33 Volume 64 September 1971 923

Section of Medicine President J G Scadding FRCP

Meeting November 24 1970

Dr G R Fearnley fibrinolytic activity plays its part. Natural (Gloucestershire Royal Hospital, activator is unstable in fluid blood outside the Gloucester) body, and has a half-life of about 20 minutes in anticoagulated blood at room temperature The Fibrinolytic System (Fearnley et al. 1952). In order to demonstrate and to measure the spontaneous fibrinolytic Fibrinolysis may be defined as the enzymatic activity of blood two conditions are necessary. liquefaction of blood or fibrin clot, and its First, all manipulations between obtaining blood postulated function in vivo is the removal of and setting up the test must be carried out at unwanted fibrin deposits or fibrin once it has low temperature in order to preserve the activator, served its purpose in, for example, healing and which is stabilized by adsorption to fibrin. inflammation. Fibrinolysis may also be regarded Second, the inhibitory effect of antiplasmin as the physiological antithesis of blood coagu- must be either removed or diminished by dilution lation, and blood may be considered as a tissue of the blood sample before the clots are formed. of the body which can exist in the fluid or the The dilute blood clot lysis-time (Fearnley gel phase dependent upon the balance between et al. 1957) consists of the time required for lysis fibrinolysis and . of a 1 in 10 dilution of blood in phosphate buffer Plasma contains an inactive enzyme precursor, clotted with thrombin and incubated at 37°C. plasminogen, which is converted to the active Clearly lysis-time and fibrinolytic activity are proteolytic enzyme, , by a number of inversely related, so that a long lysis-time means activators, of which the most studied has been low fibrinolytic activity and a short lysis-time the foreign protein streptokinase. The discovery means high activity. In our hands the normal of streptokinase, and subsequently of the limit of the dilute blood clot lysis-time is between natural urinary activator, urokinase, has led 11 and 7 hours. Long lysis-times - up to 24 hours to the concept of thrombolytic therapy, that is - are obtained photographically. Another method the dissolution of formed thrombi in vivo by the ofmeasurement, in which antiplasmin is removed, injection of such exogenous plasminogen acti- is the euglobulin lysis-time (von Kaulla 1963) vators. Normally a plasmin inhibitor, antiplasmin, in which the euglobulin fraction of plasma is neutralizes any plasmin liberated in fluid blood. precipitated, suspended in buffer, clotted with It is now recognized that blood has spontaneous thrombin and observed for lysis at 37°C. fibrinolytic activity due to a natural activator It may reasonably be asked what could be of plasminogen (Feamley & Tweed 1953) which the function in vivo, if any, of a factor which present evidence suggests is secreted mainly requires dilution for its demonstration in vitro. from the endothelium or vasa vasorum of veins The answer may lie in adsorption, since natural into the circulation (Todd 1959, Clarke et al. activator is adsorbed and stabilized by fibrin clot 1960, Chakrabarti et al. 1963). The endothelium (Fearnley 1953). This being so, it is possible to of arteries, especially the larger arteries, is conceive that an amount of activator insufficient relatively deficient in plasminogen activator; to lyse native blood clot in vitro could be con- and it may be that, whilst contiguous fibrinolysis centrated at sites of fibrin deposition on arterial from venous walls is of importance in preserving walls by adsorption from circulating blood. venous patency, on the arterial side blood This would then convert the contained plasmin-

52 924 Proc. roy. Soc. Med. Volume 64 September 1971 34 ogen to plasmin with rapid lysis of the deposits coronary artery disease plus defective fibrinolysis (Fearnley 1961). Fig 1 depicts such a function carries a poor prognosis, so that such patients schematically. A mural thrombus (a) and a are eliminated by natural selection (Chakrabarti retracted thrombus (b) will be susceptible to et al. 1968). fibrinolysis by adsorption; whereas an occlusive If natural fibrinolysis is of importance in the thrombus (c), because it halts the circulation, preservation of vascular patency, its enhancement will not be susceptible. Hence natural fibrinolysis by pharmacological means could provide a new may serve to clear unwanted fibrin deposits approachtotheprophylaxisof vascularocclusions. without impairing h2mostasis. Schor et al. (1970) have defined three separate approaches to the discovery offibrinolytic drugs - in vitro testing, in vivo testing, and combined in vivo/in vitro testing. von Kaulla (1970), using an in vitro test, has discovered several hundred - compounds which, when added to blood in the test-tube, will enhance its fibrinolytic activity by (a) lb) {e) diminishing the inhibitory effect of antiplasmin. Fig 1 Types ofthrombosis, and effect onfibrinolysis. There is, however, no guarantee that a substance In a mural thrombus (a) or a retracted thrombus (b), activator is availablefor absorption and concentration which does this in vitro will have a corresponding from circulating blood. In an occlusive thrombus (c), effect in vivo. the circulation ceases andactivator is not available. Schor has pioneered the combined in vivo/in Reproducedfrom Fearnley (1961) by kindpermission vitro method, and has discovered an analogue of quinidine which, when given to the rat by intra- If fibrinolysis plays a part in maintaining peritoneal injection, shortens the dilute blood vascular patency, a difference of fibrinolytic clot lysis-time as measured in vitro. He and others activity between patients with occlusive vascular have shown that this substance, Bisobrin, when disease and healthy controls might be expected. given intravenously, will lyse experimental Tlhis however, is not so easy to demonstrate as thrombi in rats and dogs (Schor et al. 1970), and one might think, since an unknown proportion of limited studies in patients suggest that it may the controls will have imminent or undisclosed prove to be a safe and effective synthetic throm- occlusive vascular disease. Nevertheless, in a bolytic agent (Ambrus et al. 1970). study of 107 male survivors of myocardial Our own studies have employed the in vivo infarction and 90 age-matched healthy controls method, that is the giving of drugs to patients and with normal electrocardiograms before and after the noting of their* effect on blood fibrinolytic exercise, fibrinolysis, as judged by a lysis-time activity. We have discovered a number of drugs of 7 hours or greater on two or more of three in common use which will do this but, in general, tests, was defective in 32% of the coronary resistence to the fibrinolytic effect of each has patients compared with 12% of the controls been found to develop within from a few weeks to (Table 1). When the figures are examined in a few months of treatment in most people. relation to age, it can be seen that the incidence However, some years ago we found that a combi- of defective fibrinolysis among the coronary nation of the anti-diabetic drug phenformin with patients declines sharply with age, much more the anabolic steroid ethyloestrenol, which is a sharply than among the controls, so that after progestogen and not an cestrogen as its name the age of 60 the incidence of low fibrinolytic might suggest, would produce a sustained increase activity is virtually the same in the two groups. of fibrinolytic activity in about 90 % of people in One possible explanation for this finding is that whom it is defective (Fearnley et al. 1967). In a much larger series, studied for 3 years or longer, we have found that the effect of phenformin plus Tabk I Incidence of low fibrinolytic activity in patients and controls ethyloestrenol on fibrinolysis is maintained. This (from Chakrabarti et al. 1968, by kind permission) combination of drugs also reduces serum chole- sterol and plasma even No. ofsubjects with blood lysis and, perhaps time of > 7 h mnat least two more important, produces a sustained reduction out ofthree tests of stickiness of the order of 50% Age-group Survivors of (Chakrabarti & Fearnley 1967). These findings (years) Controls myocardial infarction XI have recently been confirmed by Isacson & UnderS0 7 (19 Y.) 17 (52 Y.) 6-460 50-59 2(7%) 13(34%) 5880 Nilsson (1970). That the fibrinolytic effect of 60 and over 2 (8%.) 4 (11%) 0 phenformin plus ethyloestrenol as measured in vitro reflects removal of fibrin in vivo is suggested Allages 11(12%) 34(32%) - by the increase of fibrin degradation products *Significant(P=O05) during treatment with the drugs (Fearnley et al. 35 Section ofMedicine 925

1969). Experiments using the fibrin plate method (Petromyzon marinus) or the blackfish (Tautoga of Astrup show that the drugs increase activator onitis), but in the smooth dogfish (Mustelis canis) activity (Chakrabarti & Feamley, unpublished complete lysis of whole blood clots occurs in 2-4 data). hours. Lysis of whole blood clots implies either In conclusion, it is not suggested that an that the lytic activity is very high or that the association between defective fibrinolysis and blood contains no fibrinolytic inhibitors. This occlusive vascular disease establishes cause and second possibility is supported by Niewiarowski effect; but, since we now have a combination of & Latallo (1959), who were unable to demonstrate drugs which increases fibrinolytic activity over inhibition ofhuman plasmin by fish blood. the long term, this possibility could be put to the Fibrinolytic activity cannot be induced in fish test by a controlled prophylactic trial in survivors blood by any known plasminogen activator, so of vascular occlusions, for example myocardial in this class of animals the presence of an inactive infarction. fibrinolytic precursor is as yet unproven. REFERENCES Amongst the amphibians, the marine toad Ambrus J L, Ambrus C M, Stutzman L, Schimert G, Jacobsen R, (Bufo marinus) and the tree frog (Hyla cwrula) Schor J & Jainchill J (1970) In: Chemical Control of also show spontaneous lysis of whole blood Fibrinolysis-Thrombolysis. Ed. J M Schor. New York; p 153 Chakrabarti R, Birks P M & Fearnley G R (1963) Lancet i, 1288 clots (Hackett & LePage 1961, Hackett & Hann Chakrabarti R & Fearnley G R (1967) Lancet ii, 1012 1964). In the common frog (Rana temporaria), the Chakrabarti R, Hocking E D, Fearnley G R, Mann R D, Attwell T N & Jackson D (1968) Lancet i, 987 leopard frog (R. pipiens) and the clawed toad Clarke R L, Orandi A & Clif{ton E E (1960) Angiology 11, 367 (Xenopus lkvis), whole blood clots do not lyse but Fearnley G R (1953) Nature (Lond.) 172, 544 spontaneous lysis can be revealed if steps are (1961) Lancet i, 992 taken to remove inhibitors (Blofield 1965, 1971). Fearnley G R, Balmforth G V & Feamley E (1957) Clin. Sci. 16, 645 These findings show that in all five species a Fearnley G R, Chakrabarti R & Evans J F (1969) Lancet i, 910 Fearuley G R, Chakrabarti R & Hocking E D (1967) Lancet ii, 1008 fibrinolytic enzyme is present and suggest that, Fearnley G R, Revill R & Tweed J M (1952) Clin. Sci. 11, 309 in the latter three, inhibitors also exist. In Fearnley G R & Tweed J M (1953) Clin. Sc. 12,81 Isacson S & Nilsson I M (1970) Scand. J. Hemat. 7,404 xenopus, fibrinolysis is greatly enhanced by Schor J M, Steinberger V, Tutko E, Aboulafia S, Pachter I J human urokinase (UK), or streptokinase plus & Jacobsen R J (1970) In: Chemical Control ofFibrinolysis- Thrombolysis. Ed. J M Schor. New York; p 113 human serum (SKHS), indicating the presence of Todd A S (1959)J. Path. Bact. 78, 281 a plasminogen-like precursor. Thus in this von Kaulla K N representative of the Amphibia the fibrinolytic (1963) Chemistry ofThrombolysis: Human Fibrinolytic Enzymes. Springfield, Ill.; p 79 mechanism is similar to that ofman. (1970) In: Chemical Control of Fibrinolysis-Thrombolysis. Reptiles are unusual amongst the vertebrates in Ed. J M Schor. New York; p 1 that the normal blood clotting time is compara- tively prolonged. This is attributed to the presence of a potent circulating anticoagulant which is thought to protect these animals from intravascular thrombosis which might occur as a result of their slow circulation and sluggish way of life (Hackett & Hann 1967). There is no evidence of a fibrinolytic mechanism in these Dr Christine Hawkey animals (Hawkey 1970); possibly a clot-lysing (NuffieldInstitute ofComparative Medicine, mechanism is rendered unnecessary by the TheZoological Society ofLondon, clotting inhibitor. Regent'sPark, London NWJ) Spontaneous fibrinolysis has not been detected in the blood of domestic birds (Niewiarowski & Fibrinolysis in Animals Latallo 1959), but in some wild birds spontaneous activity after removal of inhibitors has been There is not sufficient comparative data to permit found (Hawkey 1970). Activation cannot be a full assessment of the role of the fibrinolytic achieved with UK, SK or SKHS (Cliffton & mechanism as a counterbalance to coagulation Cannamela 1951), but in some domestic birds throughout the animal kingdom. In all vertebrate the fibrinolytic mechanism is activated by saliva animals, hemostasis involves the production of from the bird-feeding vampire bat (Dikmus fibrin clots, but little is known about the part youngi) (Cartwright & Hawkey 1969). Thus a played by fibrinolysis in the subsequent fate of fibrinolytic enzyme, an inactive precursor and the fibrin in animals other than man and the inhibitors are present in at least some animals of more common laboratory mammals. this class. Three species of fish have been studied in Fibrinolytic studies on mammals have shown detail (Doolittle & Surgenor 1962). No fibrinoly- that the samecomponents arepresent as in man. In tic activity has been found in the lamprey this class of animals some quantitation of results