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Biochemistry Aug 1995Streptokinase is a plasminogen activator widely used to treat patients with myocardial infarction. However, streptokinase is not a protease, and must first bind and...
Streptokinase is a plasminogen activator widely used to treat patients with myocardial infarction. However, streptokinase is not a protease, and must first bind and interact with plasminogen to form an enzymatic complex. By measuring the binding of recombinant streptokinase fragments to plasminogen, we have sought, first, to identify a plasminogen binding region in streptokinase and, second, to explore the relation between binding (via this region) and the generation of a functional streptokinase--plasminogen activator complex. Recombinant streptokinase bound in a saturable and specific manner to human Glu-plasminogen with a dissociation constant of 4.2 x 10(-10) M. Recombinant streptokinase fragments spanning amino acids 1-127 and 1-253 could not be shown to bind to Glu-plasminogen, whereas fragments spanning amino acids 1-352, 120-352, and 244-414 bound tightly to plasminogen and each fragment completely inhibited the binding of full-length streptokinase to plasminogen. Although these latter streptokinase fragments formed a complex with plasminogen, enzymatic assays indicated that none of them was capable of generating an active site. When the streptokinase region shared by these three fragments, spanning residues 244-352, was expressed, it also bound plasminogen and competitively inhibited the formation of a functional plasminogen activator complex by full-length streptokinase. Taken together, these data indicate that streptokinase binds to plasminogen with high affinity, that a primary binding region for plasminogen is located within amino acids 244-352, and that binding via this region is necessary for the generation of a functional plasminogen activator complex.
Topics: Base Sequence; Binding Sites; Cloning, Molecular; Molecular Sequence Data; Oligodeoxyribonucleotides; Peptide Fragments; Plasminogen; Plasminogen Activators; Streptokinase
PubMed: 7640282
DOI: 10.1021/bi00032a021 -
Australian and New Zealand Journal of... Jun 1999Thrombolytic drugs reduce mortality from myocardial infarction and research is focused on newer drugs with improved clinical efficacy. The ideal thrombolytic drug should...
Thrombolytic drugs reduce mortality from myocardial infarction and research is focused on newer drugs with improved clinical efficacy. The ideal thrombolytic drug should be effective in dissolving fresh and older thrombi, be rapid in its action with complete dissolution of the thrombus, be able to be given as a bolus and be safe without hypotensive or allergic or immunogenic reactions. The types of agents being developed fall into five broad categories: * mutants or variants of single chain urokinase type plasminogen activator; * mutants or variants of tissue type plasminogen activator; * recombinant chimaeric plasminogen activators; * conjugates of plasminogen activators and anti-fibrin monoclonal antibodies; * compounds derived from haemophagous animals. Within the mutant and variant groups there may be single point mutations which increase the half life or deletion of various amino acid sequences to increase resistance to plasma protease inhibitors or cause more selective binding to fibrin. The recombinant chimaeric plasminogen activators are designer drugs where the kringle regions, the growth hormone domain region, the finger domain region and the serine protease part of the molecules are all cleaved and recombined in various ways to improve potency. The conjugates of plasminogen activators and anti-fibrin and monoclonal antibodies improve the targeting of the agent to fibrin clot. Monoclonal antibodies are commonly used against the seven aminoterminal residues of the beta chain of fibrin. These are cross linked and bound to plasminogen activators. A variety of substances from haemophageous animals are currently being studied including salivary plasminogen activator from vampire bats, venom from southern copperhead snakes and staphylokinase from bacteria. Currently available thrombolytic agents have many limitations, but the novel thrombolytic agents have yet to be tested in clinical trials. With few exceptions, they all act via the plasminogen system and it may be in the future that combinations of anti-platelet and thrombolytic agents may prove to be more efficacious than thrombolytics and aspirin alone.
Topics: Antibodies, Monoclonal; Fibrinolytic Agents; Humans; Plasminogen Activators; Point Mutation; Recombinant Proteins; Tissue Plasminogen Activator
PubMed: 10868516
DOI: 10.1111/j.1445-5994.1999.tb00739.x -
The British Journal of Dermatology Sep 1985Extracts of scale from four patients with psoriasis all contained plasminogen activator. All plasminogen activator activity was of the tissue-type (t-PA) as judged from...
Extracts of scale from four patients with psoriasis all contained plasminogen activator. All plasminogen activator activity was of the tissue-type (t-PA) as judged from the following criteria: all enzyme activity was inhibited by a monoclonal antibody against human t-PA, while there was no measurable inhibition by a monoclonal antibody against human urokinase-type plasminogen activator (u-PA); as determined by zymography the extracts contained one type of plasminogen activator which had an electrophoretic mobility identical to that of purified t-PA; this plasminogen activator was removed by passage through a Sepharose column coupled with a monoclonal antibody against t-PA, but not by passage through a column coupled with a monoclonal antibody against u-PA. On a molar basis the detection limit for u-PA was 10% of that of t-PA present.
Topics: Antibodies, Monoclonal; Electrophoresis, Polyacrylamide Gel; Humans; Plasminogen Activators; Psoriasis; Skin; Tissue Plasminogen Activator; Urokinase-Type Plasminogen Activator
PubMed: 3933538
DOI: 10.1111/j.1365-2133.1985.tb02076.x -
Arteriosclerosis (Dallas, Tex.) 1984Tissue-type plasminogen activator (t-PA), purified from the culture fluid of a stable human melanoma cell line, is a serine protease, different from urokinase, with a... (Comparative Study)
Comparative Study Review
Tissue-type plasminogen activator (t-PA), purified from the culture fluid of a stable human melanoma cell line, is a serine protease, different from urokinase, with a molecular weight of about 70,000. It is composed of one polypeptide chain, which is converted to a two-chain molecule by limited plasmic action. Activation of plasminogen to plasmin occurs by cleavage of the Arg 560-Val 561 peptide bond. Kinetic analysis has shown that the activation obeys Michaelis-Menten kinetics and that the presence of fibrin strikingly enhances the activation rate by increasing the affinity of plasminogen for fibrin-bound t-PA. The directed action of plasmin toward fibrin in vivo, might be explained by the low Michaelis constant in the presence of fibrin (0.16 microM), which allows efficient plasminogen activation on a fibrin clot, while its high value in the absence of fibrin (65 microM) prevents efficient activation in plasma. Plasmin formed on the fibrin surface would then be protected from rapid inactivation by alpha 2-antiplasmin. An important consequence of this molecular model for physiological fibrinolysis is that specific thrombolysis is only expected with the use of a specific plasminogen activator, which confines activation to the fibrin surface. Studies on the thrombolytic properties of purified t-PA in various animal species and in humans have revealed a higher specific thrombolytic activity than urokinase. Thrombolysis could be achieved without causing significant plasminogen activation, alpha 2-antiplasmin consumption, or fibrinogen breakdown. Alternatively, pro-urokinase, the zymogen precursor of urokinase, also displays a certain degree of fibrin specificity. Its mechanism of action and potential therapeutic value remain to be established.
Topics: Animals; Chemical Phenomena; Chemistry, Physical; Coronary Disease; Dogs; Drug Evaluation, Preclinical; Enzyme Precursors; Fibrinolysis; Fibrinolytic Agents; Humans; In Vitro Techniques; Plasminogen Activators; Plasminogen Inactivators; Pulmonary Embolism; Rabbits; Structure-Activity Relationship; Thrombophlebitis; Urokinase-Type Plasminogen Activator
PubMed: 6439177
DOI: 10.1161/01.atv.4.6.579 -
Recent Patents on Cardiovascular Drug... Jan 2008Urokinase (UK) [EC 3.4.99.26] is a serine protease that activates plasminogen to plasmin, which in turn degrades fibrin clots. Hence, UK finds its value as an important... (Review)
Review
Urokinase (UK) [EC 3.4.99.26] is a serine protease that activates plasminogen to plasmin, which in turn degrades fibrin clots. Hence, UK finds its value as an important anti-thromboembolic drug. Plasmin has diverse physiological roles apart from its fibrinolytic role in the regulation of blood clotting. It has been implicated in complement activation, cell migration, wound healing, and generation of localized extracellular proteolysis during tissue remodelling, pro-hormone conversion, carcinogenesis and neoplasia. Among the plasminogen activators, UK provides a superior alternative for the simple reasons of its being more potent as compared to tissue-plasminogen activator and non-antigenic by virtue of its human origin unlike streptokinase. Based on these observations, UK is a very popular cardiovascular agent. Hence, UK, as one of the most potent plasminogen activators is attracting a great deal of attention. We will summarize recent patents related to the occurrence, mechanism of action, structure and function, physico-chemical properties, in vitro production, cloning and expression, purification and applications of UK.
Topics: Animals; Cardiovascular Agents; Cardiovascular Diseases; Chemical Phenomena; Chemistry, Physical; Humans; Patents as Topic; Plasminogen Activators; Urokinase-Type Plasminogen Activator
PubMed: 18221128
DOI: 10.2174/157489008783331670 -
The New England Journal of Medicine Oct 1997
Topics: Humans; Infusions, Intravenous; Injections, Intravenous; Myocardial Infarction; Plasminogen Activators; Recombinant Proteins; Therapeutic Equivalency; Tissue Plasminogen Activator
PubMed: 9329939
DOI: 10.1056/NEJM199710163371610 -
Behring Institute Mitteilungen Aug 1983Fibrinolysis in the blood seems to be regulated by specific molecular interactions between plasminogen activator, plasmin(ogen), fibrin and alpha 2-antiplasmin....
Fibrinolysis in the blood seems to be regulated by specific molecular interactions between plasminogen activator, plasmin(ogen), fibrin and alpha 2-antiplasmin. Plasmin(ogen) contains structures, called lysine-binding sites, which mediate its interaction with fibrin and with alpha 2-antiplasmin. In plasma normally no systemic plasminogen activation by plasminogen activator occurs and plasmin, if formed, is efficiently neutralized by alpha 2-antiplasmin. When fibrin is formed in plasma a small amount of plasminogen is bound via its lysine-binding sites. Plasminogen activator present or released in the blood is strongly adsorbed to the fibrin and activates bound plasminogen in situ. The formed plasmin, which remains transiently complexed to fibrin, both by its lysine-binding site(s) and active center, is only slowly inactivated by alpha 2-antiplasmin, while plasmin which is released from digested fibrin is rapidly and irreversibly neutralized. The fibrinolytic process thus seems to be triggered by and confined to fibrin. An important consequence of this molecular model for fibrinolysis is that specific thrombolysis is only expected with the use of a specific activator, like the physiological extrinsic plasminogen activator, which confines the activation of plasminogen to the fibrin surface. Recent in vitro and in vivo studies have confirmed that the extrinsic plasminogen activator (tissue-type) might constitute a superior thrombolytic agent compared to urokinase or streptokinase.
Topics: Antifibrinolytic Agents; Fibrinolysin; Fibrinolysis; Humans; Plasminogen Activators
PubMed: 6236789
DOI: No ID Found -
Wiadomosci Lekarskie (Warsaw, Poland :... Apr 1989In the light of a survey of the pertinent literature the results of tissue plasminogen activator and pro-urokinase++ use in the thrombolytic treatment of acute... (Comparative Study)
Comparative Study Review
In the light of a survey of the pertinent literature the results of tissue plasminogen activator and pro-urokinase++ use in the thrombolytic treatment of acute myocardial infarction are discussed.
Topics: Drug Evaluation; Fibrinolytic Agents; Humans; Myocardial Infarction; Plasminogen Activators; Tissue Plasminogen Activator; Urokinase-Type Plasminogen Activator
PubMed: 2516932
DOI: No ID Found -
Archives of Ophthalmology (Chicago,... Jan 2002
Topics: Humans; Plasminogen Activators; Retinal Hemorrhage; Thrombolytic Therapy; Tissue Plasminogen Activator
PubMed: 11786077
DOI: No ID Found -
Blood Coagulation & Fibrinolysis : An... Jul 2000There has been a recent decline in interest in fibrinolysis, suggesting that its physiological basis is sufficiently understood and that therapeutic thrombolysis has... (Review)
Review
There has been a recent decline in interest in fibrinolysis, suggesting that its physiological basis is sufficiently understood and that therapeutic thrombolysis has reached its limit. The importance of the subject has not diminished since cardiovascular disease is now a leading health problem even in developing countries. Certain highlights and inconsistencies are reviewed. The clinical trials of tissue plasminogen activator (t-PA) revealed a major discrepancy between its fibrinolytic efficacy and its clinical benefit (the 't-PA paradox') that is unexplained. Dose-finding studies also showed that the fibrinolytic efficacy of t-PA required significant nonspecific plasminogen activation. Furthermore, the longstanding belief that t-PA is responsible for physiological fibrinolysis and urokinase-type PA (u-PA) for pericellular plasminogen activation is belied by extensive experimental animal data, but these findings have had little impact on traditional thinking. As a result, the mechanisms responsible for the u-PA paradigm of fibrinolysis have received little attention. Clinical experience with pro-u-PA remains limited and most clinical trials have used infusion rates at which pro-u-PA is largely converted systemically to urokinase. This is due to the unanticipated instability of pro-u-PA in plasma at pharmacological concentrations. Insufficient understanding of basic mechanisms of fibrinolysis has handicapped the design of chimeric or mutant activators. It is submitted that physiological fibrinolysis remains to be better defined, and that it is premature to conclude that therapeutic thrombolysis will be inevitably accompanied by side effects that undermine this method of inducing reperfusion.
Topics: Fibrinolysis; Humans; Mutagenesis; Plasminogen Activators; Streptokinase; Tissue Plasminogen Activator; Urokinase-Type Plasminogen Activator
PubMed: 10937799
DOI: 10.1097/00001721-200007000-00001