Co-ordinated spatial propagation of blood plasma clotting and fibrinolytic fronts.

Fibrinolysis is a cascade of proteolytic reactions occurring in blood and soft tissues, which functions to disintegrate fibrin clots when they are no more needed. In order to elucidate its regulation in space and time, fibrinolysis was investigated using an in vitro reaction-diffusion experimental model of blood clot formation and dissolution. Clotting was activated by a surface with immobilized tissue factor in a thin layer of recalcified blood plasma supplemented with tissue plasminogen activator (TPA), urokinase plasminogen activator or streptokinase. Formation and dissolution of fibrin clot was monitored by videomicroscopy. Computer systems biology model of clot formation and lysis was developed for data analysis and experimental planning. Fibrin clot front propagated in space from tissue factor, followed by a front of clot dissolution propagating from the same source. Velocity of lysis front propagation linearly depended on the velocity clotting front propagation (correlation r2 = 0.91). Computer model revealed that fibrin formation was indeed the rate-limiting step in the fibrinolysis front propagation. The phenomenon of two fronts which switched the state of blood plasma from liquid to solid and then back to liquid did not depend on the fibrinolysis activator. Interestingly, TPA at high concentrations began to increase lysis onset time and to decrease lysis propagation velocity, presumably due to plasminogen depletion. Spatially non-uniform lysis occurred simultaneously with clot formation and detached the clot from the procoagulant surface. These patterns of spatial fibrinolysis provide insights into its regulation and might explain clinical phenomena associated with thrombolytic therapy.

Assessment of oxidative/nitrative modifications of plasma proteins, selected ROTEM parameters and kinetics of fibrinogen polymerization in patients with multiple myeloma at diagnosis.

Patients with multiple myeloma (MM) are at increased risk of thrombosis. Growing evidence indicates that oxidative and nitrative modifications of proteins, including fibrinogen, may lead to changes in hemostasis. The study compares samples from patients with MM at diagnosis and healthy volunteers with regard to the oxidative/nitrative modifications of proteins, ROTEM and thrombin-catalyzed fibrin polymerization. The content of carbonyl groups in plasma proteins of patients with MM was significantly higher than in controls (2.981 vs. 1.807 nmol/mg of protein, p = 0.005), while no differences were seen in the concentrations of nitrated proteins. Maximum clot firmness readings were significantly higher in the samples of patients than in controls according to FIBTEM test (23.5 vs. 15 mm, p = 0.006). The lag time of the fibrin polymerization process and the velocity of clot lysis (V Lys) were found to be significantly higher in the group of MM patients than controls. In contrast, no marked differences were identified between studied groups in reference to maximal velocity of fibrin polymerization process (V max), maximal absorbance (A max) and plasmin amidolytic activity values. In conclusion, our study demonstrates that at the time of diagnosis, patients with MM demonstrated greater oxidative stress than healthy volunteers, which is reflected in a higher amount of carbonylated proteins. Some prothrombotic features found in ROTEM tests in MM patients were not confirmed by turbidimetry.

Preparation and characterization of self-assembled chitosan nanoparticles for the sustained delivery of streptokinase: an in vivo study.

Chitosan (CS) nanoparticles have been extensively studied as carriers for therapeutic proteins in recent years. In this study, streptokinase loaded-CS nanoparticles were prepared and the pharmacokinetic parameters of streptokinase were compared with those of naked streptokinase. The preparation method included stirring the protein with the CS solution. The optimized combination was used for animal experiments to determine the streptokinase activity in rat plasma. Blood samples were collected at specified intervals and the activity assay was performed based on amidolysis activity of the chromogenic substrate, S2251, by streptokinase-plasminogen activator complex. The results demonstrated that streptokinase-loaded CS nanoparticles have more prolonged amidolytic activity in vivo compared to the naked one.

Platelet activity and antibody titers after exposure to streptokinase or streptococcal infection.

INTRODUCTION: Streptokinase use, in acute myocardial infarction, is hindered by failure to reperfuse (60%) and early reocclusion (16%). This phenomenon may, among other causes, be due to systemic inactivation of streptokinase, as well as streptokinase-induced platelet aggregation and clot propagation from antibodies to streptokinase produced after streptokinase administration or streptococcal infections. The purpose of this study was to determine the incidence of streptokinase-induced, antibody-mediated, platelet activation and aggregation after administration of SK or development of a streptococcal infection. MATERIALS AND METHODS: We included 45 normal volunteers (Control group), as well as 45 patients who had received streptokinase (Streptokinase group) and 13 who had suffered a severe streptococcal infection (Streptococcal infection group) within the past 3 years. Extent of streptokinase-induced, antibody-mediated, platelet activation and aggregation, as well as anti-streptokinase antibody and streptokinase resistance titers (lowest streptokinase concentration to cause clot lysis within 10 min) were measured. RESULTS: Whereas streptokinase-induced, antibody-mediated, platelet activation was observed in 49% of streptokinase patients and in only 17% and 15% of streptococcal infection patients and normal volunteers (p<0.05 Streptokinase vs. Control and Streptokinase vs. Streptococcal infection), streptokinase-induced platelet aggregation was observed in 23% of streptokinase patients and streptococcal infection patients, and in none of the control patients (p<0.05). CONCLUSIONS: Streptokinase-induced, antibody-mediated, platelet activation and aggregation occur in patients with high titers of anti-streptokinase antibody and may play a role in failure of streptokinase therapy. Streptococcal infection patients behave like streptokinase patients in terms of the reactivity of their platelets to subsequent streptokinase dose in vitro.

Electrochemical assay of plasminogen activators in plasma using polyion-sensitive membrane electrode detection.

Two relatively simple electrochemical assay methods suitable for the measurement of plasminogen activators (including urokinase (u-PA), streptokinase (SK), and tissue plasminogen activator (t-PA)) in plasma samples are described. In one approach, the initial rate of decrease in the potentiometric response of a polycation-sensitive membrane electrode toward protamine is monitored after addition of a preincubated reaction mixture containing the sample and exogenous plasminogen. The plasmin formed from plasminogen by the activators catalyzes the decomposition of the arginine-rich protamine substrate, yielding smaller polycationic fragments that are not sensed by the electrode. Alternately, the sample, plasminogen, and protamine can be incubated together, and the remaining protamine in this reaction mixture can be measured at a fixed point in time by placing the electrode into the mixture and recording the electromotive force response. Working curves found with both methods for plasma samples spiked with varying levels of the activators cover the expected therapeutic activity ranges found in the plasma of patients treated with these "clot-busting" drugs.

Sixty-minute alteplase protocol: a new accelerated recombinant tissue-type plasminogen activator regimen for thrombolysis in acute myocardial infarction.

OBJECTIVES: Our aim was to design and evaluate a new and easily administered recombinant tissue-type plasminogen activator (rt-PA) regimen for thrombolysis in acute myocardial infarction (AMI) based on established pharmacokinetic data that improve the reperfusion success rate. BACKGROUND: Rapid restoration of Thrombolysis in Myocardial Infarction (TIMI) grade 3 flow is a primary predictor of mortality after thrombolysis in AMI. However, TIMI grade 3 patency rates 90 min into thrombolysis of only 50% to 60% indicate an obvious need for improved thrombolytic regimens. METHODS: Pharmacokinetic simulations were performed to design a new rt-PA regimen. We aimed for a plateau tissue-type plasminogen activator (t-PA) plasma level similar to that of the first plateau of the Neuhaus regimen. These aims were achieved with a 20-mg rt-PA intravenous (i.v.) bolus followed by an 80-mg i.v. infusion over 60 min (regimen A). This regimen was tested in a consecutive comparative trial in 80 patients versus 2.25 10(6) IU of streptokinase/60 min (B), and 70 mg (C) or 100 mg (D) of rt-PA over 90 min. Subsequently, a confirmation trial of regimen A in 254 consecutive patients was performed with angiographic assessment by independent investigators of patency at 90 min. RESULTS: The comparative phase of the trial yielded, respectively, TIMI grade 3 and total patency (TIMI grades 2 and 3) of 80% and 85% (regimen A), 35% and 50% (B), 50% and 55% (C) and 60% and 70% (D). In the confirmation phase of the trial, regimen A yielded 81.1% TIMI grade 3 and 87.0% total patency. At follow-up angiography 7 (4.1%) of 169 vessels had reoccluded. In-hospital mortality rate was 1.2%. Nadir levels of fibrinogen, plasminogen and alpha2-antiplasmin were 3.6 +/- 0.8 mg/ml, 60 +/- 21% and 42 +/- 16%, respectively (mean +/- SD). Fifty-seven patients (22.4%) suffered from bleeding; 3.5% needed blood transfusions. CONCLUSIONS: The 60-min alteplase thrombolysis in AMI protocol achieved a TIMI grade 3 patency rate of 81.1% at 90 min with no indication of an increased bleeding hazard; it was associated with a 1.2% overall mortality rate. These results are substantially better than those reported from all currently utilized regimens. Head to head comparison with established thrombolytic regimens in a large-scale randomized trial is warranted.

Circulatory concentrations of fibrinolytic species during thrombolytic therapy estimated by stirred-tank reactor analysis.

PURPOSE: A model for calculating the circulatory concentrations of fibrinolytic species is proposed and the findings are compared to reported values where available. METHODS: The model uses a CSTR analysis with fourth order Runge-Kutta solution of the differential equations to determine concentration profiles of key fibrinolytic species as a function of time during fibrinolytic therapy. Concentrations of the species are also determined for various dosage regimens of streptokinase and plasminogen. RESULTS: Data calculated by the model is in agreement with general experimental trends determined in vitro and in vivo. CONCLUSIONS: The proposed model can be used to predict concentration profiles of key fibrinolytic species during administration of streptokinase.

[Antibodies against extracellular products of group A Streptococcus. Diagnostic importance in acute rheumatic fever]. / Anticuerpos contra productos extracelulares del estreptococo grupo A. Importancia diagnóstica en la fiebre reumática aguda.

Streptococcal throat infection is a sine qua non for the development of rheumatic fever (RF) in genetically susceptible people. Demonstration of such infection is not easy. In overt RF less than 10% of patients still carry streptococci in their throat and immunologic methods are required to identify antibodies against streptococcal products (SP). Humoral response against SP was studied in children and adults with and without RF. Antistreptolysin O (ASO) showed a non-gaussian distribution, and reference value was established as percentile. Adults have a 97 percentile of 227, in children 90 percentile was 451. When RF was present all cases, except one, showed higher values. When antibodies against SP besides ASO were sought by an agglutination test (Streptozyme tm), people below 15 years of age showed low titers in 15 out of 28 cases. In contrast, high titers were the rule in children suffering RF. High ASO titer correlated with high Streptozyme value. These methods are capable to recognize an specific immune response against Group A Beta hemolytic streptococci, and are valuable tools in the diagnosis of RF.

Influence of intrinsic and extrinsic plasminogen upon the lysis of thrombi in vitro.

When the rate of lysis of artificial thrombi (prepared from plasma or whole blood) was expressed according to the concentration of tissue type plasminogen activator (t-PA) or single chain urokinase type plasminogen activator (sc-uPA) then bell-shaped dose response curves were obtained, low rates being observed at concentrations of activator greater than 500 units/ml. Bell-shaped dose response curves were not observed for rate of lysis of artificial thrombi over the concentrations of streptokinase tested (SK) or for the lysis of plasma gel clots by any of the activators tested. Further investigation indicated that the preponderant mechanism for dissolution of thrombi at 500 units/ml of t-PA was by activation of the plasminogen within the thrombus (intrinsic) since the plasminogen present in the plasma perfusing the thrombus (extrinsic) rapidly became depleted. On the other hand, at 50 units/ml t-PA the lysis was observed to be due preponderantly to the action of plasmin arising from extrinsic rather than intrinsic plasminogen. If "plasminogen enriched" thrombi were prepared in the presence of Lys plasminogen (Lys-Plg) faster rates of lysis occurred and bell-shaped biometric curves were not observed.

Prevention of in vitro fibrinogenolysis during laboratory monitoring of thrombolytic therapy with streptokinase or APSAC.

In the present study, we systematically investigated aprotinin, epsilon-aminocaproic acid (EACA) and tranexamic acid as inhibitors of fibrinogen breakdown and of the generation of fibrinogen degradation products (FgDP). The experimental setting very closely imitated the conditions in practice when collecting blood from patients receiving thrombolytic therapy with streptokinase or APSAC. The minimal concentration of aprotinin required to completely inhibit fibrinogen breakdown and FgDP generation was 200 KIU/ml blood. This was sufficient even at the highest concentrations of streptokinase and APSAC expected to occur in patients (300 U/ml and 46 nM, respectively). However, 200 KIU/ml aprotinin heavily interfered in the determinations of plasminogen and alpha 2-antiplasmin. Relatively low concentrations of EACA (200 mM) and tranexamic acid (35 mM) were sufficient to prevent FgDP generation, but they interfered in the Clauss assay of fibrinogen. A non-interfering concentration of EACA (7 mM) allowed the inhibition of lower concentrations of APSAC (20 nM) and streptokinase. We conclude that at least 200 KIU aprotinin per ml blood is necessary to effectively inhibit in vitro fibrinogenolysis under circumstances likely to be met in clinical practice during thrombolytic therapy.

Chromogenic assay for equine plasminogen.

A functional assay for equine plasminogen was established, using urokinase as the activator, a synthetic chromogenic substrate, a computer-assisted centrifugal analyzer, and acidified/neutralized plasma. One documented effect of plasma acidification appears to be inactivation of alpha-2-antiplasmin. Intra- and interassay precision testing yielded coefficients of variation of 4.1% (n = 10) and 5.6% (n = 26), respectively. Plasminogen was stable in equine plasma stored up to 1 week at 4 C and up to 5 months at -70 C. Plasminogen in nonacidified equine plasma was not activated by urokinase, streptokinase, tissue plasminogen activator, or tissue plasminogen activator plus soluble fibrin. Streptokinase also failed to activate plasminogen in acidified/neutralized equine plasma.

[A critical review of the methods used in evaluating the antistreptolysin titre as a diagnostic index]. / Revisione critica della metodologia utilizzata nella valutazione del titolo antistreptolisinico come indice diagnostico.

Authors review the most common laboratory tests showing an immunologic response caused by group A beta-hemolytic streptococcus infections. After a preliminary evaluation on characteristics and peculiarities of different methods the results obtained are compared with the usually employed systems. The survey of most common analytical methods and personal experience lead to confirm and emphasize the laboratory results supporting and confirming, without substituting, the clinical data.

Streptokinase binds to lactate dehydrogenase subunit-M, which shares an epitope with plasminogen.

The bacterial thrombolytic agent streptokinase binds to human, porcine, and chicken lactate dehydrogenase (EC 1.1.1.27; LD) isoenzyme subunit M, but not to the H or C subunits. There is amino acid sequence homology between LD and the streptokinase binding site on plasminogen to account for this interaction that results in the formation of high-molecular-mass complexes in serum that contain LD activity. Binding of highly immunogenic streptokinase with LD may lead to induction of anti-LD autoantibodies, known to occur in some patients after therapeutic administration of streptokinase for treatment of acute myocardial infarction. This interaction may also be a general mechanism for inducing autoimmunity against other proteins that share the streptokinase binding epitope.

Pharmacokinetics of streptokinase in patients based on amidolytic activator complex activity.

The purpose of this study was to determine the pharmacokinetics of intravenous streptokinase in humans. Five patients with myocardial infarction, six patients with venous thromboembolism, and two normal volunteers were studied. The patients with myocardial infarction received 500,000 U over 30 minutes, the patients with venous thromboembolism received 250,000 U over 30 minutes followed by 100,000 U/hr over 16 to 78 hours, and the normal volunteers received 100,000 U over 15 minutes. Plasma streptokinase levels were measured based on the amidolytic activity of the streptokinase-plasminogen complex on the chromogenic substrate S-2251. Pharmacokinetic parameters were: biologic half-life 82 +/- 25 minutes, total clearance 10.8 +/- 8.8 ml/min, and apparent volume of distribution 1.10 +/- 0.71 l. Streptokinase levels declined progressively during the continuous, prolonged infusion in the patients with venous thromboembolism over 60 hours of treatment. We conclude that there are distinct time-dependent changes in the pharmacokinetics of streptokinase during continuous intravenous infusion and that this phenomenon is likely to be associated with progressively decreasing thrombolytic efficacy.

[Formation of streptokinase-heparin complexes and their properties]. / Issledovanie obrazovaniia kompleksov streptokinazy u geparina i ikh svoistv.

Streptokinase and heparin developed complex at the ratio 3.2:1, w/w, respectively, and neutral values of pH, which was registered by means of thin-layer chromatography and differential UV-spectroscopy. IR-, differential UV- and CD-spectroscopic methods showed that secondary and tertiary structures of streptokinase were altered in the complex. Streptokinase containing in the complex exhibited the higher thermostability at 50 degrees as compared with native protein. After intravenous administration of the complex into rabbits more distinct alterations were observed in thrombin time, blood clot lysis and in blood plasma fibrinolytic activity as compared with native streptokinase effect.