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CARDIOVASCULAR
Institute for Environmental Medicine (K.G., T.K., S.M., V.R.M., and J.C.M.) and Departments of Pathology and Laboratory Medicine (K.B., D.C.) and Pharmacology (V.R.M.), University of Pennsylvania, Philadelphia, Pennsylvania; and Cardiovascular Research Center, Madrid, Spain (J.C.M.)
Received August 5, 2004; accepted October 28, 2004.
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Abstract |
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; Lynch et al., 1995; Hennan et al., 2002). However, no available antithrombotic agents are free of side effects and fully effective in prevention of clotting (Clagett et al., 1995; Hirsh and Hoak, 1996). Plasminogen activators [PAs, such as tissue-type plasminogen activator (tPA)] may have especially desirable properties in this regard, including the capacity to lyse newly developing clots and thus improve thromboprophylaxis by dissolving clots formed despite anticoagulants and platelet inhibitors (Lam et al., 1991; Feigen et al., 1993; Noble and McTavish, 1996; Wooster and Luzier, 1999). However, use of PA for prophylactic purposes has been hampered by their: rapid elimination from the blood, inability to distinguish between nascent pathogenic clots and their preformed hemostatic counterparts, and diffusion into tissues where they may cause neurotoxicity, aberrant vascular remodeling, and other side effects (Holvoet et al., 1993; Tsirka et al., 1995; Wang et al., 1998, 2003; Benchenane et al., 2004).
Using red blood cells (RBCs) as carriers might help to circumvent these shortcomings (Muzykantov and Murciano, 2003). In theory, RBC-bound PAs would be afforded little access to tissues and mature retracted hemostatic clots but would enjoy prolonged intravascular circulation, enabling them to incorporate into nascent clots, which they might lyse from within. We recently affirmed some of these assumptions in animal models of arterial and venous thrombosis (Murciano et al., 2003; Ouriel, 2003). Injection of RBC/tPA prior to inciting thrombus formation dissolved nascent clots, while relatively sparing pre-existing clots. In contrast, soluble tPA was cleared from blood and did not dissolve clots formed within minutes of administration even when given at considerably higher doses (Murciano et al., 2003). This encouraging initial set of results has motivated us to systematically study the potential utility of the RBC/PA delivery strategy, including optimizing composition, determining the scope of its applicability and delineating its limitations as an approach to thromboprophylaxis.
The intensity and duration of thromboprophylaxis are likely to vary widely in human diseases, ranging from intense and acute (e.g., interruption of unstable angina or recurrent transient ischemic attacks) to subtle and chronic (e.g., lessening the risk of deep venous thrombosis and pulmonary embolism postsurgery). Therefore, it is likely that the preferred characteristics of the RBC/PA as a prophylactic agent will differ as well. However, to date, nothing is known as to whether conjugating PAs to RBC alters their critically important intrinsic biological features [e.g., stimulation by fibrin or suppression by PA inhibitors (PAIs)] and/or endows the RBC/PA complexes with unpredictable beneficiary or deleterious features.
Considerable effort has been expended to improve the clinical profile of PAs themselves (Verstraete et al., 1985; Jackson et al., 1992; Benedict et al., 1995; Collen and Lijnen, 2004). For example, Retavase (rPA, a mutant tPA derivative lacking the finger, extracellular growth factor, and kringle-1 domains) is more susceptible to inhibitors and has lower affinity for fibrin than wild-type tPA. However, due to its lack of domains recognized by hepatic and other tissue receptors, rPA circulates for a longer time than tPA, which may offset these limitations (Martin et al., 1991; Bu et al., 1992; Kohnert et al., 1992; Rijken et al., 1994; Noble and McTavish, 1996; Topol et al., 2000). In this study, we examined effects of coupling to RBCs on the functional profiles of tPA versus rPA and found that it causes profound and, in some cases, unanticipated favorable alterations in the pharmacokinetics and enzymatic regulation of PAs of direct relevance to drug development.
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Materials and Methods |
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).
Coupling of Fibrinolytics to Carrier RBCs. To produce RBC/tPA and RBC/rPA conjugates, PAs and washed RBC were biotinylated by B-LC-NHS as described (Muzykantov and Murciano, 1996). Briefly, biotinylated RBCs (b-RBCs) were mixed 1:1 with naive RBC (10% Hct) to prevent aggregation and incubated with SA (106 molecules/b-RBC) for 30 min at room temperature. Unbound SA was removed by washing the cells three times. Radiolabeled biotinylated tPAs or Retavases were incubated with the RBC for 30 min at room temperature (106 molecules/b-RBC), the unbound material was removed by washing, and the cell-associated 125I-labeled b-PA was measured. The resultant b-RBC/SA/b-PA complexes (indicated as RBC/tPA or RBC/rPA) were resuspended in phosphate-buffered saline (PBS) containing 3% bovine serum albumin to final 10% hematocrit.
Kinetics of Blood Clearance of PA versus RBC/PA and ex Vivo Blood Clot Lysis. 125I-tPA, 125I-rPA, 51Cr-RBC, or dual-labeled 51Cr-RBC/125I-PA complexes were injected intravenously in anesthetized rats and mice (2–3 µg PA/animal). At selected times, aliquots of blood (200 µl) were drawn into heparin (0.05U/ml), and the radioactivity in the plasma and cell pellets was measured.
Blood fibrinolytic activity was measured using an ex vivo clot lysis assay described previously (Murciano et al., 2003). Briefly, 200-µl aliquots of blood were drawn into borosilicate glass tubes without anticoagulants either 10 or 60 min postinjection of PA, RBC/PA, or saline. The blood was rapidly mixed with a 4-µl solution containing trace amounts of 125I-fibrinogen (Fg) and incubated for 20 min at room temperature to form a clot, obtaining 125I-fibrin (Murciano et al., 2002). Clots were covered with 200 µl of saline and incubated for 6 h (unless specified otherwise) at 37°C to permit fibrinolysis, assessed by the release of dissolved 125I-fibrin fragments into supernatant.
Dissolution of Blood and Fibrin Clots by PA and RBC/PA. To model in vitro prophylactic lysis of clots from within by entrapped RBC/PA, indicated doses of RBC/PA (5 nM) were added into blood, plasma, or fibrinogen solution prior to clotting. To test fibrinolysis of whole-blood clots, aliquots of human, rat, or mouse blood (200 µl) drawn in the absence anticoagulants were rapidly mixed with 125I-Fg and allowed to clot (20 min at RT) as described above. Plasma and fibrin clots (200 µl) were prepared by adding 125I-Fg to fresh platelet-poor plasma or PBS containing 3 mg/ml fibrinogen. Samples were then clotted by adding CaCl2 and thrombin (final concentrations 20 mM and 0.2 U/ml, respectively). PBS (200 µl) was layered over the clots. Unless stated otherwise, indicated doses of soluble PA (5 nM) were added to this layer (on top of the clot) to model therapeutic dissolution of pre-existing clots. Lysis was assessed by measuring the release of radioactivity into supernatants as described above. In a separate experiment, the same dose (5 nM) of free PA or RBC/PA was preincubated with 0.75- and 1-fold molar excess of PAI-1 for 30 min at room temperature and tested for fibrin clot lysis as described above.
Enzymatic Activity of Free PA and RBC/PA. The amidolytic activity of PA and RBC/PA was determined using chromogenic substrates for tPA and plasmin (Gardell et al., 1989). In the first series of experiments, 0.05 µM PA or RBC/PA (also containing 0.05 µM PA) was incubated with PA substrate, Spectrozyme tPA (0.4 mM), at 25°C for 20 min. In the second series, PA or RBC/PA was incubated with 0.25 µM human plasminogen and plasmin substrate, Spectrozyme PL (0.5 mM), at 25°C for 20 min. In the third series, CnBr-digested fibrinogen (100 µg/ml) was added to the reaction medium. The mixtures (final volume 200 µl in all settings) were incubated for 20 min at 25°C in V-shaped plates centrifuged at 1200g for 2 min to precipitate RBCs, and the optical density was measured at 405 nm in 100-µl aliquots of supernatants. In experiments with free PA, the enzymes and substrates were dissolved in PBS. RBC/PA were suspended in PBS containing 3% bovine serum albumin.
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, 1996, 2003; Muzykantov and Taylor, 1994; Zaltzman et al., 1995; Muzykantov and Murciano, 1996, 2003). Biotinylation of tPA and rPA with up to 40-fold molar excess of B-LC-NHS did not compromise their fibrinolytic activity; greater incorporation of biotin partially inhibited the PA activity of rPA (Fig. 1A, open diamonds). Thus, PAs biotinylated with 5-fold molar excess of B-LC-NHS (Fig. 1A, inset) were used thereafter and indicated simply as tPA and rPA.
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Streptavidin was used to couple biotinylated PAs to biotinylated RBC forming b-PA/SA/b-RBC complexes (indicated hereafter as RBC/tPA and RBC/rPA). RBC coating with trace-labeled biotinylated 125I-PA was proportional to RBC biotinylation up to an input concentration of 10 µM B-LC-NHS; at higher concentrations, coupling of rPA was more extensive (Fig. 1B). Therefore, RBC biotinylation was performed using 10 µM B-LC-NHS in all subsequent experiments to equalize PA concentration per RBC. Mouse, rat, or human RBCs biotinylated at this concentration of B-LC-NHS were coated with essentially the same amount of tPA and rPA (varying within the range of 6–8 x 104 PA molecules/RBC, Fig. 1C). Less than 2% of the coupled 125I-tPA or 125I-rPA detached from the RBC over 48 h in autologous serum.
Blood Clearance of RBC/tPA and RBC/rPA. Blood levels of 51Cr-RBC/tPA were 
10 to 20% lower than 51Cr-RBC/rPA in both rats and mice 15 min postinjection (Fig. 2), but the circulation of both RBC/PAs was stable for the remainder of the experiment (3 h). Within 1 min of injection, less than 40% of the injected soluble 125I-tPA dose remained in the blood of rats and mice, and less than 10% remained at 15 min (Fig. 3, A and B, closed circles). In agreement with the literature, 125I-rPA was cleared more slowly, but by 1 h postinjection, blood levels dropped to 10 and 25% of the injected dose in rats and mice, respectively (Fig. 3, A and B, closed triangles).
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The kinetics of RBC/125I-PA was quite different. Coupling to RBC markedly prolonged circulation time of tPA and rPA (compare open versus closed symbols in Fig. 3). The enhancement of blood bioavailability of RBC/tPA was somewhat greater than RBC/rPA, based on an estimate of their respective area under curve tracings. For example, in mice, the area under curve of RBC/tPA was 8-fold greater than tPA, whereas a 5-fold difference was seen with RBC/rPA versus rPA. The greater enhancement afforded by coupling of tPA to RBC relative to rPA was due to the faster clearance of free tPA versus rPA. Therefore, the amount of RBC/125I-rPA in the blood was actually higher than seen with RBC/125I-tPA in rats and mice at any inspected time point (Fig. 3).
125Iodine was found mostly in blood plasma fraction after injection of free 125I-tPA or 125I-rPA (Fig. 4). Note that the scale shows a relative distribution of residual 125I-labeled tPA and rPA in blood fractions, whereas their absolute level in blood was very low (see Fig. 3). When RBC/125I-tPA or RBC/125I-rPA were injected, approximately 90% of the radioactivity remained cell-associated for at least 3 h (Fig. 4), indicating that both RBC/tPA and RBC/rPA circulate as stable complexes.
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Fibrinolytic Activity of Circulating RBC/tPA versus RBC/rPA. To characterize the activity of circulating PAs, we measured fibrinolysis ex vivo. To do so, 125I-labeled clots were formed from fresh blood drawn at various times after injection of free PA or RBC/PA, and the release of radioactivity was measured (Murciano et al., 2003). Clot formation was not impaired in rats or mice injected with free or RBC/PA 5 to 180 min earlier (data not shown). Clots formed from rat blood extracted 10 min after injection of free tPA or rPA underwent 40 and 60% lysis within 6 h incubation time, respectively (Fig. 5A); therefore, the extent of fibrinolysis correlated with relative blood levels of the respective free PAs (Fig. 3A). Clots formed from rat blood extracted 1 h postinjection of tPA or rPA reverted to basal levels of spontaneous fibrinolysis (Fig. 5A), consistent with the virtual complete clearance of both PAs by that time (Fig. 3A).
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In sharp contrast, lysis of clots formed from rat blood drawn either 10 min or 1 h after injection of RBC/tPA or RBC/rPA was markedly accelerated (Fig. 5A). The finding that the extent of fibrinolysis induced by both RBC/PA formulations was equivalent implies that the specific activity of RBC/tPA is greater than that of RBC/rPA because its concentration in blood was lower at the time of the assay (Fig. 3A). The same outcome was seen in mice: although the blood level of RBC/tPA was lower than RBC/rPA (Fig. 5B, left), significantly greater stimulation of ex vivo fibrinolysis was observed (Fig. 5B, right).
Fibrinolytic Activity of Free versus RBC-Coupled tPA and rPA in Vitro. A series of in vitro studies were then conducted to investigate the discrepancy between the blood level and fibrinolytic activity of circulating RBC/tPA and RBC/rPA. First, we studied the effect of adding the PAs directly to clots, thereby eliminating potentially confounding differences in blood clearance, species specificity, plasma inhibitors, and stimulatory cofactors. Free tPA and rPA caused more extensive lysis of fibrin clots than plasma clots, likely due to effect of plasma inhibitors (Fig. 6A). This data are consistent with findings that rPA is more susceptible to inhibition by PAI-1 than tPA (Martin et al., 1991; Lijnen et al., 1994). However, free tPA and rPA caused comparable lysis of fibrin clots (Fig. 6A, inset). RBC/tPA and RBC/rPA also dissolved fibrin clots more effectively than plasma clots (Fig. 6, B and C), and the activity of RBC/rPA was inhibited by plasma to a greater extent than RBC/tPA (Fig. 6B and C, left). However, in clear contrast to their soluble counterparts, RBC/tPA dissolved fibrin clots more effectively than RBC/rPA (Fig. 6, B and C, right).
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Coupling to RBC Alters Fibrin Regulation of Enzymatic Activity of rPA but Not tPA. To determine whether this alteration in RBC/rPA activity was due to interference with the catalytic site of the enzyme, we next analyzed enzymatic activity of PA and RBC/PA using small chromogenic substrates. Both soluble and RBC-coupled tPA and rPA expressed nearly identical amidolytic activity using a tPA substrate (Fig. 7A), indicating that coupling to RBC did not directly affect the catalytic site of either enzyme. Soluble and RBC-coupled tPA and rPA also exerted comparable conversion of plasminogen into plasmin tested using a plasmin substrate (Fig. 7B), indicating that coupling to RBC did not impose conformational or steric changes that hindered cleavage of its physiological substrate, plasminogen.
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Based on this outcome, we then asked whether fibrin itself might differentially regulate the activity of RBC/tPA and RBC/rPA. Soluble fibrin fragments stimulated plasmin formation by free tPA and rPA to the same extent (note scale difference between Fig. 7, B and C). In contrast, fibrin stimulated plasmin generation by RBC/tPA to a considerably greater extent than by RBC/rPA (Fig. 7C). Therefore, coupling of rPA to RBC partially suppresses the cofactor activity of fibrin toward this mutant tPA variant.
Coupling to RBC Increases tPA Resistance to PAI-1. Lastly, we tested whether coupling to RBC alters susceptibility of PA to PAI-1. To do so, soluble PA or RBC/PA was incubated with buffer or with purified PAI-1, and their capacity to lyse fibrin clots in vitro was measured. At a PAI-1/PA molar ratio of 0.75 (Fig. 8A), free tPA was less susceptible to inhibition than rPA (30 versus >75% inhibition), as expected. Both free tPA and rPA were inhibited by >70% when preincubated with equimolar PAI-1 (Fig. 8B). Coupling to RBC had minimal, but rather reducing, effect on rPA resistance to PAI-1 (Fig. 8A). In sharp contrast, coupling tPA to RBC significantly increased tPA resistance to PAI-1, relative to free tPA: RBC/tPA was inhibited by less than 30% by an equimolar ratio of PAI-1 (Fig. 8B).
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; Magnani et al., 2002; Murciano et al., 2003; Muzykantov and Murciano, 2003). This notion, initially put forth 3 decades ago (Ihler et al., 1973), has gained momentum due to the growing interest in cell-based therapies, including administration of stem cells as well as cells modified ex vivo either genetically or chemically. Linking drugs to RBC can be expected to markedly prolong their intravascular circulation and enhance their activity within blood, while concomitantly reducing their diffusion into tissues, which may be the site of dose-limiting toxicity (Muzykantov et al., 1996; Bax et al., 2000; Murciano et al., 2003).
We have exploited these concepts successfully by linking plasminogen activators such as tPA to RBC. Consistent with expectations, linking tPA to RBC markedly increased its intravascular half-life, permitting selective dissolution of nascent clots while physically restricting the penetration of the drug into pre-existing clots. This effectively changed the therapeutic profile of tPA from a means to lyse existing clots into a prophylactic agent capable of preventing thrombosis over a relatively prolonged period of time (Murciano et al., 2003).
The clinical requirements for prophylaxis vary widely depending upon the intensity and duration of the pro-thrombotic stress. For example, acute and intense local release of thrombin underlies acute coronary and cerebral occlusion after plaque fissure. On the other end of the spectrum, the risk of deep venous thrombosis and pulmonary embolism after hip or knee surgery, for example, may be associated with a less intense burst of thrombin formation, but the period of risk is far more protracted. Thus, it would not be surprising if certain RBC/PA formulations were more efficacious in one or another clinical context.
Conceivably, favorable features of plasminogen activators may differ in the context of therapeutic versus prophylactic mode of administration. For example, one can expect that abolishing of diffusion into clots imposed by coupling to RBC carrier would preclude therapeutic utility of RBC/PA (Sakharov and Rijken, 1995). However, the same feature, lack of penetration of RBC/PA into existing hemostatic clots and extravascular tissues represents a safety advantage for prophylaxis (Ouriel, 2003).
There is no information, however, as to whether or, if so, how the intrinsic features of individual fibrinolytic agents translate into prophylactic efficacy when the agent is bound to RBC or other carriers. To begin to address this issue, we compared the pharmacokinetics of RBC-bound tPA (molecular mass 60 kDa) versus its truncated variant Retavase (molecular mass 40 kDa), which has a lower affinity for fibrin and is more susceptible to plasma inhibitors but enjoys a longer half-life in the circulation than does tPA (Martin et al., 1991; Kohnert et al., 1992; Rijken et al., 1994).
Our data show that coupling of tPA to RBC cause a modestly (<20%) accelerated clearance of the complex within the first 15 min after injection, suggesting that a relatively small fraction, possibly the most heavily sensitized subpopulation of RBC/tPA, is more susceptible to elimination, primarily in the liver (data not shown). Accelerated clearance was not seen with RBC/rPA, consistent with deletion of tissue receptor recognition domains from the variant molecule.
Nevertheless, RBCs carrying either tPA or rPA are relatively stable in the bloodstream (Fig. 2) and markedly prolong circulation of the coupled PAs (Fig. 3). After injection of 51Cr-RBC/125I-tPA, clearance of both labels occurred in parallel (Figs. 2 and 3), indicating that both PAs circulated almost exclusively as stable RBC/PA complexes (Fig. 4). Consistent with conclusion, both RBC/tPA and RBC/rPA retained their fibrinolytic activity in the circulation for hours, whereas activity of their injected free counterparts disappeared within minutes (Fig. 5A).
Circulating RBC/tPA-mediated fibrinolysis ex vivo more effectively than RBC/rPA, notwithstanding its faster elimination from the circulation (Fig. 5B). This difference was not due to disruption of the catalytic site caused by biotinylation because the amidolytic activity of both RBC/tPA and RBC/rPA was preserved and comparable (Fig. 7). Nor did biotinylation and RBC conjugation disrupt the capacity of RBC/rPA to bind and convert plasminogen to plasmin (Fig. 7). Rather, the enhanced specific activity of RBC/tPA is attributable not only to its relative resistance to plasma inhibitors compared with RBC/rPA (Figs. 6 and 8) but also to the preservation of fibrin-mediated cofactor activity (Fig. 7). The mechanism underlying the loss of cofactor activity of RBC/rPA is unknown but may relate to the smaller size (40 kDa) and lack of auxiliary domains of the variant molecule compared with wild-type tPA (60 kDa), which make the former more susceptible to modification-induced functional changes.
One important conclusion to draw from this study is that the properties desirable in a PA intended for use as a soluble fibrinolytic agent may differ from those that are most desirable in a prophylactic agent designed for prolonged intravascular activity. Our data shown that genetic modifications that prolong the survival of a soluble PA in the circulation do not contribute meaningfully to its survival once bound to RBC. Rather, RBC carriage minimizes differences in blood levels while exacerbating the unfavorable fibrinolytic potency profile of rPA caused possibly by the lost of fibrin cofactor activity (Fig. 7). Furthermore, coupling of tPA to RBC enhanced its resistance to PAI-1, whereas it had no or even an opposite effect on rPA (Fig. 8), which helps to explain the increased specific activity of RBC/tPA in vivo. The molecular basis of this unexpected outcome will require additional interrogations.
Fully preserved fibrin stimulation of RBC/tPA activity is a highly desirable feature that will help to minimize potential side effects in vasculature and further augment targeting of the nascent clots, since RBC/tPA will exert relatively limited effects until it is activated by fibrin at sites of thrombosis. The enhanced resistance of RBC/tPA to PAI-1 would be expected to further its utility as a prophylactic modality.
In summary, the data presented in this paper indicate that the pharmacological profile of RBC/PA complexes results from an interplay of diverse factors, including: pharmacokinetic features provided by the RBC carrier (e.g., prolonged circulation, lack of extravasation), intrinsic features of an individual PA (e.g., resistance to inhibitors, rate, and pathway of clearance), and the newly revealed changes imposed by coupling to RBC carrier (e.g., steric limitations of interactions with plasma partners and altered resistance to inhibitors). Greater understanding of these factors will help to further optimize RBC-based drug delivery systems and facilitate the design of RBC/PA conjugates that can be tailored to manage differing requirements to treat a range of thrombotic conditions.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: PA, plasminogen activator; tPA, tissue-type plasminogen activator; RBC, red blood cell; PAI, plasminogen activator inhibitor; rPA, Retavase; SA, streptavidin; B-LC-NHS, long-chain 6-biotinylaminocaproic acid N-hydroxysuccinimide ester; b-RBC, biotinylated RBC; PBS, phosphate-buffered saline; Fg, fibrinogen.
Address correspondence to: Dr. Vladimir R. Muzykantov, IFEM, University of Pennsylvania School of Medicine, One John Morgan Building, 3620 Hamilton Walk, Philadelphia, PA 19104-6068. E-mail: muzykant{at}mail.med.upenn.edu
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