Cytotoxicity of chitosan/streptokinase nanoparticles as a function of size: An artificial neural networks study.

Predicting the size and toxicity of chitosan/streptokinase nanoparticles at various values of processing parameters was the aim of this study. For the first time, a comprehensive model could be developed to determine the cytotoxicity of the nanoparticles as a function of their size. Then, artificial neural networks were used for identifying main factors influencing self-assembly prepared nanoparticles size and cytotoxicity. Three variables included polymer concentration; pH and stirring time were used for a modeling study. A second modeling was performed to evaluate the influence of particles' size on toxicity. Experimentally data modeled using ANNs was validated against unseen data. The response surfaces generated from the software demonstrated that chitosan concentration is the dominant factor with a direct effect on size. Results also showed that the most important factor in determining the particles' toxicity is size--smaller particles showed more toxic effects, regardless of the effect of other input parameters. From the Clinical Editor: The understanding of toxicity of nanoparticles is of prime importance. In this article, the authors generated a model to visualize the relationship between nanoparticle size and its cellular toxicity, using chitosan/streptokinase nanoparticles. The data generated here would help the design of future nanoparticles of appropriate sizes for the application in the clinical setting.

Effect of streptokinase on prostacyclin synthesis and phospholipase activity in cultured pulmonary artery endothelial cells.

In this study, we examined the effects of streptokinase on arachidonic acid release and prostacyclin biosynthesis in cultured bovine pulmonary artery endothelial cells. When intact cells were incubated with streptokinase, a significant stimulatory effect on prostacyclin biosynthetic activity in cells was evident without any cellular damage at all concentrations used (1-10,000 units/ml). Streptokinase also caused a marked release of arachidonic acid. It induced rapid phospholipid hydrolysis, resulting in the release of up to 15% of incorporated [3H]arachidonic acid into the medium. After the addition of streptokinase, degradation of phosphatidylcholine and phosphatidylethanolamine was observed and lysophosphatidylcholine and lysophosphatidylethanolamine were produced. We also observed a transient rise in diacylglycerol after the addition of streptokinase. To test for phospholipase C activity, the release of incorporated [3H]choline, [3H]inositol and [3H]ethanolamine into the culture medium was determined. The level of radioactive inositol showed an increase, but the changes in choline and ethanolamine were comparatively small. An increase in inositol was detectable within 1 min after streptokinase addition and peaked after 15 min. Inositol phosphate and inositol trisphosphate were released, and these releases were suppressed by the addition of neomycin (50 microM). These results suggest that streptokinase stimulates phospholipase A2 and C activity, and that prostacyclin biosynthesis is subsequently increased in cultured endothelial cells.

The preclinical toxicology of anisoylated plasminogen streptokinase activator complex.

The preclinical toxicological evaluation of anisoylated plasminogen streptokinase activator complex (APSAC) was designed with specific regard to the potential for immunogenic effects in animals arising from the high molecular weight of the complex. Animals were treated with multiples of the human dose by use of a dose regimen spanning that proposed clinically and in conventional repeat dose toxicity studies employing the maximum practicable dose level and duration. Few adverse effects were noted, despite the large doses administered with respect to the clinical dose. The thrombolytic activity of APSAC resulted in pronounced acute effects on blood coagulation times and fibrinogen levels in rats and dogs but there was little evidence of clinically relevant systemic toxicity in either species. Evidence of a possible effect on the liver was seen 24 hours after single doses much higher than the proposed human dose in the rat (40-fold higher) and dog (9-fold higher). No hepatic effects were apparent following repeated administration. The main adverse effect was focal acute myocarditis, which was seen only in rats of the Sprague Dawley strain. Administration of human plasminogen alone or in combination with streptokinase also produced this lesion, suggesting that plasminogen may play a central role in its appearance. Experiments in anaesthetised dogs showed APSAC to be devoid of undesirable haemodynamic effects. An intravenous acute toxicity study in rats with p-anisic acid, which is released on deacylation of APSAC, showed the levels of p-anisic acid which occur in humans to be of no toxicological significance. Finally, in a series of tests designed to investigate potential genetic toxicity, no mutagenic activity was detected.

A bleeding model in rabbits demonstrate fresh clot selectivity for a genetically engineered variant of tissue-type plasminogen activator and for streptokinase.

The way in which three thrombolytic agents, tissue-type plasminogen activator (t-PA), streptokinase (SK) and a genetically engineered variant of t-PA composed of the second kringle and the protease domain (K2P), cause the dissolution of haemostatic plugs of differing ages was investigated in a novel rabbit model. Standardized incisions were made on the rabbit ear and the wounds were left to heal for 0.5 h or 24 h, before the thrombolytic agents were infused. In the absence of heparin, t-PA showed little discrimination between clots of different ages (36% and 28% lysis of the 0.5 h and 24 h wounds, respectively). In contrast, K2P and SK showed a pronounced fresh clot selectivity since they were significantly more effective in lysing fresh clots than old ones (68% and 4% lysis for K2P and 72% and 36% for SK, respectively). In the presence of heparin the potency of t-PA on fresh clots was considerably increased whilst the effect on old clots was not affected, a fresh clot selectivity for t-PA (64% lysis of fresh clots, 24% lysis of old clots) was thus effected. Heparin did not significantly affect the fresh clot selectivity of K2P or SK, although lysis of old clots was increased (from 4% to 36%) when it was given together with K2P. Furthermore, heparin did not affect the time to onset of bleeding nor was the bleeding time prolonged by its addition. The bleeding time observed for t-PA (20-25 min) was markedly shorter than that found for K2P or SK (40-50 min).(ABSTRACT TRUNCATED AT 250 WORDS)

An animal model of fibrinolytic bleeding based on the rebleed phenomenon: application to a study of vulnerability of hemostatic plugs of different age.

The primary bleeding time is prolonged when tested during the infusion of both plasminogen activators and anticoagulants, and such sites frequently exhibit rebleeding after initial hemostatic control. This study describes an animal (rabbit) model which distinguishes fibrinolytic from anticoagulant hemorrhage and further applies the model to the study of hemostatic plugs of increasing age. In this model, rebleeding occurred from hemostatically-stable ear puncture sites induced prior to infusion of streptokinase (SK) or recombinant tissue-plasminogen activator (rt-PA), but not of heparin or hirudin. This distinction was apparent even for lesions induced only 15 minutes prior to the infusion and fibrinolytic bleeding was observed in such lesions induced up to 24 hours earlier. Post-infusion sites bled more quickly than did pre-infusion sites, and there was a gradual decrease in susceptibility of such prior trauma sites for rebleeding, evidenced not only by a lower proportion of sites that rebled, but also by a longer lag time after starting SK or rt-PA before such rebleeding occurred. At the dosages tested, SK showed a trend (not statistically significant) toward more sites that rebled, while rt-PA showed a trend towards a longer duration of rebleeding. Thus, this animal model of rebleeding appears to be unique for fibrinolytic agents and allows for more detailed study of the physiological mechanisms of such bleeding and for a multifaceted comparison of the bleeding potential of plasminogen activators.

A paradox of thrombogenesis by streptokinase and its prevention by iloprost and camonagrel.

Here we report that streptokinase is responsible for forming thrombi both in vitro on blood-superfused endothelial cells of rabbit aorta and in vivo on blood-superfused collagen strips in extracorporal circulation of anesthetized cats. This short-lasting paradoxical thrombogenic phase is followed by the expected long-lasting thrombolysis. The biphasic action of streptokinase occurred in vitro at concentrations of 100-2000 U/ml and in vivo at doses of 1000-3000 U/kg i.v. Both phases are mediated by endogenous plasmin as evidenced by deleting the streptokinase-induced thrombogenesis and thrombolysis following pretreatment with epsilon-aminocaproic acid or aprotinin. On the other hand, selective block of the paradoxical thrombogenesis was achieved after pretreatment with camonagrel, a thromboxane synthase inhibitor which raises plasma levels of endogenous prostacyclin, or with iloprost, a stable analog of prostacyclin. It is suggested that endogenous or exogenous prostacyclin inhibits activation of platelets by plasmin, and hence the thrombogenesis by streptokinase is abolished, while the beneficial thrombolytic action of streptokinase is augmented.

[Pharmacology of fibrinolytics]. / Zur Pharmakologie der Fibrinolytika.

The pharmacological basis for clinical uses of the fibrinolytic agents streptokinase and urokinase is summarizing described. Since streptokinase is still the drug of choice in fibrinolytic therapy of thromboembolic disorders, its chemistry, mode of action, pharmacokinetics, toxicity, and side-effects are reported in detail.

Hemorrhagic complications of thrombolytic therapy in experimental stroke.

Recent success with thrombolytic therapy for acute myocardial infarction has stimulated interest in its use for stroke. To determine the hemorrhagic potential of thrombolytic therapy in experimental cerebral infarction, we compared a group of tissue plasminogen activator-treated rabbits (n = 4) with 2 groups of streptokinase-treated rabbits (n = 6 in each), as well as with 3 groups of heparin-treated rabbits (n = 5 in each) and untreated controls (n = 12). Focal cerebral infarction was produced in rabbits by occlusion of the right common carotid and middle cerebral arteries coupled with 2 hours of halothane-induced hypotension. Treatment with heparin or thrombolytic agents began 24 hours after occlusion. One additional group was treated with streptokinase 1 hour after occlusion (n = 6) to determine the hemorrhagic potential of thrombolytic agents in evolving infarction. Rabbits were killed 29-33 hours after occlusion, and brain sections were examined using light microscopy. The results demonstrate that microscopic hemorrhage is frequently present in infarcted tissue irrespective of treatment. Gross cerebral hemorrhage did not occur in untreated rabbits or in rabbits treated with streptokinase 1 hour after occlusion. Only rabbits treated with streptokinase, tissue plasminogen activator, or excessive doses of heparin 24 hours after occlusion, at a time when cerebral infarction was well established, exhibited gross hemorrhage in the area of infarction. These data suggest that treatment of ischemic stroke with thrombolytic agents carries an increased risk of cerebral hemorrhage unless the agents are given early after the onset of symptoms.

High dose and short-term streptokinase infusion in patients with pulmonary embolism: prospective with seven-year follow-up trial.

BACKGROUND: High dose and short-term streptokinase infusion has proved to improve survival among few patients with pulmonary embolism and cardiogenic shock, without increasing hemorrhagic complications. However its efficacy and safety in terms of long follow-up and in major number of patients requires to be established. METHODS: Patients with pulmonary embolism proved through high probability V/Q lung scan, suggestive echocardiogram, or deep venous thrombosis were enrolled. All were assigned to receive 1,500,000 IU in one-hour streptokinase infusion. The primary end point was efficacy and safety of streptokinase regimen in terms of pulmonary arterial hypertension, right ventricular dysfunction, perfusion abnormalities, recurrence, mortality and hemorrhagic complications. In long-term follow-up, we assessed functional class, recurrence, chronic pulmonary arterial hypertension, postthrombotic-syndrome and mortality. RESULTS: A total of 40 consecutive patients (47.3+/-15.3 years of age) with large or massive pulmonary embolism were enrolled. In 35 patients high dose and short-term streptokinase regimen reversed acute pulmonary arterial hypertension, clinical and echocardiographic evidence of right ventricular dysfunction and improved pulmonary perfusion without increasing hemorrhagic complications. In acute phase 5 patients died, necropsy study performed in 4 patients showed massive pulmonary embolism and right ventricular myocardial infarction, without significant coronary arterial obstruction. Risk factors for mortality and recurrence were: right ventricular global hypokinesis (p<0.0001), 6 hours or over between onset symptoms and streptokinase regimen (p=0.02), severe systolic pulmonary arterial hypertension (p=0.001) right ventricular hypokinesis (p=0.001), hypoxemia (p=0.02) and right ventricular acute myocardial infarction (p<0.0001). Right ventricular hypokinesis (p=0.02) was the only independent risk factor for recurrence. In a seven-year follow-up of the original 35 patients who survived in acute phase, 2 patients were lost and 33 are alive, in functional class I, without recurrence or chronic pulmonary arterial hypertension. CONCLUSIONS: Our report indicates that among properly selected high-risk PE patients, short-term streptokinase infusion is effective and safe.

Comparative effects of tissue plasminogen activator, streptokinase, and staphylokinase on cerebral ischemic infarction and pulmonary clot lysis in hamster models.

BACKGROUND: The effects of alteplase (rtPA), streptokinase, and staphylokinase (rSak) on focal cerebral ischemia (FCI) and on pulmonary clot lysis (PCL) were studied in hamsters. METHODS AND RESULTS: ++FCI was produced by ligation of the left middle cerebral artery (MCA) and common carotid artery (CCA) and a 10-minute occlusion of the right CCA. FCI was measured after 24 hours by 2,3, 5-triphenyltetrazolium chloride staining. (125)I-fibrin-labeled plasma clots were injected via the jugular vein, and clot lysis was determined from residual radioactivity at 90 minutes. Study drugs were given intravenously over 60 minutes. FCI increased from 1.2 (0. 27 to 2.3) mm(3) (median and 17th to 83rd percentile range, n=24) in controls to 19 to 27 mm(3) with thrombolytic agent, with maximal rates at 0.13+/-0.05 mg/kg rtPA, 0.23+/-0.09 mg/kg streptokinase, and 0.037+/-0.025 mg/kg rSak. PCL increased from 18+/-2% (mean+/-SEM, n=27) in controls to approximately 85% with thrombolytics, with maximal rates at 0.12+/-0.03 mg/kg rtPA, 0.17+/-0.05 mg/kg streptokinase, and 0.018+/-0.002 mg/kg rSak. All agents caused maximal FCI and PCL rates at similar doses without alpha(2)-antiplasmin and fibrinogen depletion. Injection of 6 mg/kg human plasminogen combined with streptokinase caused a "systemic fibrinolytic state" with fibrinogen depletion. Maximal rates of FCI were obtained with 0.097+/-0.077 mg/kg streptokinase (P=0.26 versus streptokinase alone) and of PCL with 0.010+/-0.002 mg/kg (P=0.006 versus streptokinase alone). CONCLUSIONS: Thrombolytic agents cause similar dose-related extension of FCI after MCA ligation and PCL, irrespective of the agent or systemic plasmin generation.