effective thrombolysis without marked plasminemia...

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Effective Thrombolysis Without MarkedPlasminemia After Bolus Intravenous

Administration of Vampire Bat SalivaryPlasminogen Activator in Rabbits

Stephen J. Gardell, PhD; Denise R. Ramjit, BS; Inez I. Stabilito, BS; Tsuneo Fujita, MS;

Joseph J. Lynch, PhD; Gregory C. Cuca, MS; Deepak Jain, PhD; Shiping Wang, MS;

Jwu-sheng Tung, PhD; George E. Mark, PhD; and Ronald J. Shebuski, PhD

Background. The use of recombinant tissue-type plasminogen activator (t-PA) in thrombolytictherapy is frequently associated with significant fibrinogenolysis. In contrast, recombinantvampire bat salivary plasminogen activator (Bat-PA) displays strict fibrin specificity, an

attribute that could be desirable in a fibrinolytic agent.Methods and Results. The efficacy and fibrin selectivity of Bat-PA was evaluated and compared

with that of t-PA using a rabbit model of femoral arterial thrombosis. Administration of 8.1, 14,and 42 nmol Bat-PA/kg by bolus intravenous injection restored flow in 50%o, 75%, and 80% ofthe rabbits, respectively. The incidence of reperfusion after bolus intravenous injection of 14and 42 nmol t-PA/kg was 15% and 78%, respectively. The maximal femoral artery reperfusionflows were equivalent after treatment with 42 nmol Bat-PA/kg or 42 nmol t-PA/kg, but the timeto reach maximal flow for Bat-PA was approximately one half that of t-PA. Furthermore, therapid restoration of flow by 42 nmol Bat-PA/kg, in contrast to equimolar t-PA, was accom-plished without fibrinogenolysis and with only small decreases in the plasminogen anda2-antiplasmin levels. Equipotent doses of Bat-PA and t-PA both resulted in approximate2.5-fold increases in the template bleeding times of aspirin-pretreated rabbits. The clearance ofBat-PA from rabbits exhibited biexponential elimination kinetics; approximately 80% wascleared by the relatively slow phase (half-life of 17.1 minutes). Overall, Bat-PA was clearedapproximately fourfold slower than t-PA.

Conclusions. Bolus intravenous administration of Bat-PA would facilitate prompt initiation ofthrombolytic therapy, and the avoidance of plasminemia could result in fewer and less severebleeding complications. (Circulation 1991;84:244-253)

T he salutary effect of thrombolytic therapy de-pends on the timely administration of a

fibrinolytic agent soon after the onset of theischemic event.1 Hence, there has been interest inestablishing therapeutic regimens for the facile andrapid introduction of plasminogen activators (PAs)lby intravenous2,3 or intramuscular bolus administra-

From the Departments of Biological Chemistry (S.J.G.), Phar-macology (D.R.R., I.I.S., T.F., J.J.L., R.J.S.), and Cellular andMolecular Biology (G.C.C., D.J., S.W., J-S.T., G.E.M.), MerckSharp & Dohme Research Laboratories, West Point, Pa., andRahway, N.J.

Presented in part at the Tenth International Congress onFibrinolysis, Indianapolis, Ind., August 1990.Address for correspondence: Stephen J. Gardell, PhD, Depart-

ment of Biological Chemistry, Merck Sharp & Dohme ResearchLaboratories, West Point, PA 19486.

Received November 21, 1990; revision accepted February 26, 1991.

tion.4 Bolus intravenous administration of a PA notonly hastens the initiation of therapy but may alsoimprove efficacy, as suggested by the results of recentclinical studies indicating that rapidly achieving ahigh concentration of tissue-type plasminogen activa-tor (t-PA) enhances thrombolysis.5-7t-PA is not ideally suited for dosing by bolus

intravenous administration because of its relatively

See p 436rapid clearance.8 t-PA variants that are eliminatedslower than the natural protein are currently beingdeveloped and may be eventually useful for thisdosing schedule.9"0 An alternative strategy to cir-cumvent the dosing restriction imposed by the rapidclearance of t-PA is to administer larger amounts;however, this option is generally avoided because ofthe fear of catastrophic hemorrhagic complications.

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Gardell et al Vampire Bat Salivary Plasminogen Activator 245

The spontaneous, internal bleeding complicationsthat can accompany thrombolytic therapy probablyresult from degradation of blood coagulation factorsand vascular injury. Spontaneous hemorrhagic com-plications attendant to fibrinolytic therapy have beenshown to be correlated with elevated fibrinogendegradation productsll-13 and prolonged templatebleeding times.14 Interestingly, short courses ofthrombolytic therapy appear to induce less seriousbleeding than regimens involving prolonged admin-istration of the fibrinolytic agent.15A PA that exhibits strict selectivity toward fibrin-

bound plasminogen may cause fewer and less severebleeding complications by avoiding the perilous con-sequences of plasminemia. Accordingly, t-PA waspredicted to be a safer thrombolytic agent thanstreptokinase largely because of the relative prefer-ence of t-PA for fibrin-bound plasminogen.16 How-ever, the results of numerous clinical investigationshave shown that the use of t-PA is frequently asso-ciated with a significant, although variable, degree offibrinogenolysis.1217 The pronounced systemic effectof t-PA may arise from the ability of fibrinogen in aplasma milieu to serve as a cofactor for t-PA activi-ty.18 In contrast to t-PA, the PA present in vampirebat saliva (Bat-PA) exhibits a strict requirement for afibrin cofactor19 that is not satisfied by fibrinogen(P.W. Bergum and S.J. Gardell, manuscript in prep-aration). Furthermore, we have also shown thatBat-PA activity is quiescent in human plasma butlevels of activity similar to that of t-PA are mani-fested in the presence of fibrin.20

In the present study, a rabbit model of arterialthrombosis was used to initially evaluate the use ofBat-PA as a fibrinolytic agent. The efficacy and fibrinselectivity of Bat-PA were monitored as well as itspharmacokinetics and its effect on template bleedingtimes. Bat-PA exhibits remarkable selectivity towardfibrin-bound plasminogen in vivo despite a bolusintravenous injection dosing schedule. Hence,Bat-PA represents a unique opportunity to evaluatethe importance of fibrin selectivity for the safe andefficacious implementation of thrombolytic therapy.

MethodsPlasminogen Activators

Recombinant t-PA (Activase), predominantly one-chain material, was obtained from Genentech, SouthSan Francisco, Calif. t-PA was dissolved in buffersupplied by the manufacturer to a final concentrationof 2 mg/ml (equivalent to 28 nmol/ml). RecombinantBat-PA was produced by heterologous expression ofthe Bat-PA cDNA'9 in an African Green Monkeykidney cell line (CV-1P) using a microcarrier beadprocess in spinner flasks (J.W. Tung et al, manuscriptin preparation). Briefly, a vampire bat salivary glandcDNA library'9 provided the template for polymer-ase chain reaction amplification of the cDNA encod-ing Bat-PA. The 5' oligonucleotide primer used forthe polymerization chain reaction contributed the

vertebrate consensus initiation flanking sequence,CCACC.21 Both the 5' and 3' oligonucleotide prim-ers contained appended restriction endonucleasesites that enabled subcloning of the amplified prod-uct into the intermediate vector pSP73 (Promega,Madison, Wis.). One of these Bat-PA cDNA cloneswas chosen as the source of the insert DNA; it wassequence-verified and subsequently ligated into anHIV LTR promoter expression vector (pCD23). Cal-cium phosphate-mediated transfection of CV-1Pcells constitutively expressing HIV-TAT led to theisolation of the clones stably expressing recombinantBat-PA. The expressed protein was secreted into theconditioned media and purified by passage over anaffinity column containing immobilized Erythrinatrypsin inhibitor (Amer. Diagnostica, Inc., Green-wich, Conn.).22 The purified Bat-PA preparation wasdiafiltered into a buffer containing 50 mM sodiumacetate and 0.01% Triton X-100 at pH 5.0 (SATbuffer) and stored as a lyophilized powder at -70°C.Immediately before use, the Bat-PA was resus-pended, and its concentration was determined usingan extinction coefficient (1%, 1 cm, 280 nm) of 19.0.The extinction coefficient was derived by amino acidcomposition analysis and verified by active site titra-tion using 4-methylumbelliferyl p-guanidinoben-zoate.19 For comparative purposes, the PAs used inthis study were administered on a molar basis ratherthan on a weight basis due to their dissimilar molec-ular mass values (Bat-PA and t-PA are approxi-mately 50 and 70 kDa, respectively).

Rabbit Model of Peripheral Arterial ThrombosisMale New Zealand White rabbits weighing 2.0-2.5

kg were anesthetized with sodium pentobarbital (35mg/kg) administered via the marginal ear vein. Theright carotid artery was cannulated for recording ofarterial blood pressure with a Statham P50 trans-ducer (Grass Instruments, Quincy, Mass.). A solu-tion of sodium pentobarbital was infused continu-ously (6 mg/kg/hr in a volume of 0.6 ml/hr) throughthe carotid artery cannula to maintain the anestheticlevel of the rabbit.The establishment of an occlusive thrombus in the

femoral artery was carried out essentially as de-scribed elsewhere.23 Briefly, the right femoral arterywas isolated distal from the inguinal ligament andtraumatized distal from the lateral circumflex arteryby rubbing the artery on the jaw of a forceps. Anelectromagnetic flow probe (Carolina Medical Elec-tronics, Inc., King, N.C.) was placed around the rightfemoral artery distal to the lateral circumflex arteryto monitor femoral artery blood flow. The superficialepigastric artery was cannulated for induction of athrombus. Thrombi were localized distal from thelateral circumflex artery with snares approximately 1cm apart and induced by the sequential injection of 5gl human thrombin (5 units), 5 ,ul of 0.25 M CaCl2,and 10-20 gl autologous arterial whole blood suffi-cient to distend the artery. After 30 minutes, thesnares were released, and the flow was monitored for


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246 Circulation Vol 84, No 1 July 1991

30 minutes to confirm that blood flow was completelyobstructed by the thrombus.

Rabbits were randomized into seven treatmentgroups: 1) saline, n = 12; 2) 14 nmol/kg t-PA, n = 13; 3)42 nmol/kg t-PA, n= 18; 4) 4.7 nmol/kg Bat-PA, n =7;5) 8.1 nmol/kg Bat-PA, n=6; 6) 14.0 nmol/kg Bat-PA,n=8; and 7) 42 nmol/kg Bat-PA, n=10. PAs wereadministered as bolus injections via the marginal earvein at 60 minutes after the initiation of thrombusinduction (30 minutes after removal of the femoralartery snares, as described above). Reperfusion, orrestoration of femoral arterial blood flow, was de-fined as measurable flow that persisted for more than3 minutes. At 120 minutes after the administration ofPAs, the residual thrombus within the femoral arterywas excised and blotted, and wet weight was deter-mined. Data are expressed as mean+SEM. Among-group comparisons of incidences of reperfusion andresidual thrombus mass were conducted using Fish-er's exact probability test or analysis of variance,followed by Dunnett's test for multiple group com-parisons, where appropriate.Blood for the measurement of plasma fibrinogen

concentrations and a2-antiplasmin and plasminogenactivities was drawn from the right carotid arterycannula into EDTA-precoated tubes containing 5 ,ulof 1 ,uM D-Phe-Pro-Arg-CH2Cl/ml blood (to preventfibrinogen degradation) before (time 0) and at 30-minute intervals up to 2 hours after the administra-tion of the PA. The samples were centrifuged at 4°Cat 1,500g for 15 minutes, and the platelet-free plasmawas stored in liquid nitrogen until assayed.

Rabbit Bleeding Time ModelTemplate bleeding times were measured in aspirin

(ASA)-pretreated animals essentially as describedelsewhere.24 Male New Zealand White rabbits (2-2.5kg) were anesthetized with sodium pentobarbital (35mg/kg) administered via the marginal ear vein. Theright carotid artery was cannulated for recording thearterial blood pressure and for continuous intrave-nous infusion of sodium pentobarbital, as describedpreviously. The carotid artery was also cannulated towithdraw blood samples for the determination ofplatelet counts. The lateral and medial aspects ofboth upper hind legs of the rabbits were shaved, anda depilatory agent (Nair, Carter Products, NewYork) was applied to prepare the skin for the deter-mination of template bleeding times. Bleeding timeswere performed using a spring-loaded Simplatebleeding time device (Organon Teknika, Durham,N.C.). Uniform incisions were made with the Sim-plate device while avoiding superficial veins. Bloodwas blotted from the incision with filter paper every30 seconds; care was taken to avoid contact with theincision. The bleeding time was determined by mea-suring the time from incision until blood no longerstained the filter paper.

Rabbits were administered ASA (15 mg/kg) viathe marginal ear vein 60 minutes before the admin-istration of test agent. Rabbits were randomized into

four treatment groups: 1) ASA+SAT buffer (Bat-PAvehicle), n=5; 2) ASA+42 nmol/kg t-PA, n=5; 3)ASA+14 nmol/kg Bat-PA, n=5; and 4) ASA+42nmol/kg Bat-PA, n=5. PAs or SAT were adminis-tered as bolus injections via the marginal ear vein (attime designated as 0 minutes). Template bleedingtimes were determined 5 minutes before ASA admin-istration (control), 5 minutes before the administra-tion of PA or SAT (post-ASA), and at 5, 15, 30, 60,90, 120, and 180 minutes after administration of PAor SAT. Data are expressed as mean+SEM. Com-parison of the control and post-ASA template bleed-ing times (n=20) was made by Student's paired t test.Within-group comparisons of post-PA bleeding timesto post-ASA values were made using a one-wayrepeated-measures analysis of variance, followed by aDunnett's test for multiple comparisons to the cor-responding post-ASA group. Blood was drawn fromthe carotid artery and anticoagulated with 3.8%trisodium citrate before ASA administration (con-trol), immediately before PA or SAT administration(post-ASA), and at 15 and 60 minutes after PA orSAT administration. Whole-blood platelet countswere determined using a System 9000 cell counter(Baker Instrument Corp., Allentown, Pa.).

Clearance of Bat-PA or t-PA in RabbitsBat-PA or t-PA (2.2 nmol/kg) was administered to

rabbits by bolus intravenous injection via the mar-ginal ear vein. Blood samples (0.9 ml) were collectedat various times through an indwelling catheter in thecarotid artery into a syringe containing 0.1 ml of 3.8%trisodium citrate. Samples (0.5 ml) were acidified bythe addition of 0.25 ml of 1 M acetic acid at pH 4.0and centrifuged. The plasma was removed immedi-ately and stored at -80°C until assayed for residualPA activity. PA activities were converted to func-tional molarities using standard activity curves gen-erated with known concentrations of Bat-PA or t-PA.The elimination rates of Bat-PA or t-PA after theiradministration to rabbits were analyzed with an itera-tive nonlinear regression computer program that fittedthe data to monoexponential and multiexponentialdecay functions (GraphPad Inplot, GraphPAD Soft-ware, San Diego). Pharmacokinetic parameters werecalculated from the derived coefficients and exponentsusing standard formulas for the disposition of com-pounds from plasma after bolus injection.25

Analytical MethodsPlasminogen activator assay. Fibrinogen, Glu-plas-

minogen, hirudin, Spectrozyme P1, and two-chaint-PA activity standard were from American Diagnos-tica Inc., Greenwich, Conn., and thrombin was fromSigma Chemical Co., St. Louis. Solutions (190 ,gl)placed in the wells of a microtitration plate contain-ing fibrinogen (0.132 mg/ml), Glu-plasminogen (0.66,uM), and thrombin (0.26 units/ml) in 10 mMHEPES, 150 mM NaCl, and 0.24mM Triton X-100 atpH 7.5 were incubated at 37°C for 30 minutes.Ten-microliters hirudin (200 units/ml) was added,


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TABLE 1. Recanalization of Occluded Femoral Artery in Rabbits After Intravenous Bolus Administration ofTissue-Type Plasminogen Activator, Vampire Bat Salivary Plasminogen Activator, or Saline Control

Incidence of

Treatment reperfusion Median time to Residual thrombusgroup Ratio % reperfusion (min) mass (mg)

Saline 0/12 0 > 120 9.6+0.8t-PA

14 nmol/kg 2/13 15 >120 8.2+0.742 nmol/kg 14/18 78 45 4.7±0.5t

Bat-PA4.7 nmol/kg 0/7 0 >120 8.6±1.78.1 nmol/kg 3/6 50t 109 6.3+1.614.0 nmol/kg 6/8 75* 35 6.5 ±2.042.0 nmol/kg 8/10 80* 26 2.6±0.4t

Residual thrombus mass values are mean±SEM. t-PA, tissue-type plasminogen activator; Bat-PA, vampire batsalivary plasminogen activator.

*p<0.01 vs. Saline group by Fisher's exact test; tp<0.01 vs. Saline group by one-way analysis of variance, followedby Dunnett's multiple comparison test; tp<0.05 vs. Saline group by Fisher's exact test.

and the clots were incubated for an additional 30minutes at 37°C. Thirty microliters of 3.33 mMSpectrozyme P1 and 20 ,ll PA were added, and therelease of p-nitroaniline from the chromogenic sub-strate was measured spectrophotometrically using aThermomax microplate reader (Molecular DevicesCorp., Menlo Park, Calif.). The activities (IU/ml)were were derived from a standard curve generatedwith a two-chain t-PA activity standard.

Miscellaneous ex vivo assays. Plasminogen levelswere measured using the chromogenic substrate Spec-trozyme P1 and urokinase (Calbiochem, Behring Di-agnostics, San Diego).26 Plasma for this assay wasacidified and neutralized to destroy plasmin inhibitorsbefore testing. The activity of a2-antiplasmin wasdetermined by a modification of the Coatest Antiplas-min method (KabiVitrum AB, Stockholm) using thechromogenic substrate S-2251.27 The results for plas-minogen and a2-antiplasmin are expressed as a per-centage of activity relative to control (pretreatment)activity. The fibrinogen concentration was determinedby the Clauss thrombin-clotting method28 using theElectra 800 automated clot timer (Medical LaboratoryAutomation, Mt. Vernon, N.Y.) and commerciallyavailable reagents (American Dade, Aquada, PuertoRico). Among-group comparisons of fibrinogen, plas-minogen, and a2-antiplasmin values were performedby analysis of variance, followed by Dunnett's test formultiple comparisons.

ResultsEfficacy of Bat-PA In VivoThe incidence and median time to reperfusion as

well as residual thrombus wet weights after intrave-nous bolus administration of t-PA and Bat-PA to therabbits are summarized in Table 1. No spontaneousreperfusion was observed in 12 saline-treated controlrabbits. t-PA at an intravenous bolus dose of 1 mg/kg(or 14 nmol/kg) was essentially ineffective, elicitingrestoration of blood flow in only two of 13 rabbits.

However, the administration of t-PA at 42 nmol/kgresulted in successful reperfusion in 14 of 18 (78%)rabbits, with a median time to reperfusion of 45minutes. Residual thrombus mass, determined at thetermination of the study, was reduced significantly bythe intravenous administration of t-PA (42 nmol/kg)compared with saline control (4.7+0.5 versus 9.6 +0.8mg, respectively,p<0.01). The intravenous adminis-tration of 8.1, 14, and 42 nmol Bat-PA/kg resulted indose-dependent increases in reperfusion efficacy(successful reperfusions: three of six rabbits [50%],six of eight rabbits [75%], and eight of 10 rabbits[80%], respectively), with median reperfusion timesof 109, 35, and 26 minutes, respectively. Residualthrombus mass was reduced significantly by the in-travenous administration of 42 nmol Bat-PA/kg com-

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0 30 60 90 120Time (min)

FIGURE 1. Graph showing restoration of blood flow in theoccluded femoral artery. Rabbits received 42 nmol/kg tissue-typeplasminogen activator (-), 14 nmol/kg vampire bat salivaryplasminogen activator (o]), or 42 nmol/kg vampire bat salivaryplasminogen activator () by intravenous bolus injection.

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FIGURE 2. Graph showing effect of vampire bat salivaryplasminogen activator and tissue-type plasminogen activatoradministration on the plasma level of clottable fibrinogen.Rabbits containing an occluded femoral artery received 42nmol/kg tissue-type plasminogen activator (0, n=18) or vam-pire bat salivary plasminogen activator (o, n=10) by bolusintravenous injection. Control rabbits received saline (A,n=12). *p<O.Ol vs. the corresponding saline-treatment sam-pling times.

pared with saline controls (2.6+0.4 versus 9.6±0.8mg, respectively, p<0.01).

Figure 1 depicts femoral arterial reperfusion bloodflows in rabbits treated intravenously with 42 nmolt-PA/kg, 14 nmol Bat-PA/kg, and 42 nmol Bat-PA!kg. Early femoral artery reperfusion blood flow pro-files were similar for the 42 nmol t-PA/kg and 14nmol Bat-PA/kg. The maximal femoral artery reper-fusion blood flows achieved tended to be greater inrabbits treated with t-PA at 42 nmol/kg relative tothose treated with a threefold lower dose of Bat-PA(14 nmol/kg), although the observed difference wasnot statistically significant. The administration of thehigher dose of Bat-PA (42 nmol/kg) resulted in anearlier reestablishment of femoral artery reperfusionblood flow compared with 14 nmol Bat-PA/kg and 42nmol t-PA/kg. This difference reflected the shortermedian reperfusion time for 42 nmol Bat-PA/kg (26

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minutes) compared with those of 14 nmol/Bat-PA/kg(35 minutes) and 42 nmol t-PA/kg (45 minutes)(Table 1). The maximal femoral artery reperfusionflows were equivalent in the preparations treatedwith t-PA or Bat-PA at 42 nmol/kg.

Fibrin Selectivity of Bat-PA In VivoFigure 2 shows the effects of the intravenous bolus

administration of saline, Bat-PA (42 nmol/kg), ort-PA (42 nmol/kg) on plasma levels of clottablefibrinogen. During the course of the experimentalprotocol, plasma fibrinogen concentrations de-creased inaximally by approximately 6% (from225.6±+11.7 to 212.1±9.9 mg/dl) from control valuesin saline-treated rabbits. Plasma fibrinogen concen-trations decreased maximally by approximately 14%and 69% from control values in rabbits treated withBat-PA (from 238.3±17.8 to 204.9±11.2 mg/dl) andt-PA (from 231.6±6.1 to 71.2±13.1 mg/dl), respec-tively (Figure 2). The fibrinogen concentrations foreach sampling time after the administration of t-PA,but not Bat-PA, were significantly less (p<0.01)compared with the corresponding values for thesaline-treated rabbits.

Figure 3 shows the accompanying effects of saline,Bat-PA, or t-PA administration on the plasma levelsof functional plasminogen and a2-antiplasmin. Plas-minogen and a2-antiplasmin activities were expressedas a percentage of control, preadministration values(Figure 3). During the course of the experimentalprotocol, the plasma plasminogen activities werereduced to the following minimal values: 95.1±1.6%for the saline treatment group, 81.0±2.6% for theBat-PA treatment group, and 26.5±2.6% for thet-PA treatment group (Figure 3, left panel). Theminimal values for the plasma a2-antiplasmin activi-ties were: 86.2±5.4% for the saline treatment group,73.7±3.3% for the Bat-PA treatment group, and35.2±3.1% for the t-PA treatment group (Figure 3,right panel). The reductions in plasminogen anda2-antiplasmin after the administration of t-PA were

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FIGURE 3. Graphs showing effect of vampire bat salivary plasminogen activator and tissue-type plasminogen activatoradministration on theplasma levels ofplasminogen (left panel) and a2-antiplasmin (right panel). Rabbits containing an occludedfemoral artery received 42 nmol/kg tissue-type plasminogen activator (., n=18) or vampire bat salivary plasminogen activator (-,n=10) by bolus intravenous injection. Control rabbits (A, n=12) received saline. *p<9.05 and *p<0.01 vs. correspondingsaline-treatment sampling times.


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50 60

FIGURE 4. Graphs showing clearance of vampire bat salivary plasminogen activator (BAT-PA) and tissue-type plasminogenactivator (t-PA) in rabbits after bolus intravenous administration. Blood samples were withdrawn at the indicated times,anticoagulated, and acidified. Residual plasminogen activator activities were assessed using the coupled enzymatic assay and were

converted to functional molarity by comparison with standard curves generated with known concentrations ofBAT-PA or T-PA.Left panel: 2.2 nmol/kg BAT-PA. Right panel: 2.2 nmol/kg T-PA.

statistically different (p<0.01) from each of thecorresponding sampling times in the saline-treatedrabbits. In contrast, none of the a2-antiplasmin andonly one of the plasminogen determinations from theBat-PA treatment group (60-minute sampling time)were significantly less (p<0.05) than the correspond-ing samples from the saline-treated rabbits.

Pharmacokinetics of Bat-PA and t-PA in RabbitsThe disappearance rates of Bat-PA or t-PA admin-

istered to rabbits by bolus intravenous injection weremonitored by measuring PA activities in plasma. Theactivity determinations were converted to molaritiesof active PA (nM) using standard curves generatedwith known concentrations of Bat-PA or t-PA. Theclearance of Bat-PA was best described by a biphasicelimination profile that exhibited a dominant ,B phase(Figure 4, left panel). The elimination profile of t-PA

TABLE 2. Pharmacokinetic Properties of Vampire Bat SalivaryPlasminogen Activator and Tissue-type Plasminogen ActivatorAfter Intravenous Bolus Administration in Rabbits

Parameter Bat-PA t-PA

CO (nM) 25.3±1.5 32.8+2.0

VD (ml/kg) 87.8±4.7 67.8±4.0t112a (min) 1.48±0.15 1.07±0.10

tl2,8 (min) 17.1±2.3 ...

AUC (nmol . min/l) 201±23 50.5±3.7AUCa(%) 21±3AUC,(%) 79±3 *-

Clip (ml . min1 kg-') 11.4±1.3 44.3+3.5

Values are mean+SEM of four experiments. Bat-PA, vampirebat salivary plasminogen activator; t-PA, tissue-type plasminogenactivator; C0, plasma concentration at time 0; VD, volume ofdistribution; tl2, half-life of the a or f8 elimination phases; AUC,area under the plasma concentration vs. time curves; Clp,, clear-ance rate of Bat-PA or t-PA from plasma. The plasma disappear-ance of plasminogen activator activities was monitored and con-verted to plasminogen activator concentration using standardcurves constructed with Bat-PA or t-PA.

from rabbits is much steeper than that of Bat-PA andcan adequately be described as a monophasic profile(Figure 4, right panel). Table 2 summarizes thepharmacokinetic parameters deduced from multipleexperiments (n=4) using Bat-PA or t-PA. The esti-mated plasma concentrations at time 0 for Bat-PA(25.3 nM) and t-PA (32.8 nM) were 58% and 75%,respectively, of the theoretical values, assuming uni-

form distribution into a plasma volume of 50 ml/kg.The a and /3 phases for Bat-PA elimination displayedhalf-lives of 1.5 and 17.1 minutes, respectively. Inter-estingly, approximately 80% of the Bat-PA is clearedby the relatively slow ,B elimination phase. Theoverall clearance rate for Bat-PA is approximately 11ml min-1 kg-'. The half-life for the elimination oft-PA was 1.1 minutes, whereas the clearance rate wasapproximately 44 ml min-1 * kg-'. Hence, the clear-ance rate of Bat-PA from plasma after bolus intra-venous administration to rabbits was approximatelyfourfold less than that of t-PA.

Effect of Bat-PA on Bleeding Times inAspirin-Pretreated Rabbits

The control template bleeding times performed on20 rabbits consolidated from the four experimentalgroups was 1.8+0.1 minutes (Figure 5). One hourafter bolus intravenous injection of ASA, the consol-idated group displayed template bleeding times thatwere increased slightly but significantly to 2.4+0.2minutes (p<0.02). The administration of SAT buffer(Bat-PA vehicle) to ASA-pretreated rabbits did notresult in a further elevation of the template bleedingtimes. After the administration of Bat-PA (14 nmol/kg) to ASA-pretreated rabbits (n=5), the templatebleeding times increased significantly, from 1.9+0.2to 5.2+1.4 (p<0.01) and 4.2±0.6 (p<0.05) minutesat 15 and 30 minutes, respectively. The administra-tion of t-PA (42 nmol/kg) to ASA-pretreated rabbits(n=5) caused significant elevations in the template

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L ASAi ASA + t-PA (42 nmol/kg)* ASA + Bat-PA (1 4 nmol/kg)* ASA + Bat-PA (42 nmol/kg)

5 15 30 60 90 120 180

Control iPost-ASA Post-PA (min)FIGURE 5. Bar graph showing prolongation of template bleeding times in rabbits pretreated with aspirin (ASA) and administeredvampire bat salivary plasminogen activator (Bat-PA) or tissue-type plasminogen activator (t-PA). Rabbits were administered 15mg/kgASA by bolus intravenous injection. After 1 hour, 14 nmol/kg Bat-PA, 42 nmol/kg Bat-PA, or 42 nmol/kg t-PA was also givenby bolus intravenous administration. Template bleeding times were determined as detailed in "Methods." Shown are the serialbleeding times (mean±SEM) assayed at the following times: pretreatment (Control), 60 minutes after ASA administration(Post-ASA), and at increasing times after plasminogen activator administration (Post-PA). *p<0.05 and *p<0.01 vs. correspond-ing ASA (n) sampling times.

bleeding times from post-ASA values, from 1.9±0.2to 5.2±0.4 (p<0.01), 5.1±0.7 (p<0.01), and 3.9±0.6(p<0.05) minutes at 15, 30, and 90 minutes, respec-tively. The 14 and 42 nmol/kg doses of Bat-PA andt-PA, respectively, were compared because of theirequivalence at restoring flow in the rabbit femoralarterial thrombosis model (Table 1).A dose of Bat-PA (42 nmol/kg) equimolar to that

of t-PA was also evaluated for its effect on thetemplate bleeding time. In this high-dose Bat-PAgroup (n=5), there was a marked elevation in tem-plate bleeding times earlier in the protocol comparedwith those in the low-dose Bat-PA and t-PA treat-

ment groups. Five minutes after the administration of42 nmol/kg Bat-PA, the template bleeding timeincreased from 3.1±+0.3 to 7.4±+0.8 minutes(p<0.01). The corresponding values at 5 minutes forthe low-dose Bat-PA and t-PA treatment groupswere 3.2+0.6 (p=NS versus post-ASA) and 4.1±0.6(p=NS versus post-ASA) minutes, respectively. Af-ter this early time point, the profiles for elevation intemplate bleeding times were similar for 42 nmol/kgBat-PA, 14 nmol/kg Bat-PA, and 42 nmol/kg t-PA.Furthermore, in each instance, the template bleedingtime values approached post-PA treatment values by60 minutes after PA administration.

TABLE 3. Whole-Blood Platelet Counts in Rabbit Bleeding Time Model

Sampling times (platelet counts/txl 103)Treatment A B C D

SAT buffer 444+79 431±84 409+117 409+84t-PA (42 nmol/kg) 332±64 317+60 334±55 309+53Bat-PA (14 nmol/kg) 422+83 423±70 410+60 426+78Bat-PA (42 nmollkg) 422+35 425+37 436+49 441+59

Values are mean+SEM; n=5 in each treatment group. Blood was withdrawn and platelet counts were determinedat: A, control (5 minutes before aspirin administration); B, 60 minutes after aspirin and 5 minutes before plasminogenactivator administration; C, 15 minutes after plasminogen activator administration; D, 60 minutes after plasminogenactivator administration; SAT buffer, 50 mM sodium acetate and 0.01% Triton X-100, pH 5.0; t-PA, tissue-typeplasminogen activator; Bat-PA, vampire bat salivary plasminogen activator.

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Table 3 summarizes whole-blood platelet countsdetermined in SAT buffer, t-PA, and Bat-PA treat-ment groups in the template bleeding time evalua-tion. No significant differences in platelet countswere noted during the course of the protocol withinany of the treatment groups.

DiscussionOur studies demonstrate that bolus intravenous

administration of Bat-PA will rapidly restore bloodflow in an experimentally thrombosed peripheralartery without precipitating severe plasminemia.Bat-PA was significantly more fibrin specific thant-PA and equal to or superior in thrombolytic po-tency to t-PA with respect to absolute incidence ofreperfusion, magnitude of reperfusion blood flow,and reduction in residual thrombus mass. The bolusintravenous dosing schedule was chosen because itwould facilitate prompt initiation of thrombolytictherapy and, as pointed out by others5-7 for t-PA,may have beneficial consequences on efficacy. Addi-tional studies are required to determine whether theremarkable fibrin selectivity of Bat-PA would resultin less frequent and less severe bleeding complica-tions than those encountered after fibrinolytic ther-apy with t-PA or streptokinase.The causal relation between PA-mediated gener-

ation of the lytic state and bleeding complications iscontroversial. Although t-PA causes a milder fibrin-ogenolytic state than does streptokinase, the use oft-PA as a fibrinolytic agent does not consistentlyresult in a decreased incidence of serious bleedingcomplications.29-32 A correlation between fibrinogendegradation and serious bleeding complications hasbeen noted,11-13 but in other studies, the correlationis weak.17 Hence, a fibrinolytic agent that is morefibrin specific than t-PA, such as Bat-PA, may not besafer than either t-PA or streptokinase. However, theimportance of fibrin specificity has not been convinc-ingly assessed, because the most fibrin-specific agentheretofore available, t-PA, is only fibrin specificrelative to streptokinase, and the use of t-PA forthrombolytic therapy frequently results in apprecia-ble activation of circulating plasminogen.A significant correlation between template bleed-

ing time prolongation and incidence of spontaneousbleeding during thrombolytic therapy with t-PA wasrecently reported.14 If this relation is valid, then thesimilar protracted bleeding times exhibited by ASA-treated rabbits that received t-PA or Bat-PA suggeststhat the use of either of these PAs for thrombolytictherapy would yield similar frequencies of spontane-ous bleeding complications. However, there are manyexamples in which prolonged bleeding times are notassociated with spontaneous, unprovoked bleeding.For example, patients stricken with Glanzmann'sthrombasthenia have elevated template bleedingtimes but generally bleed only as a result of physio-logical or pathological conditions that cause bleedingin normal subjects.33

The Bat-PA-induced prolongation of the templatebleeding times occurs in the absence of appreciablefibrinogenolysis and, thus, is apparently not due to aninhibitory effect of fibrinogen degradation productson platelet aggregation. The observed interferenceby Bat-PA with primary hemostasis may result fromplasmin-mediated proteolysis of cohesive fibrinogenmolecules leading to platelet disaggregation as pre-viously suggested for t-PA by Loscalzo andVaughan.34 The absence of overt plasminemia afterBat-PA administration necessitates that the plasminpurportedly responsible for platelet disaggregationbe generated in the vicinity of the hemostatic plug.Perhaps the Bat-PA activity is stimulated by incipientfibrin or the favorable presentation of the cohesivefibrinogen molecules.The clearance of Bat-PA after bolus injection in

rabbits is best described by a biphasic eliminationprofile. Interestingly, the p elimination phase is dom-inant and accounts for approximately 80% of theBat-PA clearance. In contrast, the elimination oft-PA activity after bolus administration in rabbitsbests fits a monoexponential function, as has beenreported by others.35,36 Biphasic elimination profilesfor t-PA activity in rabbits have also been de-scribed,37'38 but in these instances, in contrast toBat-PA, the rapid a phase dominates the pharma-cokinetic profile. The disparate pharmacokinetic be-havior of Bat-PA and t-PA gives rise to an approxi-mately fourfold difference in their respectiveclearance rates. The basis for the delayed eliminationof Bat-PA is unclear at this time. It was establishedthat elimination of t-PA from plasma is mediatedmainly by the liver and that the rate of clearance isinfluenced by the nature of the oligosaccharidechains38 as well as polypeptide structures resident inthe fibronectin fingerlike and epidermal growth fac-tor-like domains.39 Each of these structural determi-nants of t-PA clearance may differ in the case ofBat-PA. Regardless, it is likely that the slower clear-ance rate of Bat-PA relative to t-PA contributesdecisively to the apparent greater efficacy of Bat-PAas assessed by reperfusion incidence in our rabbitmodel of peripheral arterial thrombosis.The fibrin selectivity exhibited by Bat-PA can also

be responsible in part for the observed difference inpotency according to the "plasminogen steal" hy-pothesis forwarded by Sobel et al.40 According to thishypothesis, a fibrin-selective plasminogen activatorwould not consume circulating plasminogen, andconsequently, plasminogen bound to the occlusivethrombus would not be depleted because of thereestablishment of equilibrium between these twopools of plasminogen. As a result, plasminogen re-mains sequestered with the thrombus, where it isavailable for activation to form plasmin and result insustained intense fibrinolysis.The recanalization caused by t-PA, especially at

the 42 nmol/kg dose, may be due, in large part, to theestablishment of a systemic lytic state. Our compar-ison of Bat-PA with t-PA with regard to efficacy does


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not distinguish between the lytic effects of plasminformed on the clot or in circulation. Hence, thecomparable efficacies of t-PA and Bat-PA at thehigher doses probably reflect a lytic mechanism in thecase of t-PA that may be undesirable in light of safetyconsiderations.

Erythrocyte-rich thrombi such as those formed inour rabbit model are more susceptible to t-PA-mediated lysis than the platelet-rich variety thatmore closely mimics those found in occluded coro-nary vessels.4' The potency exhibited by Bat-PA inthe present study might likewise be diminished whenone attempts to lyse platelet-rich thrombi. Neverthe-less, the extraordinary fibrin selectivity of Bat-PAand the potency of Bat-PA relative to t-PA wouldprobably not change if thrombi enriched with plate-lets were used. Experiments are currently under wayusing other animal models of thrombotic complica-tions to evaluate the consequences of a large contin-gent of platelets within thrombi on the pharmaco-logic profile of Bat-PA.Aside from sparking teleological discussions re-

garding the machinations of a feeding vampire bat,the remarkable fibrin specificity of Bat-PA may be aserendipitous means of attaining a frequently citedtherapeutic goal: localizing the activity of a fibrin-olytic agent to the vicinity of a clot, thereby avoidingthe activation of circulating plasminogen.42 45 Thepotential merits of this goal with regard to safety andefficacy of thrombolytic therapy have not yet beenconvincingly evaluated, because effective doses of thepurported fibrin-specific plasminogen activators re-sult in frequent and sometimes severe episodes ofplasminemia. Hence, Bat-PA and its strict selectivitytoward fibrin-bound plasminogen should serve tocritically assess the possible advantages of a fibrin-specific plasminogen activator for the treatment ofthrombotic complications.

AcknowledgmentsWe thank Dr. P. Friedman for encouragement and

helpful discussions, Drs. M. Silberklang and R. Ellisfor their participation in the heterologous expressionof Bat-PA, Dr. C. Reilly for critical review of themanuscript, and Ms. D. Bonenberger for her assis-tance in the preparation of the manuscript.

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KEY WORDS * fibrinolysis * tissue-type plasminogen activator* vampire bat salivary plasminogen activator * thrombolysis


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Tung and G E MarkS J Gardell, D R Ramjit, I I Stabilito, T Fujita, J J Lynch, G C Cuca, D Jain, S P Wang, J S

administration of vampire bat salivary plasminogen activator in rabbits.Effective thrombolysis without marked plasminemia after bolus intravenous

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