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J Clin Pathol: first published as 10.1136/jcp.s3-14.1.24 on 1 January 1980. Downloaded from

J Clin Pathol, 33, Suppl (Roy Coll Path), 14, 24-30

Natural inhibitors of

D COLLEN From the Centre for and Vascular Research, Department of Medical Research, University ofLeuven, Belgium

There are two main types of inhibitors offibrinolysis: which is devoid of or esterase activity.4 5 those which inhibit (plasmin inhibitors or Complex formation occurs by strong interaction antiplasmins) and those which inhibit the activation between the light-(B)-chain of plasmin and the of plasminogen (anti-activators). Many substances inhibitor. The physiological role of CX2-antiplasmin derived from biological fluids, tissues, plants, or as an inhibitor of other than plasmin micro-organisms inhibit fibrinolysis. Some active seems negligible.8 9 site titrants of serine proteases are potent inhibitors The reaction between plasmin and aX2-antiplasmin of fibrnolytic and certain amino-acids such proceeds in at least two steps-a very fast reversible as lysine, 6-aminohexanoic acid, or second order reaction followed by a slower inhibit the digestion of . This review will deal irreversible fist order reaction'0 11 and may be mainly with the natural inhibitors of fibrinolysis represented by occurring in human blood. The properties of the k, k, other inhibitors are reviewed elsewhere.' P + A < PA - PA' Inhibitors of plasmin occurring in human blood The rate constant ki at pH 7 5 is 3 8 x 107 M-'S-'

and 1-8 x 107 M-'s-' for the two plasmin forms copyright. Human plasma exerts a very important inhibitory which have different affinities for lysine-Sepharose." action on plasmin. There are at least five well- This reaction rate is one of the fastest so far described defined proteins which inhibit plasmin in a purified for protein-protein interactions and is one order of system-namely, aX2-macroglobulin, ol-antitrypsin, magnitude higher than the reaction rate of inter-oz-, III- with its inhibitors. The dissociation constant of the complex, and C,-esterase inhibitor. The physiological reversible step is approximately 2 x 10-1' M and the inhibitor of plasmin formed in blood, however, is a rate constant of the second step 4 x 10-3s-1."1 relatively recently described plasma protein called Plasmin which has 6-aminohexanoic acid" or http://jcp.bmj.com/ o02-antiplasmin.2 This inhibitor was independently lysine'0 bound to its lysine-binding sites or substrate identified by three groups3-5 and most probably also bound to its active site1" reacts only very slowly with by a fourth.6 Upon activation of plasminogen in 0u2-antiplasmin. These findings indicate that free plasma the formed plasmin is first preferentially lysine-binding sites and a free active site in the bound to Oc2-antiplasmin. Only upon complete plasmin molecule are of great importance for the activation of plasminogen (concenttation in plasma rate of its reaction with 0x2-antiplasmin. As discussed

approximately 1.5 ,umol/l), resulting in saturation of further, these interactions are probably of great on September 24, 2021 by guest. Protected the inhibitor (concentration I ,umol/l), is the excess importance for the regulation of fibrinolysis in vivo. plasmin neutralised by oX2-macroglobulin. In the A structural analysis of the plasmin-CX2-antiplasmin presence of normal concentrations of these two complex suggested that the stable complex is inhibitors the other plasma protease inhibitors do formed by a plasmic attack at a specific leucyl- not play a role in the inactivation of plasmin. methionyl peptide bond in the COOH-terminal portion of the inhibitor. A strong, probably covalent CY2-ANTIPLASMIN (THE PHYSIOLOGICAL bond is formed between the active site seryl residue in PLASMIN INHIBITOR) plasmin and the carbonyl group of this specific cx2-Antiplasmin is a single chain glycoprotein with leucyl residue in the inhibitor.12 a molecular weight of 70 000 containing approxi- The turnover of '251-labelled oX2-antiplasmin was mately 13 % carbohydrate.4 7 The inhibitor is studied in control subjects and in patients during immunochemically different from the other known thrombolytic therapy.'3 In the control group plasma protease inhibitors. Ox2-Antiplasmin forms a 0X2-antiplasmin had a plasma half life of 2-64 ± 0-32 very stable 1:1 stoichiometric complex with plasmin, days and a fractional catabolic rate of 0 53 ± 0 09 of 24 J Clin Pathol: first published as 10.1136/jcp.s3-14.1.24 on 1 January 1980. Downloaded from

Natural inhibitors offibrinolysis 25 the plasma pool per day. During thrombolytic about 100 ms. Plasmin with occupied lysine-binding therapy the half life shortened to approximately sites and active site, however, would react at least 100 0 5 days as a result of formation of plasmin-0o2- times slower and have a half life of the order of antiplasmin complex. The long half life of the magnitude of 10 S.28 plasmin-0c2-antiplasmin complex was confirmed by Thus fibrinolysis in vivo seems to be regulated at studying the turnover of the purified complex both two levels: (1) efficient activation at the fibrin before and during thrombolytic therapy in patients surface of fibrin-bound plasminogen by fibrin-bound with thrombotic disease. , and (2) degradation of fibrin The normal concentration of oX2-antiplasmin is by fibrin-bound plasmin which is protected from between 80 % and 120 % (mean ± 2 SD) of the value rapid inactivation by x2-antiplasmin since it has both obtained for pooled normal plasma.'4-'6 The its lysine-binding sites and active site occupied. concentration of 062-antiplasmin in pooled normal Since the fibrin-bound plasmin has an estimated plasma is approximately 1 ,mol/1.4 5 7 The con- half life of about 10 s effective clot dissolution in vivo centration may decrease to below 30% in severe would also seem to require a continous replacement cases ofliver disease or intravascular '4-'6 at the fibrin surface of inactivated plasmin molecules but is normal in patients with cardiovascular, renal, (complexed with 0x2-antiplasmin) by plasminogen or malignant disease. The inhibitor is temporarily molecules.28-29 exhausted during thrombolytic therapy with strepto- Several lines of evidence indicate that this model kinase,5 17 when measured enzymatically. Residual for the regulation of fibrinolysis, which was con- antigen may, however, be found immunologically structed mainly on the basis of molecular interactions representing complexed or degraded inhibitor, or which were demonstrated in purified systems, is also both.'7 oa2-Antiplasmin is a weak acute phase operative in vivo.30 reactant.'5 Possibly some of the OZ2-antiplasmin in plasma is inactive.5 0X2-MACROGLOBULIN a2-Macroglobulin represents the slower reacting (X2-ANTIPLASMIN AND REGULATION OF plasmin inhibitor of plasma. Its role seems to be to FIBRINOLYSIS inactivate plasmin formed in excess of the inhibitory copyright. Physiological fibrinolysis appears to be regulated and capacity of 0x2-antiplasmin.35 Indeed, when the controlled by specific molecular interactions between plasma plasminogen (concentration approximately plasminogen activator and fibrin, between plasmin- 1-5 ,lmol/l) is activated the formed plasmin is initially ogen and fibrin, and between plasmin and OC2- primarily bound to CX2-antiplasmin (concentration antiplasmin. approximately 1 ,tmol/l) until after its saturation. An important property of the blood-vascular- Excess plasmin is neutralised by CX2-macroglobulin.

tissue type of plasminogen activator is its high A number of reviews on the physiology and bio- http://jcp.bmj.com/ affinity for fibrin,18 19 which has been used for its chemistry of 0X2-macroglobulin have recently isolation.20 Tissue activator is a relatively poor appeared.31-33 plasminogen activator in pure systems but fibrin strikingly stimulates the activation.20 21 OTHER PLASMA PROTEASE INHIBITORS Human plasminogen and plasmin contain There are at least three plasma protease inhibitors structures, known as lysine-binding sites, which are in addition to oX2-antiplasmin and ao2-macroglobulin responsible for their interaction not only with that inhibit plasmin in purified systems. These are amino-acids such as lysine or 6-aminohexanoic oxl-antitrypsin, antithrombin III, and Cl-inacti- on September 24, 2021 by guest. Protected acid22-24 but also with fibrin25 and with antiplasmin.4 7 vator.34 Inter-oa-trypsin inhibitor is a polyvalent In purified systems native plasminogen (with inhibitor and probably also reacts to some extent NH2-terminal glutamic acid) has a weak affinity for with plasmin.34 In the presence of normal con- fibrin whereas the partially degraded form of centrations of cX2-antiplasmin and oX2-macroglobulin, plasminogen (with NH2-terminal lysine) has a much however, none of these inhibitors play a role in the stronger affinity.26 When whole plasma is clotted neutralisation of plasmin formed in the blood.3 35 approximately 4% of the plasminogen is specifically Purified antithrombin III is a progressive, time- bound to fibrin through its lysine-binding sites.27 dependent inhibitor of purified plasmin. Heparin The rapidity of the plasmin-cx2-antiplasmin accelerates the rate of this reaction 50 to 100 fold.36 reaction is strongly dependent on the availability of On the basis of these findings it has been suggested free lysine-binding sites and active site in the . that antithrombin-heparin complex may be a major At a concentration of 1 ,umol/l of a2-antiplasmin (the inhibitor of in-vivo fibrinolysis. The role of anti- normal plasma concentration) the half life of small III-heparin complex as an inhibitor of amounts of free plasmin can be calculated to be plasmin formed in plasma has been re-evaluated J Clin Pathol: first published as 10.1136/jcp.s3-14.1.24 on 1 January 1980. Downloaded from

26 Collen in vitro and in vivo.37 38 In the absence of heparin the vessel wall and is released into the blood by and after complete activation of the plasma certain stimuli or trauma, and an exogenous plasminogen about 1 % of the formed plasmin binds pathway in which the activating substances strepto- to antithrombin III. In the presence of therapeutic kinase or may be infused for therapeutic concentrations of heparin (1-2 IU/ml plasma) this purposes. All plasminogen activators studied so far value increased to 2-5% both in vitro and in vivo. exert their action through hydrolysis of the Arg These data suggest that the antithrombin III- 560-Val 561 bond in plasminogen. heparin complex plays a very limited role in the Intrinsic activation of plasminogen may occur by inactivation of plasmin. one or more pathways involving factor XII (Hageman factor), (Fletcher factor), PLASMIN INHIBITORS FROM high molecular weight (Fitzgerald factor), Johnson and Schneider39 reported on an antiplasmin and possibly other components. However, the activity of bovine platelets which accounted for a exact mechanism of this activation as well as its major part of the antiplasmin of bovine blood. In biological role remain unknown. human blood, however, the platelets contributed The plasminogen activator found in blood only 1-3 % of the antiplasmin activity.40 41 The represents released vascular plasminogen activator, contribution to the total antiplasmin and these activators are similar or identical to the activity of whole human blood is therefore probably tissue activator but different from urokinase. small. The physiological importance of extrinsic plasmin- Antiplasmin materials in platelets have, however, ogen activators has been inferred from the association been characterised to some extent. McDonagh et al.42 between low blood fibrinolytic activity and throm- found two antiplasmin activities, one of which was botic or atherosclerotic disease. platelet factor XIII and the other one was eluted earlier on Sephadex G-200. Ganguly et al.43 44 INHIBITORS OF INTRINSIC PLASMINOGEN described low molecular weight antiplasmins, which ACTIVATION were dialysable in extracts of human platelets. All Several inhibitors of intrinsic plasminogen activation these plasmin inhibitors are at present poorly charac- occur in human plasma, C1-inactivator,46 an copyright. terised. inhibitor of factor XIIa-induced fibrinolysis,47 48 heparin-antithrombin III complex,49 and U2-macro- Inhibitors of plasminogen activation in human blood globulin.50 Since the physiological role of the intrinsic fibrinolytic pathway is not established the The existence in blood of physiological inhibitors to role of these inhibitors in the regulation and control the activation of plasminogen has been much of fibrinolysis thus remains entirely speculative.

disputed. The demonstration of activation inhibitors http://jcp.bmj.com/ in plasma is indeed hampered by two obstacles. INHIBITORS OF EXTRINSIC PLASMINOGEN Firstly, it is very difficult to measure antiactivator ACTIVATION activity in the presence ofantiplasmins. Antiactivator The presence in plasma of inhibitors of extrinsic activity may, however, be demonstrated in the plasminogen activators forming a complex which following ways: complex formation between acti- dissociates in the presence of fibrin has been vator and antiactivator, inhibition of activator- postulated already in the 1950s'9 and also in more catalysed hydrolysis of low molecular weight recent studies.51 The formation of a reversible substrates, and inhibition of cleavage of the internal activator-activator inhibitor complex which dis- on September 24, 2021 by guest. Protected peptide bond in plasminogen.45 Secondly, the sociates in the presence of fibrin has been invoked to various protease inhibitors in plasma may each have explain the rapid lysis of fibrin in plasma and the some affinity for plasminogen activators and in resistance of to degradation by plasmin.51 concert confer some inhibitory activity to the plasma The enhancing effect of fibrin on the plasminogen without actually being specific inhibitors. Despite activation may be explained by adsorption of these difficulties it seems, however, that plasma activator and plasminogen to its surface facilitating contains components which act or may act as activation.20 21 28 The evidence for the existence of a inhibitors of plasminogen activation. But the physio- specific inhibitor of extrinsic plasminogen activators logical role of most of these inhibitors is unknown. in plasma, forming a reversible complex, can thus at Plasminogen activation may occur by three best be regarded as preliminary.52 different pathways: an intrinsic or humoral pathway There is good evidence that a significant amount of in which all participating components are present in extrinsic plasminogen activator released in the blood precursor form in the blood, an extrinsic pathway in is cleared in vivo by mechanisms other than neutral- which the activator originates from tissues or from isation by plasmatic inhibitors. Indeed, whereas J Clin Pathol: first published as 10.1136/jcp.s3-14.1.24 on 1 January 1980. Downloaded from

Natural inhibitors offibrinolysis 27 plasminogen activator released in the plasma by Synthetic inhibitors of fibrinolysis used for clinical nicotinic acid injection in normal subjects has a application half life in vivo of approximately 15 min5354 its half life in plasma in vitro is approximately 75 min,53 as Certain amino-acids such as 6-aminohexanoic measured with clot lysis assays. acid (epsilon-, EACA), trans-4-aminomethylcyclohexane-1-carboxylic acid INHIBITORS OF EXOGENOUS PLASMINOGEN (AMCHA, tranexamic acid), and p-aminomethyl- ACTIVATION benzoic acid (PAMBA) inhibit fibrinolysis both Human plasma contains antibodies directed against in vitro and in vivo.66 The relation between the , which most probably result from structure of these substances and their antifibrino- previous infections with beta-haemolytic strepto- lytic effect has been studied in detail.66 Their cocci. The amount of streptokinase antibodies varies clinical usefulness has recently been reviewed.67 In over a wide range among individuals. Verstraete purified systems, however, these agents greatly et al.55 found that 352 000 units of streptokinase accelerate the activation of native plasminogen.68 69 were required to neutralise the circulating antibodies This paradox has recently been elucidated.20 70 in 95 % of a healthy population, but that the Native plasminogen (with NH2-terminal glutamic individual requirements ranged between 25 000 and acid) is slowly activated by urokinase, whereas 3 000 000 units. Since streptokinase reacts with partially degraded plasminogen (with NH2-terminal antibodies and is thereby rendered biochemically lysine, valine, or methionine) is rapidly activated.68 69 inert, sufficient streptokinase must be infused to In the presence of EACA, however, the activation neutralise the antibodies before fibrinolytic activation rate of native plasminogen is greatly increased and is obtained.56 A few days after streptokinase injection approaches that of the partially degraded plasmin- the antistreptokinase titre rises rapidly to 50 to 100 ogen. This acceleration has been ascribed to con- times the preinfusion value and remains high for in the plasminogen molecule 4 to 6 months, during which renewed treatment is formational changes impracticable. Administration of corticosteroids induced by the presence of EACA or removal of the NH2-terminal part of the molecule. The con- copyright. commonly is used as adjuvant to streptokinase to formational change is probably a result of the prevent allergic side reactions. The streptokinase- dissociation between a site in the NH2-terminal part plasmin(ogen) activator complex is virtually un- (residues45-51) and a structure in the plasminogen reactive towards ox2-antiplasmin.57 molecule which binds the amino acids The mechanism of urokinase inhibition in blood ('lysine-binding site').71 72The plasminogen molecule is poorly known. Urokinase-inhibitor assays based contains at least two such lysine-binding sites.24 73 74 on clot lysis are strongly dependent on the presence The antifibrinolytic effect of EACA and analogues of plasmin inhibitors and are therefore in no way appears to result from their interference with the http://jcp.bmj.com/ specific. Assayed with clot lysis methods the half life binding of plasmin(ogen) to fibrin.25-27 When whole of urokinase in vivo is 9 to 16 min but in vitro 27 to 61 plasma is clotted, approximately 4% of the plasmin- min,58 suggesting that clearing of the enzyme from ogen is specifically bound to fibrin through its thebloodplaysanimportantrole. ox2-Macroglobulin,59 lysine-binding sites, and this interaction is abolished al-antitrypsin,60 antithrombin 111,61 and Cx2-anti- by EACA.27 The specific affinity between plasmin- plasmin4 all inhibit urokinase slowly. ogen and fibrin seems to play an important role in

INHIBITORS OF PLASMINOGEN ACTIVATION the activation of plasminogen with tissue activator on September 24, 2021 by guest. Protected FROM PLATELETS which is also specifically adsorbed to fibrin.20 75 Platelets contain inhibitors which have been claimed Indeed, fibrin markedly potentiates the activation of primarily to inhibit fibrinolysis induced by plasminogen, but this stimulating effect is completely activators.62-65 Washed platelets, at a concentration lost in the presence of EACA.20 70 EACA and its which does not inhibit plasmin, are capable of more active analogue tranexamic acid have been inhibiting plasminogen activators.63 Murray et al.64 successfully used to reduce blood loss in essential partially purified an inhibitor of urokinase and tissue menorrhagia, prostatectomy, and activator from human platelets with an estimated in haemophiliacs. molecular weight of 45 000. Moore et al.65 isolated a fraction possessing antiurokinase activity but no Physiopathological conditions associated with antiplasmin activity, in addition to a fraction inhibition of fibrinolysis containing antiplasmin activity. The physiological significance of these inhibitors is A number of physiopathological changes in the uncertain. They may have a role in the protection of blood are associated with a delayed lysis of eu- thrombi from premature lysis. globulin clots or dilute blood clots. These include J Clin Pathol: first published as 10.1136/jcp.s3-14.1.24 on 1 January 1980. Downloaded from

28 Collen pregnancy, hyperlipidemia, smoking, wine, and beer 16 Aoki N. Natural inhibitors of fibrinolysis. Progr drinking and possibly atherosclerosis. The molecular Cardiovasc Dis 1979;21:267-86. mechanisms of inhibition of fibrinolysis by these 17 Verstraete M, Vermylen J, Schetz J. Biochemical factors is in most instances unknown. changes noted during intermittent administration of streptokinase. Thromb Haemostas 1978 ;39 :61-8. 18 Mullertz S. A plasminogen activator in spontaneously References active human blood. Proc Soc Exp Biol Med 1953; 82:291-5. Collen D. Inhibitors of fibrinolysis. In: Kline DL, 19 Mullertz S. Mechanism of activation and effect of Reddy NN, eds. Fibrinolysis. CRC Press, 1979 (in plasmin in blood. Acta Physiol Scand (Suppl) 1956; press). 38:130. 2 Hedner U, Abildgaard U. Report on the joint meeting 20 Wallen P. Activation of plasminogen with urokinase of the task forces on nomenclature and standards of and tissue activator. In: Paoletti R, Sherry S, eds. inhibitors of coagulation and fibrinolysis. Thromb Thrombosis and urokinase. London: Academic Press, Haemostas 1978 ;39 :524-5. 1977:91-102. 3Collen D. Identification and some properties of a new 21 Camiolo SM, Thorsen S, Astrup T. Fibrinogenolysis fast-reacting plasmin inhibitor in human plasma. and fibrinolysis with tissue plasminogen activator, Eur J Biochem 1976;69:209-16. urokinase, streptokinase-activated human globulin, 4Moroi M, Aoki N. Isolation and characterization of and plasmin. Proc Soc Exp Biol Med 1971;138: a2-plasmin inhibitor from human plasma. A novel 277-80. proteinase inhibitor which inhibits activator- 22 Alkjaersig N. The purification and properties of induced clot lysis. J Biol Chem 1976;251 :5956-65. human plasminogen. Biochem J 1964;93:171-82. sMullertz S, Clemmensen I. The primary inhibitor of 23 Abiko Y, Iwamoto M, Tomikawa M. Plasminogen- plasmin in human plasma. Biochem J 1976;159: plasmin system. V. A stoichiometric equilibrium 545-53. complex of plasminogen and a synthetic inhibitor. 6 Bagge L, Bjork I, Saldeen T, Wallin R. Purification and Biochim Biophys Acta 1969;185:424-31. characterization of an inhibitor of plasminogen 24 Iwamoto M. Plasminogen-plasmin system. IX. Specific activation from post-traumatic patients. Forensic Sci binding of tranexamic acid to plasmin. Thromb

1976;7:83-6. Diathes Haemorrh 1975;33:573-85. copyright. 7Wiman B, Collen D. Purification and characterization 21 Wiman B, Wallen P. The specific interaction between of human antiplasmin, the fast-acting plasmin plasminogen and fibrin. A physiological role of the inhibitor in plasma. Eur J Biochem 1977;78 :19-26. lysine binding site in plasminogen. Thromb Res 1977; 8 Edy J, Collen D. The interaction in human plasma of 10:213-22. antiplasmin, the fast-reacting plasmin inhibitor with 26 Thorsen S. Differences in the binding to fibrin of native plasmin, thrombin, trypsin and . plasminogen and plasminogen modified by proteo- Biochim Biophys Acta 1977;484:423-32. lytic degradation. Influence of omega-amino- 9 Ohlsson K, Collen D. Comparison of the reactions of carboxylic acids. Biochim Biophys Acta 1975;393: neutral granulocyte proteases with the major plasma 55-65. http://jcp.bmj.com/ protease inhibitors and with antiplasmin. Scand J 27 Rakoczi I, Wiman B, Collen D. On the biological Clin Lab Invest 1977;37:345-50. significance of the specific interaction between 10 Christensen U, Clemmensen I. Kinetic properties of fibrin, plasminogen, and antiplasmin. Biochim the primary inhibitor of plasmin from human Biophys Acta 1978;540:295-300. plasma. Biochem J 1977;163:389-91. 28 Wiman B, Collen D. On the role of a2-antiplasmin in 1 Wiman B, Collen D. On the kinetics of the reaction the regulation of fibrinolysis. In: Collen D, Wiman between human antiplasmin and plasmin. Eur J B, Verstraete M, eds. The physiological inhibitors of Biochem 1978;84:573-8. blood coagulation and fibrinolysis. Amsterdam: on September 24, 2021 by guest. Protected 12 Wiman B, Collen D. On the mechanism of the reaction Elsevier/North Holland, 1979:177-85. between human a2-antiplasmin and plasmin. J Biol 29 Wiman B, Collen D. Molecular mechanism of physio- Chem 1979;254:9291-7. logical fibrinolysis. Nature 1978;272:549-50. 13 Collen D, Wiman B. Turnover of antiplasmin, the 30 Collen D. On the regulation and control of fibrinolysis. fast-acting plasmin inhibitor of plasma. Blood 1979; Thromb Haemostas 1979;42:366. 53:313-24. 31 Harpel PC. Human a2-macroglobulin. Methods in 14 Edy J, Collen D, Verstraete M. Quantitation of the Enzymology 1976;45:639-52. plasma protease inhibitor antiplasmin with the 32 Laurell CB, Jeppsson JO. Protease inhibitors in plasma. chromogenic substrate S-2251. In: Davidson JF, In: Putnam FW, ed. The plasma proteins, 2nd ed, Rowan RM, Samama MM, Desnoyers PC, eds. vol 1. New York: Academic Press, 1975:chap 5. Progress in chemical fibrinolysis and , 33 Barret AJ, Starkey PM, Munn EA. The unique nature vol 3. New York: Raven Press, 1978:315-22. of the interaction of a,-macroglobulin with protein- 15 Teger-Nilsson AC, Friberger P, Gyzander E. Deter- ases. In: Fritz H, Tschesche H, Greene LJ, Truscheit mination of a new rapid plasmin inhibitor in human E, eds. Proteinase inhibitors (Bayer Symposia 5). blood by means of a plasmin specific tripeptide Berlin: Springer, 1974:72-7. substrate. Scand J Clin Lab Invest 1977;37:403-9. 34 Steinbuch M. Les antiproteases du plasma. Rev Franc J Clin Pathol: first published as 10.1136/jcp.s3-14.1.24 on 1 January 1980. Downloaded from

Natural inhibitors offibrinolysis 29 Transfus 1971;14:61-82. 52 Collen D. a2-Antiplasmin inhibitor deficiency. Lancet 35 Mullertz S. Different molecular forms of plasminogen 1979;1 :1039-40. and plasmin produced by urokinase in human 53 Fletcher AP, Biederman 0, Moore D, Alkjaersig N, plasma and their relation to protease inhibitors and Sherry S. Abnormal plasminogen-plasmin system lysis of fibrinogen and fibrin. Biochem J 1974;143: activity (fibrinolysis) in patients with hepatic 273-83. cirrhosis: its cause and consequences. J Clin Invest 36 Highsmith RF, Rosenberg RD. The inhibition of 1964;43 :681-95. human plasmin by human antithrombin-heparin 54 Tytgat G, Collen D, De Vreker R, Verstraete M. cofactor. J Biol Chem 1974;249:4335-8. Investigations on the fibrinolytic system in 37 Semeraro N, Colucci M, Telesforo P, Collen D. The cirrhosis. Acta Haematol 1968 ;40 :265-74. inhibition of plasmin by antithrombin-heparin 55 Verstraete M, Vermylen J, Amery A, Vermylen C. complex. I. In human plasma in vitro. Br J Haematol Thrombolytic therapy with streptokinase using a 1978;39:91-9. standard dosage scheme. Br Med J 1966;1 :454-6. 38 Collen D, Semeraro N, Telesforo P, Verstraete M. 56 Johnson AJ, McCarty WR. The lysis of artificially The inhibition of plasmin by antithrombin-heparin induced intravascular clots in man by intravenous complex. II. During thrombolytic therapy in man. infusions of streptokinase. J Clin Invest 1959;38: Br J Haematol 1978;39:101-10. 1627-43. 39 Johnson SA, Schneider CL. The existence of anti- 57 Cederholm-Williams SA, De Cock F, Lijnen HR, activity in platelets. Science 1953;117: Collen D. Kinetics of the reactions between strepto- 229-30. kinase, plasmin and a2-antiplasmin. Eur J Biochem 40 den Ottolander GJH, Leijnse B, Cremer-Elfrink 1979; (in press). HMJ. Plasmatic and platelet anti-plasmins and 58 Fletcher AP, Alkjaersig N, Sherry S, Genton E, anti-activators. Thromb Diathes Haemorrh 1967; Hirsh J, Bachmann F. The development of urokinase 18:404-15. as a thrombolytic agent. Maintenance of a sustained 41 Ekert H, Friedlander I, Hardisty RM. The role of thrombolytic state in man by its intravenous infusion. platelets in fibrinolysis. Studies on the plasminogen J Lab Clin Med 1965;65:713-31. activator and anti-plasmin activity of platelets. 59 Ogston D, Bennett B, Herbert RJ, Douglas AS. The Br J Haematol 1970;18:575-84. inhibition of urokinase by a2-macroglobulin. Clin Sci

42 McDonagh J, Kiesselbach TH, Wagner RH. Factor 1973 ;44 :73-9. copyright. XIII and antiplasmin activity in human platelets. 60 Clemmensen I, Christensen F. Inhibition of urokinase Am J Physiol 1969;216:508-13. by complex formation with human alpha I-anti- 43 Ganguly P. A low molecular weight antiplasmin of trypsin. Biochim Biophys Acta 1976;429:591-9. human blood platelets. Clin Chim Acta 1972;39: 61 Clemmensen I. Inhibition of urokinase by complex 466-8. formation with human antithrombin III in absence 44 Mui PTK, James HL, Ganguly P. Isolation and and presence of heparin. Thromb Haemost 1978; properties of a low molecular weight antiplasmin of 39:616-23. human blood platelets and serum. Br J Haematol 62 Thorsen S, Brakman P, Astrup T. Influence of platelets 1975 ;29 :627-37. on fibrinolysis: a critical review. In: Ambrus JL, ed. http://jcp.bmj.com/ 45 Miillertz S. Natural inhibitors of fibrinolysis. In: Hematologic reviews, vol 3. New York: Marcel Davidson JF, Rowan RM, Samama MM, Des- Dekker, 1972:123-79. noyers PC, eds. Progress in chemical fibrinolysis and 63 den Ottolander GJH, Leijnse B, Cremer-Elfrink thrombolysis, vol 3. New York: Raven Press, 1978: HMJ. Plasmatic and thrombocytic antiplasmins and 213-37. anti-activators. II. Thromb Diathes Haemorrh 1969; 46 Kluft C. Elimination of inhibition in euglobulin 21:26-34. fibrinolysis by use of flufenamate: involvement of 64 Murray J, Crawford GPM, Ogston D, Douglas AS. Ci-inactivator. Haemostasis 1977;6:351-69. Studies on an inhibitor of plasminogen activators in on September 24, 2021 by guest. Protected 47 Hedner U. Studies on an inhibitor of plasminogen human platelets. Br J Haemat 1974;26:661-8. activation in human serum. Thromb Diathes Haem- 65 Moore S, Pepper DS, Cash JD. The isolation and orrh 1973;30:414-24. characterization of a platelet-specific fl-globulin 48 Hedner U, Martinsson G. Inhibition of activated (,B-thromboglobulin) and the detection of anti- Hageman factor (factor XIIa) by an inhibitor of the urokinase and antiplasmin released from thrombin- plasminogen activation (PA-inhibitor). Thromb Res aggregated washed human platelets. Biochim Biophys 1978 ;12 :1015-23. Acta 1975;379:360-9. 49 Stead N, Kaplan AP, Rosenberg RD. Inhibition of 66 Okamoto S, Oshiba S, Mihara H, Okamoto U. activated factor XII by antithrombin-heparin Synthetic inhibitors of fibrinolysis: in vitro and in cofactor. J Biol Chem 1976;251:6481-8. vivo mode of action. Ann NY Acad Sci 1968;146: 50 McConnell DJ. Inhibitors of in human 414-29. plasma. J Clin Invest 1972;51:1611-23. 67 Verstraete M. Haemostatic drugs: a critical appraisal. 51 Gurewich V, Hyde E, Lipinski B. The resistance of The Hague: Nijhoff, 1977. fibrinogen and soluble fibrin monomer in blood to 68 Claeys H, Vermylen J. Physicochemical and pro- degradation by a potent plasminogen activator from enzyme properties of NH2-terminal glutamic acid cadaver limbs. Blood 1975;46:555-65. and NH2-terminal lysine human plasminogen. J Clin Pathol: first published as 10.1136/jcp.s3-14.1.24 on 1 January 1980. Downloaded from

30 Collen Influence of 6-aminohexanoic acid. Biochim Biophys Eur J Biochem 1975;50:489-94. Acta 1974;342:351-9. 73 Sottrup-Jensen L, Claeys H, Zajdel M, Petersen TE, 69 Wallen P, Wiman B. On the generation of intermediate Magnusson S. The primary structure of human plasminogen and its significance for activation. In: plasminogen: isolation of two lysine-binding frag- Reich E, Rifkin DB, Shaw E, eds. Proteases and ments and one 'mini'-plasminogen (M.W. 38 000) by biological control. Cold Spring Harbor NY: Cold -catalyzed-specific limited proteolysis. In: Spring Harbor Laboratory, 1975:291-303. Davidson JF, Rowan RM, Samama MM, Des- 70 Thorsen S. Human urokinase and porcine tissue noyers PC, eds. Progress in chemical fibrinolysis and plasminogen activator. Thesis. Copenhagen: Laege- thrombolysis, vol 3. New York: Raven Press, 1978: foreningens Forlag, 1977. 191-209. 71 Sjoholm I, Wiman B, Wallen P. Studies on the con- 74 Markus G, DePasquale JL, Wissler FC. Quantitative formational changes of plasminogen induced determination of the binding of E-aminocaproic acid during activation to plasmin and by 6-amino- to native plasminogen. J Biol Chem 1978;253: hexanoic acid. Eur J Biochem 1973 ;39 :471-9. 727-32. 72 Wiman B, Wallen P. Structural relationship between 7s Thorsen S, Glas-Greenwalt P, Astrup T. Differences in 'glutamic acid' and 'lysine' forms of human plasmin- the binding to fibrin of urokinase and tissue plas- ogen and their interaction with NH2-terminal activa- minogen activator. Thromb Diathes Haemorrh tion peptide as studied by affinity chromatography. 1972;28:65-74. copyright. http://jcp.bmj.com/ on September 24, 2021 by guest. Protected