Streptokinase

Streptokinase is a 48 kDa extracellular bacterial protein produced by several strains of Streptococcus haemolyticus group C. Its ability to induce lysis of blood clots was first demonstrated in 1933. Early therapeutic preparations administered to patients often caused immunological and other complications, usually prompted by impurities present in these products. Chromatographic purification (particularly using gel filtration and ion-exchange columns) overcame many of these initial difficulties. Modern chromatographically pure streptokinase preparations are usually supplied in freeze-dried form. These preparations (still obtained by non-recombinant means) often contain albumin as an excipient. The albumin prevents flocculation of the active ingredient upon its reconstitution. 

Streptokinase is a widely employed thrombolytic agent. It is administered to treat a variety of thrombo-embolic disorders, including  

Streptokinase induces its thrombolytic effect by binding specifically and tightly to plasminogen. This induces a conformational change in the plasminogen molecule that renders it proteolytically active. In this way, the streptokinase-plasminogen complex catalyses the proteolytic conversion of plasminogen to active plasmin. 

As a bacterial protein, streptokinase is viewed by the human immune system as an antigenic substance. In some cases, its administration has elicited allergic responses that have ranged from mild rashes to more serious anaphylactic shock (an extreme and generalized allergic response characterized by swelling, constriction of the bronchioles, circulatory collapse and heart failure). 

Therapeutic Function Enzyme Chemical Name Streptococcal fibrinolysin Common Name — 

The following description is from U.S. Patent 2,701,227 To 50 liters of distilled water there was added 10.17 kg of enzyme hydrolyzed casein (N-Z-Amine). The temperature was raised to 100°C and held until the casein digest solution was clear. The container was then cooled rapidly to 15°C and the cooled solution filtered through a coarse grade of filter paper. A small amount of toluene was added as a preservative and the solution 

Structural Formula Complex enzyme mixture Chemical Abstracts Registry No. 900201 -1 

Trade Name Manufacturer Country Year Introduced 

Bone agents, e.g, polyphosphates, pyrophosphates, diphos-phonates, iminodiphosphonates 

Tc-DTPA, Tc-EDTA, Tc-MIDA (methyliminodiacetic acid), Tc-citrate 

Tc-gluconate, Tc-glucoheptonate, Tc-Fe-ascorbate, Tc-inulin, Tc-mannitol, Tc-dimercaptosuccinic acid 

Tc-pyrophosphate, Tc-glucoheptonate, Tc-tetracycline and Tc-HEDP (hydroxy-ethylidene) diphosphonate 

Tc-dihydrothioctic acid, Tc-HIDA, Tc-isomercaptobutyric acid and Tc-pyridoxylideneglutamate 

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Approximately 500,000 Americans suffer strokes each year. Many of the 80% that survive suffer paralysis and impaired vision and speech, often needing rehabiUtation and/or long-term care. Hence, whereas treatment using rt-PA is likely to be expensive (costs are 2200/dose for treating heat attacks), the benefits of rt-PA could outweigh costs. In the case of heart attacks, the 10 times less expensive microbiaHy derived streptokinase can be used. There is currentiy no competing pharmaceutical for treatment of strokes (18,19). Consequentiy, the cost of manufacture of rt-PA may not be as dominant an issue as would be the case of other types of bioproducts. 

Therapeutics. Therapeutic materials represent a class of polypeptides that are a low volume, high value product. The production system need not be very efficient but the quaHty of the recombinant protein has to be extremely pure (33,34). Thus high cost mammalian production systems can be tolerated. However, some of the therapeutic proteins such as insulin, human growth hormone, interleukins, interferon, and streptokinase are produced microbially. 

Fig. 4. Fibrinolytic system where SCUPA is single-chain urokinase plasminogen activator rTPA is recombinant tissue plasminogen activator APSAC is acylated plasminogen streptokinase activator complex SK is streptokinase and UK is urokinase.

APSAC = acylated plasminogen streptokinase activator complex. 

Streptokinase. Streptokinase is a single-chain protein containing 415 amino acids, mol wt of 45,000 to 50,000 (261,284—286). It is produced by P-hemolytic streptococci and is not an enzyme per se. Only after streptokinase combines with plasminogen on a 1 1 basis to form a... 

The mortahty is usually reduced from 12% in the control group to 9—10% in the streptokinase group. Side effects are bleeding and hemorrhage, fever, and in rare occasions, anaphylaxis. 

Streptokinase. The fibrinolytic activity of streptokinase, isolated from strains of hemolytic Streptococci, was first demonstrated in 1933 (63). Streptokinase is a secreted protein product inasmuch as filtrates free of demonstrable bacteria were found to dissolve fibrin clots with rapidity. 

Streptokinase has a molecular weight of about 47,000 with a single chain of 415 amino acids there are no intramolecular disulfide bonds (64). The complete nucleotide sequence of the gene encoding the RNA for this protein has been reported (65,66). 

Indications for treatment with streptokinase include acute occlusion of arteries, deep vein thrombosis, and pulmonary embolism. Streptokinase therapy in coronary thrombosis, which is the usual cause of myocardial infarction (54,71,72), has proved to be valuable. In this frequently fatal condition, the enzyme is adrninistered intravenously at a dose of 1.5 million units over 60 min, or given by intracoronary infusion at a 20,000- to 50,000-unit bolus dose followed by 2000 to 4000 units/min for 60 min therapy must be instituted as soon as practicable after the diagnosis of heart attack is made. For deep vein thrombosis, pulmonary embolism, or arterial occlusion, streptokinase is infused at a loading dose of 250,000 units given over 30 min, followed by a maintenance dose of 100,000 units over a 60-min period. 

Streptokinase has an initial plasma half-life (/ 2 of 18 min, and a P half-life of 83 min (73) it is well recognized that the thrombolytic efficacy of the enzyme decreases as the age of the thrombus increases thus, thrombolysis is significantly decreased when therapy is initiated more than three hours after an occlusion (74). 

Several clinical trials have been conducted with streptokinase adrninistered either intravenously or by direct infusion into a catheterized coronary artery. The results from 33 randomized trials conducted between 1959 and 1984 have been examined (75), and show a significant decrease in mortaUty rate (15.4%) in enzyme-treated patients vs matched controls (19.2%). These results correlate well with an ItaUan study encompassing 11,806 patients (76), in which the overall reduction in mortaUty was 19% in the streptokinase-treated group, ie, 1.5 million units adrninistered intravenously, compared with placebo-treated controls. The trial also shows that a delay in the initiation of treatment over six hours after the onset of symptoms nullifies any benefit from this type of thrombolytic therapy. Conversely, patients treated within one hour from the onset of symptoms had a remarkable decrease in mortaUty (47%). The benefits of streptokinase therapy, especially in the latter group of patients, was stiU evident in a one-year foUow-up (77). In addition to reducing mortahty rate, there was an improvement in left ventricular function and a reduction in the size of infarction. Thus early treatment with streptokinase is essential. 

In a more extensive international trial, 17,187 patients were treated intravenously with streptokinase alone, aspirin alone, a combination of streptokinase and aspirin, or placebo (78). Streptokinase and aspirin were equally effective in treating acute myocardial infarction, each decreasing mortahty by 25% their combination further reduced mortahty by 42%. A significant reduction in mortahty was seen even in those patients treated up to 24 hours after the onset of symptoms. 

In addition to the intravenous route, streptokinase is also adrninistered by the direct intracoronary route. In a Dutch study with 533 patients treated intracoronarily, a significant reduction (12%) in mortahty was noted (79). Of the two methods of adininistration, the intravenous route appears to be the method of choice because of ease of adininistration and the shorter lag period before therapy is initiated the lag period observed in studies utilizing the intracoronary route is attributable to the time required for catheterization. 

The thrombolytic efficacy of streptokinase treatment may be compromised by the presence of antibodies to the enzyme in the patient s blood. 

These neutralizing antibodies may arise because of a prior streptococcal infection, or prior streptokinase treatment (80—82). Titers of antibodies sufficient to neutralize a complete dose of 1.5 million units of streptokinase may be present even one year after enzyme treatment (83). 

Anistreplase has a considerably longer a half-life than streptokinase, ie, 90 min compared to 20 min (87,88). Moreover, it does not require prolonged infusion to achieve its thrombolytic effects. Anistreplase was found to be highly effective after a single intravenous dose of 30 units over a 5-min period compared to a 60-min infusion of 1.5 million units of streptokinase (89—94). In direct comparative studies, anistreplase was as effective as intracoronary (95,96) and intravenously (96—100) adrninistered streptokinase. In a randomized, double-blind, placebo-controUed study (AIMS trial) with 1004 patients given this modified enzyme, the 30-day mortaUty rate was 12.2% for patients receiving placebo, compared to 6.4% for patients who received 30 units of anistreplase intravenously within six hours of the onset of symptoms (101). 

The side effects of anistreplase appear to be similar to those of streptokinase, including immune reactions and a systemic lytic state conductive to hemorrhage. 

Compared to streptokinase, urokinase has been less extensively studied because of its high cost, ie, about 10 times that of a comparable treatment with streptokinase. In addition to the indications described for streptokinase, urokinase is indicated for use in patients with prior streptokinase treatment, or prior Streptococcal infection. Urokinase is commonly used at a loading dose of 4400 units /kg, with a maintenance intravenous infusion dose of 4400 units/kg/h for thromboses other than acute myocardial infarction. In the latter case, a much larger dose, ie, 0.5—2.0 million units/h or a bolus dose of 1.0 million units followed by a 60-min infusion with 1.0 million units, has been found optimal (106). An intracoronary dose of 2000 units/min for two hours was used in one comparative study with intracoronary streptokinase (107). In this study, urokinase exhibited efficacy equivalent to streptokinase with fewer side effects. Other studies with intracoronary urokinase have adrninistered doses ranging from 2,000 to 24,000 units/min with a reperfusion efficacy of 60—89% (108—112). In another urokinase trial, 2.0 million units were adrninistered intravenously, resulting in a thrombolytic efficacy of 60% (113). Effectiveness in terms of reduction in mortaUty rate has not been deterrnined because of the small number of patients studied. 

Tissue Plasminogen Aetivator (tPA). While streptokinase and urokinase can effectively induce clot dissolution in the majority of patients if given early, they lack clot specificity. Treatment with these enzymes results in a systemic lytic state attributable to their degradative action on circulating fibrinogen. Tissue plasminogen activator (tPA) was developed to achieve rapid and specific thrombolysis. 

One drawback of thrombolytic therapy is a high incidence of reocclusion. In a report using a canine model, inclusion of heparin [9005-49-6] (anticoagulant therapy) in the treatment prevented this side effect (158). The combination of aspirin [50-78-2] (antiplatelet therapy) and streptokinase (thrombolytic therapy) has also shown significant therapeutic advantages (78). Although additional work is needed to estabUsh the thrombolytic advantage of various combinations, preliminary results in this area indicate promise in terms of increased efficacy and reduced side effects. 

Streptokinase dissolves such fibrin clots, thereby permitting effective antibiotic therapy (201). 

The above sterilized medium was inoculated with 11 liters of seed inoculum having a bacterial count of approximately 20 billion per cc. The tank was fermented at 37°C without pH adjustment, aeration, or other modification for 14 hours at the end of which time 320 cc of 50% dextrose was added. After this the pH was adjusted to 7.0 at 15 minute intervals with 5.0 N sodium hydroxide. The volume of sodium hydroxide required for neutralization was noted and 115% of this volume of 50% dextrose solution added after each pH adjustment. At the end of about 8 hours the bacterial count had ceased to increase and the fermentation was terminated. At this time the fermentation medium contained approximately 1,000 units of streptokinase per cc. 

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