SEMINARS IN THROMBOSIS AND HEMOSTASIS—VOLUME 17, NO. 4, 1991
Effect of Heparin on the Fibrinolytic Response to Plasminogen Activators
Cardiovascular disease accounts for up to 50% of total mortality in most industrialized countries: acute myocardial infarction (AMI) causes more than 500,000 deaths annually in the United States alone. Thrombolytic agents can improve survival in AMI populations. Reduc tions in early mortality (at about 1 month) have ranged from 20 to 30% for streptokinase (SK)1 and tissue-type plasminogen activator (t-PA)2 up to 50% for anisoylated lys-plasminogen streptokinase activator complex (APSAC, anistreplase, Eminase).3 However, lysis of the thrombus in the infarct-related artery will uncover the underlying vessel wall damage and there may be a high propensity for reocclusion. Therefore heparin has often been employed as an initial adjunct agent in thrombolytic therapy in order to attempt to control the rate of rethrombosis. There is continuing concern, however, about this use of heparin,4 particularly regarding the high rethrombosis rate sometimes experienced with t-PA and because of the possible contribution of heparin to bleed ing side effects. In addition to acting as an anticoagulant, heparin and related polysaccharides may directly influence fi brinolysis. In early experimental studies, induction of endogenous plasminogen activator release from the en dothelium and promotion of the intrinsic fibrinolytic pathway were observed in response to heparin. More recently, it has been shown that heparin can bind t-PA and, thus, directly increase catalytic activity. It is the purpose of this review to describe the biochemistry of the interaction between heparin and plasminogen activators and to assess the likely clinical significance.
From SmithKline Beecham Pharmaceuticals, Epsom, United Kingdom. Reprint requests: Dr. Fears, SmithKline Beecham Pharmaceuti cals, Great Burgh, Epsom, Surrey, KT18 5XQ, UK.
HEPARIN-PLASMINOGEN ACTIVATOR BINDING IN VITRO Studies in Buffer Systems t-PA binds stoichiometrically (molar ratio 1:1) to heparin (Table 1) and to pentosan polysulfate.8 t-PA does not bind to chondroitin sulfate5,8 or hyaluronic acid8 and there is disagreement as to whether t-PA can bind to dextran sulfate.5,6 Binding of heparin to t-PA results in an enhancement of catalytic activity, mediated primarily via an effect on Km and presumably reflecting the formation of a ternary complex. Urokinase (tcu-PA) also binds heparin with a con sequent enhancement of catalytic activity (Table 2), but there is preliminary evidence that tcu-PA and t-PA bind to a different site on heparin.10 Heparin and other sulfated polysaccharides do not appear to bind or influ ence the enzymatic activity of SK-plasminogen (Table 2). Most of the current evidence indicates that a site on the A-chain of t-PA mediates binding to heparin (Table 1); discrepant results7 might be accounted for by variations in the origin of t-PA (Table 1). Because fibrin also binds to sites on the A-chain of t-PA and this binding determines the biochemical selectivity of t-PA, it was of great interest to determine whether heparin and fibrin shared a binding site on t-PA. Initial work using both a specific monoclonal antibody and the anti-fibrinolytic amino acid ε-aminocaproic acid suggested that heparin and the fibrin mimetic cyanogen bromide digested fibrin ogen, may not bind to precisely the same site on the A-chain of t-PA.8 Although kringle 2 domain on the A-chain can bind both heparin and fibrin (when partly degraded), it is the fibronectin-like finger domain on the A-chain that accounts for much of the binding of heparin to t-PA.11 The t-PA binding site on heparin appears not to be the same as the antithrombin III binding site. 1011
Copyright © 1991 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York, NY 10016. All rights reserved.
389
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ROBIN FEARS, Ph.D., ASHIQ F. ESMAIL, Ph.D., and HELEN C. GREENWOOD, M.I.Biol.
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SEMINARS IN THROMBOSIS AND HEMOSTASIS—VOLUME 17, NO. 4, 1991
Study
Heparin Preparation
t-PA Cell origin
Observation
Andrade-Gordon and Strickland5 (1986)
Standard*
Recombinant (Bowes melanoma)
Heparin binds and enhances basal activity but attenuates fibrin enhancement
Paquesetal 6 (1986)
Unspecified
Bowes melanoma
Heparin binds and enhances basal activity but effect is competitive with fibrin
Soedaetal 7 (1987)
Standard*
Porcine heart
Heparin interaction localized to B-chain of t-PA
Fears 8 (1988)
Standard*
Recombinant (Bowes melanoma)
Heparin interaction localized to A-chain of t-PA
Stein et al9 (1989)
Standard*
Mutant variants (Mouse Ltk - )
Heparin interaction mediated by kringle 2 and fibronectin-like finger domain of t-PA
Andrade-Gordon and Strickland10 (1989)
LMW† (nitrous acid depolymerization or heparinase treatment)
Recombinant (CHO,† Bowes melanoma)
LMW heparin fractions did not interact with t-PA
Andrade-Gordon and Strickland11 (1990)
t-PA affinity fractionated
Recombinant (CHO, Bowes melanoma)
Identification of AT III† binding heparin fraction with low affinity for t-PA
* Unfractionated heparin, origin porcine intestinal mucosa or bovine lung. †LMW: low molecular weight; CHO: Chinese hamster ovary; AT III: antithrombin III.
Although heparin and fibrin may not bind to precisely the same site on t-PA, the stimulatory action of heparin on the catalytic activity of t-PA is not additive with the stimulatory action of fibrin preparations.5"8 In some experimental conditions, the degree of fibrin enhancement may be markedly diminished in the presence of heparin.5-7
Studies in Blood If the effects observed for heparin on the activation of plasminogen by t-PA in buffer were to occur in vivo, then an increase in systemic effects should be expected (heparin augmenting catalytic efficacy in the absence of fibrin) together with a diminution in thrombolytic activity (to the extent that heparin interferes with the fibrin TABLE 2. Effect of Standard Heparin on Plasminogen Activation by Other Thrombolytic Agents in Buffer Systems Study Andrade-Gordon and Strickland5 (1986)
Plasminogen Activator*
Observation
tcu-PA
Bound and activity enhanced by heparin
SK-plasminogen
No effect of heparin
Paques et al 6 (1986)
tcu-PA
Bound and activity enhanced by heparin
Fears 8 (1988)
tcu-PA
Activity enhanced by heparin and effect potentiated by fibrin
SK-plasminogen
No effect of heparin
*tcu-PA: two-chain urokinase plasminogen activator; SK: streptokinase.
binding of t-PA). Thus, the biochemical selectivity of t-PA would be attenuated. The possibility of an exacerbation of systemic activation by heparin only applies to t-PA of the standard thrombolytic agents because tcu-PA causes extensive plasminogen activation, even in the absence of heparin, and SK-plasminogen activity is unaffected. Evidence to support the possibility that heparin will impair the thrombolytic activity of t-PA was provided by one study in vitro using human blood.12 Heparin (1 U/ml) delayed the lysis of blood clots induced by 0.5 µg/ml rt-PA (Wellcome). By contrast, in another whole blood in vitro study,13 heparin (0.5 to 5 U/ml) affected neither the mass of thrombus lysed by rt-PA (1 µg/ml, Genentech) nor the rate of fluid phase plasminogen activation. We have confirmed a lack of effect of heparin on systemic plasminogen activation by t-PA when measuring the reduction in blood fibrinogen produced as a consequence of plasminogen activation. In our study, blood samples from 6 volunteers (Fig. la) were incubated with a concentration of rt-PA (4 µg/ml plasma, rt-PA from Bowes melanoma cells), sufficient to give appreciable fibrinogen depletion. Addition of a high concentration of heparin (4U/ml plasma) did not affect the response to t-PA. There was also no effect of heparin on the fibrinogenolytic response to APSAC (6 µg/ml plasma) in blood samples taken from six other volunteers (Fig. lb). Thus, there is no consensus on whether heparin can modulate the enzymatic activity of t-PA in blood. Presumably, the availability of heparin for binding to t-PA will be determined, in part, by the concentration of other heparin-binding proteins in blood, especially an-
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TABLE 1. Characterization of Heparin-Tissue-Type Plasminogen Activator (t-PA) Interaction in Buffer Systems
391
FIG. 1. Measurement of fibrinogenolysis in human whole blood in vitro. Citrated blood samples (fibrinogen concentration 2.8 mg/ml plasma) were incubated with standard heparin (4 U/ml plasma) and (a) rt-PA (Bowes melanoma, 4 (µg/ml plasma) or (b) APSAC (6 µg/ml plasma) or an equivalent volume of 0.9% sodium chloride (w/v). Results are shown as the arithmetic mean (± SEM) for samples from six volunteers incubated for up to 90 minutes at 37°C: saline alone (○), saline plus heparin (•), plasminogen activator alone (Δ), plasminogen activator plus heparin (▲).
tithrombin III. Therefore the measurement of effects in blood, in vitro, will not necessarily simulate conditions in AMI patients in whom the locally effective concentration of antithrombin III may be decreased by thrombin binding and by proteolysis.
ACTIVITY OF HEPARIN IN VIVO Animal Models of Thrombosis Adjunct heparin appears to potentiate the lytic response in a variety of published animal models (Table 3). The effect is observed with SK,14,15 u-PA,17,18 t-PA, 16-18 low molecular weight heparin preparations,18 and unfractionated heparin. It seems likely therefore that any beneficial effects of heparin can be attributed to action as an anticoagulant rather than as a cofactor in plasminogen activation.
In our studies, the effect of heparin on fibrinolytic potency in vivo has been investigated in a guinea pig pulmonary embolism model.19 Radiolabeled human whole blood clots were embolized to the lungs and human plasminogen was coadministered in order to simulate more closely the activity of thrombolytic agents in patients. In this model, the potency of t-PA was similar when given by bolus intravenous injection or by infusion over 90 minutes (10% loading dose).19 Coadministration of heparin increased the potency of t-PA (Fig. 2a). By contrast, the thrombolytic response to bolus APSAC was not affected by adjunct heparin (Fig. 2b). Furthermore, on a gravimetric basis, APSAC was approximately 10-fold more potent than t-PA in the presence of heparin (30-fold more potent in the absence of heparin). We conclude that the markedly greater potency of APSAC compared with t-PA in this model and the relative lack of dependency on concurrent anticoagulant use can be explained by the long plasma half-life of APSAC and by the induction of greater fibrinogen depletion.20
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EFFECT OF HEPARIN ON FIBRINOLYSIS—FEARS, ESMAIL, GREENWOOD
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TABLE 3. Examples of Augmented Thrombolytic Activity by Heparin in Animal Models Study 14
Heparin Preparation
Plasminogen Activator
Thrombosis Model
Standard*
SK
Dog pulmonary embolism
Schumacher et al 15 (1985)
Standard*
SK
Dog left circumflex coronary artery (electrical injury)
Cercek et al 16 (1986)
Standard*
Recombinant t-PA† (CHO† cells)
Dog femoral artery, (copper coil-induced carotid thrombus)
Susawaet al 17 (1987)
Unspecified
t-PA (melanoma cells), tcu-PA†
Dog left anterior descending coronary artery (copper coil)
Stassen et al 18 (1987)
LMW fragments (CY 216, CY 212)
Recombinant t-PA (CHO cells), scu-PA†
Rabbit jugular vein
Cade etal
(1974)
Patients with Acute Myocardial Infarction t-PA is the thrombolytic agent most commonly associated with a high rethrombosis rate in clinical trials.4 Although the pharmacokinetic defect (short plasma half-life) can be compensated for by prolonged infusion, the consequences of hypercoagulability may be exacerbated in patients with AMI in whom the underlying arterial wall damage and platelet hyperreactivity provide strong predisposing factors in reocclusion. Concomitant heparin administration did not influence the coronary artery patency rate achieved 90 minutes after initiation of t-PA therapy.21 Therefore it is unlikely that heparin is participating directly as a cofactor in plasminogen activation catalyzed by t-PA in vivo.
However, concomitant use of aspirin rather than heparin with t-PA resulted in a significantly lower patency rate when coronary arteries were evaluated over a period of 7 to 24 hours (average, 18 hours) after start of therapy.22 Therefore it appears that t-PA use is associated with a high early reocclusion rate in the absence of heparin. Other clinical experience with t-PA has revealed a high incidence of recurrent ischemic events in the hospital despite early use of both heparin and aspirin.23 The concomitant use of heparin with t-PA may also result in a relatively high incidence of intracranial haemorrhage.24 Thus, improvement of the therapeutic response to t-PA may require the development of anticoagulants with greater potency and selectivity than heparin. Because of their effects on systemic fibrinogen and
FIG. 2. Effect of heparin on thrombolysis in a pulmonary embolism model in guinea pigs. Standard heparin was administered (100 U/kg body weight) prior to embolization of clot and, again, 2 hours after administration of thrombolytic agent. Results are shown as the mean (± SEM) for groups of 6 to 12 guinea pigs given (a) t-PA or (b) APSAC. Control rates of spontaneous lysis (14 to 20%) have been subtracted.
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* Porcine intestinal mucosa. †scu-PA: single-chain urokinase plasminogen activator. See Tables 1 and 2 for other abbreviations.
other clotting factors, SK, tcu-PA, and APSAC may be less dependent on immediate heparinization to control early rethrombosis. The incidence of reocclusion within the first 24 hours has been observed to be particularly low (4%) for APSAC,25 reflecting the slow plasma clearance of total fibrinolytic activity. The relative response to heparin use is currently being compared for patients with AMI given t-PA, SK and APSAC in large mortality studies (the Second Gruppo Italiano per lo Studio della Streptochinasi nell'Infarto Miocardico International t-PA/SK Mortality Trial, the Third International Study on Infarct Survival) and the results are awaited. Acknowledgment. We thank J Kalindjian for technical assistance and N James for typing the manuscript.
12.
13.
14.
15.
16.
REFERENCES 17. 1. ISIS-2 Collaborative Group: Randomised trial of intravenous streptokinase, oral aspirin, both or neither among 17187 cases of suspected myocardial infarction: ISIS-2. Lancet 2:349-360, 1988. 2. Wilcox RG, G Von Der Lippe, CG Olsson, G Jensen, AM Skene, JR Hampton: Trial of tissue plasminogen activator for mortality reduction in acute myocardial infarction. Lancet 2:525-530, 1988. 3. AIMS Trial Study Group: Long-term effects of intravenous anistreplase in acute myocardial infarction: Final report of the AIMS study. Lancet 335:427-431, 1990. 4. Sherry S: Unresolved clinical pharmacologic questions in thrombolytic therapy for acute myocardial infarction. J Am Coll Cardiol 12:519-525, 1988. 5. Andrade-Gordon P, S Strickland: Interaction of heparin with plasminogen activators and plasminogen: Effects on the activation of plasminogen. Biochemistry 25:4033-4040, 1986. 6. Pâues E-P, H-A Stöhr, N Heimburger: Study on the mechanism of action of heparin and related substances on the fibrinolytic system: Relationship between plasminogen activators and heparin. Thromb Res 42:797-807, 1986. 7. Soeda S, M Kakiki, H Shimeno, A Nagamatsu: Localisation of the binding sites of porcine tissue-type plasminogen activator and plasminogen to heparin. Biochim Biophys Acta 916:279-287, 1987. 8. Fears R: Kinetic studies on the effect of heparin and fibrin on plasminogen activators. Biochem J 249:77-81, 1988. 9. Stein PL, A-J van Zonneveld, H Pannekoek, S Strickland: Structural domains of human tissue-type plasminogen activator that confer stimulation by heparin. J Biol Chem 264:15441-15444, 1989. 10. Andrade-Gordon P, S Strickland: Anticoagulant low molecular weight heparin does not enhance the activation of plasminogen by tissue plasminogen activator. J Biol Chem 264:15177-15181, 1989. 11. Andrade-Gordon P, S Strickland: Fractionation of heparin by
18.
19.
20.
21.
22.
23.
24.
25.
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chromatography on a tissue plasminogen activator-Sepharose column. Proc Natl Acad Sci USA 87:1865-1869, 1990. Görög P, CD Ridler, IB Kovacs: Heparin inhibits spontaneous thrombolysis and the thrombolytic effect of both streptokinase and tissue-type plasminogen activator. An in vitro study of the dislodgement of platelet-rich thrombi formed from native blood. J Int Med 227:125-132, 1990. Fry ETA, BE Sobel: Lack of interference by heparin with thrombolysis or binding of tissue-type plasminogen activator to thrombi. Blood 71:1347-1352, 1988. Cade JF, J Hirsh, E Regoeczi, M Gent, MR Buchanan, DM Hynes: Resolution of experimental pulmonary emboli with heparin and streptokinase in different dosage regimens. J Clin Invest 54:782791, 1974. Schumacher WA, EC Lee, BR Lucchesi: Augmentation of streptokinase-induced thrombolysis by heparin and prostacyclin. J Cardiovasc Pharmacol 7:739-746, 1985. Cercek B, AS Lew, H Hod, J Yano, NKN Reddy, W Ganz: Enhancement of thrombolysis with tissue-type plasminogen activator by pretreatment with heparin. Circulation 74:583-587, 1986. Susawa T, Y Yui, R Hattori, M Takahashi, T Aoyama, Y Takatsu, K Sakaguchi, N Yui, C Kawai: Heparin requirement in tissue-type plasminogen activator-induced experimental coronary thrombolysis: Comparison with urokinase-induced coronary thrombolysis. JpnCirc J 51:431-435, 1987. Stassen JM, I Juhan-Vague, MC Alessi, F De Cock, D Collen: Potentiation by heparin fragments of thrombolysis induced with human tissue-type plasminogen activator or human single-chain urokinase-type plasminogen activator. Thromb Haemost 58:947950, 1987. Esmail AF, H Ferres, JH Robinson: Efficacy and selectivity of tissue-type and urokinase-type plasminogen activators in a humanised pulmonary embolism model. Fibrinolysis 4:87-94, 1990. Ferres H: Preclinical pharmacological evaluation of anisoylated plasminogen streptokinase activator complex. Drugs 33 (Suppl 3):33-50, 1987. Topol EJ, BS George, DJ Kereiakes, DC Stump, RJ Candela, CW Abbottsmith, L Aronson, A Pickel, JM Boswick, KL Lee, SG Ellis, RM Califf: A randomised controlled trial of intravenous tissue plasminogen activator and early intravenous heparin in acute myocardial infarction. Circulation 79:281-286, 1989. Ross AM, J Hsia, W Hamilton, B Chaitman, R Roberts, NS Kleiman: Heparin versus aspirin after recombinant tissue plasminogen activator therapy in myocardial infarction: A randomised trial. J Am Coll Cardiol 15:64A, 1990. Ellis SG, EJ Topol, BS George, DJ Kereiakes, D Debowey, KN Sigmon, A Pickel, KL Lee, RM Califf: Recurrent ischaemia without warning. Circulation 80:1159-1165, 1989. Kase CS, AM O'Neal, M Fisher, GM Girgis, JI Ordia: Intracranial hemorrhage after use of tissue plasminogen activator for coronary thrombolysis. Ann Intern Med 112:17-21, 1990. Relik-Van Wely L: APSAC reocclusion study at 24 hours. In: Chamberlain DA, JP Boissel: APSAC-Clinical Benefits and Survival of Myocardial Infarct Patients Treated with Eminase. Excerpta Medica, Amsterdam, 1988, pp 10-14.
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EFFECT OF HEPARIN ON FIBRINOLYSIS—FEARS, ESMAIL, GREENWOOD
Effect of Heparin on the Fibrinolytic Response to Plasminogen Activators
Cardiovascular disease accounts for up to 50% of total mortality in most industrialized countries: acute myocardial infarction (AMI) causes more than 500,000 deaths annually in the United States alone. Thrombolytic agents can improve survival in AMI populations. Reduc tions in early mortality (at about 1 month) have ranged from 20 to 30% for streptokinase (SK)1 and tissue-type plasminogen activator (t-PA)2 up to 50% for anisoylated lys-plasminogen streptokinase activator complex (APSAC, anistreplase, Eminase).3 However, lysis of the thrombus in the infarct-related artery will uncover the underlying vessel wall damage and there may be a high propensity for reocclusion. Therefore heparin has often been employed as an initial adjunct agent in thrombolytic therapy in order to attempt to control the rate of rethrombosis. There is continuing concern, however, about this use of heparin,4 particularly regarding the high rethrombosis rate sometimes experienced with t-PA and because of the possible contribution of heparin to bleed ing side effects. In addition to acting as an anticoagulant, heparin and related polysaccharides may directly influence fi brinolysis. In early experimental studies, induction of endogenous plasminogen activator release from the en dothelium and promotion of the intrinsic fibrinolytic pathway were observed in response to heparin. More recently, it has been shown that heparin can bind t-PA and, thus, directly increase catalytic activity. It is the purpose of this review to describe the biochemistry of the interaction between heparin and plasminogen activators and to assess the likely clinical significance.
From SmithKline Beecham Pharmaceuticals, Epsom, United Kingdom. Reprint requests: Dr. Fears, SmithKline Beecham Pharmaceuti cals, Great Burgh, Epsom, Surrey, KT18 5XQ, UK.
HEPARIN-PLASMINOGEN ACTIVATOR BINDING IN VITRO Studies in Buffer Systems t-PA binds stoichiometrically (molar ratio 1:1) to heparin (Table 1) and to pentosan polysulfate.8 t-PA does not bind to chondroitin sulfate5,8 or hyaluronic acid8 and there is disagreement as to whether t-PA can bind to dextran sulfate.5,6 Binding of heparin to t-PA results in an enhancement of catalytic activity, mediated primarily via an effect on Km and presumably reflecting the formation of a ternary complex. Urokinase (tcu-PA) also binds heparin with a con sequent enhancement of catalytic activity (Table 2), but there is preliminary evidence that tcu-PA and t-PA bind to a different site on heparin.10 Heparin and other sulfated polysaccharides do not appear to bind or influ ence the enzymatic activity of SK-plasminogen (Table 2). Most of the current evidence indicates that a site on the A-chain of t-PA mediates binding to heparin (Table 1); discrepant results7 might be accounted for by variations in the origin of t-PA (Table 1). Because fibrin also binds to sites on the A-chain of t-PA and this binding determines the biochemical selectivity of t-PA, it was of great interest to determine whether heparin and fibrin shared a binding site on t-PA. Initial work using both a specific monoclonal antibody and the anti-fibrinolytic amino acid ε-aminocaproic acid suggested that heparin and the fibrin mimetic cyanogen bromide digested fibrin ogen, may not bind to precisely the same site on the A-chain of t-PA.8 Although kringle 2 domain on the A-chain can bind both heparin and fibrin (when partly degraded), it is the fibronectin-like finger domain on the A-chain that accounts for much of the binding of heparin to t-PA.11 The t-PA binding site on heparin appears not to be the same as the antithrombin III binding site. 1011
Copyright © 1991 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York, NY 10016. All rights reserved.
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ROBIN FEARS, Ph.D., ASHIQ F. ESMAIL, Ph.D., and HELEN C. GREENWOOD, M.I.Biol.
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SEMINARS IN THROMBOSIS AND HEMOSTASIS—VOLUME 17, NO. 4, 1991
Study
Heparin Preparation
t-PA Cell origin
Observation
Andrade-Gordon and Strickland5 (1986)
Standard*
Recombinant (Bowes melanoma)
Heparin binds and enhances basal activity but attenuates fibrin enhancement
Paquesetal 6 (1986)
Unspecified
Bowes melanoma
Heparin binds and enhances basal activity but effect is competitive with fibrin
Soedaetal 7 (1987)
Standard*
Porcine heart
Heparin interaction localized to B-chain of t-PA
Fears 8 (1988)
Standard*
Recombinant (Bowes melanoma)
Heparin interaction localized to A-chain of t-PA
Stein et al9 (1989)
Standard*
Mutant variants (Mouse Ltk - )
Heparin interaction mediated by kringle 2 and fibronectin-like finger domain of t-PA
Andrade-Gordon and Strickland10 (1989)
LMW† (nitrous acid depolymerization or heparinase treatment)
Recombinant (CHO,† Bowes melanoma)
LMW heparin fractions did not interact with t-PA
Andrade-Gordon and Strickland11 (1990)
t-PA affinity fractionated
Recombinant (CHO, Bowes melanoma)
Identification of AT III† binding heparin fraction with low affinity for t-PA
* Unfractionated heparin, origin porcine intestinal mucosa or bovine lung. †LMW: low molecular weight; CHO: Chinese hamster ovary; AT III: antithrombin III.
Although heparin and fibrin may not bind to precisely the same site on t-PA, the stimulatory action of heparin on the catalytic activity of t-PA is not additive with the stimulatory action of fibrin preparations.5"8 In some experimental conditions, the degree of fibrin enhancement may be markedly diminished in the presence of heparin.5-7
Studies in Blood If the effects observed for heparin on the activation of plasminogen by t-PA in buffer were to occur in vivo, then an increase in systemic effects should be expected (heparin augmenting catalytic efficacy in the absence of fibrin) together with a diminution in thrombolytic activity (to the extent that heparin interferes with the fibrin TABLE 2. Effect of Standard Heparin on Plasminogen Activation by Other Thrombolytic Agents in Buffer Systems Study Andrade-Gordon and Strickland5 (1986)
Plasminogen Activator*
Observation
tcu-PA
Bound and activity enhanced by heparin
SK-plasminogen
No effect of heparin
Paques et al 6 (1986)
tcu-PA
Bound and activity enhanced by heparin
Fears 8 (1988)
tcu-PA
Activity enhanced by heparin and effect potentiated by fibrin
SK-plasminogen
No effect of heparin
*tcu-PA: two-chain urokinase plasminogen activator; SK: streptokinase.
binding of t-PA). Thus, the biochemical selectivity of t-PA would be attenuated. The possibility of an exacerbation of systemic activation by heparin only applies to t-PA of the standard thrombolytic agents because tcu-PA causes extensive plasminogen activation, even in the absence of heparin, and SK-plasminogen activity is unaffected. Evidence to support the possibility that heparin will impair the thrombolytic activity of t-PA was provided by one study in vitro using human blood.12 Heparin (1 U/ml) delayed the lysis of blood clots induced by 0.5 µg/ml rt-PA (Wellcome). By contrast, in another whole blood in vitro study,13 heparin (0.5 to 5 U/ml) affected neither the mass of thrombus lysed by rt-PA (1 µg/ml, Genentech) nor the rate of fluid phase plasminogen activation. We have confirmed a lack of effect of heparin on systemic plasminogen activation by t-PA when measuring the reduction in blood fibrinogen produced as a consequence of plasminogen activation. In our study, blood samples from 6 volunteers (Fig. la) were incubated with a concentration of rt-PA (4 µg/ml plasma, rt-PA from Bowes melanoma cells), sufficient to give appreciable fibrinogen depletion. Addition of a high concentration of heparin (4U/ml plasma) did not affect the response to t-PA. There was also no effect of heparin on the fibrinogenolytic response to APSAC (6 µg/ml plasma) in blood samples taken from six other volunteers (Fig. lb). Thus, there is no consensus on whether heparin can modulate the enzymatic activity of t-PA in blood. Presumably, the availability of heparin for binding to t-PA will be determined, in part, by the concentration of other heparin-binding proteins in blood, especially an-
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TABLE 1. Characterization of Heparin-Tissue-Type Plasminogen Activator (t-PA) Interaction in Buffer Systems
391
FIG. 1. Measurement of fibrinogenolysis in human whole blood in vitro. Citrated blood samples (fibrinogen concentration 2.8 mg/ml plasma) were incubated with standard heparin (4 U/ml plasma) and (a) rt-PA (Bowes melanoma, 4 (µg/ml plasma) or (b) APSAC (6 µg/ml plasma) or an equivalent volume of 0.9% sodium chloride (w/v). Results are shown as the arithmetic mean (± SEM) for samples from six volunteers incubated for up to 90 minutes at 37°C: saline alone (○), saline plus heparin (•), plasminogen activator alone (Δ), plasminogen activator plus heparin (▲).
tithrombin III. Therefore the measurement of effects in blood, in vitro, will not necessarily simulate conditions in AMI patients in whom the locally effective concentration of antithrombin III may be decreased by thrombin binding and by proteolysis.
ACTIVITY OF HEPARIN IN VIVO Animal Models of Thrombosis Adjunct heparin appears to potentiate the lytic response in a variety of published animal models (Table 3). The effect is observed with SK,14,15 u-PA,17,18 t-PA, 16-18 low molecular weight heparin preparations,18 and unfractionated heparin. It seems likely therefore that any beneficial effects of heparin can be attributed to action as an anticoagulant rather than as a cofactor in plasminogen activation.
In our studies, the effect of heparin on fibrinolytic potency in vivo has been investigated in a guinea pig pulmonary embolism model.19 Radiolabeled human whole blood clots were embolized to the lungs and human plasminogen was coadministered in order to simulate more closely the activity of thrombolytic agents in patients. In this model, the potency of t-PA was similar when given by bolus intravenous injection or by infusion over 90 minutes (10% loading dose).19 Coadministration of heparin increased the potency of t-PA (Fig. 2a). By contrast, the thrombolytic response to bolus APSAC was not affected by adjunct heparin (Fig. 2b). Furthermore, on a gravimetric basis, APSAC was approximately 10-fold more potent than t-PA in the presence of heparin (30-fold more potent in the absence of heparin). We conclude that the markedly greater potency of APSAC compared with t-PA in this model and the relative lack of dependency on concurrent anticoagulant use can be explained by the long plasma half-life of APSAC and by the induction of greater fibrinogen depletion.20
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TABLE 3. Examples of Augmented Thrombolytic Activity by Heparin in Animal Models Study 14
Heparin Preparation
Plasminogen Activator
Thrombosis Model
Standard*
SK
Dog pulmonary embolism
Schumacher et al 15 (1985)
Standard*
SK
Dog left circumflex coronary artery (electrical injury)
Cercek et al 16 (1986)
Standard*
Recombinant t-PA† (CHO† cells)
Dog femoral artery, (copper coil-induced carotid thrombus)
Susawaet al 17 (1987)
Unspecified
t-PA (melanoma cells), tcu-PA†
Dog left anterior descending coronary artery (copper coil)
Stassen et al 18 (1987)
LMW fragments (CY 216, CY 212)
Recombinant t-PA (CHO cells), scu-PA†
Rabbit jugular vein
Cade etal
(1974)
Patients with Acute Myocardial Infarction t-PA is the thrombolytic agent most commonly associated with a high rethrombosis rate in clinical trials.4 Although the pharmacokinetic defect (short plasma half-life) can be compensated for by prolonged infusion, the consequences of hypercoagulability may be exacerbated in patients with AMI in whom the underlying arterial wall damage and platelet hyperreactivity provide strong predisposing factors in reocclusion. Concomitant heparin administration did not influence the coronary artery patency rate achieved 90 minutes after initiation of t-PA therapy.21 Therefore it is unlikely that heparin is participating directly as a cofactor in plasminogen activation catalyzed by t-PA in vivo.
However, concomitant use of aspirin rather than heparin with t-PA resulted in a significantly lower patency rate when coronary arteries were evaluated over a period of 7 to 24 hours (average, 18 hours) after start of therapy.22 Therefore it appears that t-PA use is associated with a high early reocclusion rate in the absence of heparin. Other clinical experience with t-PA has revealed a high incidence of recurrent ischemic events in the hospital despite early use of both heparin and aspirin.23 The concomitant use of heparin with t-PA may also result in a relatively high incidence of intracranial haemorrhage.24 Thus, improvement of the therapeutic response to t-PA may require the development of anticoagulants with greater potency and selectivity than heparin. Because of their effects on systemic fibrinogen and
FIG. 2. Effect of heparin on thrombolysis in a pulmonary embolism model in guinea pigs. Standard heparin was administered (100 U/kg body weight) prior to embolization of clot and, again, 2 hours after administration of thrombolytic agent. Results are shown as the mean (± SEM) for groups of 6 to 12 guinea pigs given (a) t-PA or (b) APSAC. Control rates of spontaneous lysis (14 to 20%) have been subtracted.
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* Porcine intestinal mucosa. †scu-PA: single-chain urokinase plasminogen activator. See Tables 1 and 2 for other abbreviations.
other clotting factors, SK, tcu-PA, and APSAC may be less dependent on immediate heparinization to control early rethrombosis. The incidence of reocclusion within the first 24 hours has been observed to be particularly low (4%) for APSAC,25 reflecting the slow plasma clearance of total fibrinolytic activity. The relative response to heparin use is currently being compared for patients with AMI given t-PA, SK and APSAC in large mortality studies (the Second Gruppo Italiano per lo Studio della Streptochinasi nell'Infarto Miocardico International t-PA/SK Mortality Trial, the Third International Study on Infarct Survival) and the results are awaited. Acknowledgment. We thank J Kalindjian for technical assistance and N James for typing the manuscript.
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REFERENCES 17. 1. ISIS-2 Collaborative Group: Randomised trial of intravenous streptokinase, oral aspirin, both or neither among 17187 cases of suspected myocardial infarction: ISIS-2. Lancet 2:349-360, 1988. 2. Wilcox RG, G Von Der Lippe, CG Olsson, G Jensen, AM Skene, JR Hampton: Trial of tissue plasminogen activator for mortality reduction in acute myocardial infarction. Lancet 2:525-530, 1988. 3. AIMS Trial Study Group: Long-term effects of intravenous anistreplase in acute myocardial infarction: Final report of the AIMS study. Lancet 335:427-431, 1990. 4. Sherry S: Unresolved clinical pharmacologic questions in thrombolytic therapy for acute myocardial infarction. J Am Coll Cardiol 12:519-525, 1988. 5. Andrade-Gordon P, S Strickland: Interaction of heparin with plasminogen activators and plasminogen: Effects on the activation of plasminogen. Biochemistry 25:4033-4040, 1986. 6. Pâues E-P, H-A Stöhr, N Heimburger: Study on the mechanism of action of heparin and related substances on the fibrinolytic system: Relationship between plasminogen activators and heparin. Thromb Res 42:797-807, 1986. 7. Soeda S, M Kakiki, H Shimeno, A Nagamatsu: Localisation of the binding sites of porcine tissue-type plasminogen activator and plasminogen to heparin. Biochim Biophys Acta 916:279-287, 1987. 8. Fears R: Kinetic studies on the effect of heparin and fibrin on plasminogen activators. Biochem J 249:77-81, 1988. 9. Stein PL, A-J van Zonneveld, H Pannekoek, S Strickland: Structural domains of human tissue-type plasminogen activator that confer stimulation by heparin. J Biol Chem 264:15441-15444, 1989. 10. Andrade-Gordon P, S Strickland: Anticoagulant low molecular weight heparin does not enhance the activation of plasminogen by tissue plasminogen activator. J Biol Chem 264:15177-15181, 1989. 11. Andrade-Gordon P, S Strickland: Fractionation of heparin by
18.
19.
20.
21.
22.
23.
24.
25.
393
chromatography on a tissue plasminogen activator-Sepharose column. Proc Natl Acad Sci USA 87:1865-1869, 1990. Görög P, CD Ridler, IB Kovacs: Heparin inhibits spontaneous thrombolysis and the thrombolytic effect of both streptokinase and tissue-type plasminogen activator. An in vitro study of the dislodgement of platelet-rich thrombi formed from native blood. J Int Med 227:125-132, 1990. Fry ETA, BE Sobel: Lack of interference by heparin with thrombolysis or binding of tissue-type plasminogen activator to thrombi. Blood 71:1347-1352, 1988. Cade JF, J Hirsh, E Regoeczi, M Gent, MR Buchanan, DM Hynes: Resolution of experimental pulmonary emboli with heparin and streptokinase in different dosage regimens. J Clin Invest 54:782791, 1974. Schumacher WA, EC Lee, BR Lucchesi: Augmentation of streptokinase-induced thrombolysis by heparin and prostacyclin. J Cardiovasc Pharmacol 7:739-746, 1985. Cercek B, AS Lew, H Hod, J Yano, NKN Reddy, W Ganz: Enhancement of thrombolysis with tissue-type plasminogen activator by pretreatment with heparin. Circulation 74:583-587, 1986. Susawa T, Y Yui, R Hattori, M Takahashi, T Aoyama, Y Takatsu, K Sakaguchi, N Yui, C Kawai: Heparin requirement in tissue-type plasminogen activator-induced experimental coronary thrombolysis: Comparison with urokinase-induced coronary thrombolysis. JpnCirc J 51:431-435, 1987. Stassen JM, I Juhan-Vague, MC Alessi, F De Cock, D Collen: Potentiation by heparin fragments of thrombolysis induced with human tissue-type plasminogen activator or human single-chain urokinase-type plasminogen activator. Thromb Haemost 58:947950, 1987. Esmail AF, H Ferres, JH Robinson: Efficacy and selectivity of tissue-type and urokinase-type plasminogen activators in a humanised pulmonary embolism model. Fibrinolysis 4:87-94, 1990. Ferres H: Preclinical pharmacological evaluation of anisoylated plasminogen streptokinase activator complex. Drugs 33 (Suppl 3):33-50, 1987. Topol EJ, BS George, DJ Kereiakes, DC Stump, RJ Candela, CW Abbottsmith, L Aronson, A Pickel, JM Boswick, KL Lee, SG Ellis, RM Califf: A randomised controlled trial of intravenous tissue plasminogen activator and early intravenous heparin in acute myocardial infarction. Circulation 79:281-286, 1989. Ross AM, J Hsia, W Hamilton, B Chaitman, R Roberts, NS Kleiman: Heparin versus aspirin after recombinant tissue plasminogen activator therapy in myocardial infarction: A randomised trial. J Am Coll Cardiol 15:64A, 1990. Ellis SG, EJ Topol, BS George, DJ Kereiakes, D Debowey, KN Sigmon, A Pickel, KL Lee, RM Califf: Recurrent ischaemia without warning. Circulation 80:1159-1165, 1989. Kase CS, AM O'Neal, M Fisher, GM Girgis, JI Ordia: Intracranial hemorrhage after use of tissue plasminogen activator for coronary thrombolysis. Ann Intern Med 112:17-21, 1990. Relik-Van Wely L: APSAC reocclusion study at 24 hours. In: Chamberlain DA, JP Boissel: APSAC-Clinical Benefits and Survival of Myocardial Infarct Patients Treated with Eminase. Excerpta Medica, Amsterdam, 1988, pp 10-14.
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EFFECT OF HEPARIN ON FIBRINOLYSIS—FEARS, ESMAIL, GREENWOOD
