Bioconjugate
ChemBtzy SEPTEMBER/OCTOBER 1991 Volume 2, Number 5 @I Copyright 1991 by the American Chemical Society
REVIEWS Enhancement of the Thrombolytic Potency of Plasminogen Activators by Conjugation with Clot-Specific Monoclonal Antibodies M.Dewerchin and D. Collen’ Center for Thrombosis and Vascular Research, University of Leuven, B-3000 Leuven, Belgium. Received April 11, 1991 Thrombolytic therapy has become a routine treatment of acute myocardial infarction in man. Thrombolytic agents are plasminogen activators which activate the fibrinolytic system in blood (Figure 1) by converting the inactive proenzyme plasminogen to the active enzyme plasmin, a serine protease that degrades the fibrin in the blood clot and thereby causes dissolution of the thrombus. Plasmin, however, also degrades fibrinogen, the circulating precursor of fibrin. Extensive fibrinogen degradation may predispose one to bleeding although such correlation has not been unequivocally observed in clinical trials. Streptokinase and urokinase (two-chain urokinase-type plasminogen activator, tcu-PA, UK) are plasminogen activators belonging to the group of the “classic”thrombolytic agents. These indiscriminately activate fibrin-bound as well as circulating plasminogen and consequently induce a systemic fibrinolytic state, characterized by extensive depletion of armtiplasmin, the physiologicplasmin inhibitor in blood, and degradation of fibrinogen and other circulating coagulation factors. With the ‘hew generation” thrombolytic agents tissue-type plasminogen activator (tPA) on one hand and single-chain urokinase-type plasminogen activator (scu-PA) on the other hand, plasminogen activation is assumed to occur preferentially at the fibrin surface. This fibrin-specificplasminogen activation, resulting in localized generation of active plasmin in the vicinity of the clot, would render these fibrin-specific thrombolytic agents superior to the classic agents. However, clinical experience has revealed that at the high doses of t-PA or scu-PA required for efficient coronary thrombolysis, the fibrin specificity is only relative and systemic activation of the fibrinolytic system with the risk of bleeding complications may still occur ( I ) .
* Address for correspondence: D.Collen, M.D.,Center for Thrombosis and Vascular Research, K.U. Leuven, Campus Gasthuisberg, 0 & N, Herestraat 49, B-3000 Leuven, Belgium. lQ43-I002/91 f2902-O293$O2.5Qf0
PLASMINOGEN ACTIVATOR
PLASM I NOGEN
1
FIBRINOGEN d DEGRADATION PRODUCTS
+
4 PLASMIN
$.
FIBRIN
F I B R I N DEGRADATION PRODUCTS
Figure 1. Schematic representation of the fibrinolyticsystem. Several approaches to develop more potent and more clot-selective thrombolytic agents are being explored. These include the construction of mutants and variants of the physiologic plasminogen activators t-PA or scu-PA and of recombinant chimeric molecules containing the protease part of urokinase and fibrin-binding structures of t-PA or of plasminogen (for review, see refs 2 and 3). In this review, we will focus on an alternative approach toward the development of improved thrombolytic agents, consisting of antibody-mediated targeting of plasminogen activators to the thrombus. The concept of using monoclonal antibodies as carrier molecules for therapeutic agents has proven its value in the area of tumor therapy (4). The same strategy was first applied to cardiovascular disease by Bode et al. (51,who showed successful targeting of a plasminogen activator to fibrin, one of the components of a thrombus, using a conjugate of urokinase with a monoclonal antibody directed against fibrin but not crossreacting with fibrinogen. Apart from fibrin-like material, a thrombus also contains platelet-rich material, representing an alternative target site in the blood clot. Thus, targeting of plasminogen activators might also be achieved by monoclonal antibodies that discriminate between activated platelets in the thrombus and circulating resting platelets. 0 1991 Amerlcan Chemical Society
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Table I. In Vitro Fibrinolytic Prowrties of Antifibrin Antibody/Plasminogen Activator Conjugates In vitro fibrinolytic potency
plasminogen activator moiety rt-PA B-chain t-PA B-chain UK LMW-UK
antibody moiety
SCU-PA‘ LMW-scu-PA rscu-PAf rtcu-PAR rtcu-PA/Th rscu-PA-32ki
IgGi F(ab’)z IgGl Fab’ IgGi IgGi Fab’ Fab’ CH2-truncated IgG IgGi IgGi IgGl F(ab’)z
rtcu-PA-32k
F(ab’I2
rtcu-PA-BSk/T F(ab’)2 rtcu-PA-33k j rtcu-PA-33Kk
acFv‘ scFv
designation t-PA-59D8 t-PA-59D8 64C8-UK 64C5Fab’-UK 64C5-UK 59D8-UK 59D8-UK scu-PA-59DBFab’ scu-PA-59D8 rscu-PA/MA-l5C5 rtcu-PA/MA-lBC5 rtcu-PA/MA-l5C5/T rscu-PA-32k/ MA-15C5-F(ab’)~ rtcu-PA-32kI MA-l5C5:F(ab’)z rtcu-PA-32kl MA-15C5-F(ab’)2/T KizGoSsz K12G&S2
fibrin monomer assay“ enhancement conjugation epitope factofl chemical @-chain 10 recombinant @-chain chemical @-chain 100 chemical @-chain 95 chemical @-chain 250 chemical @-chain 100-250 chemical @-chain 250 chemical @-chain 230 recombinant @-chain 10-50 chemical D-dimer chemical D-dimer chemical D-dimer chemical D-dimer -
plmma clot lysisb enhancement fibrinogen factofl breakdownd refs 3.2 reduced 12 ineffective 17,18 5 2-4 14 -
-
15
4.5
-
reduced
-
1.6-1.8
-
6.4 2.2 >5(l 5
increased reduced increased
12,15 15 16 19 6 6 22 24
-
reduced
?
chemical
D-dimer
-
0.6
increased
25
chemical
D-dimer
-
>2,
?
25
recombinant D-dimer recombinant D-dimer
-
13 2.5
reduced increased
26 26
-
a The fibrin monomer assay is a solid phase assay in which radiolabeled fibrin monomer, covalently linked to Sepharose, is pretreated with the different plasminogen activators after which it is incubated with plasminogen in buffer. The release of radiolabeled degradation fragments into the supernatant reflects lyeie of bound fibrin. b The plasma clot lysis assay is an in vitro system composed of a [125I]fibrin-labelednormal human plasma clot immersed in normal human plasma. The release of radiolabeled degradation productcl into the supernatant following incubation with the different plasminogen activators reflects the fibrinolytic potency of the agents. Ratio of the activity obtained with conjugated plasminogenactivator over the activity obtained with the correspondingunconjugated plasminogenactivator. d Fibrinogen breakdown observed with conjugated plasminogen activator as compared to that observed with the corresponding unconjugated plasminogen activator at equieffective dose. e scu-PA single-chain urokinase-type plasminogen activator produced by the human kidney cell line TCL-598. f rscuPA: recombinant scu-PA, obtained by expression of cDNA in Escherichia coli. 8 rtcu-PA and rtcu-PA Conjugates: two-chain derivatives obtained by treatment of rscu-PA or rscu-PA conjugates with plasmin. rtcu-PA/T and rtcu-PA/T conjugates: two-chain derivatives obtained by treatment of rscu-PA or rscu-PA conjugates with thrombin. i rscu-PA-32k a low molecular weight form of rscu-PA with M,32 OOO and NH2 terminal amino acid LeulU. j rscu-PA-33k: a low molecular weight form of rscu-PA with M,33 OOO and NHz terminal amino acid Ala182. rtcu-PA-33k in K12G&z: two-chain derivative obtained by treatment of the single-chain u-PA form of K12C‘&&2with plasmin. acFv: single-chain variable domain fragment of an antibody.
ANTIBODY-MEDIATED TARGETING TO FIBRIN Up to now, two different types of anti-human fibrin antibodies have been used for the construction of antifibrin antibody/plasminogen activator conjugates: the murine monoclonal antibodies 59D8 and 64C5, specifically recognizing the amino terminal residues of the /+chain of human fibrin which become exposed after release of fibrinopeptide B brought about by thrombin (5), and the murine monoclonal antibody MA-15C5, directed against fragment D dimer of cross-linked human fibrin (6). Both types of antibodies specifically bind to fibrin but not to fibrinogen (7-9)and have been used successfully for thrombus imaging in animal models ( 1 0 , I I ) . Functional properties of conjugates of these antibodies with different types of plasminogen activators are summarized in Tables I and 11. Covalent Conjugates Targeted to the Fibrin BChain. Chemical coupling of recombinant t-PA (rt-PA) with the antifibrin 8-chain antibody 59D8 (12) was found to result in a 10-fold enhancement of its potency toward fibrin monomer in a purified system and a 3.2-fold increase of its potency toward a human plasma clot in a plasma milieu in vitro (Table I). Invivo, the t-PA/59D8 conjugate was shown to possess a 2.8-fold higher thrombolytic potency toward a human plasma clot than rt-PA, by using a jugular vein thrombosis model in the rabbit (13) (Table 11). The enhanced in vitro and in vivo thrombolytic potency of conjugated rt-PA was accompanied by a decrease in the consumption of a2-antiplasmin, plasminogen, and fibrinogen, indicating an increased fibrin selectivity (12, 13).
Similar results were obtained with chemical conjugates of single-chain or two-chain urokinase-type plasminogen activator with the antibodies 59D8 or 64C5 or with fragments of the antibodies (5, 14-16). The purified conjugates had 100-250-fold higher fibrinolytic potencies than their respective unconjugated counterparts toward fibrin monomer and 1.6-4.5-fold enhanced potencies toward human plasma clots in vitro (Table I). In the rabbit jugular vein thrombosis model, a conjugate of scu-PA with 59D8-Fab’ had a 29-fold enhanced in vivo thrombolytic potency as compared to unconjugated scu-PA (Table 11). Schnee et al. have constructed a recombinant version of the t-PA/59D8 conjugate (17). Therefore, the cDNA encoding the variable domain region of the 59D8 heavychain gene was cloned and recombined in an expression vector with the CH1 and hinge sequence of mouse immunoglobulin y2b and with the cDNA coding for the catalytic B-chain of t-PA. This construct was transfected into a heavy-chain loss-variant cell line derived from hybridoma 59D8. The resulting fusion protein produced by these cells retained antifibrin antibody activity as well as plasminogen activating potential (17). However, the recombinant t-PA/59D8 conjugate was ineffective in plasmaclot lysis (18). In contrast, a recombinant conjugate comprising the 59D8 variable region combined with the mouse immunoglobulin y2b CH1, hinge, and CH2 domains and with a low molecular weight form of scu-PA (LMWscu-PA) was 10-50-fold more efficient than urokinase in the fibrin monomer assay (19) (Table I). Covalent Conjugates Targeted to Fibrin Fragment D Dimer. Targeting of recombinant scu-PA (rscu-PA)
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Table 11. Thrombolytic and Pharmacokinetic Properties of Antifibrin Antibody/Plasminogen Activator Conjugates in Animal Modelr in Vivo enhancement factorc specific plasminogen thrombo- thromboa!r clearance activator antibody lytic lytic fibrinogen antiplasmin prolongation moiety moiety designation conjugation epitope species potency activityb breakdownd depletiond factor refs chemical &chain rabbite 2.8 reduced rt-PA I S 1 tPA-SgD8 reduced 13 29 chemical @chain rabbit equal SCU-PA Fab scu-PA16 59D8Fab’ chemical D-dimer rabbit 8 3 equal ~scu-PA IF1 ISCU-PA/ 4-8 20 qual equal MA-15C5 bahoonf 3 0.1 15 equal 23 0.4 chemical rabbit 0.1 equal 1.7-3 equal 20 IS1 rscu-PA/ baboon equal 0.9 0.03 MA-1C8 equal 15 23 reduced 4 0.5 chemical D-dimer rabbit rtcu-PAI I& rtcu-PA/ reduced 6 20 MA-15C5 chemical D-dimer rabbit 1.4 0.3 equal equal 4 20 rtcu-PAITh IgGl rtcu-PA/ MA-l5C5/T ~~
~
~~
Thrombolytic potency, derived from the doseresponse data based on percent lysis obtained versus the doee (in mg of com und per kg of body weight) adminatered, representing potency at com arable dose. b Specificthrombolytic activit ,derived from the dose-response based on lysis obtained versus steady-state asma anti en /&el (in pg u-PA or t-PA antigen per mL p6ama) representing at comparable s t e a G 2 plasma antigen levels. C Ratio of t i e activity htained with conjugated plasmin0 en activator over the activity o tain with the correspondmg uncon’ugated plasminogen activator. d Fibrinogen breakdown or a*-antiplasmin dep!etion observed with con ated plasminogen activator as com ared to that observed with the correspondingunconj ated plasminogen activator at equieffectivedose. e In the ra%t jugular vein model a [1aI]fibrin-la%eled human plasma clot was produced inn se ment %he ju@ar vein. Thrombol sis was quantitated 30 or 60 min after the end of a 4-h intravenous infusion of the different plasminogen activators,Bby determination of the residual radoactivity in the ‘ugularvein segment. f In the baboon femoral vein model, a thrombus was produced in a segment of a femoral vein using fresh baboon blood mixed with a trace amount of laI-lab@ed human fibrin0 en. Thrombolysis was quantitated 30 min after the end of a 2-h intravenous infusion of the differentplasmino en activators by determination of the resifual radioactivity in the femoral vein s e p t . I rtcu-PA and rtcu-PA conjugates: two-chain derivatives o%tained by treatment of rscu-PA or mu-PA conj ates with plasmin. h rtcu-PA/ and rtcu-PA/T conjugates: two-chain derivatives obtained by treatment of rscu-PA or rscu-PA conjugatae with thro%in.
rtez
to fibrin fragment D dimer similarly resulted in an enhancement of the fibrinolytic and thrombolytic potency of the plasminogen activator (Tables I and 11). Chemical conjugation of rscu-PA with MA-15C5 yielded a functionally intact conjugate, both with respect to the in vitro enzymatic properties of the rscu-PA moiety and the antigen-binding capacity of the antibody moiety (6).The conjugate, rscu-PA/MA-l5C5, had a 6.4-fold higher fibrinolytic potency than rscu-PA in a human plasma clot lysis assay in vitro (6) and an 8-fold enhanced thrombolytic efficiency toward a human plasma clot in the rabbit jugular vein thrombosis model (20).The enhanced invitro and in vivo thrombolytic potency of the conjugate as compared to unconjugated rscu-PA was not observed when the fibrin clots were produced with rabbit plasma or when rscu-PA was conjugated with a control antibody, MA-1C8 (6,20)(Table 11). Treatment of scu-PA with plasmin results in conversion of the single-chain molecule to tcu-PA (urokinase), a twochain derivative in which the constituting A- and B-chains are still linked by a disulfide bond. In contrast to SCU-PA, plasmin-derived tcu-PA indiscriminately activates circulating as well as fibrin-bound plasminogen. The plasminderived two-chain form of the conjugate rtcu-PA/MA15C5 had a 2.2-fold higher fibrinolytic potency in the in vitro plasma clot lysis assay than unconjugated rtcu-PA (6) and a 4-fold enhanced thrombolytic potency in the rabbit jugular vein model (20).Fibrinogen breakdown or az-antiplasmin depletion during in vitro and in vivo thrombolysis was less pronounced with rtcu-PA/MA-l5CB than with rtcu-PA (Tables I and 11), suggesting that the conjugate has an increased fibrin specificity. In contrast, fibrinogen breakdown during in vitro clot lysis was somewhat more pronounced with the single-chain form of the conjugate than with unconjugated rscu-PA (Table I). No increased fibrinogen breakdown or az-antiplasmin depletion was however observed with rscu-PA/MA-l5C5 during in vivo thrombolysis (Table 11). Treatment of scu-PA with thrombin converts this singlechain molecule to tcu-PAIT, a two-chain derivative in which the A- and B-chains are still disulfide linked, but
gta
which is cleaved at a site different from the one cleaved by plasmin. Thrombin-derived tcu-PA/T is virtually inactive in in vitro assays but can be converted to active tcu-PA with plasmin. This conversion by plasmin occurs however with a 500-fold lower catalytic efficiency as compared to that of scu-PA (21). In contrast to rtcuPA/T, the thrombin-derived two-chain form of rscu-PA/ MA-15C5, rtcu-PA/MA-lBCB/T, was found to have a significant fibrinolytic activity in in vitro or in vivo plasma clot lysis and this activity occurred in the absence of fibrinogen degradation or az-antiplasmin depletion (20, 22) (Tables I and 11). These results may be explained by targeting of the conjugate to the fibrin clot and reactivation by plasmin at the fibrin surface (22). In the rabbit jugular vein thrombosis model, interference of endogenous rabbit fibrin or fibrinogen with clot lysis by rscu-PA/MA-l5C5 is avoided since MA-15C5 does not cross-react with rabbit fibrin (8, 11). Although fibrin fragments do not trigger activation of the fibrinolytic system by rscu-PA/MA-l5C5 in plasma in vitro (6),they might well reduce its thrombolytic efficacy by blocking the antigen binding site of the antibody moiety. Therefore, the thrombolytic properties of rscu-PA/MA-l5C5 were investigated in baboons with an autologous femoral vein clot, after demonstration of cross-reactivity of MA-15C5 with cross-linked baboon fibrin (23). In this system, the thrombolytic potency of rscu-PA/MA-l5C5 was still 3-fold higher than that of rscu-PA (Table 11). Conjugation of rscu-PA or its two-chain derivatives with MA-15C5 significantly decreased their plasma clearance rate (Table 11),resulting in considerably higher steadystate plasma u-PA antigen levels than observed with unconjugated u-PA (20,23).Therefore, specific thrombolytic activities (reflecting potency at comparable steady-state plasma antigen levels) were determined for the different conjugates in both the rabbit and baboon model. Unlike the thrombolytic potencies (potency at comparable dose), the specific thrombolytic activities of the conjugates in most cases were decreased as compared to those of the corresponding unconjugated moieties (Table 11). However, the targeting effect of MA-15C5 in combination with the
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prolonged half-life appears to compensate for the reduced specific thrombolytic activity, resulting in significantly enhanced thrombolytic potencies. Indeed, with a control conjugate (rscu-PA/MA-lC8) with similarly decreased plasma clearance rates, both the thrombolytic potency and specificthrombolytic activities were markedly reduced (20, 23). In order to reduce the molecular size of the conjugates, a low molecular weight form of rscu-PA, rscu-PA-32k (comprising amino acids L e ~ l L L e u ~was ~ ~chemically ), coupled to F(ab’)z fragments of MA-15C5 (24). This yielded a conjugate, rscu-PA-32k/MA-15C5-F(ab’)z,with M,130 W a n d with similar in vitro biochemical properties for its single-chain form and its plasmin- or thrombinderived two-chain derivatives as compared to intact rscuPA/MA-l5C5 and its two-chain derivatives (24,25). The in vitro fibrinolytic potency of rscu-PA-32k/MA-l5C5F(ab’)zor of the thrombin-derived rtcu-PA-32k/MA-l5C5F(ab’)a/T was respectively increased 5- and 2-fold over that of the corresponding unconjugated counterpart (Table I). With the plasmin-derived two-chain form rtcu-PA32k/MA-15C5-F(ab’)z, however, no enhancement of the in vitro fibrinolytic potency over plasmin-derived rtcuPA-32k was observed (Table I). Alternatively, a recombinant 57 kDa single-chain chimeric plasminogen activator, K12GoS32, has been constructed (26),consisting of the variable region fragment (Fv) of MA-15C5 and of a 33 kDa low molecular weight form of rscu-PA (rscu-PA-33k, comprising amino acids Ala132-Leu411). The synthetic single-chain Fv fragment was obtained by linking the carboxy terminal end of the variable domain of the 6-chain of MA-15C5 to the amino terminal end of the variable domain of the MA-15C5 y-chain by means of a seven amino acid residue synthetic linker (27). Purified K12GoS32, produced in an insect cell expression system, retained the in vitro enzymatic properties of low molecular weight rscu-PA (rscu-PA-32k) and the fragment D dimer binding capacity of MA-15C5 (26). In the in vitro plasma clot lysis assay, the single-chain u-PA form of K12GoS32 was found to have a 13-fold higher fibrinolytic potency than rscu-PA-32k. The plasminderived two-chain form of the chimera had a 2.5-fold higher in vitro fibrinolytic potency than plasmin-derived rtcuPA-32k (26) (Table I). Bispecific Antibodies. In another attempt to concentrate plasminogen activators to the surface of a fibrin clot, bifunctional antibodies have been prepared that are capable of binding both fibrin and t-PA (28-31) or both fibrin and u-PA (31,32). Such bispecific antibodies have been obtained by chemical coupling (29,32)or by somatic cell fusion (28, 30, 31). In the presence of bispecific antibody, u-PA or t-PA indeed were found to have 5-50fold enhanced potencies for lysing fibrin monomer (28, 29,32) and 2-&fold higher fibrinolytic potencies toward human plasma clots in vitro (29,32). In the rabbit jugular vein thrombosis model, clot lysis with plasminogen activator administered in combination with bispecific antibody was 1.6-4-fold higher than lysis with plasminogen activator alone (28, 30). In addition, after pretreatment of fibrin with bispecific antibody, subsequently administered plasminogen activator was concentrated at the fibrin surface, both in the in vitro fibrin monomer lysis assay (29,32)and in the rabbit model in vivo (29). The use of bispecific antibodies thus offers the possibility of avoiding administration of exogenous plasminogen activator, by inducing thrombolysis via concentration of endogenous plasminogen activator at the site of the thrombus. In summary, antibody-mediated targeting of plasmi-
Dewerchin and Collen
nogen activators to fibrin appears to have the potential to increase the concentration of plasminogen activator in the vicinity of a thrombus, resulting in localized generation of active plasmin and enhanced clot lysis. Urokinase-type plasminogen activator displays fibrin specificity only in its single-chain form (33)and its fibrin specificity is not dependent on the presence of the NH2-terminal143 amino acids (34). With t-PA,the functional domains responsible for ita fibrin affinity reside within the NH2-terminal region (A-chain)of the molecule (35). Thus, conjugation of single chain u-PA molecules (scu-PA or low molecular weight forms of scu-PA comprising amino acids Ala13“Leu411 or LeulU-Leu411) or of the intact t-PA molecule with antifibrin antibodies yields plasminogen activators possessing fibrin specificityby two mechanisms. The results obtained with Fab’ or scFv conjugates (Tables I and 11) and with bispecific antibodies indicate that univalent antibody binding is sufficient for effective targeting. The specificity of the targeting effect is,demonstrated (a) by the finding that the enhancement of the fibrinolytic potency in vitro is inhibited by addition of purified antigen (5,6,15,26), (b)by the lack of targeting to fibrin clots that do not crossreact with the targeting antibody (6,20),and (c) by the absence of targeting when using conjugates with a control monoclonal antibody (5, 6, 12, 13, 20, 23). ANTIBODY-MEDIATED TARGETING TO PLATELETS Recently, evidence has accumulated that platelet-rich zones of a thrombus, particularly arterial thrombi, are very resistant to thrombolysis (36). In addition, platelet deposition may contribute to coronary artery reocclusion followingsuccessfulthrombolytic therapy ( I ) . Thus, monoclonal antibodies specific for activated plateleta might represent an alternative means to target plasminogen activators to a thrombus. Bode et al. (37) chemically coupled two-chain urokinase to 7E3, a monoclonal antibody selectively binding to platelet membrane glycoprotein IIb/IIIa, which is the platelet receptor for fibrinogen and other adhesive proteins (38). The resulting conjugate had a markedly enhanced potency toward platelet-rich human plasma clots in vitro (Table 111). We have chemically coupled rscu-PA to different monoclonalantibodies specificallyrecognizing surface proteins on activated platelets (39)(Tables I11and IV): MA-TSPI1, directed against human thrombospondin, a platelet a-granule glycoprotein expressed on the surface of stimulated platelets but minimally on the surface of resting platelets; and MA-PMI-2, MA-PMI-1, and MA-LIBS-1, recognizing different epitopes on the platelet surface glycoprotein IIb/IIIa that become exposed only when, upon platelet activation, fibrinogen has bound to the receptor. The purified conjugates retained rscu-PA activity, and binding to ADP-stimulated human platelets in a plasma milieu in vitro was significantly higher than binding to resting platelets (39). However, none of the conjugates appeared to have a higher fibrinolytic potency than rscuPA toward platelet-rich human plasma clots in a plasma milieu in vitro, and lysis was associated with extensive fibrinogenolysis (Table 111). Nevertheless, the conjugates rscu-PA/MA-PMI-1 and rscu-PA/MA-LIBS-1 were found to have a 2.3-3-fold increase in vivo thrombolytic potency as compared to rscu-PA and a 5-7-fold higher potency than a control conjugate rscu-PA/MA-lC8, when tested in hamsters with pulmonary embolism consisting of a platelet-rich human plasma clot (TableIV). The enhanced thrombolytic potency of these rscu-PA/antiplatelet an-
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Table 111. I n Vitro Fibrinolytic Properties of Antiplatelet Antibody/Plasminogen Activator Conjugates in vitro fibrinolytic potency plasminogen toward platelet-rich clots activator antibody enhancement fibrinogen moiety moiety designation conjugation epitope factorb breakdown refs urokinase Fab' UK-7E3Fab' chemical GPIIb/IIIa 1.3-10 37 rscu-PA IgG2a rscu-PA/MA-TSPI-1 chemical thrombospondin
