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Thrombolytic properties of Desmodus (Vampire Bat) salivary plasminogen activator DSPAα1, alteplase and streptokinase following intravenous bolus injection in a rabbit model of carotid artery thrombosis
Fibrirwlysis (1993) ‘I, 284-290 @I!%3 Longman GroupUK Ltd
Thrombolytic Properties of Desmodus (Vampire Bat) Salivary Plasminogen Activator DSPAaI, Alteplase and Streptokinase Following Intravenous Bolus Injection in a Rabbit Model of Carotid Artery Thrombosis
P. Muschick, D. Zeggert, P. Donner, W. Witt SUMMARY. Thrombolytic properties of recombinant Lksmodus salivary plasminogen activatora (DSPA,i) were compared with alteplase (t-PA) and streptokinase (SK) following intravenous bolus injection in rabbits. Thrombosis was induced by a copper coil inserted in the common carotid artery. Heparin (2OOWkgi.v.+i.m.) and aspirin (5mg/kg i.v.) were given in addition to the thrombolytics. Effective thrombolysis at 1,2,6 and 20nmoVkg DSP&i occurred in l/6,4/6,4/7 and 7/7 animals (controls O/7), at 2,6 and 2OnmoI/kg t-PA in l/6, S/6 and 6/7 animals and at 3000,10000 and 3OOOOIU/kg SK in 2/4,4/6, and l/7 animals respectively. t-PA at 2Onmoykg significantly decreased plasma levels of fibrinogen to 72.7f4.2% (p
Due to the importance of preserving left ventricular function following the onset of myocardial infarction a great deal of interest has been generated in establishing therapeutic regimens for i.v. bolus administration of plasminogen activators (PA) to achieve rapid and successful recanalisation. l-3 However, high PA-plasma levels, which may occur with bolus administration of PA’s with a short half-life such as alteplase (t-PA), can lead to unspecific (fibrinindependent) plasminogen conversion to plasmin and depletion of coagulation factors in the circulating blood. This may limit efficacy4 and give rise to bleeding episodes.5 Therefore, PA’s with a high fibrin specificity and a longer half-life may have advantages for i.v. bolus treatment. Effective plasma levels of t-PA in patients with myocardial infarction show some degree of clot-specificity in that t-PA induces a less pronounced plasminemia and coagulopathy than streptokinase (SK).6 However, depletion of coagulation factors with t-PA is still substantial and may explain to some extent the
similarity of t-PA and SK regarding the level of hemorrhagic side-effects. The partial fibrin specificity of t-PA, on the other hand and its short half-life appears to be subject to early reocclusion more frequently than streptokinase-mediated reperfusion.7s In the present study, a rabbit copper coil model of carotid thrombosis was employed to compare the thrombolytic properties of t-PA, Desmodus salivary plasminogen activator,1 (DSPA,,) and streptokinase following bolus administration. DSPA,i is one of four plasminogen activators originally derived from the saliva of the vampire bat Desmodus rotundus. It consists of the finger/EGF/ kringle/protease structural domains as one of the two larger bat plasminogen activator forms alpha 1 and alpha 2. The alpha 2- form, also named bat-PA(H), was first isolated and characterised by Garde11 et a1.9 DSP&i has been shown to possess a superior fibrin specificity to DSPAa2. lo DSPA,i is now being produced by recombinant technology in a eukaryotic cell line.” It has previously been shown to be a potent plasminogen activator in vitro and in vivo with a markedly higher fibrin specificity than t-PA. 12*‘3
P. Musehick, D. Zeggert, P. Dormer, W. Witt, Research Laboratories of Schering AG, Berlin, Germany. 284
Fibrinolysis
The type of animal model employed is essential to the investigation of novel agents effective in the treatment of arterial thrombosis. The characteristics of the model should be common to those of the clinical disease. In the case of arterial thrombosis such characteristics include the development of a thrombus with platelet involvement, poor collateral circulation of the vessel, vessel injury and a pulsatile vessel motion. Postmortem microscopic examination of serial sections of coronary thrombi of patients with acute myocardial infarction has revealed that a thrombus formed at the plaque fissure is rich in platelets. Proximal and distal extensions of the thrombus are composed of erythrocyte-rich material l4 It has recently been hypothesized by Jang et a1.‘6 that the resistance of thrombi to lysis may result from their platelet-rich nature, as opposed to more fibrin-rich, platelet-poor thrombi. In some experimental models of arterial thrombosis, thrombogenesis is induced by introduction of a copper coil into a coronary or femoral artery.“~” Histological examination of thrombi removed from copper coils reveal platelet-rich thrombi which also contain red blood cells and fibrin.15 In the present study these models have been adapted to the rabbit carotid artery by advancing the copper coil via the external carotid. Doppler blood flow velocity was measured continuously in the common carotid artery proximal to the site of thrombus generation. Effective thrombolysis following bolus administration or short infusion of t-PA has been reported in animalsn’+*i and humans.2~3~22~23We investigated whether DSPA,i was suitable for this route of administration. The rapidity and success rate of recanalisation by t-PA, DSPA,, and streptokinase and their effects on selected haemostasis parameters were measured.
MATERIALS AND METHODS Male New Zealand White rabbits (2.2-3.7kg) were anaesthetised by intramuscular injection of 25mg/kg ketamine hydrochloride (Parke Davis and Company, Berlin) and 5 mg/kg xylazine hydrochloride (Bayer, Leverkusen). The marginal ear vein was cannulated and a constant infusion of 33.3mglkglh ketamine and 1.1 mg/kg/h xylazine was started. A tracheotomy was performed following administration of O.O6mg/kg hexcarbacholine bromide (Hormon Chemie, Munich). The animal was ventilated with room air by a positive pressure rodent ventilator (45 strokes/min and 21 ml stroke volume). The p02 was maintained at just above 100mmHg throughout the experiment. The right femoral artery was cannulated for blood sampling and the jugular vein for intravenous bolus application of the thrombolytics. After these initial procedures, the carotid artery and its cranial bifurcation were exposed and mean and pulsatile blood flow velocity in the common
285
carotid artery were measured. The external carotid artery was occluded and blood flow was measured until a stable value was obtained after approximately 10 to 30min. The established flow varied from 10 to 50% of the initial flow value. Following this, the internal and common carotid artery were occluded by external compression with blunt forceps. The external carotid artery was incised and a copper coil (diameter l.Smm, 5 turns) was inserted and advanced over the bifurcation into the common carotid artery. The external carotid artery was ligated proximal to the bifurcation and the forceps released to restore blood flow. Thrombus formation within the coil led to a progressively increasing stenosis and subsequently to occlusive thrombus formation. Occlusion occurred within 30min in approximately 50% of the animals. In rabbits with persistent patency at 30min, the vessel segment around the copper coil was damaged by a compression with blunt forceps once or twice, whereby occlusion was consistently obtained. One hour after occlusion, therapy was started by bolus injection of the thrombolytic agents in combination with heparin (2OOU/kg i.v./i.m.) and aspirin (5mg/kg i.v.). Reperfusion was defined as mean blood flow of more than 10% of the maximal baseline flow measured following external carotid artery occlusion at the beginning of the experiment. Observation of recanalisation was continued for two hours after bolus injection of the thrombolytics. The maximal blood flow obtained, time to lysis and patency at 2h were recorded. At the end of the experiment, the vessel segment containing the copper coil and the adjacent regions were opened longitudinally for visual inspection. To obtain plasma for determination of haemostasis factors and plasminogen activator antigen levels blood samples were taken from the femoral artery prior to thrombolytic treatment and at 30, 60 and 120 min during the observation period of 2 h following bolus administration. Plasma was obtained from 0.9 ml of whole blood to which 0.1 ml of 3.16% sodium citrate was added. 9Ol.~lof this plasma was added to 10~1 of 1OmM PPACK (Calbiochem, La Jolla, CA, USA) to stabilise the plasma against proteolytic degradation. All plasma samples were kept frozen at - 80°C. Test Compounds Application Recombinant DSPA,, was purified from the supernatant of transfected Chinese hamster ovary (CHO) cells as previously describedI and dissolved in PBSbuffer at a concentration of lmg/ml. For administration of 1,2,6 and 20nmol/kg (apparent molecular weight 52 kD) DSPA,r-solution was further diluted with 0.9% sodium chloride to 4.5ml total volume. A commercial t-PA preparation registered for clinical use (Actilyse@) was purchased from Thomae (Biberach, Germany). t-PA was dissolved in sterile
286 Thrombolytic Properties of Desmodus Salivary Plasminogen Activator DSPA,,
water in a concentration of 1 mg/ml and diluted with 0.9% sodium chloride to 4Sml total volume for administration of 2, 6 and 20nmoVkg (apparent molecular weight 65 kD). Streptokinase (Kabikinasee’) was purchased from Pfrimmer Kabi GmbH (Erlangen, Germany). Streptokinase was diluted with 0.9% sodium chloride to 4Sml total volume for administration of 3000,10000 and 30000 IU/kg. Control animals received 4Sml 0.9% sodium chloride, aspirin and heparin as indicated above.
binary response criterion treatment groups were compared versus control using the one-sided. Fishertest (a=O.O5 per comparison). Statistical analysis of the effects of the thrombolytic agents on haemostatic factors (comparison of thrombolytics with vehicle, and comparison between equimolar doses of the thrombolytics) was carried out using the two-tailed Students’ t-test. Plasminogen activator antigen levels were analysed by means of descriptive statistics (meankstandard error).
Haemostasis Factors
RESULTS
Plasma fibrinogen was measured as clottable protein according to the method of Clauss.23 Plasminogen and aZantiplasmin were determined on microtitre plates employing the chromogenic substrate S2251 (Kabi Vitrum, Munich, Germany) and values were expressed as percentage of prevalue plasma levels. For comparison among groups, the percent changes from initial values were calculated.
Thrombolysiskeperfusion and Reocclusion The incidence of reperfusion and patency at the end of a 2 h observation period as well as time to lysis are summarized in Table 1. DSP&r at the intravenous bolus dose of 1nmoYkg reperfused l/6 animals treated to >lO% of the initial blood flow (Fig. 1). The administration of 2, 6 and 20nmoYkg resulted in successful reperfusion in 66%, 57% and 100% of the animals. t-PA at a dose of 2nmoVkg was ineffective in restoring blood flow, however, t-PA at 6 and 20 nmoY kg resulted in successful reperfusion in 83% and 86% of the animals. Low and high dose streptokinase (3000 and 30OOOIU/kg) caused thrombolysis in 2/4 and l/6 animals, whereas the intermediate dose (lOOOOIU/kg) led to thrombolysis in 4/6 animals. This effect of the intermediate dose of streptokinase is comparable with effects of intermediate doses of DSPA,, and t-PA. A further increase of the dose of streptokinase (from 10000 to 30OOOIU/kg) resulted in a marked decrease in efficacy. None of the arteries reperfused with SK showed a high grade reperfusion (~80% initial flow). The time to lysis did not show a clear dose-dependency and did not differ between DSPA,r and t-PA.
Plasminogen Activator Antigens DSP&r antigen levels in rabbit plasma were determined using a sandwich ELISA employing biotinylated rabbit anti-DSPA,r antibodies. t-PA antigen concentrations were also determined by ELISA employing a commercially available kit (Biopool, Umea, Sweden). Statistics The mean blood flow was calculated as area under the flow velocity curve divided by duration of the observation period (AUC/120 min). Thrombolysis was defined as a mean flow after thrombolysis of at least 10% of the baseline blood flow. With regard to this
Table 1 Comparison of frequency of reperfusion, patency at 2h and time to lysis with DSPA,,, t-PA and SK. Figures given in parenthesis represent percent of total animals reperfused, percentage with high grade reperfusion and percentage of patency at 2h
Reperfusion mean blood flow >lO%
Dose of thrombolytic agent
o/7
Control DSPA,, [nmol/kg]
t-PA [nmol/kg]
SK [W/kg]
Reperfusion mean blood flow >80%
(0%)
-
(100%)
2/7
Patency at 2 h
1 2 6 20
l/6 *4/6 +4/7 *7/7
2 6 20
l/6 ‘516 W7
116
3OOtl
2J4 *4/6 l/6
2l4 316 116
(*significant vs control, ~~0.05, Fishers exact test)
Time to lysis min (+SEM)
(5) (17%)
28
(3) (10)
Fibrinolysis
287
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SK at the higher doses had a tendency to a slow lysis time compared to DSPA,, and t-PA. Reocclusions occurred with all three agents in spite of the adjunctive treatment with heparin and aspirin (compare patency at 2h with reperfusion in Table 1). Haemostasis Factors
Fibrinogen levels measured at baseline and during the observation period decreased significantly with t-PA at the 6 and 20 nmol/kg dose (Fig. 2). In contrast neither DSPA,r nor SK depleted plasma fibrinogen significantly. Plasminogen and aZantiplasmin levels measured at baseline and 30min after starting thrombolysis (see Fig. 3) were not significantly decreased by DSPA,r and t-PA at the 1, 2 and 6nmoYkg dose while the 20nmoYkg dose of both thrombolytics significantly decreased olZantiplasmin to 85.3?2.5% (p < 0.01) and 54.6f4.7% (p C 0.001) of the initial value respectively. The decrease by t-PA was much more pronounced than that caused by DSPA,i (p < 0.001). Plasminogen plasma levels 30min after starting thrombolysis were not significantly decreased by DSPA,r at all doses studied, however, the highest dose of t-PA led to significant plasminogen degradation (p < 0.001). In SK treated animals only fibrinogen levels were determined which did not decrease significantly with any dose. Plasminogen Activator Antigen Levels
The plasma antigen levels of both DSPA,i and t-PA
of flow before
10
30
[kU/kd SK
[=Mkgl t-PA
bW431 DSPAa, Fig. 1 Mean blood flow/min
6
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thrombus
generation.
(Fig. 4) measured 30,60 and 120min decreased with time in a dose-dependent manner. The plasma level of DSPA,i exceeded that of t-PA for equimolar doses at all time points measured.
DISCUSSION DSPA,i is an effective thrombolytic agent in this rabbit copper coil model of carotid artery thrombosis. DSPA,i is at least as efficacious as t-PA in this model, while there is evidence to suggest that DSPA,i may be more potent by a factor of 2-3 than t-PA calculated on a molar basis. The higher potency of DSPA,, could be explained by a slower clearance compared to t-PA as suggested by higher plasma levels 30 to 120min post dosing. Not only the frequency of reperfusion but also the time to clot lysis characterises the clinical efficiacy of thrombolytic therapy. There were no differences between DSPA,r and t-PA in this respect whereas clot lysis with SK appeared to be somewhat slower. SK showed a bell-shaped dose-response relationship and was only effective in 4/6 animals in the intermediate dose of lOOOOIU/kg i.v. bolus, due to rapid clearance from the blood and the lack of fibrin binding. *’ All arteries which reperfused with SK achieved only low grade patency (<80% of initial flow) indicating that bolus SK was a poorly efficacious thrombolytic treatment in the rabbit model. With regard to the haemostatic parameters, DSPA,r differed from t-PA, as it was demonstrated to cause no significant fibrinogenolysis nor plasminoaZantiplasmin plasma levels gen depletion.
288 Thrombolytic Properties of Destnodus Salivary Plasminogen Activator DSPA,,
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An additional aim of the present study was to determine whether the present animal model was useful for the evaluation of novel fibrinolytic agents. The bolus intravenous dosing schedule was chosen to facilitate prompt initiation of thrombolytic therapy. Spontaneous flow restorations occurred infrequently. One short spontaneous flow restoration of 3min duration was observed in 1 out of 7 (0.9% sodium chloride/heparin/aspirin treated) control rabbits, however this did not meet the criteria defined above as thrombolysis and was as such counted as ‘no reperfusion’. Because of the small distance of the copper coil from the point of insertion the influence of methodological factors in the coil models described by Kordenat et al.” and Bergmann et a126e.g. the speed with which the copper coil is inserted and certain other variables can be excluded in the present model. However, the geometry of the coil and the number of turns greatly influence the time to occlusion of the vessel. In the present study a coil with 5 turns at a distance of approximately 0.2mm and a diameter of 1.8mm was chosen as under these conditions the coil fits snugly against the vessel wall with no acute occlusion of the vessel occurring after coil insertion. In addition, intimal injury of the artery by careful compression with blunt forceps is a prerequisite for a thrombotic occlusion in some rabbits. In comparison with previous data2’ obtained in a rabbit model of peripheral arterial thrombosis following bolus application of t-PA and bat-PA (DSP&2)
a
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B t0
100
d
125
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60 60 40 tn c
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30’ 60’ 120’ time post-dosing [min]
Fig. 2 Time course of plasma fibrinogen levels following i.v. bolus injection of DSPA,,, t-PA and SK in rabbits. The figure shows percentage change from initial values in plasma fibrinogen levels at 30,60, and 120min post-dosing. (mean+SEM n=f%7; asterisks refer to significance versus control (*p
2
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120
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20 nmol/kg DSPA,,
decreased to a lesser extent than with t-PA. The absence of fibrinogenolysis with any dose of SK again indicates the poor efficacy of this non clot selective agent when given i.v. bolus. The plasma half-life of DSPA,i exceeded that of t-PA at all doses.
t-PA
t-PA
Fig. 3 Plasminogen and a2-antiplasmin levels at 30min postdosing. The figure shows percentage change from initial values. (meanHEM, n=6-7, *p
Fibrinolysis
289
1200 E 2
6 1
1000
c
a, 5
\
800
k .--0, s F g a
P
600
400
200
0 0
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60 DSPA
90
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0
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Fig. 4 Plasma antigen levels (@ml)
of DSPA,,
60
90
120
t-PA
a,
post-dosing
[min]
and t-PA following iv. bolus injection at 30,60, and 120min (mean+SEM,
and in a dog model with coronary occlusion*” the median time to lysis (time to reperfusion) was longer and the incidence of reperfusion was lower for t-PA and bat-PA than in the present model. Both t-PA and DSPA,, were about 7 times more potent in our copper coil models compared to t-PA and DSPA,i (bat-PA) in the above mentioned model. One possible explanation for this could be a difference in clot structure in the two models. More likely adjunctive therapy with heparin and aspirin has increased the effectiveness of t-PA and DSPA,, in our study. In summary, the carotid artery thrombosis model presented here is a method suitable to study fibrinolytic agents in rabbits. The new bat plasminogen activator DSPA,, is an effective thrombolytic agent in experimental arterial thrombosis. It is more potent and more clot (fibrin) specific than t-PA. The prolonged half-life of DSPA,i compared to t-PA together with its superior clot specificity may allow a bolus treatment with this agent to achieve a rapid and safe thrombolysis in myocardial infarction and possibly in stroke.
2.
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ACKNOWLEDGEMENTS
8.
The authors would like to thank Eva Maria Jahn for secreterial assistance, Karola Hohlmann for statistical analysis, Gabi Gehrmann and Petra Kolsch for the measurement of hemostasis factors. We thank our colleagues at Berlex Biosciences, Brisbane, CA, USA, who provided us with the DSPA,, used in this study.
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REFERENCES 1. Sheelan F H, Doerr R, Schmidt W G et al. Early recovery
11.
n=6-7).
of left ventricular function after thrombolytic therapy for acute myocardial infarction: an important determinant of survival. J Am Coll Cardiol 1988; 12: 289300. Tranchesi B, Chamone D F, Cobbaert C, Van de Werf F, Vanhove P, Verstraete M. Coronary recanalisation rate after intravenous bolus of alteplase in acute myocardial infarction. Am J Cardiol 1991; 68: 161-165. Tebbe U, Tanswell P, Seifried E, Feuerer W, Scholz K H, Herrmann K S. Single-bolus injection of recombinant tissuetype plasminogen activator in acute myocardial infarction. Am J Cardiol 1989; 64: 448-453. Sobel B A, Nachowiak D A, Frey E T A, Bergmann S R, Torr S R. Paradoxical attenuation of fibrinolysis attributable to ‘plasminogen steal’ and its implications for coronary thrombolysis. Coronary Artery Disease 1990; 1: 111. Bovill E G, Terrin M L, Stump D C et al. Hemorrhagic events during therapy with recombinant tissue-type plasminogen activator, heparin, and aspirin for myocardial infarction-results of the thrombolysis in myocardial infarction (TIMI), phase II trial. Ann Intern Med 1991; 115: 256-26.5. Rae A K, Pratt C, Berke A et al. Thrombolysis in myocardial infarction (TIMI) trial-phase I: the fibrinolytic system in patients treated with recombinant tissue plasminogen activator and streptokinase. J Am Coll Cardiol 1988; 11: 11. Chesbro H K, Knatterud G, Roberts R et al. Thrombolysis in myocardial infarction (TIMI) trial phase I: comparison between intravenous tissue plasminogen activator and intravenous streptokinase: clinical findings through hospital discharge. Circulation 1987; 76: 142-154. Sherry S, Marder V J. Streptokinase and recombinant tissue plasminogen activator (rt-PA) are equally effective in treating myocardial infarction. Am Intern Med 1991; 114: 417-423. Gardell S J, Duong L T, Diebl R E et al. Isolation characterisation and cDNA cloning of the vampire bat salivary plasminogen activator. J Biol Chem 1989; 264: 17947-17952. Witt W, Baldus B, Bringmann P, Schleuning W D. The new plasminogen activators DSPA,i and aa from the vampire bat Desmodus rotundus. Fibrinolysis 1992; 6: Suppl. 2: 28 (abst.). Baldus B., Gehrmann G, Bringmann P, Donner P. Kinetics
290 Thrombolytic Properties of Desmodus Salivary Plasminogen Activator DSPA,,
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of Glu-plasminogen activation do not explain the fibrinolytic potency of recombinant desmodus rotundus salivary plasminogen activator (DSPA,,). Thromb Haemost 1991; 65: 884 (abst.). Bringmann P, Donner P, Baldus B, Alagon A, Schleuning W D. Isolation and characterisation of plasminogen activators of vampire bat Desmodus rotundus. Fibrinolysis 1990; 4: 97 (abst). Witt W, Baldus B, Bringmann P, Cashion L, Donner P and Schleuning W D. Thrombolytic properties of desmodus rotundus (vampire bat) salivary plasminogen activator in experimental pulmonary embolism in rats. Blood 1992; 79: 1213-1217. Friedman M F, van der Bovenkamp E J. The pathogenesis of a coronary thrombus. Am J Pathol 1966; 48: 19-44. Bush L R, Mellot M J, Kanovsky S M, Holahan M A, Patrick D H. A model of femoral artery thrombolysis in dogs. Fibrinolysis 1989; 3: 107-114. Jan I K, Gold H K, Ziskind A A, et al. Different sensivity of erythrocyte-rich and platelet-rich arterial thrombi to lysis with recombinant tissue-type plasminogen activator. Circulation 1989; 79: 920-928. Kordenat R K, Kezdi P. Experimental intracoronary thrombosis and selective in situ lysis by catheter technique. Am Heart J 1972; 83: 360-364. Badylak S F, Voytik S, Klabunde R E, Kenkin JH, Leski M. Bolus dose response characteristics of single chain urokinase plasminogen activator and tissue plasminogen activator in a dog model of arterial thrombosis. Thromb Res 1988; 52: 295-312. Clozel J P, Tschopp T, Luedin E, Holvoet P. Time course of thrombolysis induced by intravenous bolus infusion of tissue
Received: 8 September 1992 Accepted after revision: 7 December 1992 Offphnt orders to: W. Witt, PhD, Cardiovascular Pharmacology, Schering AG, Miillerstr. 170-178, 1000 Berlin 65, Germany. Phone: (30)-4684088, Fax: (30)-46916704.
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plasminogen activator in a rabbit jugular vein thrombosis model. Circulation 1989; 79: 125-133. Agnelli G, Buchanan B R, Fernandez F, Hirsh J. The thrombolytic and hemorrhagic effects of tissue type plasminogen activator: influence of dosage regimes in rabbits. Thromb Res 1985: 40: 764-777. Agnelli G. Rationale for bolus t-PA therapy to improve efficacy and safety. Chest 1990; 97: 1615-1675. Neuhaus K-L, Feuerer W, Jeep-Tebbe S, Niederer W, Vogt A, Tebbe U. Improved thrombolysis with a modified dose regimen of recombinant tissue-type plasminogen activator. J Am Coll Cardiol 1989; 14: 1566-1569. Top01 E J. Ultrathrombolysis. J Am Coll Cardiol 1990; 15: 922-924. Clauss V A. Gerinnungsphysiologische Schnellmethode zur Bestimmung des Fibrinogens. Acta Haematol 1957; 17: 127. Agnelli G, Buchanan M R, Fernandez F, van Ryn J, Hirsh J. Sustained thrombolysis with DNA-recombinant tissue type plasminogen activator in rabbits. Blood 1985; 66: 399401. Bergmann S R, Fox K A A, Ter-Pogossian M M, Sobel B E, Collen D. Clot-selective coronary thrombolysis with tissuetype plasminogen activator. Science 1983; 220: 1181-1183. Garde11 S J, Ramjit D R, Stabilito I I et al. Effective thrombolysis without marked plasminemia after intravenous administration of vampire bat salivary plasminogen activator in rabbits. Circulation 1991; 84 No. 1: 244-253. Mellott M J, Stabilito I I, Holahan M A. Vampire bat salivary plasminogen activator promotes rapid and sustained reperfusion without concomitant systemic plasminogen activation in a canine model of arterial thrombosis. Arterioscler Thromb 1992; 12: 212-221.
Thrombolytic Properties of Desmodus (Vampire Bat) Salivary Plasminogen Activator DSPAaI, Alteplase and Streptokinase Following Intravenous Bolus Injection in a Rabbit Model of Carotid Artery Thrombosis
P. Muschick, D. Zeggert, P. Donner, W. Witt SUMMARY. Thrombolytic properties of recombinant Lksmodus salivary plasminogen activatora (DSPA,i) were compared with alteplase (t-PA) and streptokinase (SK) following intravenous bolus injection in rabbits. Thrombosis was induced by a copper coil inserted in the common carotid artery. Heparin (2OOWkgi.v.+i.m.) and aspirin (5mg/kg i.v.) were given in addition to the thrombolytics. Effective thrombolysis at 1,2,6 and 20nmoVkg DSP&i occurred in l/6,4/6,4/7 and 7/7 animals (controls O/7), at 2,6 and 2OnmoI/kg t-PA in l/6, S/6 and 6/7 animals and at 3000,10000 and 3OOOOIU/kg SK in 2/4,4/6, and l/7 animals respectively. t-PA at 2Onmoykg significantly decreased plasma levels of fibrinogen to 72.7f4.2% (p
Due to the importance of preserving left ventricular function following the onset of myocardial infarction a great deal of interest has been generated in establishing therapeutic regimens for i.v. bolus administration of plasminogen activators (PA) to achieve rapid and successful recanalisation. l-3 However, high PA-plasma levels, which may occur with bolus administration of PA’s with a short half-life such as alteplase (t-PA), can lead to unspecific (fibrinindependent) plasminogen conversion to plasmin and depletion of coagulation factors in the circulating blood. This may limit efficacy4 and give rise to bleeding episodes.5 Therefore, PA’s with a high fibrin specificity and a longer half-life may have advantages for i.v. bolus treatment. Effective plasma levels of t-PA in patients with myocardial infarction show some degree of clot-specificity in that t-PA induces a less pronounced plasminemia and coagulopathy than streptokinase (SK).6 However, depletion of coagulation factors with t-PA is still substantial and may explain to some extent the
similarity of t-PA and SK regarding the level of hemorrhagic side-effects. The partial fibrin specificity of t-PA, on the other hand and its short half-life appears to be subject to early reocclusion more frequently than streptokinase-mediated reperfusion.7s In the present study, a rabbit copper coil model of carotid thrombosis was employed to compare the thrombolytic properties of t-PA, Desmodus salivary plasminogen activator,1 (DSPA,,) and streptokinase following bolus administration. DSPA,i is one of four plasminogen activators originally derived from the saliva of the vampire bat Desmodus rotundus. It consists of the finger/EGF/ kringle/protease structural domains as one of the two larger bat plasminogen activator forms alpha 1 and alpha 2. The alpha 2- form, also named bat-PA(H), was first isolated and characterised by Garde11 et a1.9 DSP&i has been shown to possess a superior fibrin specificity to DSPAa2. lo DSPA,i is now being produced by recombinant technology in a eukaryotic cell line.” It has previously been shown to be a potent plasminogen activator in vitro and in vivo with a markedly higher fibrin specificity than t-PA. 12*‘3
P. Musehick, D. Zeggert, P. Dormer, W. Witt, Research Laboratories of Schering AG, Berlin, Germany. 284
Fibrinolysis
The type of animal model employed is essential to the investigation of novel agents effective in the treatment of arterial thrombosis. The characteristics of the model should be common to those of the clinical disease. In the case of arterial thrombosis such characteristics include the development of a thrombus with platelet involvement, poor collateral circulation of the vessel, vessel injury and a pulsatile vessel motion. Postmortem microscopic examination of serial sections of coronary thrombi of patients with acute myocardial infarction has revealed that a thrombus formed at the plaque fissure is rich in platelets. Proximal and distal extensions of the thrombus are composed of erythrocyte-rich material l4 It has recently been hypothesized by Jang et a1.‘6 that the resistance of thrombi to lysis may result from their platelet-rich nature, as opposed to more fibrin-rich, platelet-poor thrombi. In some experimental models of arterial thrombosis, thrombogenesis is induced by introduction of a copper coil into a coronary or femoral artery.“~” Histological examination of thrombi removed from copper coils reveal platelet-rich thrombi which also contain red blood cells and fibrin.15 In the present study these models have been adapted to the rabbit carotid artery by advancing the copper coil via the external carotid. Doppler blood flow velocity was measured continuously in the common carotid artery proximal to the site of thrombus generation. Effective thrombolysis following bolus administration or short infusion of t-PA has been reported in animalsn’+*i and humans.2~3~22~23We investigated whether DSPA,i was suitable for this route of administration. The rapidity and success rate of recanalisation by t-PA, DSPA,, and streptokinase and their effects on selected haemostasis parameters were measured.
MATERIALS AND METHODS Male New Zealand White rabbits (2.2-3.7kg) were anaesthetised by intramuscular injection of 25mg/kg ketamine hydrochloride (Parke Davis and Company, Berlin) and 5 mg/kg xylazine hydrochloride (Bayer, Leverkusen). The marginal ear vein was cannulated and a constant infusion of 33.3mglkglh ketamine and 1.1 mg/kg/h xylazine was started. A tracheotomy was performed following administration of O.O6mg/kg hexcarbacholine bromide (Hormon Chemie, Munich). The animal was ventilated with room air by a positive pressure rodent ventilator (45 strokes/min and 21 ml stroke volume). The p02 was maintained at just above 100mmHg throughout the experiment. The right femoral artery was cannulated for blood sampling and the jugular vein for intravenous bolus application of the thrombolytics. After these initial procedures, the carotid artery and its cranial bifurcation were exposed and mean and pulsatile blood flow velocity in the common
285
carotid artery were measured. The external carotid artery was occluded and blood flow was measured until a stable value was obtained after approximately 10 to 30min. The established flow varied from 10 to 50% of the initial flow value. Following this, the internal and common carotid artery were occluded by external compression with blunt forceps. The external carotid artery was incised and a copper coil (diameter l.Smm, 5 turns) was inserted and advanced over the bifurcation into the common carotid artery. The external carotid artery was ligated proximal to the bifurcation and the forceps released to restore blood flow. Thrombus formation within the coil led to a progressively increasing stenosis and subsequently to occlusive thrombus formation. Occlusion occurred within 30min in approximately 50% of the animals. In rabbits with persistent patency at 30min, the vessel segment around the copper coil was damaged by a compression with blunt forceps once or twice, whereby occlusion was consistently obtained. One hour after occlusion, therapy was started by bolus injection of the thrombolytic agents in combination with heparin (2OOU/kg i.v./i.m.) and aspirin (5mg/kg i.v.). Reperfusion was defined as mean blood flow of more than 10% of the maximal baseline flow measured following external carotid artery occlusion at the beginning of the experiment. Observation of recanalisation was continued for two hours after bolus injection of the thrombolytics. The maximal blood flow obtained, time to lysis and patency at 2h were recorded. At the end of the experiment, the vessel segment containing the copper coil and the adjacent regions were opened longitudinally for visual inspection. To obtain plasma for determination of haemostasis factors and plasminogen activator antigen levels blood samples were taken from the femoral artery prior to thrombolytic treatment and at 30, 60 and 120 min during the observation period of 2 h following bolus administration. Plasma was obtained from 0.9 ml of whole blood to which 0.1 ml of 3.16% sodium citrate was added. 9Ol.~lof this plasma was added to 10~1 of 1OmM PPACK (Calbiochem, La Jolla, CA, USA) to stabilise the plasma against proteolytic degradation. All plasma samples were kept frozen at - 80°C. Test Compounds Application Recombinant DSPA,, was purified from the supernatant of transfected Chinese hamster ovary (CHO) cells as previously describedI and dissolved in PBSbuffer at a concentration of lmg/ml. For administration of 1,2,6 and 20nmol/kg (apparent molecular weight 52 kD) DSPA,r-solution was further diluted with 0.9% sodium chloride to 4.5ml total volume. A commercial t-PA preparation registered for clinical use (Actilyse@) was purchased from Thomae (Biberach, Germany). t-PA was dissolved in sterile
286 Thrombolytic Properties of Desmodus Salivary Plasminogen Activator DSPA,,
water in a concentration of 1 mg/ml and diluted with 0.9% sodium chloride to 4Sml total volume for administration of 2, 6 and 20nmoVkg (apparent molecular weight 65 kD). Streptokinase (Kabikinasee’) was purchased from Pfrimmer Kabi GmbH (Erlangen, Germany). Streptokinase was diluted with 0.9% sodium chloride to 4Sml total volume for administration of 3000,10000 and 30000 IU/kg. Control animals received 4Sml 0.9% sodium chloride, aspirin and heparin as indicated above.
binary response criterion treatment groups were compared versus control using the one-sided. Fishertest (a=O.O5 per comparison). Statistical analysis of the effects of the thrombolytic agents on haemostatic factors (comparison of thrombolytics with vehicle, and comparison between equimolar doses of the thrombolytics) was carried out using the two-tailed Students’ t-test. Plasminogen activator antigen levels were analysed by means of descriptive statistics (meankstandard error).
Haemostasis Factors
RESULTS
Plasma fibrinogen was measured as clottable protein according to the method of Clauss.23 Plasminogen and aZantiplasmin were determined on microtitre plates employing the chromogenic substrate S2251 (Kabi Vitrum, Munich, Germany) and values were expressed as percentage of prevalue plasma levels. For comparison among groups, the percent changes from initial values were calculated.
Thrombolysiskeperfusion and Reocclusion The incidence of reperfusion and patency at the end of a 2 h observation period as well as time to lysis are summarized in Table 1. DSP&r at the intravenous bolus dose of 1nmoYkg reperfused l/6 animals treated to >lO% of the initial blood flow (Fig. 1). The administration of 2, 6 and 20nmoYkg resulted in successful reperfusion in 66%, 57% and 100% of the animals. t-PA at a dose of 2nmoVkg was ineffective in restoring blood flow, however, t-PA at 6 and 20 nmoY kg resulted in successful reperfusion in 83% and 86% of the animals. Low and high dose streptokinase (3000 and 30OOOIU/kg) caused thrombolysis in 2/4 and l/6 animals, whereas the intermediate dose (lOOOOIU/kg) led to thrombolysis in 4/6 animals. This effect of the intermediate dose of streptokinase is comparable with effects of intermediate doses of DSPA,, and t-PA. A further increase of the dose of streptokinase (from 10000 to 30OOOIU/kg) resulted in a marked decrease in efficacy. None of the arteries reperfused with SK showed a high grade reperfusion (~80% initial flow). The time to lysis did not show a clear dose-dependency and did not differ between DSPA,r and t-PA.
Plasminogen Activator Antigens DSP&r antigen levels in rabbit plasma were determined using a sandwich ELISA employing biotinylated rabbit anti-DSPA,r antibodies. t-PA antigen concentrations were also determined by ELISA employing a commercially available kit (Biopool, Umea, Sweden). Statistics The mean blood flow was calculated as area under the flow velocity curve divided by duration of the observation period (AUC/120 min). Thrombolysis was defined as a mean flow after thrombolysis of at least 10% of the baseline blood flow. With regard to this
Table 1 Comparison of frequency of reperfusion, patency at 2h and time to lysis with DSPA,,, t-PA and SK. Figures given in parenthesis represent percent of total animals reperfused, percentage with high grade reperfusion and percentage of patency at 2h
Reperfusion mean blood flow >lO%
Dose of thrombolytic agent
o/7
Control DSPA,, [nmol/kg]
t-PA [nmol/kg]
SK [W/kg]
Reperfusion mean blood flow >80%
(0%)
-
(100%)
2/7
Patency at 2 h
1 2 6 20
l/6 *4/6 +4/7 *7/7
2 6 20
l/6 ‘516 W7
116
3OOtl
2J4 *4/6 l/6
2l4 316 116
(*significant vs control, ~~0.05, Fishers exact test)
Time to lysis min (+SEM)
(5) (17%)
28
(3) (10)
Fibrinolysis
287
130 120
-
$
110
G:
loo-
ag
90
3
ao- .-
1
70
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60 -
:g
50
2
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-
-
-
-
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10 -
0’ &i?
I
’
CONTROL
1
2
6
20
2
following
thrombolysis
calculated
?? , I
20
3
as percent
SK at the higher doses had a tendency to a slow lysis time compared to DSPA,, and t-PA. Reocclusions occurred with all three agents in spite of the adjunctive treatment with heparin and aspirin (compare patency at 2h with reperfusion in Table 1). Haemostasis Factors
Fibrinogen levels measured at baseline and during the observation period decreased significantly with t-PA at the 6 and 20 nmol/kg dose (Fig. 2). In contrast neither DSPA,r nor SK depleted plasma fibrinogen significantly. Plasminogen and aZantiplasmin levels measured at baseline and 30min after starting thrombolysis (see Fig. 3) were not significantly decreased by DSPA,r and t-PA at the 1, 2 and 6nmoYkg dose while the 20nmoYkg dose of both thrombolytics significantly decreased olZantiplasmin to 85.3?2.5% (p < 0.01) and 54.6f4.7% (p C 0.001) of the initial value respectively. The decrease by t-PA was much more pronounced than that caused by DSPA,i (p < 0.001). Plasminogen plasma levels 30min after starting thrombolysis were not significantly decreased by DSPA,r at all doses studied, however, the highest dose of t-PA led to significant plasminogen degradation (p < 0.001). In SK treated animals only fibrinogen levels were determined which did not decrease significantly with any dose. Plasminogen Activator Antigen Levels
The plasma antigen levels of both DSPA,i and t-PA
of flow before
10
30
[kU/kd SK
[=Mkgl t-PA
bW431 DSPAa, Fig. 1 Mean blood flow/min
6
Y
thrombus
generation.
(Fig. 4) measured 30,60 and 120min decreased with time in a dose-dependent manner. The plasma level of DSPA,i exceeded that of t-PA for equimolar doses at all time points measured.
DISCUSSION DSPA,i is an effective thrombolytic agent in this rabbit copper coil model of carotid artery thrombosis. DSPA,i is at least as efficacious as t-PA in this model, while there is evidence to suggest that DSPA,i may be more potent by a factor of 2-3 than t-PA calculated on a molar basis. The higher potency of DSPA,, could be explained by a slower clearance compared to t-PA as suggested by higher plasma levels 30 to 120min post dosing. Not only the frequency of reperfusion but also the time to clot lysis characterises the clinical efficiacy of thrombolytic therapy. There were no differences between DSPA,r and t-PA in this respect whereas clot lysis with SK appeared to be somewhat slower. SK showed a bell-shaped dose-response relationship and was only effective in 4/6 animals in the intermediate dose of lOOOOIU/kg i.v. bolus, due to rapid clearance from the blood and the lack of fibrin binding. *’ All arteries which reperfused with SK achieved only low grade patency (<80% of initial flow) indicating that bolus SK was a poorly efficacious thrombolytic treatment in the rabbit model. With regard to the haemostatic parameters, DSPA,r differed from t-PA, as it was demonstrated to cause no significant fibrinogenolysis nor plasminoaZantiplasmin plasma levels gen depletion.
288 Thrombolytic Properties of Destnodus Salivary Plasminogen Activator DSPA,,
::1i__-_l 0’
E
s g g li
30’
60’
120’
125
100
75
150 E f F 5 li
An additional aim of the present study was to determine whether the present animal model was useful for the evaluation of novel fibrinolytic agents. The bolus intravenous dosing schedule was chosen to facilitate prompt initiation of thrombolytic therapy. Spontaneous flow restorations occurred infrequently. One short spontaneous flow restoration of 3min duration was observed in 1 out of 7 (0.9% sodium chloride/heparin/aspirin treated) control rabbits, however this did not meet the criteria defined above as thrombolysis and was as such counted as ‘no reperfusion’. Because of the small distance of the copper coil from the point of insertion the influence of methodological factors in the coil models described by Kordenat et al.” and Bergmann et a126e.g. the speed with which the copper coil is inserted and certain other variables can be excluded in the present model. However, the geometry of the coil and the number of turns greatly influence the time to occlusion of the vessel. In the present study a coil with 5 turns at a distance of approximately 0.2mm and a diameter of 1.8mm was chosen as under these conditions the coil fits snugly against the vessel wall with no acute occlusion of the vessel occurring after coil insertion. In addition, intimal injury of the artery by careful compression with blunt forceps is a prerequisite for a thrombotic occlusion in some rabbits. In comparison with previous data2’ obtained in a rabbit model of peripheral arterial thrombosis following bolus application of t-PA and bat-PA (DSP&2)
a
‘*O I
B t0
100
d
125
b
100
& 5 8 C
E d P
60 60 40 tn c
3 2
0’
30’ 60’ 120’ time post-dosing [min]
Fig. 2 Time course of plasma fibrinogen levels following i.v. bolus injection of DSPA,,, t-PA and SK in rabbits. The figure shows percentage change from initial values in plasma fibrinogen levels at 30,60, and 120min post-dosing. (mean+SEM n=f%7; asterisks refer to significance versus control (*p
2
6 20 DSPA,,
120
K 100 % ?.%I 60 E E B 60 E $ a”
4
40
20 nmol/kg DSPA,,
decreased to a lesser extent than with t-PA. The absence of fibrinogenolysis with any dose of SK again indicates the poor efficacy of this non clot selective agent when given i.v. bolus. The plasma half-life of DSPA,i exceeded that of t-PA at all doses.
t-PA
t-PA
Fig. 3 Plasminogen and a2-antiplasmin levels at 30min postdosing. The figure shows percentage change from initial values. (meanHEM, n=6-7, *p
Fibrinolysis
289
1200 E 2
6 1
1000
c
a, 5
\
800
k .--0, s F g a
P
600
400
200
0 0
30
60 DSPA
90
120
0
30 time
Fig. 4 Plasma antigen levels (@ml)
of DSPA,,
60
90
120
t-PA
a,
post-dosing
[min]
and t-PA following iv. bolus injection at 30,60, and 120min (mean+SEM,
and in a dog model with coronary occlusion*” the median time to lysis (time to reperfusion) was longer and the incidence of reperfusion was lower for t-PA and bat-PA than in the present model. Both t-PA and DSPA,, were about 7 times more potent in our copper coil models compared to t-PA and DSPA,i (bat-PA) in the above mentioned model. One possible explanation for this could be a difference in clot structure in the two models. More likely adjunctive therapy with heparin and aspirin has increased the effectiveness of t-PA and DSPA,, in our study. In summary, the carotid artery thrombosis model presented here is a method suitable to study fibrinolytic agents in rabbits. The new bat plasminogen activator DSPA,, is an effective thrombolytic agent in experimental arterial thrombosis. It is more potent and more clot (fibrin) specific than t-PA. The prolonged half-life of DSPA,i compared to t-PA together with its superior clot specificity may allow a bolus treatment with this agent to achieve a rapid and safe thrombolysis in myocardial infarction and possibly in stroke.
2.
3.
4.
5.
6.
7.
ACKNOWLEDGEMENTS
8.
The authors would like to thank Eva Maria Jahn for secreterial assistance, Karola Hohlmann for statistical analysis, Gabi Gehrmann and Petra Kolsch for the measurement of hemostasis factors. We thank our colleagues at Berlex Biosciences, Brisbane, CA, USA, who provided us with the DSPA,, used in this study.
9.
10.
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11.
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Received: 8 September 1992 Accepted after revision: 7 December 1992 Offphnt orders to: W. Witt, PhD, Cardiovascular Pharmacology, Schering AG, Miillerstr. 170-178, 1000 Berlin 65, Germany. Phone: (30)-4684088, Fax: (30)-46916704.
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