1 Institute for Environmental Medicine, Departments of 3 Pathology, 4 Surgery, and 6 Pharmacology, University of Pennsylvania, Philadelphia 19104; 5 Centocor, Malvern, Pennsylvania 19355; and 2 Division of Vascular Surgery, University of Maryland, Baltimore, Maryland 21201
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ABSTRACT |
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Using tracings of 125I-labeled fibrin(ogen) in rodents, we examined the hypothesis that platelets impede the lysis of pulmonary emboli. 125I-Microemboli (ME, 3-10 micron diameter) lodged homogeneously throughout the lungs after intravenous injection in both rats and mice (60% of injected dose), caused no lethality, and underwent spontaneous dissolution (50 and 100% within 1 and 5 h, respectively). Although lung homogenates displayed the most intense fibrinolytic activity of all the major organs, dissolution of ME was much slower in isolated perfused lungs (IPL) than was observed in vivo. Addition of rat plasma to the perfusate facilitated ME dissolution in IPL to a greater extent than did addition of tissue-type plasminogen activator alone, suggesting that permeation of the clot by plasminogen is the rate-limited step in lysis. Platelet-containing ME injected in rats lysed much more slowly than did ME formed from fibrin alone. 125I-Thrombi, formed in the pulmonary vasculature of mice in response to intravascular activation of platelets by injection of collagen and epinephrine, were essentially resistant to spontaneous dissolution. Moreover, injection of the antiplatelet glycoprotein IIb/IIIa antibody 7E3 F(ab')2 facilitated spontaneous dissolution of pulmonary ME and augmented fibrinolysis by a marginally effective dose of Retavase (10 µg/kg) in rats. These studies show that platelets suppress pulmonary fibrinolysis. The mechanism(s) by which platelets stabilize ME and utility of platelet inhibitors to facilitate their dissolution deserves further study.
pulmonary embolism; plasminogen activators; platelets; fibrinolysis; animal model; glycoprotein IIb/IIIa
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INTRODUCTION |
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THE LUNG SERVES as a natural filter for thromboemboli of diverse sizes (12). Thrombotic occlusion of the main or lobar pulmonary arteries by large clots (massive pulmonary embolism, PE) is a common cause of morbidity and mortality (15, 20). In contrast, pulmonary deposition of microemboli (ME) often does not cause acute symptoms (silent ME). Nevertheless, pulmonary ME is a relatively common accompaniment to acute lung injury (adult respiratory distress syndrome), hyperoxia, bleomycin toxicity, sepsis, trauma, surgery on the lower extremities, and angioplasty (2, 21, 30, 33, 36, 39, 40, 44, 52, 53). Microembolism may lead to inflammation and edema acutely and contribute to the development of pulmonary fibrosis and hypertension over time (4, 9, 22, 27, 31).
The treatment of PE, as with other venous thromboembolic conditions, is typically restricted to agents such as heparin that retard fibrin clot formation, with a relatively limited use of agents that promote fibrinolysis. Despite the effectiveness of such therapy, PE remains a common cause of mortality and morbidity. Thus there is a continuing need for more effective means to prevent and treat both PE and ME, based on a more thorough understanding of the mechanisms that regulate clot dissolution in the lung.
For example, the role of platelets in PE and ME has received relatively
little study. Recently, it has been shown that platelet activation by
antibodies in mice that are transgenic for expression of the signal
transducing receptor FcRIIA leads to occlusive platelet-fibrin
thrombi in the pulmonary vasculature (50).
Inhibition of platelet function by aspirin, nitric oxide
donors, and inhibitors of other signal transduction pathways may
improve the outcome of thrombotic pulmonary vascular occlusion
secondary to intravascular thrombosis in several animal models
(16, 23, 34, 51). Venous thrombi and PE are also the most
common sequelae of intravascular platelet activation among patients
suffering from heparin-induced thrombocytopenia (55). Yet
human studies to date have provided ambiguous results concerning the
efficacy of antiplatelet agents in the treatment of PE and ME
(35, 48). However, the recent introduction into clinical
medicine of potent intravenous inhibitors of the platelet
glycoprotein IIb/IIIa complex (GP IIb/IIIa), the receptor
required for platelet aggregation, suggests the need and provides the
opportunity to reinvestigate this question.
Platelets have been proposed to suppress clot lysis in large arteries via diverse mechanisms, including mechanical cross-linking, promotion of clot stability (24), and release of fibrinolytic inhibitors (13, 25, 26). However, the extent to which platelets affect dissolution of thromboemboli in the pulmonary arterial circulation, the vasculature most commonly affected in humans, is unknown as is the efficacy of newly available potent inhibitors of platelet function. We hypothesized that incorporation of platelets into nascent pulmonary thromboemboli would make them less susceptible to fibrinolysis and that inhibition of platelet GP IIb/IIIa function may attenuate this effect.
We developed rodent models that allow quantitative assessment of pulmonary microemboli dissolution and examined the role of platelets in pulmonary fibrinolysis. The results of our study show that 1) platelets inhibit lysis of pulmonary ME and 2) antiplatelet agents facilitate both spontaneous and pharmacological fibrinolysis in the lungs.
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MATERIALS AND METHODS |
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Materials. The following materials were used: Iodogen from Pierce (Rockford, IL), Na125I from NEN Life Sciences (Boston, MA), fatty acid-free bovine serum albumin (BSA) from Boehringer-Mannheim (Indianapolis, IN), human thrombin and epinephrine from Sigma (St. Louis, MO), horse collagen (type I) from Chrono-Log (Havertown, PA), human recombinant tissue-type plasminogen activator (tPA, Activase) from Genentech (San Francisco, CA), fibrinogen (Fg; FIB 1, 1310) from Enzyme Research Laboratories (South Bend, IN), Retavase (a mutant form of human tPA that lacks kringle and growth factor domains), and murine monoclonal antibody that cross-reacts with human and rat platelet glycoprotein IIb/IIIa complex [MAb 7E3 F(ab')2] from Centocor (Malvern, PA). Adult Sprague-Dawley male rats (200-350 g) and BALB/c mice were used following a protocol approved in writing by the University of Pennsylvania Institutional Animal Care and Use Committee. Human blood was obtained by phlebotomy from healthy volunteers, following a protocol approved in writing by the University of Pennsylvania Institutional Review Board for Studies Involving Human Subjects. Fg and Retavase were radiolabeled with 125I using Iodogen per the manufacturer's instructions, and the free iodine was removed using a Bio-Spin 30 column (Bio-Rad Laboratory, Hercules, CA).
Preparation and characterization of 125I-labeled thrombi. Plasma was isolated by centrifugation of fresh human blood anticoagulated in citrate (0.32% final concentration). 125I-Fg (~30 × 106 cpm/ml) was added to fresh plasma or Fg solution 30 mg/ml in Krebs-Ringer bicarbonate buffer (KRB): 119 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.3 mM CaCl2, 25 mM NaHCO3, and 1.2 mM KH2PO4-KRB. Before adding 125I-Fg, we supplemented plasma with unlabeled Fg to a final concentration of 10 mg/ml to avoid potential differences in Fg levels among donors, to increase clot density, to prevent damage during homogenization, and to increase the yield of 125I-ME.
CaCl2 (20 mM final concentration) and human thrombin (Sigma; 0.2 U/ml final concentration) were added to plasma or fibrin containing 125I-Fg to induce clotting. After 1-h incubation at room temperature, the clots were incubated overnight at 4°C to promote retraction. Clot stability and susceptibility to lysis by streptokinase and tPA did not change over the next 48 h at 4°C (data not shown); clots stored for longer times began to undergo spontaneous lysis. Therefore, all clots were used within 24 h of formation. All subsequent steps were performed at 4°C. The procedure described above produces 125I-labeled clots consisting of extensively cross-linked fibrin (1), which were dissected into either large emboli (125I-clots, 5 mm in diameter) or homogenized to form a suspension of microemboli (125I-ME). Before homogenization, clots were decanted on a plastic lid and cut into small pieces. The pieces were resuspended in 2 ml KRB and homogenized at 26,000 rpm for 1 min in a PT-3100 Polytron homogenizer (Brinkmann Instruments, Westbury, NY). After the first homogenization step, the samples were centrifuged for 15 min at 2,000 g. The supernatant was removed, and the residual particles were pooled, resuspended in KRB, and rehomogenized. This procedure was repeated twice, and the resulting 125I-ME were suspended in 8 ml KRB-BSA (3 mg/ml). The suspension was allowed to sediment at 4°C for 5 min to eliminate large particles. Four milliliters from the supernatant were used for the final preparation of the 125I-ME suspension; it was divided into 200-µl aliquots that were stored at 4°C and used within 48 h. Random aliquots were selected to characterize the size distribution of the microparticles using a ZM Coulter counter (Coulter Electronics, Hialeah, FL). Approximately 80% of ME ranged from 1.5 to 3.5 µm in diameter, and <5% exceeded 10 micron in diameter (1). Large, single clots and 125I-ME formed from platelet-rich plasma were always used on the day they were prepared. The accumulation and dissolution of 125I-ME stored at 4°C remained stable for at least 3 days (not shown). Thus, in all experiments, fibrin or plasma 125I-ME were injected within 3 days of preparation. Our preliminary studies revealed no difference in lysis of clots formed from human plasma or from pure human Fg by tPA or when fresh rat or murine plasma was used as a source of plasminogen and its activators, under the experimental circumstances employed in our studies (not shown). In vivo studies showed no significant difference in the extent of pulmonary deposition or rate of spontaneous dissolution of 125I-ME prepared from human plasma or pure Fg in rats and mice (not shown). No acute changes in the respiratory or circulatory status in mice or rats injected with human ME was observed suggestive of an allergic reaction to heterologous protein. All experiments were terminated within 1-5 h postinjection.In vivo experiments. The external jugular vein was accessed in 300- to 350-g male rats via a neck incision under anesthesia and catheterized with a 14-gauge angiocatheter. This allowed injection of single large 125I-clots (~5 mm diameter), which adapted the diameter of the catheter and vein due to their high elasticity. 125I-ME (200-µl aliquots containing 30-100,000 cpm) were resuspended several times just before extraction into a 27.5-gauge syringe and injected via either jugular (through the pectoral muscle) or the tail vein. Only tail vein injection was used in mice. In a separate series, 125I-Fg was coinjected with the indicated dose of collagen and epinephrine (125I-Fg/coll/epi mixture) via the jugular or tail vein in both species.
At the indicated times after injection of 125I-clots, 125I-ME or 125I-Fg/coll/epi mixture, animals were killed, and the major organs were harvested immediately, rinsed in saline, and dried on a filter paper; the radioactivity in each tissue was measured (Perkin-Elmer, Gaithersburg, MD). The injected dose (ID, cpm/animal) of injected radioactivity was calculated after subtracting the residual radioactivity remaining in the tube, syringe, and injection site.Perfusion of isolated rat lung. Lungs were isolated from anesthetized rats (200-250 g) and perfused using recirculating perfusate (KRB, pH 7.4, containing 10 mM glucose and 3% fatty acid-free BSA, KRB-BSA) as described (37). Lungs were ventilated with a humidified gas mixture containing 5% CO2 and 95% air with a SAR-830 rodent ventilator (CWE, Ardmore, PA) at 60 cycles/min, 2 ml tidal volume, and 2 cm H2O end-expiratory pressure. The lungs were perfused at a flow rate of 10 ml/min at 37°C, followed by nonrecirculating perfusion for 5 min with KRB-BSA.
In vitro assay of degradation of 125I-labeled clots. Diluted homogenates of organs (1 g/10 ml KRB) were obtained from intact anesthetized rats. Homogenate (200 µl) was then added to 125I-fibrin-labeled human plasma clots (200 µl) enriched with 10 mg/ml of human Fg. Clots were incubated at 37°C, and radioactivity in the supernatants, representing degradation of the fibrin clot, was measured at the indicated time points.
Statistics. Data were analyzed using the unpaired, nonparametric Mann-Whitney test. Differences between groups were considered significant when P < 0.05.
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RESULTS |
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The purpose of our studies was to examine the effect of platelets on the lysis of pulmonary emboli and to examine the impact of platelet inhibitory therapy on the rate of clot dissolution in the pulmonary vasculature. To address this issue, our first task was to develop rodent models in which the effect of platelets on fibrinolysis could be examined quantitatively in vivo.
Pulmonary deposition of large emboli and ME in vivo.
We first compared the biodistribution of radiolabeled Fg,
125I-Fg, as well as 125I-clots and
125I-ME prepared from 125I-Fg, 5 min after
intravenous injection in intact rats (Fig.
1). Most 125I-Fg remained in
the plasma or was taken up by the liver. Little radioactivity
accumulated in the lungs (Fig. 1A), consistent with the
expected hepatic clearance of large (molecular weight >60 kDa)
radiolabeled proteins.
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Distribution and spontaneous dissolution of 125I-clots and 125I-ME in rat lungs. 125I-clots were heterogeneously distributed in the lungs and tended to lodge in a single lobe. For example, 70-90% of injected radioactivity was found in the left lobe (the largest in this species) in six out of ten animals in one experiment. In these animals, <0.5% of ID was found in the other lobes. However, in two other animals, 50-80% of injected 125I-clots were found in the right lower lobe (the second largest), with no more than 0.5% in the other lobes. In the remaining two rats, 125I-clots were found in two lobes, likely due to disintegration and embolization from the heart or pulmonary artery bifurcation. In the latter cases, a single clot was identified in the left and the lower right lobes.
In contrast, 125I-ME were distributed homogeneously throughout the lobes. Table 2 shows the lobar distribution of 125I-ME in rat lungs from several separate experiments. The data are expressed as percentages of ID per gram, to compensate for differences in lobe size. The results clearly show that ME are evenly distributed among the lobes in rats. These data also show that the pulmonary deposition of 125I-ME in intact rats is highly reproducible. These data are consistent with the results of a previous study, in which we documented the homogenous distribution of 125I-ME in lobes of the lung after tail vein injection in mice followed by gamma-radioautography (1).
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Dissolution of ME ex vivo in perfused rat lungs. Pulmonary emboli represent a substrate for plasmin generated by plasminogen activators both circulating in plasma and expressed in lung tissue. To dissect the relative contributions of pulmonary and plasma-derived fibrinolytic factors in spontaneous dissolution of ME, we characterized lysis of 125I-ME lodged in isolated perfused lungs (IPL) and in vitro lysis of 125I-clots by lung tissue homogenates.
First, we compared the rate of degradation of 125I-ME lodged in rat lungs in vivo with their dissolution in isolated rat lungs perfused with KRB (Fig. 4). Initial pulmonary deposition of 125I-ME 10 min postinjection was comparable in each model (61 ± 5% in intact rats, 75 ± 2% in the isolated lungs). Spontaneous fibrinolysis was very effective in vivo: almost no residual 125I-ME was detected in the lungs 3 h postinjection in intact rats (2.3 ± 1.1%). In contrast, fibrinolysis in the isolated lungs was not as effective (45 ± 10% residual 125I-ME by 3 h).
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Effect of exogenous tPA on dissolution of pulmonary emboli in intact rats. In the next series of experiments, we characterized the dissolution of PE in rats by exogenously added plasminogen activators. In pilot experiments, we found that intravenous injection of a relatively high dose of tPA (Activase, 500 µg/kg) 10 min after injection of large 125I-clots caused ~50% fibrinolysis. (Residual radioactivity in the lungs 1 h after 125I-clots injection was 38.4 ± 5.8 vs. 81.5 ± 4.5%ID in control rats). However, the high mortality and low reproducibility that we observed, although reflecting the clinical experience with massive PE, limited the utility of this approach as a model.
Therefore, we explored the more reproducible ME model to examine the effect of plasminogen activators in greater detail with Retavase (a variant of human recombinant tPA that lacks kringle and growth factor domains and demonstrates slower clearance from the circulation in humans). Figure 6A shows that even a relatively low dose of Retavase (30 µg/kg) injected in rats 10 min post-125I-ME administration reduced the residual pulmonary radioactivity by 50% (17% of injected 125I at 1 h). Higher doses (100 µg/kg and above) caused 90-95% dissolution of pulmonary 125I-ME in both rats and mice by 1 h.
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Involvement of platelets and effect of antiplatelet GP IIb/IIIa.
Having established reproducible, nonlethal, models of pulmonary
embolization in rodent, we then turned to examine the role of platelets
in controlling the rate of clot dissolution. To do so, we first
compared the dissolution of 125I-ME prepared from
platelet-poor plasma (the standard preparation used throughout this
study) with that of 125I-ME prepared from platelet-rich
plasma. Figure 7A shows that incorporation of platelets into 125I-ME significantly
retarded the lysis (compare residual 125I 1 h post-ME
injection). Thus even in this model of ME, which are intrinsically
highly susceptible to endogenous fibrinolysis, the impact of platelets
is evident within the often critical first hour.
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DISCUSSION |
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The risk of developing clinically significant pulmonary embolization in humans depends on underlying disease, the balance between coagulation and fibrinolysis, and preexisting pulmonary function, among other factors. Some of these factors cannot be adequately addressed in currently available animal models. Even relatively straightforward parameters, such as size and composition of the clot(s), are challenging to investigate in vivo. To carry out such investigations, one needs an in vivo model that permits accurate and reproducible measurements of fibrin deposition and dissolution caused by emboli of known size and content lodged in a vascular bed of interest. Ideally, these models should be applicable to a variety of animal species ranging from small rodents such as mice (which permits experiments to be conducted in diverse and defined genetic backgrounds) to primates (to evaluate species-specific pathways, as well as potential side effects relevant to human patients).
Models that are based on vascular trauma or injection of endotoxin to initiate intravascular fibrin formation are useful (10, 11, 18, 47, 56, 57) but difficult to control in terms of clot size, prevalence, and destination. Injection of preformed clots overcomes some of these limitations (3, 6, 29). We have used this latter approach to study PE in laboratory rodents and have established a model that enables us to study clot dissolution in a reproducible and quantitative manner. Theoretically, measurement of plasma radioactivity may permit a continuous nonlethal monitoring of fibrinolysis within the lung (44). However, this approach is limited by potential alterations in clearance mechanisms, the results of unintended changes in hepatic perfusion, renal elimination, and peripheral receptor saturation. In contrast, direct measurement of the residual radioactivity in the lungs on postmortem examination provides unambiguous data on fibrinolysis in the organ of interest.
Our data show that it is possible to model both massive PE and ME in rats and mice by varying the size of the infused fibrin clots. Injection of large (5-6 mm diameter) clots, as in humans, is associated with considerable lethality. Either embolization of the right ventricle or passage of clots through a patent foramen ovale leading to embolization of the coronary or cerebral vasculature could contribute to lethality. However, the location of large clots cannot be controlled, and their destination appears to track the size of the primary lobar vessels. In contrast, ME are nonlethal and produce a highly reproducible, homogenous deposition of fibrin in lungs in mice and rats alike. Using this model, we demonstrated that clot dissolution is impaired in mice genetically deficient in either tPA or urokinase-type plasminogen activator (1).
The purpose of this study was to begin to examine the physical, biochemical, and cellular factors that contribute to the rate of clot lysis in the pulmonary circulation. We found that large 125I-labeled pulmonary clots do not undergo significant spontaneous dissolution in intact rats (Fig. 2), whereas pulmonary 125I-ME undergo complete dissolution in intact rats and mice within the first 5 h (Fig. 3). The 30-min lag phase before appreciable lysis of ME is seen argues against mechanical elimination as a major contributor to the observed reduction in lung radioactivity. More likely, the lag time reflects the requirement for endogenous plasminogen activators to be activated, plasmin to be generated, and especially permeation of plasmin into the clot and fibrinolysis proper to occur (43).
Dissolution of PE in intact animals represents the outcome of a complex interplay between systemic (blood) and local (pulmonary) factors. Our data indicate that total fibrinolytic activity in rat lung in vitro is greater than in other organs (Fig. 5). This result corroborates studies by others using lungs from diverse other species (12, 57). A substantial fraction of total plasminogen activator activity in the lungs is generated within the alveolar space (38, 46). The pulmonary vasculature also expresses plasminogen activators (28, 49). Thus the lungs seem to be well equipped to function as blood filters that trap and dissolve emboli (12).
However, despite the presence of considerable plasminogen activator activity in lung tissue, the dissolution of 125I-ME by isolated rat lungs was relatively inefficient in the absence of plasma. This result implies that the rate-limiting step in pulmonary fibrinolysis may be the diffusion of plasminogen from the plasma to the inner layers of the fibrin clot. This speculation would also help to explain the relatively poor spontaneous and pharmacological dissolution of large clots lodged in rat lungs, notwithstanding the intravenous infusion of high concentrations of tPA. The surface-to-volume ratio of the larger clots is much lower than it is for ME, which would be expected to impair plasminogen supply to internal portions of the clot (43). However, other factors (e.g., local and general hypoxia due to vascular occlusion, redistribution of blood flow, etc.) may also impair dissolution of large clots.
Conversely, the higher surface-to-volume ratio of ME compared with massive PE permits more effective delivery of plasminogen activators, as well as permeation of the ME by plasminogen. We previously reported that intravenous infusion of recombinant human urokinase markedly accelerates dissolution of pulmonary 125I-ME in mice (1). In the present study, even a single injection of a relatively low dose of Retavase (<100 µg/kg) was highly effective (Fig. 6). However, no effect was observed when even a high (500 µg/kg) dose of Retavase was administered just 10 min before 125I-ME injection, likely the result of the extremely fast elimination of the drug from the bloodstream. Thus even tPA variants that have been modified to prolong their lifetime in the blood, such as Retavase (31), are unlikely to provide prophylaxis even when given in high doses or modifications that cannot be used for thromboprophylaxis.
Our studies provide additional support for the concept that platelets
represent an abundant and important component of PE (54).
Fg and platelets colocalize in venules as well as in arterioles after
ischemia/ reperfusion injury (32). Clots are
dynamic, and the outcome of our prior studies also suggest that fibrin ME incorporate both hematopoietic cells and additional fibrin after
they adhere to the vessel wall in vivo (1). Platelets not
only accelerate thrombin production but also are a source of
plasminogen activator inhibitor 1 (PAI-1) and induce endothelial cells
in the vicinity of PE to express this inhibitor (25, 26) through the elaboration of transforming growth factor- and other constituents (13). Vitronectin, also secreted by
activated platelets, may help to stabilize thrombi after vascular
injury (24). Platelets also cause clot retraction
generating a physical barrier to the perfusion of plasminogen and
plasminogen activators (5, 8).
We found that both inclusion of platelets during the formation 125I-ME in vitro and intravascular activation of platelets by collagen/epinephrine leading to thrombus production in vivo render PE more resistant to lysis (Fig. 7). Consistent with these observations, MAb 7E3 F(ab')2, a monoclonal antibody that inhibits Fg binding to platelet GP IIb/IIIa, facilitates the lysis of 125I-ME in rat lungs and augments the efficacy of a suboptimal dose of Retavase in vivo (Fig. 8). These data imply that platelets promote the resistance of clots to fibrinolysis. Recent in vitro findings by others revealed that an anti-GP IIb/IIIa MAb inhibits tissue factor-induced thrombin production, platelet factor-4 release, formation of procoagulant microparticles (42), and accelerates fibrinolysis (8, 19). Taken together with these in vitro data, our results obtained in intact animals imply that it may be possible to accelerate pulmonary fibrinolysis or even lessen the propensity to form occlusive thrombi by impairing platelet function. However, the kinetics of GP IIb/IIIa occupancy (41), as well as thrombocytopenic and potential bleeding effects of MAb 7E3 (17), must be carefully tested to evaluate the safety of this maneuver.
The results of this study, therefore, extend understanding of potential involvement of platelets in fibrinolysis to the pulmonary circulation, the most commonly affected organ in man. There is considerable vascular heterogeneity in the expression of plasminogen activators and receptors (14), and vascular beds also differ in terms of local mechanisms involved in formation and dissolution of clots. Therefore, the present results warrant further study addressing mechanism of platelet-mediated retardation of pulmonary fibrinolysis, as well as potential risk/benefit balance of MAb 7E3 interventions in more detail. For example, our pilot experiments showed that the rate of spontaneous dissolution of 125I-Fg deposits after 125I-Fg/coll/epi mixture injection does not differ in PAI-1-deficient mice from that in control littermates, despite the notion that PAI-1 is one of the thrombi-stabilizing factors in platelets that suppresses lysis in large vessels. We also observed that injection of MAb 7E3 in rats treated with 125I-Fg/coll/epi mixture did not change pulmonary level of 125I (that may reflect extravascular deposition of radiolabeled materials due to pulmonary edema) but changed the character of lung hemorrhages from relatively large patches to smaller, less profound and more homogenous pattern.
In summary, this paper presents what, to our knowledge, is the first quantitative analysis addressing the role of platelets on the dissolution of PE. The data suggest that there may be circumstances in which administration of antiplatelet therapy combined with a lower dose of a fibrinolytic agent may represent a useful and safer approach to treat some PE. This paradigm clearly requires more rigorous pharmacological, toxicological, and effectiveness studies to define its benefits and limitations in this vascular bed. Additional studies, including those in the animal models described in this work, may help to identify the settings in which combined therapy would be effective and safe.
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ACKNOWLEDGEMENTS |
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The authors thank Dr. A. Fishman (University of Pennsylvania) for helpful discussion.
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FOOTNOTES |
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This study was supported by American Lung Association Research Grant RG062; American Heart Association (AHA) Bugher-Stroke Award 0170035N; National Heart, Lung, and Blood Institute Grants RO1 HL-6644201 (to V. R. Muzykantov) and HL-60169 (to D. B. Cines); Spanish Government Research Fellowship (to J.-C. Murciano); and AHA Grant-in-Aid 0060251U (to K. Bdeir).
Address for reprint requests and other correspondence: V. R. Muzykantov, IFEM, Univ. of PA Medical Center, 1 John Morgan Bldg., 36th St. & Hamilton Walk, Philadelphia, PA 19104-6068 (E-mail: muzykant{at}mail.med.upenn.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajplung.00112.2001
Received 27 March 2001; accepted in final form 12 October 2001.
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