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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Departments of Pathology and
Laboratory Medicine and Pharmacology, University of Pennsylvania School
of Medicine, Philadelphia, PA; the Department of Medicine, Thomas
Jefferson Medical College, Philadelphia, PA; and the Department of
Clinical Biochemistry, Hadassah Hospital, Hebrew University, Jerusalem,
Israel.
The role of urokinase-type plasminogen activator (uPA) and its
receptor (uPAR) in fibrinolysis remains unsettled. The contribution of
uPA may depend on the vascular location, the physical properties of the
clot, and its impact on tissue function. To study the contribution of
urokinase within the pulmonary microvasculature, a model of pulmonary
microembolism in the mouse was developed. Iodine 125 (125I)-labeled fibrin microparticles injected
intravenously through the tail vein lodged preferentially in the lung,
distributing homogeneously throughout the lobes. Clearance of
125I-microemboli in wild type mice was rapid and
essentially complete by 5 hours. In contrast, uPA Tissue-type plasminogen activator (tPA) and
urokinase-type plasminogen activator (uPA) are primarily responsible
for the activation of plasminogen in vivo.1 Divergent
roles for tPA and uPA in fibrinolysis have been
postulated.2 tPA has been implicated in fibrin degradation
within the vasculature, whereas uPA has been assumed to act primarily
in the context of cell surfaces to promote cell adhesion and migration
through matrices and lysis of fibrin deposited at extravascular
sites.3 It has been reported, for example, that
uPA Yet these observations are difficult to reconcile with those of others
suggesting that endogenous uPA does contribute to intravascular fibrinolysis. For example, fibrin deposits have been identified in the
intestine, hepatic sinusoid, and ulcerated skin of
uPA This seemingly conflicting evidence as to the role of uPA in
fibrinolysis may reflect important differences in the pathophysiology of the events being studied. For example, investigations showing no
role for uPA in endogenous fibrinolysis involved either direct vascular
injury accompanied by endothelial cell desquemation4 or
occlusion of larger vessels,2 whereas fibrin has been
detected within the microvasculature of uPA Similarly, the role of uPAR in fibrinolysis remains unclear. Several
groups reported that uPAR promotes the activity of uPA on cell surfaces
and in solution in vitro.8-10 Yet, inactivation of the
uPAR gene in mice does not appear to affect endogenous fibrinolysis11,12 or lysis of clots injected through the
jugular vein.13 However, the finding of fibrin deposits in
hepatic sinusoids of uPAR To examine the role of endogenous uPA and uPAR in fibrinolysis in
the absence of overt vascular injury, we developed an experimental model of pulmonary microembolism. This model offers the advantage that
the vessel wall is spared mechanical or ischemic injury as oxygen is
supplied to the pulmonary microvasculature primarily by ventilation
rather than by perfusion. The relatively rapid rate of resolution of
the microemboli also limits secondary effects induced by the clot
itself on tissue oxygenation and tissue metabolism. The results of this
study show for the first time that endogenous uPA is as important as
tPA in fibrinolysis in the microvasculature of the lung. The data also
indicate that clot lysis is accelerated by providing uPA in association
with its receptor.
Preparation of radio-labeled fibrinogen
Preparation of fibrin microparticles
The clots were homogenized at 2600 rpm for 1 minute using a PT-10/35
Polytron homogenizer (Brinkmann Instruments, Westbury, NY). After the
first homogenization step, the samples were centrifuged at
2000g for 15 minutes. The supernatant was removed, and the residual particles were pooled and resuspended in KRB. This procedure was repeated two more times. After the final homogenization, the fibrin
microparticles were suspended in 8 mL KRB-bovine serum albumin (BSA)
(3 mg/mL). The suspension was sedimented at 4°C for 5 minutes to
eliminate the largest particles, and 4 mL from the supernatant was used
as the final preparation of microparticles. The suspension of
125I-microparticles was divided into 200-µL aliquots that
were stored at 4°C and used within 48 hours. Random aliquots were
selected to characterize the size distribution of the microparticles
using a ZM Coulter counter (Coulter Electronics, Hialeah, FL). To study the extent of fibrin cross-linking in the microparticles, the final
pellet was solubilized as previously described.15 Briefly, samples were resuspended in 40 mmol/L sodium phosphate in 9 mol/L urea,
3% sodium dodecyl sulfate (SDS), and 3% Mice The following mice were used in the study: mice heterozygous for uPAR deficiency (gift of J. Degen and T. Bugge, University of Cincinnati, Cincinnati, OH; uPA / and
tPA/ mice on a C57/black-6 background, WT C57/black-6
mice, and WT Balb/c mice (Jackson Immunoresearch Laboratories, West
Grove, PA); and uPA/ mice on a 25% C57 black-6/75%
Swiss background and their littermate controls (gift of P. Carmeliet,
Leuven, Belgium). uPAR/ mice and WT littermate controls
were generated and genotyped as previously described.13
All mice weighed 20-35 g at the time of the study. No difference in the
intrinsic lysis of pulmonary microemboli was observed between WT mice
in either genetic background or on a totally unrelated (Balb/c)
background or between male and female mice from the same
genetic background.
Distribution and lysis of 125I-fibrin microparticles 125I-fibrin microparticles (200 µL aliquots containing 15-30 000 cpm) were resuspended by pipetting several times just prior to extraction into a 27.5-gauge syringe. The mice were injected via the tail vein and returned to their cages until sacrifice. At 10 and 30 minutes and 1, 3, and 5 hours after injection, the mice were anesthetized using metofane, and blood (300-500 µL) was withdrawn by retro-orbital puncture into a heparinized microcapillary tube. The mice were then killed by cervical dislocation. The major organs were harvested immediately, rinsed in saline, and dried on Whatman paper, and the radioactivity in each tissue was measured (EG&G Wallace, Gaithersburg, MD). The exact dose (cpm) injected into each mouse was calculated by subtracting the residual radioactivity remaining in the tube, syringe, and injection site (tail) after administration. The tail counts from each mouse were used to verify the adequacy of injection. In some experiments, the lungs were harvested from treated and untreated mice 10 minutes after injection of microemboli and fixed by immersion overnight at 21°C in 10% neutral buffered formalin. Serial sections were stained exactly as described16 using 5 µg/mL mouse primary monoclonal antibody (mAb) to human fibrin (No. 350, American Diagnostica, Greenwich, CT) or a nonimmune immunoglobulin G1 (IgG1) control. In another set of experiments, the lungs were exposed to x-ray film to determine the distribution of radioactivity. In a third series of experiments, the individual lobes from each lung were isolated and counted for radioactivity.Lung injury by microparticles Lung injury was assessed by measuring neutrophil infiltration and tissue edema. Neutrophil infiltration in the lungs of mice injected with microparticles was measured as described.17 Briefly, the mice were killed and perfused with approximately 10 mL phosphate-buffered saline (PBS) via the right ventricle until there was no visible blood in the exiting fluid. The lungs were then removed immediately and placed in ice-cold 0.1 mol/L K2HPO4 buffer (pH 7.0). Lung tissues were homogenized using a Teflon homogenizer for 30 seconds in 1 mL ice-cold K2HPO4 buffer and centrifuged at 40 000g for 30 minutes at 4°C. The pellet was resuspended in 1 mL buffer and sonicated for 90 seconds. The slurry was centrifuged for an additional 10 minutes at 12 000g, and the myeloperoxidase (MPO) activity in the supernatant was measured. To do so, 0.1 mL of each sample was added to 0.3 mL Hank buffered salt solution (HBSS) containing 0.25% BSA, 50 µL O-Dianisidine (1.25 mg/mL) (Sigma), and 50 µL hydrogen peroxide (H2O2) (0.05%). The reaction was stopped after 15 minutes by adding 50 µL 1% sodium azide (Sigma), and the absorbance at 460 nm was measured. We defined 1 unit MPO activity as the quantity that converted 1 µmol H2O2 per minute at 25°C. Pulmonary vascular permeability was assessed by measuring the "wet" to "dry" (wet:dry) ratio of the lung. To do so, the lungs were dissected free from the heart, trachea, and main bronchi, blotted, weighed, and dried for 48 hours at 60°C. The ratio of wet lung weight to dry lung weight was calculated.Lysis of plasma clots by scuPA/suPAR Recombinant suPAR was generated and characterized as described previously.8,18 We added 25 nmol/L recombinant human scuPA (gift of J. Henkin, Abbott Laboratories, Chicago, IL) or preformed equimolar complexes of suPAR and scuPA (25 nmol/L each) to 200 µL 125I-fibrin-labeled human plasma clots placed in 400 µL human, mouse, or rat plasma at 37°C, and the radioactivity in the supernatant was measured at the indicated time points.20 Supplementation of plasma clots with 10 mg/mL fibrinogen had no effect on the rate of lysis by urokinase.8,19Recovery experiments We anesthetized uPA/ or WT mice by an
intraperitoneal injection of 50 mg/kg pentobarbital sodium. A
polyethylene tube (PE 10; Harvard Apparatus, Holliston, MA) siliconized
with Sigmacote (Sigma) and washed with PBS was cannulated into the
jugular vein. The mice received an infusion of scuPA alone, scuPA
complexed with suPAR, or PBS using a PHD 2000 multisyringe pump
(Harvard Apparatus) at a rate of 15 µL/min for 5 minutes.
125I-microparticles were then injected into the tail vein
as described, and the rate of infusion was continued at a rate of 5 µL/min for an additional 60 minutes. The mice were kept under
anesthesia throughout the experiment. At the completion of the
infusion, the mice were killed, and the tissues were harvested as
described above and counted for radioactivity. The endogenous
degradation of 125I-microemboli in WT mice infused with PBS
for 1 hour versus 10 minutes was defined as 100% fibrinolysis. The
radioactivity in the lungs of uPA/ and WT mice injected
with 125I-microemboli at 10 minutes were identical and
defined as baseline. The fibrinolytic activity of PBS, scuPA, or
scuPA/suPAR infused into uPA/ mice for 1 hour was
expressed relative to these 2 parameters.
To follow the biodistribution of scuPA, the same experimental format was followed except that the mice were injected with unlabeled fibrin microparticles and infused with either 0.125 mg/kg/h 125I-scuPA or 125I-scuPA complexed with equimolar concentrations of suPAR. To follow the concentration of scuPA in the circulation, the mice were injected intravenously with a single dose of 0.1 mg/kg 125I-scuPA or 125I-scuPA/suPAR. At 5-, 10-, 15-, and 20-minute intervals, 100 µL blood was withdrawn by retro-orbital puncture into a heparinized microcapillary tube and counted for radioactivity. In other experiments, blood was withdrawn for counting at specified times (15, 30, and 45 minutes) during and at the completion of the 1-hour infusion with 125I-scuPA or 125I-scuPA/suPAR.
Characterization of the pulmonary microembolus model The purpose of this study was to investigate the involvement of urokinase and the urokinase receptor in intravascular fibrinolysis in vivo. To do so, our first goal was to develop a sensitive and reproducible murine model to analyze endogenous fibrinolytic activity. We reasoned that in previous studies, clot size, vascular location, and vascular injury may have contributed to limiting susceptibility of clots to lysis by uPA. To assess the contribution of these variables, we prepared relatively homogenous preparations of radio-labeled microparticles by adding thrombin to human plasma that had been supplemented with 125I-fibrinogen. The clots were then homogenized, and the larger particles were removed by sedimentation to obtain a homogenous suspension of fibrin microparticles.The size distribution of the microparticle preparation is shown in
Figure 1A. Approximately 80% of the
particles ranged in diameter from 1.5-3.25 µm. The fibrin within
these microparticles was extensively cross-linked, as judged by the
amount of the
Trace-labeled fibrin microparticles were then injected into the tail
veins of WT mice to study their organ distribution and effect on
pulmonary function. Approximately 40% of the injected 125I-labeled fibrin microparticles, but less than 3% of
radio-labeled fibrinogen, were recovered from the lungs of the mice 10 minutes after intravenous injection (Figure
2A). The injected microparticles were
distributed homogeneously throughout the lobes of the lung, measured
both as radioactivity per gram of tissue (not shown) and as judged by
autoradiography (Figure 2B). The fibrin clots became lodged in
pulmonary capillaries (Figure 3), as none
of the occluded vessels were surrounded by smooth muscle cells. The affected vessels ranged in size from 5-32 µm, suggesting that in vivo
aggregation of the microemboli had occurred in some of them. Some
fibrin clots were found to contain a variable number of erythrocytes
and other circulating hematopoetic cells. Mice infused with 8-fold more
microparticles than were used in subsequent experiments showed no
alteration in the wet:dry ratio or total myeloperoxidase activity in
the lungs at 6 hours compared with mice injected with PBS alone (Table
1). Thus, microembolization was not
accompanied by a significant increase in vascular permeability or
leukocyte emigration.
Resolution of pulmonary microemboli in PA-deficient mice We next examined the clearance of radio-labeled fibrin microparticles from the lungs of WT mice. The animals were killed at various times after intravenous injection, and the radioactivity remaining in the lungs was measured. The data in Figure 4A shows the time-dependence of spontaneous clot lysis in WT mice. Within 1 hour, 70% of the radioactivity was cleared from the lungs, and near total resolution was evident by 3 hours. The rapid resolution may reflect activation of human plasminogen incorporated into the microemboli during their preparation.
We then compared the rate of clot lysis in tPA scuPA- and scuPA/suPAR-mediated clot lysis We then asked 2 interrelated questions: First, would scuPA restore clot lysis in uPA/ mice to normal, excluding an
indirect effect of interrupting the gene (eg, on tissue remodeling or
vascular responsiveness to a thrombotic stress)? Second, would suPAR
promote the PA activity of scuPA in vivo as we previously observed in
human plasma in vitro19? We chose uPA/ mice
to test our hypothesis that suPAR would stimulate fibrinolysis because
their rate of spontaneous clot lysis was slower.
To address these questions, uPA
The extent of clot lysis was greater when uPA The effect of suPAR on the pharmacokinetic and fibrinolytic parameters of scuPA We and others have reported that uPAR promotes adhesion of scuPA to vitronectin.20-23 Therefore, we asked whether suPAR enhanced fibrinolysis because more scuPA was present in the circulation during the 1-hour infusion when complexed with suPAR or because the receptor promoted the accumulation of scuPA within the microemboli. The results shown in Figure 6A indicate that suPAR accelerated the disappearance of scuPA from the blood when given intravenously as a single dose. Thus, suPAR did not increase the plasma concentration of scuPA by retarding its clearance from the plasma. There were also no statistically significant differences in the steady-state whole blood concentrations of 125I-scuPA given as a constant infusion at 15, 30, 45, and 60 minutes in the presence or absence of suPAR (not shown). More 125I-scuPA accumulated in the lungs of mice bearing microemboli compared to clot-free mice, as expected (Figure 6B). The uptake of 125I-scuPA was enhanced by suPAR in pulmonary tissue in mice free of emboli, but it did not cause a statistically significant increase (P = .13) in mice bearing microemboli (Figure 6B). Taken together, these data indicate that suPAR does not promote pulmonary fibrinolysis either by prolonging the half-life of scuPA in the circulation or by causing it to accumulate preferentially within the clots.
Therefore, we asked whether the increased lysis of
125I-microemboli in uPA
We developed a model to study fibrinolysis in the microvasculature of small laboratory animals using 125I-fibrin microparticles that embolize to the lung after tail vein injection. This method enabled us to measure the contribution of uPA, tPA, and uPAR to endogenous fibrinolysis in the pulmonary microvasculature. The results of this study demonstrate that mice lacking uPA have an impaired capacity to lyse pulmonary microemboli. The defect in uPA-deficient mice is comparable in severity to that seen in mice lacking tPA and can be rescued completely by providing urokinase exogenously. Our finding that endogenous urokinase contributes to fibrinolysis
differs from a previous study in which spontaneous lysis of occlusive
thrombi was normal in uPA Three other explanations for the involvement of urokinase in the resolution of microemboli as compared with large occlusive thrombi are possible. The first relates to potential structural differences between the 2 types of clots. Microemboli have a higher surface:volume ratio than do large occlusive emboli. This may increase the relative interface between the clot and the endothelium or leukocytes, which may facilitate the contribution of cell-bound uPA. Secretion of uPA by the occluded vasculature or binding of urokinase by vascular cells in proximity to the clot may have a greater opportunity to interact with the surface of the microemboli compared to larger clots. In turn, this makes the larger clots more dependent on fibrin-mediated activation of plasma tPA. Second, large occlusive clots may induce mechanical trauma or tissue
ischemia. Hypoxia down-regulates uPA synthesis while promoting the
synthesis of PAI-1.5 Emboli that lodge primarily in the
pulmonary microvasculature, which receives its oxygen supply primarily
through ventilation rather than perfusion, resolve rapidly in contrast
to occlusive thrombi affecting blood supply to other organs. Thus,
inhibition of uPA synthesis by vascular occlusion, even in WT mice,
might obscure evident differences in the behavior of
uPA A third possibility lies in the relatively unexplored issue of
vascular heterogeneity.25 Synthesis of tPA by
vascular endothelial cells, for example, is regionally
restricted.26 Studies that did not show a phenotype in
uPA The difference in clot lysis between tPA The absence of any impairment of fibrinolysis in uPAR Another possibility is that the uPA-uPAR complex is not active in vivo.
We and others8-10 have reported that the binding of scuPA
to its receptor promotes its enzymatic activity and protects the enzyme
against inactivation by PA inhibitors in vitro. Stimulation of scuPA
activity was demonstrable with certain chromogenic
substrates32 as well as when human plasma clots were used
as the substrate.20 The suPAR-stimulated
scuPA-mediated fibrinolysis in murine plasma in vitro and in vivo
(Figure 5) excludes the possibility that the absence of a phenotype in
the uPAR It has previously been reported that suPAR inhibits and promotes fibrinolysis in vitro depending on the experimental conditions.18,34 This study demonstrates that uPAR, in this case in the form of a soluble receptor, stimulates fibrinolytic activity in vivo. In this study, suPAR did not increase fibrinolysis by scuPA either by prolonging its time in the circulation or by increasing its local concentration in the lung. Indeed, suPAR accelerated initial uptake of scuPA from the circulation and did increase the accumulation of scuPA within the lungs of mice bearing microemboli. Taken together, the data suggest that uPA mediates endogenous fibrinolysis in the pulmonary microvasculature and that suPAR promotes fibrinolysis by stimulating the specific activity of urokinase. However, the ability of suPAR to promote clot lysis by protecting uPA from inactivation by PAI-135 and other serpins10 or through other mechanisms will require additional study.
We wish to thank Christopher Yaen for excellent technical assistance on this project.
Submitted December 20, 1999; accepted May 11, 2000.
Supported in part by grants HL60169 and HL47839 (D.C. and A.H.) from the National Institutes of Health, Bethesda, MD, and research grant RG-062 (V.R.M.) from the American Lung Association. J.-C.M. is supported by a North Atlantic Treaty Organization (NATO) fellowship, Washington, DC.
K.B. and J.-C.M. contributed equally to this work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Abd Al-Roof Higazi, Department of Pathology and Laboratory Medicine, 513A Stellar-Chance, 422 Curie Blvd, Philadelphia, PA 19104; e-mail: higazi{at}mail.med.upenn.edu.
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© 2000 by The American Society of Hematology.
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  S. Zaitsev, K. Danielyan, J.-C. Murciano, K. Ganguly, T. Krasik, R. P. Taylor, S. Pincus, S. Jones, D. B. Cines, and V. R. Muzykantov Human complement receptor type 1-directed loading of tissue plasminogen activator on circulating erythrocytes for prophylactic fibrinolysis Blood, September 15, 2006; 108(6): 1895 - 1902. [Abstract] [Full Text] [PDF]
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 W. M. Armstead, D. B. Cines, and A. A.-R. Higazi Plasminogen Activators Contribute to Impairment of Hypercapnic and Hypotensive Cerebrovasodilation After Cerebral Hypoxia/Ischemia in the Newborn Pig Stroke, October 1, 2005; 36(10): 2265 - 2269. [Abstract] [Full Text] [PDF]
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A. A.-R. Higazi, F. Ajawi, S. Akkawi, E. Hess, A. Kuo, and D. B. Cines Regulation of the single-chain urokinase-urokinase receptor complex activity by plasminogen and fibrin: novel mechanism of fibrin specificity Blood, February 1, 2005; 105(3): 1021 - 1028. [Abstract] [Full Text] [PDF]
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D.N. Atochin, J.C. Murciano, Y. Gursoy-Ozdemir, T. Krasik, F. Noda, C. Ayata, A.K. Dunn, M.A. Moskowitz, P.L. Huang, and V.R. Muzykantov Mouse Model of Microembolic Stroke and Reperfusion Stroke, September 1, 2004; 35(9): 2177 - 2182. [Abstract] [Full Text] [PDF]
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 C. Vadseth, J. M. Souza, L. Thomson, A. Seagraves, C. Nagaswami, T. Scheiner, J. Torbet, G. Vilaire, J. S. Bennett, J.-C. Murciano, et al. Pro-thrombotic State Induced by Post-translational Modification of Fibrinogen by Reactive Nitrogen Species J. Biol. Chem., March 5, 2004; 279(10): 8820 - 8826. [Abstract] [Full Text] [PDF]
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T. Nassar, S.'e. Akkawi, A. Shina, A. Haj-Yehia, K. Bdeir, M. Tarshis, S. N. Heyman, and A. A.-R. Higazi In vitro and in vivo effects of tPA and PAI-1 on blood vessel tone Blood, February 1, 2004; 103(3): 897 - 902. [Abstract] [Full Text] [PDF]
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K. Bdeir, A. Kuo, B. S. Sachais, A. H. Rux, Y. Bdeir, A. Mazar, A. A.-R. Higazi, and D. B. Cines The kringle stabilizes urokinase binding to the urokinase receptor Blood, November 15, 2003; 102(10): 3600 - 3608. [Abstract] [Full Text] [PDF]
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J.-C. Murciano, S. Muro, L. Koniaris, M. Christofidou-Solomidou, D. W. Harshaw, S. M. Albelda, D. N. Granger, D. B. Cines, and V. R. Muzykantov ICAM-directed vascular immunotargeting of antithrombotic agents to the endothelial luminal surface Blood, May 15, 2003; 101(10): 3977 - 3984. [Abstract] [Full Text] [PDF]
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 J. Willeit, S. Kiechl, T. Weimer, A. Mair, P. Santer, C. J. Wiedermann, and J. Roemisch Marburg I Polymorphism of Factor VII-Activating Protease: A Prominent Risk Predictor of Carotid Stenosis Circulation, February 11, 2003; 107(5): 667 - 670. [Abstract] [Full Text] [PDF]
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T. Nassar, A. Haj-Yehia, S.'e. Akkawi, A. Kuo, K. Bdeir, A. Mazar, D. B. Cines, and A. A.-R. Higazi Binding of Urokinase to Low Density Lipoprotein-related Receptor (LRP) Regulates Vascular Smooth Muscle Cell Contraction J. Biol. Chem., October 18, 2002; 277(43): 40499 - 40504. [Abstract] [Full Text] [PDF]
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 S. Idell, A. Mazar, D. Cines, A. Kuo, G. Parry, S. Gawlak, J. Juarez, K. Koenig, A. Azghani, W. Hadden, et al. Single-Chain Urokinase Alone or Complexed to Its Receptor in Tetracycline-induced Pleuritis in Rabbits Am. J. Respir. Crit. Care Med., October 1, 2002; 166(7): 920 - 926. [Abstract] [Full Text]
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 J.-C. Murciano, D. Harshaw, D. G. Neschis, L. Koniaris, K. Bdeir, S. Medinilla, A. B. Fisher, M. A. Golden, D. B. Cines, M. T. Nakada, et al. Platelets inhibit the lysis of pulmonary microemboli Am J Physiol Lung Cell Mol Physiol, March 1, 2002; 282(3): L529 - L539. [Abstract] [Full Text] [PDF]
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| Copyright © 2000 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
-mercaptoethanol and
heated at 37°C for 15 minutes to completely dissolve the clots. The
samples were boiled for 5 minutes and were subjected to electrophoresis on 10% SDS-PAGE (polyacrylamide gel electrophoresis). Solubilized fibrin (10 µg) or an equal number of counts of radio-labeled clots were loaded into each lane. Unlabeled fibrin clots were stained with
Coomassie blue dye, while samples labeled with 125I
were exposed to Kodak BioMax ML film (Eastman Kodak Company, Rochester, NY).
-chain polymer and 
-
) or
125I-fibrinogen (
) were injected into WT mice via the
tail vein. Blood was drawn 10 minutes later, and the major organs were
harvested, washed, and counted for radioactivity. The mean plus or
minus SD of 3 experiments, each performed in 3 mice, is shown. Data are
expressed as the percentage of injected dose per organ. (B) The lungs
were removed from WT mice 10 minutes and 1 hour after injection of
125I-microparticles and were analyzed by autoradiography.
); uPA
); or
tPA
). At the indicated times, the lungs were
harvested, washed, and counted for radioactivity. The data are
expressed relative to the radioactivity in the lungs 10 minutes after
injection. This latter value was the same in all 3 groups. The data
represent the mean plus or minus SEM of 3 independent experiments each
involving 4-5 mice per time point. Data from the uPA
) (0.25 or 0.5 mg scuPA/kg/h) via the jugular vein. Five minutes later,
125I-fibrin microparticles were injected, and the infusion
of scuPA or scuPA/suPAR was continued for another hour. The lungs were
harvested, washed, and counted for radioactivity. The data are
expressed relative to fibrinolytic activity in WT mice infused with PBS
in the same manner for 1 hour, as defined in "Materials and
methods." The means plus or minus SEM of 2 experiments, each
involving data from 3-5 mice in each experimental group, are
shown.
) or 125I-scuPA/suPAR (
) or 125I-scuPA /suPAR
(
) for 1 hour. The isolated lungs were washed, and the radioactivity
was measured. The results shown represent the mean plus or minus SEM of
2 experiments, each involving 3 mice. M.E. indicates microemboli.
, rat plasma indicated
by 
) at 37°C. The radioactive fibrin degradation products released
into the supernatant were measured at the indicated times. At each time
point, fibrinolysis was calculated by subtracting the endogenous
release of radioactivity and was expressed as the percent of total
lysis. The data shown represent the mean plus or minus SD of triplicate
determinations from 2 experiments.