British Medical Bulletin (1978) VoL 34, No. 2, pp. 191-199
THROMBOLYTIC THERAPY
VVKakkar&MFSadly and urokinase form the mainstays of treatment; (ii) proteolytic enzymes causing direct proteolysis of fibrin within the thrombus; the main enzymes in this category are plasmin and brinase. a Plasminogen Activators
THROMBOLYTTC THERAPY
Urokinase Urokinase is a (3 globulin which activates plasminogen, probably by first-order kinetics. In 1965, Lesuk et al. prepared crystalline urokinase, which was shown to be a single porypeptide chain (mol. wt. 54000). In 1966, White et al. were able to isolate two types of urokinase, S t and S?. Sj (mol. wt. 31600) was more active, having a specific activity of 218000units/mg protein; S2 (mol. wt. 54000) did not appear to dissociate into smaller molecules. Recent data have suggested that the high-molecular-weight form of the enzyme is a precursor of the low-molecular-weight form and that proteolytic, possibly autocatalytic, cleavage may convert the former to the latter. The elucidation of the exact sequence of events in the activation has been difficult, since degraded forms of plasminogen are produced during activation by the generated plasmin. The rate of activation of Glu-plasminogen by catalytic amounts of urokinase is considerably slower than that of Lys-plasminogen (Claeys & Vermylen, 1974; Thorsen & Mullertz, 1974); this led to the suggestion that step iii (fig. 1) was also catalysed by urokinase and represented a rate-limiting first step in the reaction sequence (Walther et al. 1975). Evidence now suggests that urokinase is capable of reacting in steps i and ii only, and not of catalysing reactions iii and iv (fig. 1); this is thought to be due solely to the effect of the plasmin formed during activation (Violand & Castellino, 1976). Urokinase differs from natural plasma activator both in its enzyme specificity and in its antigenic characteristics (Aoki & von Kaulla, 1971); this suggests that it is secreted by the kidney rather than excreted as plasma activator. The overwhelming drawback for its clinical use on a large scale is the cost of the purified product. If cost were not important, urokinase would at present be the drug of choice for thrombolysis. It does not cause allergic reactions or stimulate antibody formation. Although it has been purified sufficiently to free it from significant thromboplastic contamination, the early stages of a urokinase infusion may be associated with a "transient coagulant phase" in which the formation of cryofibrinogen, shortening of the recalcifkation time and elevation of factor-VHI level are observed (Prentice et al. 1972). These phenomena were thought to be a direct consequence of plasminogen activation,
V V KAKKAR FRCS FRCSE M F SCULLY BSc PhD Thrombosis Research Unit King's College Hospital Medical School London Types of thrombolytic agents a Plasminogen activators b Proteolytic enzymes Mechanism of thromborysis a Plasminogen b Plasmin c Thrombus dissolution d Extent and rate of lysis Dosage and method of administration a Streptokinase b Urokinase Laboratory control of treatment Clinical studies a Deep-vein thrombosis b Pulmonary embolism c Acute myocardial infarction d Arterial thrombosis e Occlusion of retinal vessels / Other disorders Complications of thrombolytic therapy New approaches to thrombolytic therapy Concluding remarks References
Thrombolytic therapy has a unique advantage in the treatment of patients suffering from thrombotic disease, since it is capable of inducing the dissolution of intravascular fibrin and thus causing the reduction or elimination of thrombi. The rapidity of thrombus removal distinguishes this form of treatment from anticoagulant therapy, in which normal physiological processes are allowed to restore the obstructed circulation. By quickly removing the obstruction, it should be possible to reduce the morbidity, and perhaps the mortality, arising from acute thrombo-embolic episodes. A description of essential components of the fibrinolytic system has already been presented in detail in a previous issue of this Bulletin (Kernoff & McNicol, 1977). The present paper gives an account of recent advances in the understanding of basic mechanisms involved in the activation of the fibrinolytic system and of the current status of clinical applications of thrombolytic therapy, as well as a brief description of how newly gained knowledge may be useful in developing safer and more effective forms of treatment.
FIG. I. Peptide bond cleavage during activation of plasminogen
1 Types of ThrombolytJc Agents A number of thrombolytic agents have now been characterized and clinically tested with variable success; these may be classified into two groups according to their mechanism of action: (0 plasminogen activators, which cause the conversion of plasminogen to plasmin; in this group streptokinase
Activator >• CHu-plasmin
(i)
Lys-plasminogen
Activator • Lys-plasmin
(ii)
Glu-plasminogen Glu-plasmin
191 Vol. 34 No. 2
Glu-plasminogen
Plasmin
»• Lys-plasminogen
Plasmin ->• Lys-plasmin
(iii) Crv)
THROMBOLYTTC THERAPY since they occurred with concentrations of urokinase too low to have contained the necessary amount of thromboplastic activity. Recent studies have also shown that infusion of urokinase in low doses is associated with altered platelet behaviour, i.e., increased tendency for platelet aggregation in animals and in human volunteers (M Dubiel, V V Kakkar and M F Scully, unpublished observations, 1977). The main theoretical advantage of urokinase is that it has a greater preferential affinity for gel-phase plasminogen within a thrombus than for soluble-phase plasma plasminogen. Consequently, infusion of urokinase should cause less systemic hyperplasminaemia, with its attendant dangers of haemorrhagic complications.
VVKakkar&MFScully given systemically and does not exceed the inhibitor capacity, thrombolysis can be achieved without general proteolysis because of the apparent higher affinity of the substance for fibrin than for other blood proteins (Freedman & Roschlau, 1970). 2 Mechanism of Thrombolysis a Plasminogen Purified plasminogen is a single-chain monomeric protein with multiple isoelectric forms, pi 6.1-8.5 (Summaria et al. 1972; Wallen & Wiman, 1972). Of these, only the more acidic (pi 6.1-7.1) are obtained when plasminogen is isolated from plasma or serum in the presence of trypsin inhibitor (Rickli & Cuendet, 1971; Collen et al. 1972). The acidic isoenzymes have electrophoretic mobilities similar to those of plasminogen detected in unfractionated plasma (P globulin) (Walle"n & Wiman, 1972; Thorsen & Mullertz, 1974) and are probably identical to native circulating plasminogen (Collen et al. 1975). They have L-glutamic acid solely as the TVterminal amino acid (Wallen & Wiman, 1972; Summaria et al. 1973) and differ from the more basic forms of plasminogen isolated from Cohn fraction IH2.3, which have L-lysine as the JV-terminal amino acid (Wallen & Wiman, 1972; Summaria et al. 1973) and migrate as y globulins on electrophoresis (Summaria et al. 1969; Wallen & Wiman, 1972). These basic forms (now termed Lys-plasminogen) probably represent a partially degraded form of plasminogen, since they have a lower molecular weight than native plasminogen (Gluplasminogen), from which they can be prepared by treatment with plasmin (Qaeys et al. 1973). Recently, the amino acid primary sequence of plasminogen has been elucidated and the data show three distinct regions of interest (Claeys et al. 1976; Magnusson et al. 1977): (i) a small segment of about 75 amino acids at the iV-terminal end of the molecule which is released during the formation of Lys-plasminogen; (ii) the central region of the molecule containingfive"Kringle" structures each of which consists of about 80 amino acids formed into a loop by cystine bridges. These structures (two of which are also found in prothrombin) appear to contain the lysine-binding sites of plasminogen (Sottrup-Jensen et al. 1978) and may therefore be involved in the adsorption of plasminogen by fibrin (Wiman & Wallen, 1977); (iii) the C-terminal part of the molecule which has been shown to contain the serine residue essential for enzyme activity (Summaria et al. 1967). Although the mechanisms of activation of plasminogen by urokinase and streptokinase differ, it now appears that similar peptide bonds are cleaved (fig. 1). Firstly, a specific arginyl-valyl bond is broken in the interior of either Qlu- or Lys-plasminogen, which results in the formation of the two-chain plasmin molecule (Robbins et al. 1967; Robbins et al. 1973a; Wiman & Wallen, 1973). A secondary event is the cleavage of a lysyl bond in the N terminus of Glu-plasminogen or the heavy chain of Gluplasmin, with the release of an TV-terminal peptide (Robbins et al. 1973a; Wiman & Wallin, 1973). The hydrolysis of these bonds has been shown to be mediated by plasmin (Sodetz et al. 1974; Sodetz & Castellino, 1975; Summaria etal. 1975).
Streptokinase Streptokinase is a single-chain exocellular protein (mol. wt. 47000) produced by (J-haemolytic streptococci. Johnson & McCarty (1959) were first to show that under controlled biochemical conditions streptokinase was effective in lysing artificially induced intravascular thrombi in human volunteers. With improved purification techniques and the elimination of other streptococcal proteins, commercial preparations of the drug have reached a high degree of purity, and the pyrogenic and allergic side-effects that were encountered in the early stages of its development have largely been eliminated. Unlike urokinase, it does not appear to have a direct proteolytic action upon plasminogen. Instead a 1:1 stoicheiometric complex between streptokinase and plasminogen is formed (McClintock & Bell, 1971). Because of this interaction, a conformational alteration occurs in plasminogen, with the resulting development of an active site in the molecule. The active complex can then act inter- or intramolecularly, converting the plasminogen to plasmin. It has recently been demonstrated that during these events the streptokinase is proteolytically modified into several fragments of lower molecular weight (Reddy & Markus, 1972). The complex becomes capable of catalysing reactions i and ii and free plasmin catalyses reactions iii and iv (fig. 1), though the mechanism is complicated, since plasmin complexed to streptokinase is unable to cleave the TV-terminal peptide (Bajaj & Castellino, 1977). Streptokinase is by far the most widely used thrombolytic agent, mainly because it is a potent and effective enzyme, is relatively easy to prepare, and is inexpensive in comparison with urokinase. However, it has three main disadvantages: it shares with other streptococcal proteins the ability to act as an antigen in humans; its administration may cause pyrogenic or toxic reactions; and it is not highly selective in causing activation of plasminogen bound within a thrombus in preference to circulating plasminogen. b Proteolytic Enzymes Unlike the activators (urokinase and streptokinase), proteolytic enzymes (trypsin, plasmin and brinase) have a direct effect not only on fibrin but also on other substrates such as fibrinogen, prothrombin and factors V and VIII. Brinase, which is obtained from Aspergillus oryzae (Stefanini & Mario, 1958), has recently been claimed to be effective in lysing intravascular fibrin without undue bleeding. It is known that there are naturally occurring inhibitors against brinase in blood and the body tissues and that these are found in the axand a2-globulin fractions (Bergkvist, 1963). If brinase is
b Plasmin The proteolytic enzyme plasmin is a two-chain, disulphidelinked molecule which can be formed from both types of 192 fir. Afed. Bun. 1978
THROMBOLYTIC THERAPY
that plasmin formed therein is immediately bound to its inhibitor to form a circulating plasmin-antiplasmin complex. Because of the natural affinity of plasmin for fibrin, this complex dissociates on coming into contact with fibrin and liberates free plasmin in the vicinity of the thrombus. However, no evidence has ever been produced for penetration of plasmin into a clot or thrombus and, furthermore, the invitro dissolution of blood clots by pure solutions of plasmin has been shown to occur only very slowly. Although exogenous lysis may contribute, it is unlikely to be the sole mechanism of thrombus dissolution. The third and most recent hypothesis has been put forward by Chesterman et al. (1972), and is based on the selective binding of activator to fibrin. Plasminogen in the blood perfusing the thrombus is then activated in the presence of high levels of activator, and the plasmin which is generated is thus protected against the effects of circulating antiplasmins. It is possible that these different hypotheses contribute in varying degrees to the mechanism of thrombus dissolution.
plasminogen (Robbins et al. 1967; Wiman & Wallen, 1973). The larger chain (the heavy A chain) is derived from the JV-terminal part of the molecule, while the smaller chain (the light B chain) contains the reactive serine and histidine residues present in the active centre (Summaria et al. 1967; Robbins et al. 1973b). The N-terminal amino acid of plasmin is L-lysine (Lys-plasmin), though under suitable experimental conditions it is possible to isolate plasmin with L-glutamic acid as end-group (Sodetz et al. 1974). Plasmin has a trypsinlike specificity (Groskopf et al. 1969), hydrolysing proteins and peptides at arginyl and lysyl peptide bonds and also hydrolysing basic amino acid esters and amides. Plasmin can also activate factor XII (Hageman factor) and hence influence coagulation and the kallikrein-kinin and complement systems. It has the capacity to activate factor VII and components of complement directly, as well as to induce platelet aggregation and release reaction (Stormorken, 1975). It now appears that, of several protease inhibitors present in plasma, a new fast-reacting inhibitor highly specific for plasmin plays a major part in plasmin inhibition (Collen, 1976; Moroi & Aoki, 1976; Miillertz & Clemmensen, 1976). Only upon complete activation of plasminogen, when the fast-reacting antiplasmin component of plasma becomes saturated, do the other plasma plasmin inhibitors, Aetal. (1974) Br. Med. J. 1, 343-347 Urokinase Pulmonary Embolism Trial (1973) Circulation, suppl. no. II to vol. 47 Urokinase-Streptokinase Pulmonary Embolism Trial (1974) / . Am. Med. Assoc. 229,1606-1613 Verstraete M, Vermylen J, Amery A & Vermylea C (1966) Br. Med. J. 1, 454-456 Verstraete M, Vermylen J, Holleman W & Barlow G H (1977) Thromb. Res. 11, 227-236 Violand B N & Castellino F J (1976) / . Biol. Chem. 251, 39063912 Wallen P & Wiman B (1972) Biochim. Biophys. Acta, 257,122-134 Walther P J, Hill R L & McKee P A (1975) / . Biol. Chem. 250, 5926-5933 White W F, Barlow G H & Mozen M M (1966) Biochemistry {Easton) 5, 2160-2169 Wiman B & Wallen P (1973) Eur. J. Biochem. 36, 25-31 Wiman B & Wallen P (1977) Thromb. Res. 10, 213-222 Zekorn D (1967) In: Abstracts of the Symposium on Thrombotytic Therapy, Copenhagen, pp. 17-23 Ziemsla J M, Marchlewski S, Meissner A J, Rudowski W, Kolakowski L, Lopaciuk S & Latallo Z S (1978) Aktuel. Probl. Angiol. 37,187-190
Stormorken H (1975) Thromb. Diath. Haemorrh. 34, 378-385 Summaria L, Hsieh B, Groskopf W R, Robbins K C & Barlow G H (1967) / . Biol. Chem. 242, 5046-5052 Summaria L, Hsieh B, Groskopf W R &. Robbins K C (1969) Proc. Soc. Exp. Biol. Med. 130, 737-743 Summaria L, Aizadon L, Bernabe P & Robbins K C (1972) / . Biol. Chem. 247, 4691^*702
199 Vol. 34 No. 2
VVKakkar&MFScully
BRITISH MEDICAL BULLETIN
1978 VOL. 34 No. 2
International Journal of
Epidemiology Official Journal of the International Epidemiologlcal Association Theobject of this quarterly journal, nowln its seventh year of publication, Is to encourage exchange of ideas among those involved In the research, teaching and application of epidemiology in different countries, and to promote an international attitude to epidemiologlcal problems. The Journal publishes original articles, reviews of advances In method, occasional comments and hypotheses, as well as book reviews, letters and a W H O publications list. Contributions from all interested in the field are welcome. Editor: Professor A E Bennett, Department of Clinical Epidemiology and Social Medicine, St George's Hospital Medical School, Cranmer Terrace, London SW17 ORE, England. Published by Oxford University Press. Subscription enquiries to the Journals Manager, Oxford University Press, Press Road, Neasden, London NWIO ODD. Annual subscription: Members of IEA: USA $30.00 or equivalent. Single copies: £3.00 (USA $8.25). Non-members of IEA: £18.00 (USA $42.00). Single copies: £6.00 (USA $14.00).
The Quarterly Journal of Medicine Official Organ of the Association for Physicians of Great Britain and Ireland One of the leading medical journals published in the UJC. (Established 1907, New Series 1932). It covers the whole field of medicine but gives some emphasis to internal medicine. Its aim is to report advances in both diagnosis and treatment, and many of its papers are concerned with the development of growing points of current interest. The basic orientation is clinical, but the journal welcomes contributions with a patho-physiological approach. In the face of the present tendency to fragment the subject, The Quarterly Journal of Medicine attempts to prpvide general physicians with a synoptic view. Many of the contributions provide a desirable link between clinical and basic science. In addition to original papers, the journal also publishes occasional requested review articles on subjects of special interest. Quarterly, 1978 prices: £22.00 (UK £17.50,US $44.00) p.a. Single issues £6.00 ($13.50).
Oxford University Press Journals Press Road, Neasden, London NW10 ODD
200
Br. Med. Bull. 1978
THROMBOLYTIC THERAPY
VVKakkar&MFSadly and urokinase form the mainstays of treatment; (ii) proteolytic enzymes causing direct proteolysis of fibrin within the thrombus; the main enzymes in this category are plasmin and brinase. a Plasminogen Activators
THROMBOLYTTC THERAPY
Urokinase Urokinase is a (3 globulin which activates plasminogen, probably by first-order kinetics. In 1965, Lesuk et al. prepared crystalline urokinase, which was shown to be a single porypeptide chain (mol. wt. 54000). In 1966, White et al. were able to isolate two types of urokinase, S t and S?. Sj (mol. wt. 31600) was more active, having a specific activity of 218000units/mg protein; S2 (mol. wt. 54000) did not appear to dissociate into smaller molecules. Recent data have suggested that the high-molecular-weight form of the enzyme is a precursor of the low-molecular-weight form and that proteolytic, possibly autocatalytic, cleavage may convert the former to the latter. The elucidation of the exact sequence of events in the activation has been difficult, since degraded forms of plasminogen are produced during activation by the generated plasmin. The rate of activation of Glu-plasminogen by catalytic amounts of urokinase is considerably slower than that of Lys-plasminogen (Claeys & Vermylen, 1974; Thorsen & Mullertz, 1974); this led to the suggestion that step iii (fig. 1) was also catalysed by urokinase and represented a rate-limiting first step in the reaction sequence (Walther et al. 1975). Evidence now suggests that urokinase is capable of reacting in steps i and ii only, and not of catalysing reactions iii and iv (fig. 1); this is thought to be due solely to the effect of the plasmin formed during activation (Violand & Castellino, 1976). Urokinase differs from natural plasma activator both in its enzyme specificity and in its antigenic characteristics (Aoki & von Kaulla, 1971); this suggests that it is secreted by the kidney rather than excreted as plasma activator. The overwhelming drawback for its clinical use on a large scale is the cost of the purified product. If cost were not important, urokinase would at present be the drug of choice for thrombolysis. It does not cause allergic reactions or stimulate antibody formation. Although it has been purified sufficiently to free it from significant thromboplastic contamination, the early stages of a urokinase infusion may be associated with a "transient coagulant phase" in which the formation of cryofibrinogen, shortening of the recalcifkation time and elevation of factor-VHI level are observed (Prentice et al. 1972). These phenomena were thought to be a direct consequence of plasminogen activation,
V V KAKKAR FRCS FRCSE M F SCULLY BSc PhD Thrombosis Research Unit King's College Hospital Medical School London Types of thrombolytic agents a Plasminogen activators b Proteolytic enzymes Mechanism of thromborysis a Plasminogen b Plasmin c Thrombus dissolution d Extent and rate of lysis Dosage and method of administration a Streptokinase b Urokinase Laboratory control of treatment Clinical studies a Deep-vein thrombosis b Pulmonary embolism c Acute myocardial infarction d Arterial thrombosis e Occlusion of retinal vessels / Other disorders Complications of thrombolytic therapy New approaches to thrombolytic therapy Concluding remarks References
Thrombolytic therapy has a unique advantage in the treatment of patients suffering from thrombotic disease, since it is capable of inducing the dissolution of intravascular fibrin and thus causing the reduction or elimination of thrombi. The rapidity of thrombus removal distinguishes this form of treatment from anticoagulant therapy, in which normal physiological processes are allowed to restore the obstructed circulation. By quickly removing the obstruction, it should be possible to reduce the morbidity, and perhaps the mortality, arising from acute thrombo-embolic episodes. A description of essential components of the fibrinolytic system has already been presented in detail in a previous issue of this Bulletin (Kernoff & McNicol, 1977). The present paper gives an account of recent advances in the understanding of basic mechanisms involved in the activation of the fibrinolytic system and of the current status of clinical applications of thrombolytic therapy, as well as a brief description of how newly gained knowledge may be useful in developing safer and more effective forms of treatment.
FIG. I. Peptide bond cleavage during activation of plasminogen
1 Types of ThrombolytJc Agents A number of thrombolytic agents have now been characterized and clinically tested with variable success; these may be classified into two groups according to their mechanism of action: (0 plasminogen activators, which cause the conversion of plasminogen to plasmin; in this group streptokinase
Activator >• CHu-plasmin
(i)
Lys-plasminogen
Activator • Lys-plasmin
(ii)
Glu-plasminogen Glu-plasmin
191 Vol. 34 No. 2
Glu-plasminogen
Plasmin
»• Lys-plasminogen
Plasmin ->• Lys-plasmin
(iii) Crv)
THROMBOLYTTC THERAPY since they occurred with concentrations of urokinase too low to have contained the necessary amount of thromboplastic activity. Recent studies have also shown that infusion of urokinase in low doses is associated with altered platelet behaviour, i.e., increased tendency for platelet aggregation in animals and in human volunteers (M Dubiel, V V Kakkar and M F Scully, unpublished observations, 1977). The main theoretical advantage of urokinase is that it has a greater preferential affinity for gel-phase plasminogen within a thrombus than for soluble-phase plasma plasminogen. Consequently, infusion of urokinase should cause less systemic hyperplasminaemia, with its attendant dangers of haemorrhagic complications.
VVKakkar&MFScully given systemically and does not exceed the inhibitor capacity, thrombolysis can be achieved without general proteolysis because of the apparent higher affinity of the substance for fibrin than for other blood proteins (Freedman & Roschlau, 1970). 2 Mechanism of Thrombolysis a Plasminogen Purified plasminogen is a single-chain monomeric protein with multiple isoelectric forms, pi 6.1-8.5 (Summaria et al. 1972; Wallen & Wiman, 1972). Of these, only the more acidic (pi 6.1-7.1) are obtained when plasminogen is isolated from plasma or serum in the presence of trypsin inhibitor (Rickli & Cuendet, 1971; Collen et al. 1972). The acidic isoenzymes have electrophoretic mobilities similar to those of plasminogen detected in unfractionated plasma (P globulin) (Walle"n & Wiman, 1972; Thorsen & Mullertz, 1974) and are probably identical to native circulating plasminogen (Collen et al. 1975). They have L-glutamic acid solely as the TVterminal amino acid (Wallen & Wiman, 1972; Summaria et al. 1973) and differ from the more basic forms of plasminogen isolated from Cohn fraction IH2.3, which have L-lysine as the JV-terminal amino acid (Wallen & Wiman, 1972; Summaria et al. 1973) and migrate as y globulins on electrophoresis (Summaria et al. 1969; Wallen & Wiman, 1972). These basic forms (now termed Lys-plasminogen) probably represent a partially degraded form of plasminogen, since they have a lower molecular weight than native plasminogen (Gluplasminogen), from which they can be prepared by treatment with plasmin (Qaeys et al. 1973). Recently, the amino acid primary sequence of plasminogen has been elucidated and the data show three distinct regions of interest (Claeys et al. 1976; Magnusson et al. 1977): (i) a small segment of about 75 amino acids at the iV-terminal end of the molecule which is released during the formation of Lys-plasminogen; (ii) the central region of the molecule containingfive"Kringle" structures each of which consists of about 80 amino acids formed into a loop by cystine bridges. These structures (two of which are also found in prothrombin) appear to contain the lysine-binding sites of plasminogen (Sottrup-Jensen et al. 1978) and may therefore be involved in the adsorption of plasminogen by fibrin (Wiman & Wallen, 1977); (iii) the C-terminal part of the molecule which has been shown to contain the serine residue essential for enzyme activity (Summaria et al. 1967). Although the mechanisms of activation of plasminogen by urokinase and streptokinase differ, it now appears that similar peptide bonds are cleaved (fig. 1). Firstly, a specific arginyl-valyl bond is broken in the interior of either Qlu- or Lys-plasminogen, which results in the formation of the two-chain plasmin molecule (Robbins et al. 1967; Robbins et al. 1973a; Wiman & Wallen, 1973). A secondary event is the cleavage of a lysyl bond in the N terminus of Glu-plasminogen or the heavy chain of Gluplasmin, with the release of an TV-terminal peptide (Robbins et al. 1973a; Wiman & Wallin, 1973). The hydrolysis of these bonds has been shown to be mediated by plasmin (Sodetz et al. 1974; Sodetz & Castellino, 1975; Summaria etal. 1975).
Streptokinase Streptokinase is a single-chain exocellular protein (mol. wt. 47000) produced by (J-haemolytic streptococci. Johnson & McCarty (1959) were first to show that under controlled biochemical conditions streptokinase was effective in lysing artificially induced intravascular thrombi in human volunteers. With improved purification techniques and the elimination of other streptococcal proteins, commercial preparations of the drug have reached a high degree of purity, and the pyrogenic and allergic side-effects that were encountered in the early stages of its development have largely been eliminated. Unlike urokinase, it does not appear to have a direct proteolytic action upon plasminogen. Instead a 1:1 stoicheiometric complex between streptokinase and plasminogen is formed (McClintock & Bell, 1971). Because of this interaction, a conformational alteration occurs in plasminogen, with the resulting development of an active site in the molecule. The active complex can then act inter- or intramolecularly, converting the plasminogen to plasmin. It has recently been demonstrated that during these events the streptokinase is proteolytically modified into several fragments of lower molecular weight (Reddy & Markus, 1972). The complex becomes capable of catalysing reactions i and ii and free plasmin catalyses reactions iii and iv (fig. 1), though the mechanism is complicated, since plasmin complexed to streptokinase is unable to cleave the TV-terminal peptide (Bajaj & Castellino, 1977). Streptokinase is by far the most widely used thrombolytic agent, mainly because it is a potent and effective enzyme, is relatively easy to prepare, and is inexpensive in comparison with urokinase. However, it has three main disadvantages: it shares with other streptococcal proteins the ability to act as an antigen in humans; its administration may cause pyrogenic or toxic reactions; and it is not highly selective in causing activation of plasminogen bound within a thrombus in preference to circulating plasminogen. b Proteolytic Enzymes Unlike the activators (urokinase and streptokinase), proteolytic enzymes (trypsin, plasmin and brinase) have a direct effect not only on fibrin but also on other substrates such as fibrinogen, prothrombin and factors V and VIII. Brinase, which is obtained from Aspergillus oryzae (Stefanini & Mario, 1958), has recently been claimed to be effective in lysing intravascular fibrin without undue bleeding. It is known that there are naturally occurring inhibitors against brinase in blood and the body tissues and that these are found in the axand a2-globulin fractions (Bergkvist, 1963). If brinase is
b Plasmin The proteolytic enzyme plasmin is a two-chain, disulphidelinked molecule which can be formed from both types of 192 fir. Afed. Bun. 1978
THROMBOLYTIC THERAPY
that plasmin formed therein is immediately bound to its inhibitor to form a circulating plasmin-antiplasmin complex. Because of the natural affinity of plasmin for fibrin, this complex dissociates on coming into contact with fibrin and liberates free plasmin in the vicinity of the thrombus. However, no evidence has ever been produced for penetration of plasmin into a clot or thrombus and, furthermore, the invitro dissolution of blood clots by pure solutions of plasmin has been shown to occur only very slowly. Although exogenous lysis may contribute, it is unlikely to be the sole mechanism of thrombus dissolution. The third and most recent hypothesis has been put forward by Chesterman et al. (1972), and is based on the selective binding of activator to fibrin. Plasminogen in the blood perfusing the thrombus is then activated in the presence of high levels of activator, and the plasmin which is generated is thus protected against the effects of circulating antiplasmins. It is possible that these different hypotheses contribute in varying degrees to the mechanism of thrombus dissolution.
plasminogen (Robbins et al. 1967; Wiman & Wallen, 1973). The larger chain (the heavy A chain) is derived from the JV-terminal part of the molecule, while the smaller chain (the light B chain) contains the reactive serine and histidine residues present in the active centre (Summaria et al. 1967; Robbins et al. 1973b). The N-terminal amino acid of plasmin is L-lysine (Lys-plasmin), though under suitable experimental conditions it is possible to isolate plasmin with L-glutamic acid as end-group (Sodetz et al. 1974). Plasmin has a trypsinlike specificity (Groskopf et al. 1969), hydrolysing proteins and peptides at arginyl and lysyl peptide bonds and also hydrolysing basic amino acid esters and amides. Plasmin can also activate factor XII (Hageman factor) and hence influence coagulation and the kallikrein-kinin and complement systems. It has the capacity to activate factor VII and components of complement directly, as well as to induce platelet aggregation and release reaction (Stormorken, 1975). It now appears that, of several protease inhibitors present in plasma, a new fast-reacting inhibitor highly specific for plasmin plays a major part in plasmin inhibition (Collen, 1976; Moroi & Aoki, 1976; Miillertz & Clemmensen, 1976). Only upon complete activation of plasminogen, when the fast-reacting antiplasmin component of plasma becomes saturated, do the other plasma plasmin inhibitors, Aetal. (1974) Br. Med. J. 1, 343-347 Urokinase Pulmonary Embolism Trial (1973) Circulation, suppl. no. II to vol. 47 Urokinase-Streptokinase Pulmonary Embolism Trial (1974) / . Am. Med. Assoc. 229,1606-1613 Verstraete M, Vermylen J, Amery A & Vermylea C (1966) Br. Med. J. 1, 454-456 Verstraete M, Vermylen J, Holleman W & Barlow G H (1977) Thromb. Res. 11, 227-236 Violand B N & Castellino F J (1976) / . Biol. Chem. 251, 39063912 Wallen P & Wiman B (1972) Biochim. Biophys. Acta, 257,122-134 Walther P J, Hill R L & McKee P A (1975) / . Biol. Chem. 250, 5926-5933 White W F, Barlow G H & Mozen M M (1966) Biochemistry {Easton) 5, 2160-2169 Wiman B & Wallen P (1973) Eur. J. Biochem. 36, 25-31 Wiman B & Wallen P (1977) Thromb. Res. 10, 213-222 Zekorn D (1967) In: Abstracts of the Symposium on Thrombotytic Therapy, Copenhagen, pp. 17-23 Ziemsla J M, Marchlewski S, Meissner A J, Rudowski W, Kolakowski L, Lopaciuk S & Latallo Z S (1978) Aktuel. Probl. Angiol. 37,187-190
Stormorken H (1975) Thromb. Diath. Haemorrh. 34, 378-385 Summaria L, Hsieh B, Groskopf W R, Robbins K C & Barlow G H (1967) / . Biol. Chem. 242, 5046-5052 Summaria L, Hsieh B, Groskopf W R &. Robbins K C (1969) Proc. Soc. Exp. Biol. Med. 130, 737-743 Summaria L, Aizadon L, Bernabe P & Robbins K C (1972) / . Biol. Chem. 247, 4691^*702
199 Vol. 34 No. 2
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BRITISH MEDICAL BULLETIN
1978 VOL. 34 No. 2
International Journal of
Epidemiology Official Journal of the International Epidemiologlcal Association Theobject of this quarterly journal, nowln its seventh year of publication, Is to encourage exchange of ideas among those involved In the research, teaching and application of epidemiology in different countries, and to promote an international attitude to epidemiologlcal problems. The Journal publishes original articles, reviews of advances In method, occasional comments and hypotheses, as well as book reviews, letters and a W H O publications list. Contributions from all interested in the field are welcome. Editor: Professor A E Bennett, Department of Clinical Epidemiology and Social Medicine, St George's Hospital Medical School, Cranmer Terrace, London SW17 ORE, England. Published by Oxford University Press. Subscription enquiries to the Journals Manager, Oxford University Press, Press Road, Neasden, London NWIO ODD. Annual subscription: Members of IEA: USA $30.00 or equivalent. Single copies: £3.00 (USA $8.25). Non-members of IEA: £18.00 (USA $42.00). Single copies: £6.00 (USA $14.00).
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