less reactivity for SK143-386 and SK60-386, respectively, p < 0.0001). The results of this study for the first time pointed to SK143-386 and SK60-386, as improved SK derivatives with increased fibrin-selectivity and decreased antigenicity as well as acceptable bioactivity profiles in a pharmacopoeia-based analysis, which deserve more detailed pharmacological studies.

Streptokinase antibodies in patients presenting with acute coronary syndrome in three rural New Zealand populations.

BACKGROUND: New Zealand Maori have some of the highest rates of Group A streptococcal infection (GAS) in the world. GAS elevates titres of antistreptokinase (SK) neutralising antibodies and may induce resistance to SK. METHODS: Anti-SK titres were measured in 180 patients presenting with symptoms consistent with an acute coronary syndrome to three New Zealand rural hospitals, selected because they provide care for patients from communities with different socio-economic and ethnic mixes (Maori proportions varying between 6% and 67%). FINDINGS: Compared with the community with the lowest proportion of Maori, patients in the community with the highest proportion of Maori had mean anti-SK titres that were 2.8 times higher (p=0.05). They were 2.5 times more likely to have a high anti-SK titre (33% vs 13% p=0.035). INTERPRETATION: Alternatives to reperfusion with SK should be the first-choice therapy in hospitals serving communities with high rates of GAS such as some predominantly Maori and Pacific Island communities.

Production of recombinant streptokinase in E. coli and reactivity with immunized mice.

Streptokinase (SK) is a potent plasminogen activator with widespread clinical use as a thrombolytic agent. In this study, we produce high level expression of recombinant streptokinase in E. coli by expression vector pET32a. Genomic DNA of streptokinase gene (SKC) was extracted, then amplified by polymerase chain reaction (PCR) method and sub-cloned to prokaryotic expression vector pET32a. Escherichia coli BL21 (DE3) pLysS were transformed with pET32a-skc and gene expression was induced by IPTG. The expressed protein was purified by affinity chromatography by Ni-NTA resin. High concentration of the recombinant protein obtained from the single-step purification by affinity-chromatography (Ni-NTA). The yield of recombinant streptokinase was nearly 470 mg L(-1) of initial culture. Our data showed that production of recombinant streptokinase improved by pET32a in Escherichia coli.

Seroreactivity against streptococcal DRS (distantly related to SIC) protein is a predictor for end-stage renal failure.

We hypothesized that immunoreactivity against antigens from nephritic strains of Streptococcus pyogenes may be elevated in patients with end-stage renal failure (ESRF). Additionally, we investigated whether a difference in seroreactivity exists between nonindigenous and indigenous (Aboriginal/Torres Strait Islander) patients. To examine these possibilities, antibodies against potentially nephritogenic proteins, streptokinase (Ska1) (from M1), streptococcal pyrogenic exotoxin type B (SpeB) (from M1), the streptococcal inhibitor of complement-mediated cell lysis (SIC) (from M1) and its two variants, closely related to SIC (CRS) (from M57) and distantly related to SIC (DRS) (from M12) were determined in 66 patients and 31 healthy controls by enzyme-linked immunosorbent assays. A significantly higher proportion of patients compared to controls were seropositive to Ska1 (P = 0.004), DRS (P = 0.0003), CRS (P = 0.001), and SIC (P = 0.018). Regression analysis showed that seroreactivity to DRS (r(2) = 0.85, P = 0.001) predicted the development of ESRF and that being diabetic was positively associated with being an ESRF patient (r(2) = 0.37, P < 0.0001) and being indigenous (r(2) = 0.47, P < 0.0001). These results suggest that these ESRF patients were exposed to strains of S. pyogenes that secrete Ska1, DRS, CRS, and SIC and may have pathological significance. No significant difference was observed between the indigenous patients and nonindigenous patients.

[Process of platelets activation by streptokinase].

This study concerns the influence of streptokinase and antistreptokinase antibodies on rabbits platelets in blood plasma depleted of plasminogen. The immune complex streptokinase-antibody causes platelets activation, whereas other investigated immune complexes didnot express such activity. Platelets aggregation wasnot detected in any case. It was determined that streptokinase induces platelets activation in the rabbit plasma with high titre of antistreptokinase antibodies in absence of plasminogen.

Anti-streptococcal antibodies in the diagnosis of acute and post-streptococcal disease: streptokinase versus streptolysin O and deoxyribonuclease B.

AIMS: To establish population normal values and compare the diagnostic value of antibodies against streptokinase (ASK), streptolysin O (ASO) and deoxyribonuclease B (ADNaseB) singularly and in combinations in acute and post-streptococcal disease. METHODS: A retrospective analysis of serological results was performed to define population norms. Subjects with acute culture-confirmed infection and post-streptococcal disease were assessed using population norms, as were matched controls. The sensitivity and specificity of each antibody assay and of combinations of the different assays were calculated. RESULTS: Age specific population normal values were derived from 2,321 specimens. None of the three antibodies alone or in combination was a reliable marker of acute streptococcal infection. The sensitivity and specificity of a single antibody titre in post-streptococcal disease ranged from 70.5 to 72.7% and 86.4 to 93.2%, respectively. The combination of ASO and ADNaseB was the most sensitive and specific combination for identifying post-streptococcal disease (sensitivity 95.5%, specificity 88.6%). CONCLUSIONS: In the diagnosis of acute and post-streptococcal disease, the addition of ASK does not increase the sensitivity or specificity of serological testing. A combination of ASO and ADNaseB is required in post-streptococcal disease to achieve maximum sensitivity and specificity.

Do recipients of kidneys from donors treated with streptokinase develop anti-streptokinase antibodies?

Streptokinase is used for preflush for non-heart-beating donors (NHBDs) in our center. The aim of this study was to evaluate whether the use of thrombolytic streptokinase results in the production of anti-streptokinase antibodies in the recipients after renal transplantation. Recipient sera taken prior to and at 1 and 6 months posttransplant were tested for the presence of antibodies to streptokinase using an enzyme-linked immunosorbent assay assay. No differences were detected between a group of 18 recipients who had kidneys from thrombolytic-treated NHBDs and a further group of 18 who received NHBD kidneys without such treatment.

Heat shock protein and gliadin peptide promote development of peptidase antibodies in children with autism and patients with autoimmune disease.

Searching for a mechanism underlying autoimmunity in autism, we postulated that gliadin peptides, heat shock protein 60 (HSP-60), and streptokinase (SK) bind to different peptidases resulting in autoantibody production against these components. We assessed this hypothesis in patients with autism and in those with mixed connective tissue diseases. Associated with antigliadin and anti-HSP antibodies, children with autism and patients with autoimmune disease developed anti-dipeptidylpeptidase I (DPP I), anti-dipeptidylpeptidase IV (DPP IV [or CD26]) and anti-aminopeptidase N (CD13) autoantibodies. A significant percentage of autoimmune and autistic sera were associated with elevated immunoglobulin G (IgG), IgM, or IgA antibodies against three peptidases, gliadin, and HSP-60. These antibodies are specific, since immune absorption demonstrated that only specific antigens (e.g., DPP IV absorption of anti-DPP IV), significantly reduced IgG, IgM, and IgA antibody levels. For direct demonstration of SK, HSP-60, and gliadin peptide binding to DPP IV, microtiter wells coated with DPP IV were reacted with SK, HSP-60, and gliadin. They were then reacted with anti-DPP IV or anti-SK, anti-HSP, and antigliadin antibodies. Adding SK, HSP-60, and gliadin peptides to DPP IV resulted in 27 to 43% inhibition of the DPP IV-anti-DPP IV reaction, but DPP IV-positive peptides caused 18 to 20% enhancement of antigen-antibody reactions. We propose that (i) superantigens (e.g., SK and HSP-60) and dietary proteins (e.g., gliadin peptides) in individuals with predisposing HLA molecules bind to aminopeptidases and (ii) they induce autoantibodies to peptides and tissue antigens. Dysfunctional membrane peptidases and autoantibody production may result in neuroimmune dysregulation and autoimmunity.

Inhibition of streptokinase-induced, antibody-mediated platelet aggregation with tirofiban after exposure to streptokinase or streptococcal infection.

STUDY OBJECTIVE: To evaluate the effect of tirofiban (a glycoprotein IIb-IIIa inhibitor) in preventing streptokinase-induced, antibody-mediated platelet aggregation after administration of streptokinase or development of a streptococcal infection. DESIGN: Prospective analysis. SETTING: Research center of a Canadian hospital. PARTICIPANTS: Forty-five healthy volunteers, 45 patients who had received streptokinase within the past 3 years, and 13 patients who had a severe streptococcal infection also within the past 3 years. INTERVENTION: Blood samples were drawn to measure the extent of inhibition of streptokinase-induced, antibody-mediated platelet activation and aggregation by tirofiban. MEASUREMENTS AND MAIN RESULTS: Platelet aggregation was measured by using a turbidimetric method. The extent of inhibition by tirofiban was measured by incubating tirofiban for 2 minutes before adding streptokinase 5000 U/ml. Also, tirofiban was added 2 minutes before adding adenosine 5'-diphosphate (ADP) 2 microM/L into the last tube as a comparison. Strepto-kinase-induced, antibody-mediated platelet aggregation was observed in 10 (22%) of the 45 patients treated with streptokinase, in 3 (23%) of the 13 patients with streptococcal infection, and in none of the 45 healthy volunteers. Tirofiban inhibited streptokinase-induced, antibody-mediated platelet aggregation by 89 +/- 14% (p<0.001). Similarly, ADP-induced platelet aggregation was inhibited by 92 +/- 6% (p<0.001) with tirofiban. CONCLUSION: Streptokinase-induced, antibody-mediated platelet aggregation occurred in 13 (22%) of 58 patients who received streptokinase or were exposed to a streptococcal infection in the past 3 years. Such patients may not benefit from streptokinase therapy. In these patients, tirofiban significantly decreased the extent of antistreptokinase antibody-mediated platelet aggregation. Hence, patients undergoing streptokinase therapy may benefit from tirofiban as adjunctive therapy.

A rapid agglutination assay to detect anti-streptokinase antibodies.

BACKGROUND: Streptokinase resistance may cause suboptimal thrombolytic therapy. AIM: To develop a rapid latex-bead assay to detect streptokinase antibodies. METHODS: Sera were obtained from 16 patients presenting with acute myocardial infarction (MI) before treatment with streptokinase and 1 and 6 months post treatment, and from 100 controls. Sera were assayed for anti-streptokinase antibodies using a functional streptokinase-neutralising assay. RESULTS: Streptokinase-neutralising activity was low in controls (54 +/- 5U/ml) and patients prior to treatment (101 +/- 18), increasing to 2,110 +/- 823 and 1,017 +/- 169 at 1 and 6 months (mean +/- SEM). The latex assay had a sensitivity of 94% and a specificity of 93% for detecting individuals with > 350U/ml of streptokinase resistance, which is sufficient to neutralise the drug clinically. CONCLUSIONS: Estimation of streptokinase resistance using an enzyme immunoassay and a latex bead assay correlated well with serum neutralising activity. This assay can rapidly identify patients who have a high level of streptokinase-neutralising activity.