Streptokinase Activator Research Articles

Exogenous activation and inhibition of plasminogen/plasmin activity during in vitro maturation of bovine cumulus-oocyte complexes: A biological and spectroscopic approach.

This study deals with the effect of plasminogen/plasmin on the in vitro maturation (IVM) of bovine cumulus-oocyte complexes (COCs). Exogenous plasminogen activator streptokinase (SK) added to the IVM medium revealed similar values of cumulus expansion and oocyte nuclear maturation compared to controls (standard IVM medium). However, a decrease in both determinations was observed in COCs matured with the supplementation of ɛ-aminocaproic acid (ɛ-ACA), a specific plasmin inhibitor. After in vitro fertilization, no differences were observed in either cleavage or blastocyst rates between SK and control groups; however, ε-ACA treatment caused a decrease in both developmental rates. Zona pellucida (ZP) digestion time decreased in the SK group while it increased in the ε-ACA group. Raman microspectroscopy revealed an increase in the intensity of the band corresponding to the glycerol group of sialic acid in the ZP of oocytes matured with SK, whereas ZP spectra of oocytes treated with ɛ-ACA presented similarities with immature oocytes. The results indicate that although treatment with SK did not alter oocyte developmental competence, it induced modifications in the ZP of oocytes that could modify the folding of glycoproteins. Plasmin inhibition impairs oocyte maturation and has an impact on embryo development, thus evidencing the importance of this protease during IVM.

Immunoglobulin Attenuates Streptokinase-Mediated Virulence inStreptococcus dysgalactiae Subspeciesequisimilis Necrotizing Fasciitis

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). We investigated the causes of a very severe clinical manifestation of SDSE-NF by assessing both host and pathogen factors. 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. 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.

An Interview with Robert Califf.

In the late 1970s, when Robert Califf was in medical school, the heart was still a kind of medical terra incognita. Most cardiologists believed that a class of untimely heartbeats, known as premature ventricular contractions (PVCs), could predict sudden cardiac death. During his third year at Duke University Medical School, Califf, and his colleagues, showed that impaired left ventricular function was the true culprit. A few years later, as new clot-busting agents were being developed, Califf, by then a Duke faculty member, and colleagues predicted that one of the drugs, tissue plasminogen activator (tPA), would be most effective when followed immediately by angioplasty. They ran a multicenter clinical trial—the first of a series collectively known as Thrombolysis and Angioplasty in Myocardial Infarction (TAMI)—which showed the opposite: waiting to do the artery-opening procedure led to better outcomes. Inspired by the power of clinical trials to topple even his own beliefs, Califf embarked, with colleagues, on an ambitious series of groundbreaking trials comparing tPA and another clot-buster, streptokinase. The study—known as the Global Utilization of Streptokinase and Tissue Plasminogen Activator for Occluded Coronary Arteries (GUSTO)—established the superiority of tPA and also Califf's reputation as one of the most creative clinical scientists of his generation. In 1995, he founded the Duke Clinical Research Institute, which has grown to become one of the world's largest academic research organizations, having enrolled more than 1.2 million participants in 65 countries and with over 1300 employees. Admired for his ability to bring people from academia and clinical medicine together with members of industry—a connection that caused some to object when he was later nominated by President Obama for the post of Commissioner of the Food and Drug Administration (FDA)—Califf is also one of the top 10 most cited medical authors. Until recently, he continued to see patients …

Molecular processes in the streptokinase thrombolytic therapy

The aim of this research is to evaluate the current streptokinase thrombolytic treatment and to identify or improve new techniques that will base new approaches with a higher efficiency in this area of expertise. In order to be as realistic as possible a new method was set up using magnetic vectorized nanoparticles streptokinase and human blood thrombus. The experimental data confirm the maximum 83% thrombus lyses whenever increase streptokinase concentration. It is very probable to happen because of the presence of high concentration of antiplasmin in the blood that neutralizes around half of the thrombolytic potential of the sanguine plasminogen. The experiment shows also that only free serum plasminogen are available for streptokinase action in order to generate plasmin.

Streptokinase versus recombinant tissue-type plasminogen activator for thrombolysis of mechanical prosthetic heart valve thrombosis

Aims and Objectives: This study was designed to compare streptokinase (STK) versus recombinant tissue-type plasminogen activator (rt-PA) for thrombolysis in patients with prosthetic heart valve thrombosis (PHVT). Background: Optimal management of PHVT remains controversial. Thrombolytic therapy is a reasonable alternative to surgery. Numerous thrombolytic protocols with STK, urokinase, and rt-PA have been used over the past 40 years. So far there is a lack of consensus on the ideal thrombolytic agent for PHVT. There are no major studies comparing efficacy of STK versus rt-PA. Methods and Results: Sixty patients who underwent thrombolysis for PHVT over 1 year were studied. Forty-eight patients were thrombolysed with STK, whereas 12 patients received rt-PA. Overall 68.33% (41/60) patients showed complete response (CR), whereas 85% (51/60) showed some improvement in hemodynamics, with no significant difference in efficacy according to age, sex, duration from surgery, New York Heart Association Class III–IV, presence of atrial fibrillation, type of valve, or history of stroke. Overall 10% patients developed major complications with mortality of 5%. CR to thrombolytic therapy was numerically better with STK (72.9% vs. 50%; P = 0.12) and mortality was significantly higher in rt-PA (16.6% vs. 2%, P = 0.038). Mean duration of successful thrombolysis was significantly longer with STK as compared to rt-PA (22.96 ± 19.2 vs. 3 h). Conclusion: STK use as compared to rt-PA was associated with numerically more successful thrombolysis and significant lower mortality, hence may be optimal for elective thrombolysis.

Abstract 12419: Preclinical Safety and Efficacy of a Novel Thrombolytic Agent Administered by Rapid Bolus Injection: Clot Specific Streptokinase (CSSK/SMRX-11)

Introduction: Clot specific Streptokinase (CSSK), also known as SMRX-11, is a novel recombinant protein, produced and purified from a yeast system (Pichia pastoris). Novel fibrin binding domains attached to each end of streptokinase mask the ability of the streptokinase component of SMRX-11 to interact with blood plasminogen. As a result, SMRX-11 remains inactive in the blood circulation and does not convert blood plasminogen into plasmin until the fibrin binding domains are cleaved by clot-bound plasmin. This is in contrast to native Streptokinase (SK), which indiscriminately converts plasminogen into plasmin. The molecular weight of the glycosylated SMRX-11 protein is approximately 80kD, with a specific activity of 80,000 Units/mg of protein. Results. In vitro experiments comparing SMRX-11 to other fibrinolytics (SK and tPA), demonstrated that SMRX-11 lyses experimentally-preformed human blood clots without reducing residual plasma fibrinogen levels. Initial in vitro studies compared the activity of SMRX-11 to lyse human whole blood clots to native Streptokinase (SK) and tissue Plasminogen Activator (tPA). SMRX-11 had comparable clot lysis activity to SK, with tPA eliciting a slightly greater percent clot lysis. Additional in vitro studies determined the relative fibrinogenolytic effects of SMRX-11 versus tPA. SMRX-11 demonstrated superior fibrinogen sparing compared to tPA with minimal consumption of alpha-2 antiplasmin relative to native SK. In vivo, SMRX-11 (0.7-1.4 mg/kg as an iv bolus), lysed experimentally-induced femoral arterial thrombi in cynomolgus monkeys with relatively minor fluctuation in plasma fibrinogen, forearm bleeding time or hemodynamics. In GLP safety studies, treatment with SMRX-11 at doses of 1, 3 and 10 mg/kg/day for 5 consecutive days was generally well-tolerated in male and female cynomolgus monkeys. Thus, the low dose of 1 mg/kg was considered the No-Observed Adverse Effect Level (NOAEL) for daily repeat-dose administration of SMRX-11 administered via bolus i.v. injection in cynomolgus monkeys. Conclusion. SMRX-11 represents a novel pharmacophore with the appropriate preclinical safety and efficacy profile to warrant clinical investigation for acute treatment of myocardial infarction and stroke.

Streptococcus pyogenes triggers activation of the human contact system by streptokinase.

Severe invasive infectious diseases remain a major and life-threatening health problem. In serious cases, a systemic activation of the coagulation cascade is a critical complication that is associated with high mortality rates. We report here that streptokinase, a group A streptococcal plasminogen activator, triggers the activation of the human contact system. Activation of contact system factors at the surface of the Streptococcus pyogenes serotype M49 is dependent on streptokinase and plasminogen. Our results also show that secreted streptokinase is an efficient contact system activator, independent from a contact surface. This results in the processing of high-molecular-weight kininogen and the release of bradykinin, a potent vascular mediator. We further investigated whether the ability of 50 different clinical S. pyogenes isolates to activate the contact system is associated with an invasive phenotype. The data reveal that isolates from invasive infections trigger an activation of the contact system more potently than strains isolated from noninvasive infections. The present study gives new insights into the mechanisms by which S. pyogenes triggers the human contact system and stresses the function of soluble and surface located plasmin exploited as a group A streptococcal virulence factor through the action of streptokinase.

Abstract 4466: Surface-functionalized nanoparticles for inhalation delivery of chemotherapeutics to lung cancer

Abstract Aerosol delivery of chemotherapeutics is an attractive strategy for treating lung cancer as it offers high local drug levels and limited systemic exposure. However, many chemotherapeutic agents have poor aqueous solubility, which presents significant formulation challenges. Encapsulation of such drugs in polymeric nanoparticles (NPs) can overcome solubility issues and improve their delivery to tumor cells. In addition, NPs enable sustained drug release and prolonged drug exposure in the deep lungs and limit drug entrapment in the nasopharyngeal region. In our studies, we investigated NPs formulated from the biodegradable polymer, poly(D,L-lactide-co-glycolide) (PLGA), for inhalation delivery of paclitaxel. NPs were aerosolized using ultrasonic atomization. Aerosol output (20 μg drug/min) and mass median aerodynamic diameter (1.57 μm) of the aerosol droplets were measured using a cascade impactor. Particle deposition studies in mice showed that inhalation delivery of NP-encapsulated paclitaxel results in high drug concentration (14 μg/g) in the lungs following a 33 minute exposure. Following deposition in the lungs, penetration of NPs within the tumor occurs predominantly through diffusion. The tumor extracellular matrix (ECM) is a dense and tortuous network of biopolymers which significantly hinder effective intratumoral distribution of NPs. The presence of leaky blood vessels in the tumor also leads to the formation of cross-linked fibrin clot in the ECM. We hypothesized that degrading fibrin in the tumor ECM will lead to enhanced intratumoral distribution of NPs. In order to improve the tumor penetration, we surface functionalized NPs with fibrinolytic enzymes, streptokinase and tissue plasminogen activator (tPA). NPs were characterized for particle size (∼240 nm), zeta potential (∼-20 mV) and drug loading (∼10% w/w). We used a plasmin generation assay to quantify the amount of active enzyme on the surface of NPs. We also compared the diffusion of NPs across fibrin gels using an in vitro transwell assay. We found that enzyme-functionalized NPs diffused through fibrin gels more efficiently than non-functionalized NPs. In vivo studies in tumor bearing mice also demonstrated a greater intratumoral distribution of enzyme-functionalized NPs compared to non-functionalized NPs. In conclusion, our studies show that inhalation delivery of NP-encapsulated paclitaxel results in high drug concentrations in the lungs. Further, functionalization of NPs with fibrinolytic enzymes can improve their transport through tumor ECM. Future studies will examine the pharmacokinetics and anticancer efficacy of aerosolized enzyme-functionalized NPs in an orthotopic mouse model of lung cancer. Citation Format: Ameya R. Kirtane, Timothy Wiedmann, Jayanth Panyam. Surface-functionalized nanoparticles for inhalation delivery of chemotherapeutics to lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4466. doi:10.1158/1538-7445.AM2014-4466

Support vector machine (SVM) based multiclass prediction with basic statistical analysis of plasminogen activators

BackgroundPlasminogen (Pg), the precursor of the proteolytic and fibrinolytic enzyme of blood, is converted to the active enzyme plasmin (Pm) by different plasminogen activators (tissue plasminogen activators and urokinase), including the bacterial activators streptokinase and staphylokinase, which activate Pg to Pm and thus are used clinically for thrombolysis. The identification of Pg-activators is therefore an important step in understanding their functional mechanism and derives new therapies.MethodsIn this study, different computational methods for predicting plasminogen activator peptide sequences with high accuracy were investigated, including support vector machines (SVM) based on amino acid (AC), dipeptide composition (DC), PSSM profile and Hybrid methods used to predict different Pg-activators from both prokaryotic and eukaryotic origins.ResultsOverall maximum accuracy, evaluated using the five-fold cross validation technique, was 88.37%, 84.32%, 87.61%, 85.63% in 0.87, 0.83,0.86 and 0.85 MCC with amino (AC) or dipeptide composition (DC), PSSM profile and Hybrid methods respectively. Through this study, we have found that the different subfamilies of Pg-activators are quite closely correlated in terms of amino, dipeptide, PSSM and Hybrid compositions. Therefore, our prediction results show that plasminogen activators are predictable with a high accuracy from their primary sequence. Prediction performance was also cross-checked by confusion matrix and ROC (Receiver operating characteristics) analysis. A web server to facilitate the prediction of Pg-activators from primary sequence data was implemented.ConclusionThe results show that dipeptide, PSSM profile, and Hybrid based methods perform better than single amino acid composition (AC). Furthermore, we also have developed a web server, which predicts the Pg-activators and their classification (available online at http://mamsap.it.deakin.edu.au/plas_pred/home.html). Our experimental results show that our approaches are faster and achieve generally a good prediction performance.

Site-restricted plasminogen activation mediated by group A streptococcal streptokinase variants

SK (streptokinase) is a secreted plasminogen activator and virulence factor of GAS (group A Streptococcus). Among GAS isolates, SK gene sequences are polymorphic and are grouped into two sequence clusters (cluster type-1 and cluster type-2) with cluster type-2 being further classified into subclusters (type-2a and type-2b). In the present study, we examined the role of bacterial and host-derived cofactors in SK-mediated plasminogen activation. All SK variants, apart from type-2b, can form an activator complex with Glu-Plg (Glu-plasminogen). Specific ligand-binding-induced conformational changes in Glu-Plg mediated by fibrinogen, PAM (plasminogen-binding group A streptococcal M protein), fibrinogen fragment D or fibrin, were required for type-2b SK to form a functional activator complex with Glu-Plg. In contrast with type-1 and type-2a SK, type-2b SK activator complexes were inhibited by α2-antiplasmin unless bound to fibrin or to the GAS cell-surface via PAM in combination with fibrinogen. Taken together, these data suggest that type-2b SK plasminogen activation may be restricted to specific microenvironments within the host such as fibrin deposits or the bacterial cell surface through the action of α2-antiplasmin. We conclude that phenotypic SK variation functionally underpins a pathogenic mechanism whereby SK variants differentially focus plasminogen activation, leading to specific niche adaption within the host.

Cangrelor for patients undergoing percutaneous coronary intervention: evidence from a meta-analysis of randomized trials

Cangrelor is a new parenteral adenosine diphosphate P2Y12 receptor inhibitor with rapid, profound and reversible inhibition of platelet activity. The aim of this meta-analysis was to evaluate efficacy and safety of this new agent in patients undergoing percutaneous coronary intervention (PCI). We searched PubMed, Cochrane Library, EMBASE, Web of Science and CINAHL databases from the inception through April 2013. Randomized controlled trials (RCTs) comparing cangrelor with control (clopidogrel/placebo) were selected. We used the random-effects models to calculate the risk ratio. The primary efficacy outcome was risk of myocardial infarction, and the primary safety outcome was TIMI major bleeding at 48 h. Three RCTs included a total of 25,107 participants. Effects of Cangrelor were not different against comparators for myocardial infarction (MI) (Risk ratio [RR] 0.94, 95% confidence interval [CI] 0.78-1.13) and all-cause mortality (RR 0.72, 95% CI 0.36-1.43). However, cangrelor significantly reduced the risk of ischemia-driven revascularization (RR 0.72, 95% CI 0.52-0.98), stent thrombosis (RR 0.60, 95% CI 0.44-0.82) and Q wave MI (RR 0.53, 95% CI 0.30-0.92) without causing extra major bleeding (Thrombolysis in Myocardial infarction criteria) and severe or life-threatening bleeding (Global utilization of streptokinase and tissue plasminogen activator for occluded coronary arteries criteria). Separate analysis against only clopidogrel also showed similar findings except Q wave MI outcome. Use of cangrelor during PCI might reduce the risk of ischemia-driven revascularization and stent thrombosis, without causing extra major bleeding.

Diffusion Limited Delivery of Streptokinase and tPA to a Thrombus

The delivery of the thrombolytics streptokinase and tissue plasminogen activator (tPA) from intravenous administration is studied computationally, in particular focusing on the convection, diffusion, and reaction in a fully occluded vessel. The thrombolytic was convectively transported approximately two occluded vessel diameters and was diffusively transported the remaining distance to the thrombus. The time required for complete reaction of the thrombolytic and the total amount of thrombolytic that reacts at the thrombus were found to be primarily functions of the length of the occluded vessel. The diameter and branching angle of the clogged artery are of secondary importance and the effect of flow rate along the main artery is negligible. Except for the shortest occluded vessels, intravenous delivery of the thrombolytic to the thrombus is a diffusion-limited process. Following a 1 h intravenous administration of the thrombolytic, it was found that 4–5 h are typically needed to achieve the maximum delivery of streptokinase to the thrombus; 2–3 h are needed for tPA. Less than 10−5% of the administered thrombolytic is actually delivered to the thrombus when intravenous administration is utilized. These results suggest that a drastic change in the mechanism of delivery is necessary.

A Natural Inactivating Mutation in the CovS Component of the CovRS Regulatory Operon in a Pattern D Streptococcal pyogenes Strain Influences Virulence-associated Genes*

A skin-tropic invasive group A Streptococcus pyogenes (GAS) strain, AP53, contains a natural inactivating mutation in the covS gene (covS(M)) of the two-component responder (CovR)/sensor (CovS) gene regulatory system. The effects of this mutation on specific GAS virulence determinants have been assessed, with emphasis on expression of the extracellular protease, streptococcal pyrogenic exotoxin B (SpeB), capsular hyaluronic acid, and proteins that allow host plasmin assembly on the bacterial surface, viz. a high affinity plasminogen (Pg)/plasmin receptor, Pg-binding group A streptococcal M protein (PAM), and the human Pg activator streptokinase. To further illuminate mechanisms of the functioning of CovRS in the virulence of AP53, two AP53 isogenic strains were generated, one in which the natural covS(M) gene was mutated to WT-covS (AP53/covS(WT)) and a strain that contained an inactivated covR gene (AP53/ΔcovR). Two additional strains that do not contain PAM, viz. WT-NS931 and NS931/covS(M), were also employed. SpeB was not measurably expressed in strains containing covR(WT)/covS(M), whereas in strains with natural or engineered covR(WT)/covS(WT), SpeB expression was highly up-regulated. Alternatively, capsule synthesis via the hasABC operon was enhanced in strain AP53/covS(M), whereas streptokinase expression was only slightly affected by the covS inactivation. PAM expression was not substantially influenced by the covS mutation, suggesting that covRS had minimal effects on the mga regulon that controls PAM expression. These results demonstrate that a covS inactivation results in virulence gene alterations and also suggest that the CovR phosphorylation needed for gene up- or down-regulation can occur by alternative pathways to CovS kinase.

Hydatid cyst of liver complicated with Budd-Chiari syndrome and portal vein thrombosis

Hydatid cyst of liver complicated with Budd-Chiari syndrome and portal vein thrombosis

A bacterial pathogen co-opts host plasmin to resist killing by cathelicidin antimicrobial peptides.

The bacterial pathogen Group A Streptococcus (GAS) colonizes epithelial and mucosal surfaces and can cause a broad spectrum of human disease. Through the secreted plasminogen activator streptokinase (Ska), GAS activates human plasminogen into plasmin and binds it to the bacterial surface. The resulting surface plasmin protease activity has been proposed to play a role in disrupting tissue barriers, promoting invasive spread of the bacterium. We investigated whether this surface protease activity could aid the immune evasion role through degradation of the key innate antimicrobial peptide LL-37, the human cathelicidin. Cleavage products of plasmin-degraded LL-37 were analyzed by matrix-assisted laser desorption ionization mass spectrometry. Ska-deficient GAS strains were generated by targeted allelic exchange mutagenesis and confirmed to lack surface plasmin activity after growth in human plasma or media supplemented with plasminogen and fibrinogen. Loss of surface plasmin activity left GAS unable to efficiently degrade LL-37 and increased bacterial susceptibility to killing by the antimicrobial peptide. When mice infected with GAS were simultaneously treated with the plasmin inhibitor aprotinin, a significant reduction in the size of necrotic skin lesions was observed. Together these data reveal a novel immune evasion strategy of the human pathogen: co-opting the activity of a host protease to evade peptide-based innate host defenses.

Stroke as an emergency: Semmelweis redivivus?

Simply told, the story of tPA or ‘tissue-type plasminogen activator’ begins like this. In 1952, Danish biochemists isolated a fibrinolytic substance from normal animal tissues. It had been detected a few years before and was known to act through activation of plasminogen. In the mid-1970s, groups in New York and Leuven found that several malignant tumours also secrete a plasminogen activator, generally in proportion to their degree of invasive behaviour. The Leuven group obtained a highly productive melanoma cell line from an American patient and eventually purified it. The Belgian group also observed that the human plasminogen activator had a specific affinity for fibrin: in the absence of fibrin it had only a weak-activating effect on plasminogen, through competitive binding and ‘silencing’ of tPA by α2-antiplasmin. This selective action represented a great advantage—at least theoretically—over the non-specific plasminogen activators urokinase and streptokinase that were available at the time. Indeed in the early 1980s, animal experiments in Leuven and St Louis showed that tPA could dissolve thrombi of venous and arterial origin without systemic fibrinolysis. The first humans to be treated with tPA (from the melanoma cell line, in hindsight with low doses) were two patients in Rotterdam with thrombosis of renal and iliofemoral veins in the region of a transplanted kidney (Weimar et al ., 1981); one of them survives to this day. The clinical experiment resulted from a chance conversation at a scientific meeting between a nephrologist from Rotterdam and a biochemist from the Leuven group. After pilot experiments in patients with coronary thrombosis had proved successful, a proper clinical trial was on the agenda but would require far greater quantities of the substance than could be extracted from the tumour cell line. In collaboration with the Leuven group, scientists at the Genentech corporation cloned the complementary DNA for …

Combined Low-Frequency Ultrasound and Streptokinase Intravascular Destruction of Arterial Thrombi In Vivo

To prevent a distal embolization in the course of ultrasound (US) angioplasty, we combined US thrombus disruption in peripheral artery in vivo with simultaneous administration of streptokinase (SK). Acute thrombosis was induced in the femoral arteries of 23 dogs. Two hours after thrombus formation, thrombus destruction was performed using US (36 kHz) and by a combined US+SK (75,000 U/kg) administration. The results showed that thrombi were disrupted completely by 1.5 ± 0.5 min US. A combined US+SK action resulted in activation of fibrinolysis, as indicated by the increase in the content of fibrinogen and fibrin degradation products and D-dimers by a factor of 1.5–2.0 after 120 min from start of treatment compared with the SK lysis. The duration of clot destruction did not change; the distal embolization was not indicated; platelet aggregation activity dropped after thrombus destruction. In summary, intravascular thrombus destruction by a combined US and SK action in vivo is accompanied by enhancing the enzymatic fibrinolysis and lowering the platelet aggregation activity that assists in preventing the distal embolization of the resulting clot debris.

Management of myocardial infarction in children with Kawasaki disease

Kawasaki disease is an acute, systemic vasculitis of unknown cause affecting mainly neonates (infants) and young children. Despite treatment during the acute phase with intravenous immunoglobulin and aspirin, up to 5% of those affected will develop coronary aneurysms, predisposing them to thrombotic complications that could result in myocardial infarction and/or death. There are treatment protocols in place for the management of myocardial infarction in adults, but the practical nature of medication is unclear in children. To date, there are no clinical trials or specific recommendations on the dosing of thrombolytic therapy for the treatment of myocardial infarction in Kawasaki pediatric patients. However, there are reports of the use of thrombolytic agents, including streptokinase, urokinase and tissue plasminogen activator, as well as the monoclonal platelet glycoprotein (GP)IIb/IIIa receptor inhibitor, abciximab, that have been used to treat myocardial infarction in children with Kawasaki disease. The outcomes in these reports are varied. This review provides a summary of the available data on the management of children with Kawasaki disease suffering from myocardial infarction or thrombotic complications that can potentially lead to myocardial infarction.

Probing the primary structural determinants of streptokinase inter-domain linkers by site-specific substitution and deletion mutagenesis

The bacterial protein streptokinase (SK) contains three independently folded domains (α, β and γ), interconnected by two flexible linkers with noticeable sequence homology. To investigate their primary structure requirements, the linkers were swapped amongst themselves i.e. linker 1 (between α and β domains) was swapped with linker 2 (between β and γ domains) and vice versa. The resultant construct exhibited very low activity essentially due to an enhanced proteolytic susceptibility. However, a SK mutant with two linker 1 sequences, which was proteolytically as stable as WT-rSK retained about 10% of the plasminogen activator activity of rSK When the native sequence of each linker was substituted with 9 consecutive glycine sequences, in case of the linker 1 substitution mutant substantial activity was seen to survive, whereas the linker 2 mutant lost nearly all its activity. The optimal length of linkers was then studied through deletion mutagenesis experiments, which showed that deletion beyond three residues in either of the linkers resulted in virtually complete loss of activator activity. The effect of length of the linkers was then also examined by insertion of extraneous pentapeptide sequences having a propensity for adopting either an extended conformation or a relatively rigid conformation. The insertion of poly-Pro sequences into native linker 2 sequence caused up to 10-fold reduction in activity, whereas its effect in linker 1 was relatively minor. Interestingly, most of the linker mutants could form stable 1:1 complexes with human plasminogen. Taken together, these observations suggest that (i) the functioning of the inter-domain linkers of SK requires a critical minimal length, (ii) linker 1 is relatively more tolerant to insertions and sequence alterations, and appears to function primarily as a covalent connector between the α and β domains, and (iii) the native linker 2 sequence is virtually indispensable for the activity of SK probably because of structural and/or flexibility requirements in SK action during catalysis.

The significance of anti-streptokinase antibodies.

Antibodies to streptokinase (SK) are widespread in the population, but reports of their effect on the action of SK are conflicting. Specific anti-SK IgG was purified from the sera of 10 patients, five with low titres of anti-SK IgG and five with high titres. The effect of increasing specific anti-SK IgG antibodies on the action of SK was evaluated in vitro using a fluorimetric assay for plasmin and by a fibrin plate lysis assay. The inhibition of SK by whole plasma from a further group of patients was also assessed by the fibrin plate assay. There was a positive correlation between the serum antibody concentration and the quantity of specific anti-SK eluted (r = 0.797; P < 0.005). The addition of specific anti-SK IgG caused a dose-related decrease in SK activity (fluorimetric assay r = -0.93; P = 0.02; fibrin plate assay r = -0.98; P < 0.001). The addition of patient plasma to the fibrin plate assay also resulted in decreased lysis, which was dependent upon antibody titre (r = -0.95; P < 0.0001). Significant in vitro reduction of the activity of SK by specific antibody was demonstrated, and this was similar with plasma containing comparable amounts of antibody. The findings suggest that treatment with SK would be unlikely to induce an effective thrombolytic state when antibody titres are high (such as those seen within 2 years of an initial dose of SK).