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CARDIOVASCULAR
Institute for Environmental Medicine (K.G., J.L., V.R.M.), Department of Pathology and Laboratory Medicine (D.B.C.), and Department of Pharmacology and Targeted Therapeutics Program, Institute for Translational Medicine and Therapeutics (V.R.M.); University of Pennsylvania, Philadelphia, Pennsylvania; Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain (J.-C.M.); Howard Hughes Medical Institute, University of Michigan, Ann Arbor, Michigan (R.W.); and Los Alamos National Laboratory, Bio Division, Los Alamos, New Mexico (K.G.)
Received September 20, 2006; accepted January 5, 2007.
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Abstract |
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2-macroglobulin and 1-antitrypsin) and pathologically high levels of glucose. However, coupling to RBC did not protect a truncated tPA mutant, Retavase, from plasma inhibitors. Chemical removal of the RBC glycocalyx negated tPA protection from inhibitors: tPA coupled to glycocalyx-stripped RBC bound twice as much 125I-PAI-1 as did tPA coupled to naive RBC, and susceptibility of the bound tPA to inhibition by PAI-1 was restored. Thus, the RBC glycocalyx protects RBC-coupled tPA against inhibition. Resistance to high levels of inhibitors in vivo contributes to the potential utility of RBC/tPA for thromboprophylaxis.
; Topol et al., 1999; Jackson et al., 2000; Hennan et al., 2002; Hong et al., 2003; Lange and Hillis, 2004). Both unfavorable pharmacokinetics and low benefit/risk ratio have precluded the use of plasminogen activators (PAs) in clot prevention. The effectiveness of PAs is limited by clot penetration and their rapid inactivation by plasma inhibitors and clearance from the blood (Verstraete et al., 1985; Lucore and Sobel, 1988; Thomas et al., 1993; Narita et al., 1995; Rijken et al., 2004). Treatment requires administration of PAs in doses several orders of magnitude above physiological levels to attain therapeutic efficacy, posing a considerable risk of hemorrhage (Ridker et al., 1994). To date, modifications in the design and delivery of plasminogen activators to attain greater affinity for fibrin, longer circulation time, and greater resistance to inhibitors have been of rather modest clinical benefit (Keyt et al., 1994; Benedict et al., 1995; Liberatore et al., 2003; Rijken et al., 2004; Inoue et al., 2005).
To be of use in thromboprophylaxis, a fibrinolytic agent should selectively lyse potentially occlusive clots during their formation, without affecting hemostatic clots or exerting extravascular toxicity (Wang et al., 1998; Melchor and Strickland, 2005). Moreover, to be practical, delivery must be feasible and PA activity must be expressed for hours to days. However, all existing fibrinolytics are relatively short-lived (<30 min) and small (<10 nm in diameter) agents, capable of diffusion into hemostatic clots and the tissues, including the CNS. None can be used as prophylaxis, even in the patients known to be at high risk of imminent or recurrent thrombosis.
Previous studies showed that coupling tPA to red blood cells (RBC) produces long-circulating enzymatically active complexes, thus converting tPA from a therapeutic agent with considerable safety concerns into an effective and safe thromboprophylactic agent (Murciano et al., 2003; Ganguly et al., 2005, 2006). Injection of either preformed RBC/tPA (Murciano et al., 2003; Ganguly et al., 2005, 2006) or tPA derivatives targeted to complement receptor-1 on circulating RBC (Zaitsev et al., 2006) prevents subsequent formation of occlusive venous and arterial clots, with no overt harmful effects on the carrier RBC (Murciano et al., 2003; Ganguly et al., 2005, 2006), activation of coagulation (Murciano et al., 2003) or impaired postsurgical hemostasis (Zaitsev et al., 2006).
Of particular interest, previous studies showed that RBC/tPA lysed human plasma clots more effectively than soluble tPA added to the clots at equal doses despite the obvious diffusional/spatial disadvantage of RBC/tPA (Ganguly et al., 2005). One possible explanation for this surprising result is that coupling to RBC renders tPA less susceptible to plasma PA inhibitors, including the most physiologically relevant, plasminogen activator inhibitor 1 (PAI-1) (Loskutoff et al., 1989). Sensitivity to inhibitors is one of the factors that may control the longevity of RBC/tPA activity in vivo and, therefore, the durability of thromboprophylaxis. Plasma levels of PAI-1 are severalfold higher than tPA in healthy subjects and rise further in response to platelet activation (Potter van Loon et al., 1992; Zhu et al., 1999) and vascular injury (Schneiderman et al., 1992; Cesari and Rossi, 1999). High levels of PAI-1 contribute to the resistance of arterial clots (Potter van Loon et al., 1992; Zhu et al., 1999) and pulmonary emboli to therapeutic lysis (Chapman et al., 1990). In this study, we investigated the mechanism by which tPA coupled to RBC may be protected against inhibitors.
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Materials and Methods |
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2-macroglobulin, and 1-antitrypsin were from Calbiochem (San Diego, CA); human thrombin, neuraminidase, hyaluronidase, and heparinase were from Sigma-Aldrich (St. Louis, MO); human fibrinogen was from Enzyme Research Laboratories Inc. (South Bend, IN); iodogen and long-chain 6-biotinylaminocaproic acid N-hydroxysuccinimide ester were from Pierce Chemical (Rockford, IL). Noncleavable human plasminogen [NC-Pg (R561A); hereafter called NC-Pg] was expressed in Drosophila S2 cells stably transfected with cDNA encoding a plasmin-resistant mutant of plasminogen (a kind gift from Dr. F. J. Castellino, University of Notre Dame, South Bend, IN). Proteins were radiolabeled with Na(125I) (PerkinElmer Life and Analytical Sciences, Boston, MA) using iodogen according to the manufacturer's instructions. Free 125I was removed using a Biospin 6 column (Bio-Rad, Hercules, CA). Wild-type (WT; C57BL/6J) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Transgenic mice overexpressing a murine PAI-1 minigene (PAI-1Tg+) under control of a cytomegalovirus promoter, generated as described previously (Eitzman et al., 1996) and crossed greater than 20 generations to C57BL/6J, were the kind gifts of Dr. Ginsburg (University of Michigan, Ann Arbor, MI).
Coupling of tPA to Carrier RBC. RBC were isolated by centrifugation from fresh anticoagulated human and mouse blood. Approximately 6 x 104 molecules of tPA were attached per RBC without loss of enzymatic activity or alterations in RBC biocompatibility as described previously (Muzykantov and Murciano, 1996; Murciano et al., 2003; Ganguly et al., 2005, 2006). Briefly tPA and RBCs (10% hematocrit) were biotinylated with 6-biotinylaminocaproic acid N-hydroxysuccinimide ester at 5-fold molar excess (b5tPA) or 10 µM (b10RBC). Biotinylated RBC were mixed with naive RBC (1:1) to prevent agglutination and then incubated with SA (106 molecules/RBC). Unbound SA was removed by washing the cells three times followed by incubation with radiolabeled b5tPA. Unbound tPA was removed by washing, and cell-associated tPA was measured in a gamma counter (PerkinElmer Life and Analytical Sciences) (Murciano et al., 2003; Ganguly et al., 2005, 2006). The b10RBC/b5tPA conjugates are henceforth designated as RBC/tPA. In separate experiments, RBCs were incubated for 3 h at 37°C with a mixture of glycocalyx-degrading enzymes: neuraminidase (5 mU/ml), hyaluronidase (5 U/ml), and heparinase (5 U/ml) before biotinylation. Cells were washed three times to remove excess enzyme, biotinylated, and incubated with SA and b5tPA as described above.
In Vitro Lysis of Blood Clots Obtained from Mice. Blood was drawn from WT mice in borosilicate tubes without anticoagulant, 125I-Fg (2 µl), and either soluble tPA or tPA coupled to RBC (RBC/tPA) (10 nM each) was added and incubated for 20 min at room temperature. The release of soluble 125I-fibrin fragments into the supernatant over the next 6 h at 37°C was measured in a gamma counter (Murciano et al., 2003; Ganguly et al., 2005).
Ex Vivo Fibrinolytic Activity of RBC/tPA versus Soluble tPA Circulating in Wild-Type and PAI-1-Overexpressing Transgenic Mice. Wild-type (C57BL/6J) and PAI-1 Tg+ mice were divided into two groups and injected with either soluble tPA (0.5 mg/kg) or RBC/tPA (0.2 mg/kg) via the jugular vein. Blood (200-µl aliquots) was drawn 5 min and 15 min postinjection into borosilicate tubes without anticoagulant, and 2 µl of 125I-Fg was added immediately. The blood was allowed to clot over the next 20 min at room temperature and fibrinolysis was determined by release of radiolabeled fibrin degradation products as described above (Murciano et al., 2003; Ganguly et al., 2005, 2006).
Amidolytic Activity of Soluble PA and RBC/PA in the Presence of PAI-1. Soluble tPA, rPA, RBC/tPA, and RBC/rPA (0.05 µM) were preincubated with an equimolar concentration of PAI-1 for 30 min at room temperature followed by incubation with Spectrozyme tPA (0.4 mM). The preparations were incubated with Spectrozyme (final volume 200 µl) in V-shaped plates for 20 min at 25°C and centrifuged at 1200g for 2 min to precipitate RBC. The optical density was measured at 405 nm in 100-µl aliquots of the supernatants (Ganguly et al., 2005).
Effect of Plasma PA Inhibitors on Lysis of Fibrin Clots by Soluble PA and RBC/PA. Soluble and RBC/PA (both tPA and rPA) (5 nM) were preincubated with PAI-1, 2-macroglobulin, and 1-antitrypsin in varying molar ratios (1.0:0.5–4.0) for 30 min at room temperature. Fibrin clots were prepared by adding a trace amount of 125I-Fg (2 µl) to a 3-mg/ml human fibrinogen-containing plasminogen. CaCl2 and thrombin (final concentrations 20 mM and 0.2 U/ml, respectively) were added, and PBS (200 µl) was layered over the clots. Fibrinolytic agents were added to the clots to simulate their proposed in vivo application (Murciano et al., 2003; Ganguly et al., 2005, 2006). Soluble tPA mixed in PBS (200 µl) was added externally onto the clot surface to model therapeutic dissolution of preexisting clots, whereas RBC/tPA was incorporated into the clot to model prophylactic lysis of nascent clots (Ganguly et al., 2005). Clot lysis was measured by the release of the soluble 125I-fibrin in the supernatant.
Binding of 125I-PAI-1 to tPA Coupled to Naive or Glycocalyx-Stripped RBC. RBC/tPA conjugates prepared using naive or glycocalyx-stripped RBC were incubated with 125I-PAI-1 at a 1:1 M ratio relative to RBC-coupled tPA for 30 min at room temperature. Unbound 125I-PAI-1 was removed by washing, and RBC-bound radioactivity was used to calculate the number of PAI-1 molecules bound to RBC/tPA.
Binding of RBC/PA and Glycocalyx-Stripped RBC/PA to Plasminogen. We measured binding of RBC/PA to immobilized proteins as described previously (Ganguly et al. 2005). Pg and NC-Pg (0.2 µM/well) were immobilized on 24-wells polystyrene plate overnight at 4°C. Protein-coated wells were blocked with PBS-BSA (3%) and incubated with RBC/PA for 20 min at room temperature on a horizontal shaker at a low speed (2g). In a separate series, RBC/PA was incubated in Pg-coated wells in the presence of 0.1 M 
-aminocaproic acid, a lysine analog that blocked the lysine-binding sites on immobilized Pg. Binding of RBC was quantified by measuring hemoglobin at 405 nm after lysing the bound cells in water as describe previously (Ganguly et al., 2006).
Fibrinolytic Activity of Soluble tPA versus RBC/tPA in Presence of a High Level of Glucose. Soluble tPA or RBC/tPA was incubated with 0, 120, 200, 400, and 600 mg/dl glucose in PBS-BSA for 24 h at 4°C. Lysis of 125I-fibrin clots was determined as described above. In a separate series of experiments, naive RBC/tPA or glycocalyx-stripped RBC/tPA was incubated with glucose before addition within fibrin clots.
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), probably because of limitations on diffusion and spatial freedom of tPA imposed by the carrier RBC. Yet, RBC/tPA caused more extensive in vitro lysis of clots formed from human plasma than soluble tPA. These seemingly discrepant observations implied that coupling to RBC in some way enhances the resistance of tPA to plasma inhibitors, such as PAI-1.
We first evaluated the relative resistance of RBC/tPA versus tPA to inhibition in vivo by injecting these formulations intravenously in WT and PAI-1 Tg+ mice. The plasma level of PAI-1 averages <0.8 ng/ml in WT mice versus 30 ng/ml in PAI-1 Tg+ mice (Eitzman et al., 1996); hence, this study also tested whether enhanced resistance of RBC/tPA to PAI-1 is evident in the pathological settings associated with elevated levels of the inhibitor. Blood was drawn and allowed to clot in the presence of 125I-fibrinogen, and then clot lysis was determined (Fig. 1). Previous studies showed that the half-life of radiolabeled RBC/tPA in mice is >3 h versus <15 min for soluble tPA (Ganguly et al., 2005). Therefore, mice were injected with either 0.5 mg/kg soluble tPA or 0.2 mg/kg RBC/tPA, and blood was drawn at 5 and at 15 min to compensate for the rapid clearance of soluble tPA.
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To exclude the pharmacokinetic effects, we compared lysis by the same concentration (10 nM) of RBC/tPA versus tPA added directly to blood drawn from WT mice before clotting in vitro. Despite being subject to fewer spatial constraints, soluble tPA caused less than 50% as much fibrinolysis as RBC/tPA in the blood of WT mice (41.1 ± 9.3 versus 83.3 ± 2.4%, respectively). These data 1) affirm the greater fibrinolytic potency of RBC/tPA compared with soluble tPA in mouse blood in vitro and in vivo, and 2) indicate that coupling to RBC protects tPA against physiological and pathological concentrations of PAI-1.
RBC/tPA Is Less Susceptible to Diverse PA Inhibitors Than Soluble tPA. We next compared the susceptibility of soluble tPA versus RBC/tPA and soluble rPA versus RBC/rPA to various purified plasma PA inhibitors. Equimolar concentrations of PAI-1 caused almost complete loss of tPA-mediated chromogenic (Fig. 2A) and fibrinolytic (Fig. 2B) activity, whereas neither activity of RBC/tPA was affected under the same experimental conditions (Fig. 2, left). However, coupling of rPA to RBC provided little protection of its amidolytic activity (Fig. 2C) and no protection of its fibrinolytic activity (Fig. 2D) from inhibition by PAI-1.
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Likewise, equimolar concentrations of two plasma serpins with broad spectrum of inhibition, 2-macroglobulin and 1-antitrypsin, suppressed the fibrinolytic activity of soluble tPA but not RBC/tPA (Fig. 3). Again, coupling of rPA to RBC did not protect its fibrinolytic activity against inhibition by 2-macroglobulin (53.2 ± 2.1 and 52.5 ± 12.7% inhibition of rPA and RBC/rPA, respectively) or 1-antitrypsin (24.9 ± 8.7 and 48.8 ± 12.1% inhibition of rPA and RBC/rPA, respectively).
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tPA coupled to RBC or glycocalyx-stripped RBC exhibited similar amidolytic (e.g., A405 equal 0.90 ± 0.03 in RBC/tPA versus 0.91 ± 0.26 in stripped RBC/tPA in the Spectrozyme assay) and fibrinolytic activity (Fig. 4A). However, coupling tPA to glycocalyx-stripped RBC did not confer the enzyme with enhanced resistance to PAI-1, 2-macroglobulin, or 1-antitrypsin (Fig. 4A). In support of this inference, binding of 125I-labeled PAI-1 to tPA coupled to glycocalyx-stripped RBC almost doubled compared with binding to tPA coupled to control RBC (Fig. 4B). These data suggest that the RBC glycocalyx protects cell-bound tPA against plasma serpins by masking their auxiliary binding sites (Carrell et al., 1991; Lawrence et al., 1995; Wilczynska et al., 1995).
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-aminocaproic acid or by replacing Pg with its noncleavable analog NC-Pg (Fig. 5), indicating that binding requires interactions between Lys binding sites in plasminogen activators and Lys residues in the substrate. However, RBC/tPA and stripped RBC/tPA bound to plasminogen to a similar extent (28.3 ± 3.1 x 103 versus 25.2 ± 4.9 x 103 RBCs/well, respectively).
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Coupling to RBC Protects tPA against Inhibition by High Levels of Glucose. Pathologically high levels of glucose have also been shown to inhibit tPA activity (Alvarez-Sabin et al., 2004). Therefore, we next tested whether coupling to RBC alters susceptibility of tPA to elevated levels of glucose. Addition of pathologically high levels of glucose (600 mg/dl) directly to fibrin clots along with tPA did not affect either spontaneous lysis or lysis induced by RBC/tPA or soluble tPA (Fig. 6A, inset). These data corroborate findings that tPA inhibition via nonenzymatic glycosylation/glycation requires a relatively prolonged (i.e., hours) exposure (Lapolla et al., 2005).
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1-antitrypsin, 2-macroglobulin, and C1 inhibitor (Madison et al., 1990; Chandler et al., 1995). PAI-1 traps tPA in the form of an inactive, stabilized acyl enzyme intermediate (1:1 complex) (Carrell et al., 1991; Lawrence et al., 1995) that is cleared from plasma with a half-life of 3 to 5 min by the low-density lipoprotein-related receptor and the very low-density lipoprotein receptor (Bu et al., 1992), leading to degradation of the enzyme in lysosomes. Increased expression of PAI-1 by ischemic endothelium and release from activated platelets contributes to the resistance of platelet-rich arterial thrombi to lysis (Zhu et al., 1999).
In part to compensate for rapid inactivation and clearance, tPA must be given at concentrations several orders of magnitude higher than physiological concentrations to mediate reperfusion after myocardial infarction or stroke (Ridker et al., 1994). The need for such high doses for efficacy is accompanied by a considerable risk of hemorrhage. Genetic reengineering of tPA to remove critical auxiliary sites that help mediate serpin binding (amino acids 296–304) increases the resistance of the molecule to PAI-1 (Madison et al., 1990) and prolongs its circulation in plasma (e.g., TNKase mutant) (Keyt et al., 1994), but not to the extent needed to provide thromboprophylaxis. Moreover, TNKase, like other tPA variants, is a relatively small (
60-kDa) protein capable of diffusing into preexisting hemostatic clots and through blood-brain barrier perturbed by ischemia into brain parenchyma, hence increasing the risk of hemorrhages and other adverse effects.
Results of the previous studies have shown that coupling tPA to carrier RBC increases its intravascular life span by several orders of magnitude compared with soluble tPA (Murciano et al., 2003; Ganguly et al., 2005) and that it restricts PA extravasation into brain (Murciano et al., 2003). During the course of this study, RBC/tPA was found to be less active than soluble tPA in lysing clots formed from fibrinogen, probably because of steric constraints on permeation, but it showed clear fibrin specificity (Ganguly et al., 2005), and surprisingly, it was more active than soluble tPA in lysing clots made from human plasma (Ganguly et al., 2005). These findings suggested a fundamental change in the relationship between tPA-mediated plasminogen activation and inhibition by cognate serpins that results from coupling the enzyme to the cell surface.
The present study shows that the protection afforded by RBC binding is dependent on the glycocalyx of the cell. Enzymatic degradation of the glycocalyx did not affect basal amidolytic (0.90 ± 0.03 in RBC/tPA versus 0.91 ± 0.26 in stripped RBC/tPA) or fibrinolytic activity of RBC-coupled tPA (Fig. 4A). However, removal of the glycocalyx led to the loss of protection of tPA by RBC, both in terms of PAI-1 binding and tPA activity. These results do not exclude that a change in conformation of tPA imposed by conjugation contribute to protection but favor a more specific effect of the glycocalyx on PAI-1 binding to the auxiliary sites on tPA, access to tPA catalytic site, or restrictions on conformational changes in PAI-1 that occur after binding (Carrell et al., 1991; Lawrence et al., 1995; Wilczynska et al., 1995; Egelund et al., 2001).
The RBC glycocalyx affects the interaction of RBC-bound tPA with its inhibitors, activators (e.g., fibrin), and substrates (physiological substrate, plasminogen, and small synthetic substrates used to measure amidolytic activity) in a complex, but precise way. Our previous studies showed that coupling to RBC does not affect the stimulation of tPA activation by fibrin (Ganguly et al. 2005). The results of the present study indicate that the RBC glycocalyx does not affect RBC/tPA binding to plasminogen (Fig. 5). Therefore, it seems that coupling to RBC does not compromise tPA interactions with its substrates and activators; yet, coupling protects it from the inhibitors.
It is known that cellular glycocalyx may affect the activity of plasmin (Pluskota et al., 2004). Therefore, in theory, the enhanced activity of RBC/tPA seen in the presence of inhibitors could be due to protection of plasmin. However, coupling to RBC did not protect a truncated form of tPA, rPA, against plasma inhibitors (Fig. 2). This selectivity argues for a direct effect of the RBC glycocalyx on tPA. This interpretation is supported by the fact that the RBC glycocalyx protects the amidolytic activity of tPA as well.
It is not clear yet why RBC does not protect rPA. In theory, this result can be explained by 1) lack of auxiliary sites for interactions with glycocalyx or/and inhibitors on the rPA molecule that lacks the finger, growth factor, and Kringle-1 domains present in tPA; and/or 2) unique conformational changes caused in rPA as a result of conjugation. It is an interesting corollary in this context that despite the fact that RBC/tPA and RBC/rPA possess equal activity in the absence of inhibitors (indicating that they interact equally well with plasminogen and convert it to plasmin), only RBC/tPA possess sufficient affinity to plasminogen to afford specific binding via lysine-mediated mechanism (Fig. 5).
Understanding the mechanism by which the glycocalyx protects tPA from PAI-1 and other serpins will require additional studies. The most simplistic explanation that the glycocalyx physically masks the auxiliary binding site(s) for PAI-1 seems less likely based on the selective effect in this tPA feature with no effect on the enzyme access to plasminogen or fibrin. Rather, specific "protection" of tPA by RBC glycocalyx against serpins may be explained by interference with charge-mediated interactions between the protease and protein inhibitor. It seems likely that PAI-1 contacts with the surface loop of tPA via residues 350 to 355, which contains several negatively charged amino acids (Glu-Glu-Ile-ILe-Met-Asp) (Madison et al., 1990). The interaction of tPA and PAI-1 may therefore be stabilized through salt bridges formed between cationic amino acids tPA and anionic residues in PAI-1 (Madison et al., 1990; Egelund et al., 2001). Negatively charged components of RBC glycocalyx such as ionized sialic acid groups may impede these interactions by forming an electrostatic as well as a physical barrier between protease and serpins.
The observation that the RBC glycocalyx also protects tPA against inactivation by nonenzymatic glycosylation may support the notion that protection is due to charge-mediated masking of vulnerable sites on tPA molecules by the glycocalyx. Interactions between RBC/tPA and the glycocalyx may mask the lysine residues in tPA molecules that are vulnerable to Maillard product formation and glycation (Lapolla et al., 2005).
The finding that RBC-bound tPA is more resistant to PAI-1 and other serpins and high levels of glucose translates into additional practical advantages for this modality. These include reduction in the effective dose with attendant lessening of the risk of hemorrhage, extended duration of thromboprophylaxis, enhanced effectiveness toward platelet-rich arterial clots and prevention of thrombosis in diabetics. The results of this study also reveal a relatively unappreciated role for the glycocalyx in modulating tPA activity, which may take place on the surface of endothelial, hematopoietic, and other vascular cell types, that deserves greater scrutiny.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: PA, plasminogen activator; RBC, red blood cell(s); PAI-1, plasminogen activator inhibitor-1; tPA, tissue-type plasminogen activator; rPA, recombinant plasminogen activator; Pg, plasminogen; SA, streptavidin; NC-Pg, noncleavable plasminogen; WT, wild type; PBS, phosphate-buffered saline; BSA, bovine serum albumin; Fg, fibrinogen.
Address correspondence to: Dr. Vladimir R. Muzykantov, Institute for Environmental Medicine, 1 John Morgan Bldg., University of Pennsylvania Medical Center, 3620 Hamilton Walk, Philadelphia, PA 19104-6068. E-mail: muzykant{at}mail.med.upenn.edu
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