Donate Help Contact The AHA Sign In Home
American Heart Association
Circulation
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Circulation. 1995;92:1883-1890

This Article
Right arrow Abstract Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sakharov, D. V.
Right arrow Articles by Rijken, D. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sakharov, D. V.
Right arrow Articles by Rijken, D. C.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH

(Circulation. 1995;92:1883-1890.)
© 1995 American Heart Association, Inc.

Articles

Superficial Accumulation of Plasminogen During Plasma Clot Lysis

Dmitry V. Sakharov, PhD; Dingeman C. Rijken, PhD

From Gaubius Laboratory, TNO-PG, Leiden, Netherlands.

Correspondence to Dr D.C. Rijken, Gaubius Laboratory, TNO-PG, PO Box 2215, 2301 CE Leiden, Netherlands. E-mail dc.rijken@pg.tno.nl.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Background Binding of plasminogen to partially degraded fibrin is an important step in fibrinolysis, influencing its rate and fibrin specificity. Little is known about the spatial distribution of plasminogen and of plasminogen-binding sites inside thrombi during lysis. In the present study, we investigated this problem, which is important for a better understanding of the local regulation of fibrinolysis and the rate-limiting factors of therapeutic thrombolysis.

Methods and Results An experimental system was used that allowed continuous visualization and quantification by fluorescence microscopy of the spatial distribution of fluorescein-labeled plasminogen inside and outside model thrombi. Strong superficial accumulation of plasminogen was observed during lysis of a plasma clot induced by tissue-type or urokinase-type plasminogen activators in the surrounding plasma. A distinctly visible plasminogen-accumulating shell moved continuously with the reducing surface of the clot. The accumulation decreased in conditions of exhaustive activation of plasminogen in the outer plasma. It was found in a purified system that a thin superficial layer ({approx}50 µm wide) of a plasmin-treated fibrin clot exposes about 2.5 plasminogen-binding sites per fibrin monomer with a Kd of 2.2 µmol/L. At a physiological concentration of plasminogen (1.5 µmol/L) in the outer medium, plasminogen was concentrated about 10-fold in this layer. The binding was dose-dependently inhibited by {varepsilon}-aminocaproic acid.

Conclusions We conclude that the generation of potent surface-associated plasminogen-binding sites during thrombolysis results in a strikingly high plasminogen concentration at the dynamically changing surface of a lysing clot. The necessity of a continuous plasminogen supply from the plasma supports the use of fibrin-specific and plasminogen-sparing agents for thrombolytic therapy.


Key Words: binding sites • plasminogen • plasminogen activators • fibrinolysis • thrombolysis


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Dissolution of insoluble fibrin is a key event in thrombolysis. The fibrin matrix of thrombi not only represents the substrate for lysis but also plays an active role because it adsorbs and modulates functional properties of components of the fibrinolytic and antifibrinolytic systems.1 2 3 4 Plasminogen, the precursor of the main fibrinolytic enzyme plasmin, can bind weakly to specific sites exposed on the fibrin network.5 6 This binding contributes to an efficient activation of plasminogen by plasminogen activators.7 8 9 10 11 12 Limited digestion of fibrin by plasmin leads to the generation of new binding sites for plasminogen,13 14 15 16 17 18 19 thereby creating positive feedback for activation of fibrinolysis. Although it is generally believed that this feedback is important for local regulation of fibrinolysis, how this system is organized in a three-dimensional (3D) thrombus has never before been investigated.

Rather contradictory data have been reported regarding quantitative parameters of the binding of plasminogen to partially degraded fibrin. Suenson et al13 reported a Kd value of 10 µmol/L calculated on the basis of the assumption that one binding site is exposed per fibrin monomer; they also reported a Kd of 1.2 µmol/L with 0.48 binding site per fibrin monomer for a gel obtained after thrombin-induced polymerization of fragment X of fibrinogen.14 Tran-Thang et al18 found a Kd of 0.3 µmol/L with 5 binding sites exposed per 100 fibrin molecules. The appearance of low-affinity binding sites was reported with a Kd of 48 µmol/L and 1.1 binding sites per fibrin monomer.17 These data were obtained in different models of 3D fibrin gels. Using an experimental system with fibrin monomer immobilized on a plastic surface, Fleury and Anglés-Cano19 obtained a Kd of 0.66 µmol/L and 2.7 binding sites per fibrin monomer for plasminogen binding to plasmin-treated fibrin. The apparent inconsistency in these data could be, at least partially, a consequence of a nonuniform distribution of plasminogen-binding sites throughout the 3D fibrin clots treated with plasmin from the outside.17 18

In this study, we have investigated the spatial distribution of plasminogen in a plasma clot during its lysis induced by tissue-type and two-chain urokinase-type plasminogen activators (TPA and TCUPA, respectively) in the surrounding plasma and the distribution of plasminogen-binding sites in a plasmin-treated fibrin clot. The results, demonstrating strong and strictly superficial accumulation of plasminogen during clot lysis, have implications in discussions of the use of fibrin-specific versus nonspecific agents for thrombolytic therapy.20 21 22


*    Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Preparations
Glu-plasminogen was a product of Biofine. No contamination by the Lys form of plasminogen or proteins was found in the preparation by SDS-PAGE. To remove possible traces of lysine-like compounds, the plasminogen stock solution was gel-filtered on a Sephadex G-25 column equilibrated with PBS before all experiments. The TPA preparation (Actilyse) was supplied by Boehringer Ingelheim; human urinary TCUPA (Ukidan) was purchased from Serono. Chromogenic substrates, human fibrinogen, and plasmin were products of Kabi. According to manufacturer's specification, specific activity of plasmin was not <15 CU/mg. Fibrinogen was made plasminogen-free by lysine-Sepharose chromatography.23 Fluorescein isothiocyanate (FITC) and porcine carboxypeptidase B were from Sigma Chemical Co. Fluorescein-5-maleimide (FM) was from Pierce. We used also {varepsilon}-aminocaproic acid (EACA, Merck), thrombin (Leo), hirudin (Pentapharm), and aprotinin (Bayer). Pooled citrated platelet-poor plasma was used in all experiments involving plasma.

Labeling of Proteins With Fluorescein Derivatives
Plasminogen (0.5 mL, 40 µmol/L) was gel-filtered in a centrifuge by use of a microcolumn with Sephadex G-25 (3.5 mL) equilibrated with a buffer containing 30 mmol/L sodium carbonate and 140 mmol/L NaCl, pH 9.2. FITC (1 mg/mL in dimethyl sulfoxide) was added to the plasminogen preparation under intensive stirring to a final concentration of 50 µg/mL. After 1 hour of incubation in the dark, unreacted FITC was removed by gel filtration on the same column with Sephadex G-25 reequilibrated with Tris-buffered saline (TBS) containing 20 mmol/L Tris-HCl and 140 mmol/L NaCl, pH 7.4. The preparation was stored frozen in small packages that were thawed only once before each experiment. The A495/A280 ratio in preparations of FITC-plasminogen was about 0.7, corresponding to about 2 FITC molecules per molecule of plasminogen. The same procedure was used for FITC labeling of TCUPA (initial concentration, 250 000 IU/mL) and BSA (3 mg/mL). TPA was labeled with FM, presumably attached to the sulfhydryl group in position 83,24 as described elsewhere.25 After labeling, both preparations of fluorescence-labeled plasminogen activators retained about 70% of their amidolytic activity toward specific chromogenic substrates (S-2444 for TCUPA and S-2288 for TPA) and about 55% of their plasminogen activating activity, as measured with plasmin-specific substrate S-2251. The labeled TPA activated plasminogen approximately 150-fold more efficiently in the presence of a fibrin-like stimulator.26

Experimental System Design
The experimental system for visualization of the spatial distribution of FITC-labeled molecules inside a model thrombus in conditions of diffusive permeation was described elsewhere.27 In brief, a plasma or fibrin clot was situated between two parallel glass slides separated by a 0.2-mm-high spacer. The geometry of the system allowed us to perfuse the clots with plasma or buffer in such a way that the perfusing solution flowed freely around the clot; no hydraulic pressure over the clot that could force infiltration of the perfusate into the clot was applied. Non–cross-linked plasma clots were formed by the addition of thrombin (final concentration, 1.4 NIH U/mL) to plasma. About 1 µL of the mixture was immediately applied to the chamber and was sucked in between the two pieces of glass by capillary forces. After 2 minutes, a clot with an approximate diameter of 2 to 3 mm was formed, attached firmly to the parallel pieces of glass, and the rest of the chamber volume (about 25 µL) was filled with plasma containing hirudin (10 ATU/mL). Hirudin was added to prevent clotting of the outer plasma by the thrombin inside the clot. To obtain cross-linked clots, clotting was performed in the presence of 30 mmol/L CaCl2, and the clots were further incubated during 3 hours at 37°C in recalcified plasma with 300 ATU/mL hirudin. For experiments with compacted clots, 50 µL of plasma was clotted in a separate tube by addition of thrombin to a final concentration of 1.4 NIH U/mL; the clot was compacted by a pipette tip and then placed between two parallel sheets of glass. After compaction, the clot volume had been reduced approximately 50- to 100-fold. Fibrin clots were formed from plasminogen-free fibrinogen (final concentration, 9.2 µmol/L) dissolved in TBS, containing 40 mg/mL BSA (TBS-BSA), by exactly the same procedure as non–cross-linked plasma clots and were washed with TBS-BSA.

In experiments with pressure-driven permeation, a modification of the experimental model was used that had a design very similar to that of the system described by Blomback et al.28 Plasma was clotted with 1.4 NIH U/mL thrombin in a chamber with dimensions of 0.2x5x25 mm formed between two parallel sheets of glass and closed on the two long sides by spacers. After clotting, the chamber was situated vertically, and pressure-driven permeation was performed by continuous application of small portions of plasma onto the upper surface of the clot. Thus, the gradient of applied pressure was approximately 1 cm H2O/cm clot (0.75 mm Hg/cm clot). In these conditions, plasma filtered through the clot with an approximate speed of 500 µm/min and dropped down from the lower part of the chamber.

Investigation of the Spatial Distribution of Fibrinolytic Components During Lysis of a Plasma Clot
To investigate the spatial distribution of FITC-plasminogen during lysis, chambers containing plasma clots were perfused at 37°C with plasma containing TPA or TCUPA and FITC-plasminogen added as a tracer to a final concentration of 0.3 µmol/L. When indicated, carboxypeptidase B (final concentration, 50 µg/mL) or EACA (final concentration, 1 mmol/L) was added to the perfusing solution. {alpha}2-Plasmin inhibitor was determined as described by Kluft et al.29 In experiments with fluorescent derivatives of plasminogen activators, FITC-TCUPA or FM-TPA was added to perfusing plasma.

Consecutive images of the spatial distribution of fluorescence during the lysis of plasma clots were obtained by making fluorescence photomicrographs of the same clot in definite time intervals with a fluorescence microscope (Microphot FXA, Nikon) using black-and-white film (400 ASA, 36x24 mm). Perfusions were interrupted for not >3 minutes for photographing.

Accumulation of FITC-Plasminogen in Plasmin-Treated Fibrin Clot
When indicated, fibrin clots were incubated during 70 minutes at 37°C with plasmin added to the outer medium to a final concentration of 0.1 CU/mL in TBS-BSA ({approx}5 µg/mL). As a result of the treatment with plasmin, clot diameters reduced by {approx}0.8 to 1.0 mm. Fibrin clots, either treated with plasmin or not, were washed with TBS-BSA containing 100 KIU/mL aprotinin and then incubated with FITC-plasminogen in the same buffer at room temperature during 24 hours, which was sufficiently long to equilibrate the clots with FITC-plasminogen. Only a negligible portion of FITC-plasminogen added to the chamber was eventually accumulated in the clots, so the concentration of FITC-plasminogen in the outer medium remained practically constant. For inhibition experiments, various concentrations of unlabeled plasminogen or EACA were added with FITC-plasminogen. After a 24-hour equilibration, clots were photographed as described above.

Quantitative Analysis of FITC-Plasminogen Binding
After development, the film with negative images of the clots was scanned along the central long axis of each picture by use of a scanning densitometer (TLC Scanner CS-910, Shimadzu) with scanning beam dimensions of 0.5x0.05 mm. To obtain quantitative data about local concentrations of FITC-plasminogen, a calibration curve was constructed (not shown), establishing the correlation between the concentration of FITC-plasminogen in the chamber and the densitometer signal. In this article, this calibration curve is reflected by the nonlinear character of the y axis of the scanning densitometry plots.

The characteristic profile of the spatial distribution of FITC-plasminogen in a lysing clot was a rather sharp peak associated with the clot boundary. The highest point of this peak was taken for determination of the concentration of FITC-plasminogen in the superficial layer.

For construction of binding curves of FITC-plasminogen in the purified system, the local concentration of bound plasminogen was calculated by subtracting the FITC-plasminogen concentration in the outer medium from the local concentration of FITC-plasminogen in the superficial layer. Thus, the outer concentration of the ligand was considered to be equal to the concentration of free ligand inside the clot after equilibration. A GRAPHPAD computer program (ISS) incorporating nonlinear regression analysis was used to calculate quantitative parameters for FITC-plasminogen superficial binding and its inhibition by unlabeled plasminogen and by EACA.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
*Results
down arrowDiscussion
down arrowReferences
 
Using an experimental system that allowed us to follow and to quantify the spatial distribution of fluorescence inside and outside a model thrombus,27 we performed two types of experiments: (1) the dynamics of the distribution of fluorescein-labeled plasminogen, TPA, or TCUPA was documented during progressive lysis of a plasma clot surrounded by plasma containing the labeled proteins and (2) after plasmin treatment of a fibrin clot in a buffer system, the lytic process was stopped by addition of aprotinin, and the spatial distribution of plasminogen inside the clot was documented after a 24-hour equilibration of the clot with FITC-labeled plasminogen.

Investigation of the Dynamics of the Distribution of Fluorescein-Labeled Fibrinolytic Components During Lysis of the Plasma Clot
Fig 1Down(A through C) presents three consecutive views of a plasma clot during ongoing lysis induced by TPA added to the surrounding plasma to a concentration of 1 µg/mL. Tracer FITC-plasminogen present in the outer plasma was strongly accumulated in a thin ({approx}10 µm wide) superficial layer of the clot, allowing continuous visualization of the distinct boundary. Along with progressive lysis, the boundary moved while the radius of the clot decreased. Under the experimental conditions, no large plasminogen activation occurred in plasma, as judged by measurements of {alpha}2-plasmin inhibitor activity, which was never reduced to <65% of its initial level during the course of the experiment (data not shown).



View larger version (73K):
[in this window]
[in a new window]
  		Figure 1. Photomicrographs showing superficial accumulation of fluorescein isothiocyanate (FITC)–plasminogen during lysis of a non–cross-linked plasma clot. FITC-plasminogen was present in perfusing plasma as a tracer (0.3 µmol/L). A through D, Images of a plasma clot after 30, 50, 70, and 100 minutes of perfusion at 37°C with plasma containing tissue-type plasminogen activator (TPA, 1 µg/mL). At 20, 40, 60, and 80 minutes, new portions of plasma with tracer FITC-plasminogen were used for the perfusion with freshly added TPA. At 72 minutes, carboxypeptidase B was added to the perfusing plasma to a final concentration of 50 µg/mL. E through G, Images of a plasma clot after 4, 30, and 36 minutes of perfusion with plasma containing two-chain urokinase-type plasminogen activator (TCUPA, 500 IU/mL). At 32 minutes, a new portion of plasma with tracer FITC-plasminogen was used for the perfusion with freshly added TCUPA. H, A plasma clot after 60 minutes of perfusion with plasma containing tracer FITC-plasminogen without exogenous plasminogen activator. Bar=250 µm.

Addition of carboxypeptidase B to the outer plasma during the process of ongoing lysis almost completely eliminated the bright plasminogen-containing shell at the surface of the clot (Fig 1DUp). Thus, on the basis of known specificity of the carboxypeptidase, it is likely that plasminogen-binding sites are represented by C-terminal lysines of partially degraded fibrin. Lysis speed decreased drastically after the carboxypeptidase-induced removal of superficially bound plasminogen, as seen from comparison of Fig 1CUp and 1DUp. The clot size remained practically unchanged in the presence of the carboxypeptidase, which was added 72 minutes after the start of lysis. In a parallel experiment without addition of carboxypeptidase (not shown), lysis continued with a constantly high speed during at least 120 minutes after the start of lysis. Similarly, addition of EACA in the millimolar range stopped lysis and simultaneously destroyed the FITC-plasminogen shell on the surface of a clot (not shown).

To find out whether the observed superficial accumulation of plasminogen also occurs during lysis induced by non–fibrin-specific plasminogen activators, we performed similar experiments with TCUPA. Fig 1EUp and 1FUp shows images of a plasma clot after 4 and 30 minutes of lysis induced by 500 IU/mL of TCUPA in the surrounding plasma. The patterns of spatial distribution of plasminogen were essentially the same superficial accumulation as for TPA-induced lysis. However, the accumulation was significantly lower after 30 minutes of lysis than after 4 minutes, as manifested by reduced brightness of the clot boundary after 30 minutes of lysis (Fig 1FUp). A possible explanation for the observed reduction of accumulation is exhaustive activation of plasminogen to plasmin in the surrounding plasma and consecutive formation of complexes of plasmin with various inhibitors ({alpha}2-plasmin inhibitor, {alpha}2-macroglobulin, etc) that may have a reduced ability to interact with fibrin. Indeed, {alpha}2-plasmin inhibitor was reduced to zero after 30 minutes in the presence of 500 IU/mL TCUPA in plasma, indicating major exhaustion of the plasminogen pool. In line with this explanation, the high accumulation was restored immediately after addition to the same clot of a new portion of FITC-plasminogen–containing plasma with freshly added TCUPA (Fig 1GUp).

According to our quantitative estimations, the ratio of the local intensity of fluorescence in the superficial layer to the fluorescence intensity in the surrounding plasma was about 3 to 4 in the absence of exhaustive activation of plasminogen (as in Fig 1AUp through 1C, 1E, and 1G) and decreased to 1.3 to 1.7 when plasminogen in the outer plasma was depleted (as in Fig 1FUp). The technique did not allow us to discriminate between labeled plasminogen and labeled plasmin-inhibitor complexes.

As Fig 1HUp shows, no remarkable accumulation of FITC-plasminogen was found in a control experiment performed in the absence of exogenous plasminogen activator in the outer plasma.

Fig 2Down shows that spatial distributions of fluorescent derivatives of the two plasminogen activators during lysis of a plasma clot were different. TPA (Fig 2ADown) exhibited a distribution similar to that of plasminogen, being dynamically accumulated in a thin superficial layer. In contrast, TCUPA did not interact with the lysing clot (Fig 2BDown). These results indicate that superficial accumulation of plasminogen during lysis is not a consequence of a specific spatial distribution of plasminogen activator because the superficial accumulation of plasminogen took place with both activators (Fig 1Up).



View larger version (45K):
[in this window]
[in a new window]
  		Figure 2. Photomicrographs showing spatial distribution of fluorescein-labeled plasminogen activators during lysis of a plasma clot. A, Fluorescein-5-maleimide tissue-type plasminogen activator (12 µg/mL); B, fluorescein isothiocyanate two-chain urokinase-type plasminogen activator (2000 IU/mL). Both pictures were taken after 30 minutes of perfusion with plasma containing labeled plasminogen activator. Bar=250 µm.

The following factors may seriously affect the speed of thrombolysis in vivo: pressure-driven permeation of solutes into the thrombus,30 31 32 33 34 Ca2+-dependent cross-linking of the fibrin network by factor XIIIa,35 36 and compaction of a clot as a result of platelet-induced retraction.37 38 The experiments shown in Fig 3Down were performed to find out whether the superficial accumulation of plasminogen is affected by these factors.



View larger version (71K):
[in this window]
[in a new window]
  		Figure 3. Photomicrographs showing the influence of filtration, cross-linking, and compaction of a plasma clot on the superficial accumulation of fluorescein isothiocyanate (FITC)–plasminogen during lysis. FITC-plasminogen (0.3 µmol/L) and tissue-type plasminogen activator (TPA, 0.5 µg/mL) were added to the perfusing plasma. A, Perfusate was filtered in the direction indicated by the arrow through a non–cross-linked plasma clot during 20 minutes. Asterisk indicates the location of the residual clot. Bar=500 µm. B, Cross-linked plasma clot after 60 minutes of lysis in conditions of diffusive penetration. Bar=250 µm. C, Compacted plasma clot after 40 minutes of lysis in conditions of diffusive penetration. Bar= 100 µm. D, Control compacted clot in the same conditions but without TPA in the outer plasma.

In a modification of our experimental system with pressure applied across the clot, the lysis was much faster than under conditions of purely diffusive permeation. The surface of the lysing clot was rather irregular; more rapid lysis took place in the directions of predominant flow. These results are in agreement with reports describing channeling of lysis under conditions of pressure-driven permeation in both in vitro31 34 and in vivo models.39 As Fig 3AUp demonstrates, plasminogen accumulated on this surface in a way similar to that observed under conditions of purely diffusive transport. It is noteworthy that the front of the lysis moved considerably more slowly than the front of the penetration of FITC-labeled plasminogen, which completely passed through the part of the clot shown in Fig 3AUp. In a control experiment without the addition of a plasminogen activator, the original surface of the clot remained unchanged during perfusion and did not accumulate substantial amounts of FITC-plasminogen (not shown).

Fig 3BUp and 3CUp represents patterns of FITC-plasminogen accumulation during lysis of a cross-linked plasma clot and a compacted plasma clot, respectively. Both Ca2+-dependent action of factor XIIIa and compaction of a plasma clot are known to increase resistance to lysis.35 36 37 38 Indeed, in both cases we observed a decrease in lysis speed, which was especially pronounced for compacted clots (data not presented). Strictly superficial accumulation of FITC-plasminogen was observed during the lysis of both cross-linked and compacted plasma clots. Some accumulation of FITC-plasminogen took place on the surface of a compacted clot in the absence of added plasminogen activator (Fig 3DUp). This accumulation may be attributed to low-affinity interaction of plasminogen with the intact fibrin,5 6 which is present in a compacted clot in a very high concentration ({approx}1 mmol/L). However, the patterns of plasminogen accumulation in both the presence and absence of TPA were clearly different; in the former case (Fig 3CUp), the accumulation was significantly higher and localized to a more narrow zone on the clot surface.

It is likely that the strictly superficial character of the binding of plasminogen observed in a variety of conditions reflects a similar superficial location of plasminogen-binding sites generated during the lytic process. Because plasma contains a number of components that may affect this binding, we used a purified system for its quantitative characterization.

Distribution of FITC-Plasminogen in the Plasmin-Treated Fibrin Clot
After plasmin treatment of a fibrin clot in a buffer system, the lytic process was stopped by the addition of aprotinin, and the spatial distribution of plasminogen inside the clot was documented after a 24-hour equilibration of the clot with FITC-labeled plasminogen. Fig 4ADown demonstrates a strong accumulation of plasminogen in the superficial layer of the clot. Quantitative representation of these data obtained by means of scanning densitometry (Fig 4ADown') indicates that FITC-plasminogen was concentrated in this superficial layer up to about 15 µmol/L, thus providing a 10-fold local accumulation compared with its concentration in the outer medium, corresponding to the physiological level of plasminogen in plasma (1.5 µmol/L). The width of the plasminogen-accumulating layer was about 50 µm, estimated as the width of the peak at half-maximal concentration (7.5 µmol/L). As Fig 4BDown shows, an intact fibrin clot not treated with plasmin did not significantly accumulate FITC-plasminogen on its surface. In this case, FITC-plasminogen was distributed inside the clot uniformly, being slightly ({approx}1.4-fold) accumulated in the whole body of the clot compared with its concentration in the outer medium (Fig 4BDown'). This finding is consistent with the known low affinity of Glu-plasminogen for intact fibrin.5 6 In a similar experiment, FITC-labeled BSA wasevenly distributed inside and outside the clot, regardless of whether the latter was pretreated with plasmin (data not shown).



View larger version (54K):
[in this window]
[in a new window]
  		Figure 4. Spatial distribution of fluorescein isothiocyanate (FITC)–plasminogen in fibrin clots after a 24-hour equilibration with 1.5 µmol/L FITC-plasminogen in TBS-BSA containing 100 KIU/mL aprotinin. A, C, and D, Fluorescence photomicrographs showing clots preincubated with plasmin (0.1 CU/mL, 70 minutes, 37°C) added to the outer medium. B, Fluorescence photomicrograph showing an untreated clot. The specificity of the superficial binding of FITC-plasminogen in a plasmin-treated clot is demonstrated on the microphotographs of clots C and D, which were incubated with FITC-plasminogen in the presence of 20 µmol/L of unlabeled plasminogen or in the presence of 1.5 mmol/L {varepsilon}-aminocaproic acid, respectively. (The reduction in the size of clots A, C, and D compared with clot B is a consequence of their partial lysis by plasmin.) A' and B', quantitative representations of the spatial distribution of FITC-plasminogen inside and outside clots A and B, respectively, as measured by means of scanning densitometry. Arrow indicates the direction of scanning.

The specificity of the binding of FITC-plasminogen in the superficial layer of a plasmin-treated fibrin clot is demonstrated by the results in Fig 4CUp and 4DUp. The binding was effectively inhibited by an excess of unlabeled plasminogen and by EACA.

The quantitative parameters of the superficial binding of plasminogen to plasmin-treated fibrin clots were derived from the results presented in Fig 5Down. The values of Kd=2.2 µmol/L and Bmax=23 µmol/L for the concentration of binding sites for FITC-plasminogen superficial binding were found by nonlinear regression analysis of the concentration dependence of FITC-plasminogen binding (Fig 5ADown). The obtained value for Bmax corresponds to about 2.5 plasminogen-binding sites per fibrin monomer exposed locally in the superficial layer. A low-affinity binding was also detected for which it was possible to determine only the ratio of the concentration of binding sites to the dissociation constant Bmax/Kd=0.18. The value IC50=2.6 µmol/L was obtained for inhibition of FITC-plasminogen superficial binding by unlabeled plasminogen (Fig 5BDown), corresponding to a Ki=2.3 µmol/L. The Kd for FITC-plasminogen binding and the Ki for its inhibition with unlabeled plasminogen were very similar, implying that the procedure of labeling with FITC did not noticeably disturb the binding properties of plasminogen.



View larger version (15K):
[in this window]
[in a new window]
  		Figure 5. Scatterplots showing binding of fluorescein isothiocyanate (FITC)–plasminogen to the superficial layer of plasmin-treated fibrin clots. Experimental points for each of the curves were obtained in three similar independent experiments. A, Variation of FITC-plasminogen concentration. The parameters of the binding curve were calculated by means of nonlinear regression analysis of the data using the formula Pgbound=AxPgfree/(B+Pgfree)+CxPgfree. The following values were obtained: A=23 µmol/L and B=2.2 µmol/L, representing the concentration of binding sites and the Kd for high-affinity binding, respectively, and C=0.18, representing the ratio of the concentration of binding sites to the dissociation constant for low-affinity binding (r=.99). B, Inhibition of FITC-plasminogen binding at 0.3 µmol/L by unlabeled plasminogen ({bullet}) and by {varepsilon}-aminocarpoic acid (EACA, {blacktriangleup}). Nonlinear regression analysis gave the best fits of the data with sigmoid curves with IC50=2.6 µmol/L and a Hill coefficient of -0.91 (r=.98) for unlabeled plasminogen and with IC50=58 µmol/L and a Hill coefficient of -0.94 (r=.96) for EACA.

Fig 5BUp also demonstrates the dose-dependent inhibition of the superficial binding of FITC-plasminogen by EACA, indicating that lysine-binding sites of plasminogen were involved in its superficial accumulation. Nonlinear regression analysis of these data gave a value of IC50=58 µmol/L. Taking into account that tranexamic acid, another inhibitor of lysine binding site-mediated interactions of plasminogen, is about fivefold more potent than EACA,5 40 41 our data are in agreement with the results of Suenson et al,13 who found that 10 µmol/L tranexamic acid inhibits 50% of Glu-plasminogen binding to plasmin-treated fibrin.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
up arrowResults
*Discussion
down arrowReferences
 
It was first reported by Suenson et al13 and later confirmed and extended by others15 16 17 18 19 that, when fibrin is "nicked" with plasmin, the binding of plasminogen to fibrin increases. Although there is much discrepancy between the data of different research groups regarding quantitative parameters of this binding, it is generally believed that this positive feedback might represent one of the key steps in the local regulation of fibrinolysis. The supposed local character of this mechanism suggests that biochemical approaches dealing with overall binding values without assessment of the distribution of binding sites in a 3D body of a thrombus may not be sufficient for comprehensive understanding of the phenomenon. Clarification of the spatial distribution of plasminogen-binding sites and plasminogen in a thrombus during its lysis is necessary for better understanding of the local regulation of fibrinolysis and its rate-limiting factors.

To investigate this problem, we have used an experimental system incorporating low-magnification fluorescence microscopy for the visualization of the spatial distribution of fluorescent molecules in a model thrombus.27 In contrast to previously described experimental systems in which the binding of labeled plasminogen to model thrombi was investigated after separation of unbound material,13 14 15 16 17 18 19 42 our system allowed us to observe the spatial distribution of plasminogen in the thrombus without disturbing the binding equilibrium. We also were able to follow the dynamic spatial distribution of FITC-plasminogen inside the plasma clot during its lysis.

The main finding of this study is a strong superficial accumulation of plasminogen accompanying lysis of the clot surrounded by plasma containing a plasminogen activator. This accumulation led to formation of a distinctly visible plasminogen-accumulating shell on the surface of the plasma clot; the shell moved continuously with the reducing surface of the clot. According to our estimations, the local concentration of plasminogen at the surface of the clot was at least threefold higher than in the surrounding plasma in the absence of exhaustive activation of plasminogen. In a variety of experimental conditions explored, lysis was always accompanied by the superficial accumulation of plasminogen. As shown in experiments with TPA- and TCUPA-induced lysis (Figs 1Up and 2Up), the fibrin-binding properties of a plasminogen activator influenced its own spatial distribution during lysis of a plasma clot, but the spatial distribution of plasminogen apparently did not depend on the type of the plasminogen activator. The superficial accumulation of plasminogen was observed during lysis of non–cross-linked, cross-linked, and compacted plasma clots. The pattern of the superficial accumulation was basically the same under conditions of diffusive and convective transport of solutes into clots.

Both carboxypeptidase B and EACA were shown to remove plasminogen from the surface of the lysing clot. Presumably, the carboxypeptidase eliminates plasminogen-binding sites (C-terminal lysines) from the fibrin surface, while EACA occupies lysine-binding sites of plasminogen. Although mechanisms of action of the two agents are different, they cause similar effects manifested by simultaneous elimination of the plasminogen shell on the surface of the lysing clot and cessation of lysis. These findings give rise to a supposition that superficial plasminogen accumulation may be a necessary condition for effective lysis.

The demonstration of the strictly superficial accumulation of plasminogen gives new insight into the mechanism of thrombolysis that might be driven primarily by the activation of the plasminogen accumulated on the surface of the thrombus during its lysis. This is likely because the plasminogen concentration on the surface of a clot is several times higher than in the surrounding plasma and because the binding of plasminogen to fibrin contributes to efficient plasminogen activation.7 8 9 10 11 12 In addition, the high accumulation of plasminogen is expected to provide a significant local excess of plasminogen over {alpha}2-antiplasmin in the thin superficial layer of the thrombus, higher than in surrounding plasma, where the molar ratio of plasminogen to {alpha}2-antiplasmin is usually about 1.5 to 243 and much higher than in a contracted blood clot.44 These factors can provide conditions for a local activation of the fibrinolytic system strictly confined in space by the superficial location of newly generated plasminogen-binding sites.

The surface of a clot is probably the only place where a really high concentration of the plasminogen-binding sites can be generated. Indeed, Glu-plasminogen at a physiological concentration binds only moderately to a fibrin gel treated throughout with plasmin; binding in the range of 10% to 40% of the plasminogen added inside such a gel was demonstrated by several authors.13 15 16 36 C-terminal lysine residues generated by plasmin cleavage of fibrin chains are probably much more accessible on the surface of a plasmin-treated fibrin gel where molecules are less involved in the maintenance of gel structure and where the lateral packing of fibrin bundles could be disorganized. Plasminogen "bridging" of fibrin fibers45 on the surface of the lysing clot may be an additional mechanism for generation of the plasminogen-rich shell.

Quantitative parameters of the superficial binding of plasminogen were measured in a purified system. As a result of a plasmin treatment of a fibrin clot, potent binding sites for plasminogen were generated only in a thin superficial layer, not exceeding 50 µm. At a physiological concentration of plasminogen in the outer medium (1.5 µmol/L), this layer bound about 10-fold more, ie, 15 µmol/L plasminogen. Analysis of binding isotherms revealed a Kd of 2.2 µmol/L with about 2.5 plasminogen-binding sites per fibrin monomer exposed locally in this superficial layer of the plasmin-treated fibrin clot. The results on the inhibition of this binding by EACA confirmed the involvement of lysine-binding sites of plasminogen. We presume that the high plasminogen-binding ability of the binding sites characterized in a purified system can be modulated by numerous plasma components, such as carboxypeptidases, {alpha}2-plasmin inhibitor, histidine-rich glycoprotein, and other proteins capable of binding with either plasminogen or fibrin. This may explain some quantitative differences in accumulation of FITC-plasminogen observed in the purified system and in plasma milieu. Smaller accumulation in the latter case may also be attributed to the dynamic nature of lysis and to possible differences in fibrin structure in plasma and fibrin clots.46

The superficial concentration of the fibrinolytic potential demonstrated should be beneficial for lysis of small thrombi with a high surface-to-volume ratio and might be much less effective in the case of massive thrombi. We suppose that this mechanism of lysis can effectively function in vivo in the prevention of thrombosis, creating potent positive feedback for dissolution of small nascent thrombi and early fibrin deposits by use of plasminogen activators that are normally present in plasma in low concentration or locally released from endothelium.47 48

The local accumulation of plasminogen implies the necessity of the supply of plasminogen from the surrounding plasma to the surface of the thrombus for its optimal lysis. Our experiments with lysis induced by a high concentration (500 IU/mL) of a non–fibrin-specific plasminogen activator (TCUPA) show that this supply may be restricted in conditions of systemic activation of plasminogen, leading to a reduction of accumulation of plasminogen in the superficial layer (Fig 1EUp through 1G). This is consistent with findings that reduced levels of plasminogen in plasma correlate with decreased thrombolysis38 49 50 51 52 53 and that administration of additional plasminogen might be advantageous for effective lysis when endogenous plasminogen in plasma is depleted.45 49 50 51 54 55 The necessity of a continuous plasminogen supply thus explains why fibrin-specific plasminogen activators sparing plasminogen in the circulation are more potent for thrombolytic therapy than nonspecific activators.21 22 This phenomenon was explained previously by Sobel et al,50 who proposed that reduction of the plasminogen concentration in plasma by nonspecific plasminogen activators leads to depletion of plasminogen already present in the clot by a "plasminogen steal" effect. We believe that both explanations are relevant and underline that plasminogen in the circulation should be spared as much as possible during thrombolytic therapy.


*    Acknowledgments
 
We are grateful to Drs E.J.P. Brommer, F. Haverkate, and J.H. Verheijen for their critical reading of the manuscript.

Received February 19, 1995; revision received April 12, 1995; accepted April 16, 1995.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
up arrowResults
up arrowDiscussion
*References
 

  1. Rijken DC, Hoylaerts M, Collen D. Fibrinolytic properties of one-chain and two-chain human extrinsic (tissue-type) plasminogen activator. J Biol Chem. 1982;257:2920-2925. [Free Full Text]
  2. Fears R. Binding of plasminogen activators to fibrin: characterization and pharmacological consequences. Biochem J. 1989;261:313-324. [Medline] [Order article via Infotrieve]
  3. Sakata Y, Aoki N. Crosslinking of {alpha}2-antiplasmin inhibitor to fibrin by fibrin-stabilizing factor. J Clin Invest. 1980;65:290-297.
  4. Wagner OF, de Vries C, Hohmann C, Veerman H, Pannekoek H. Interaction between plasminogen activator inhibitor type 1 (PAI-1) bound to fibrin and either tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA). J Clin Invest. 1989;84:647-655.
  5. Thorsen S. Differences in the binding to fibrin of native plasminogen and plasminogen modified by proteolytic degradation: influence of {omega}-aminocarboxylic acids. Biochim Biophys Acta. 1975;393:55-65. [Medline] [Order article via Infotrieve]
  6. Lucas MA, Fretto LJ, McKee PA. The binding of human plasminogen to fibrin and fibrinogen. J Biol Chem. 1983;258:4249-4256. [Free Full Text]
  7. Hoylaerts M, Rijken DC, Lijnen HR, Collen D. Kinetics of the activation of plasminogen by human tissue plasminogen activator: role of fibrin. J Biol Chem. 1982;257:2912-2919. [Abstract/Free Full Text]
  8. Lucas MA, Straight DL, Fretto LJ, McKee PA. The effects of fibrinogen and its cleavage products on the kinetics of the plasminogen activation by urokinase and subsequent plasmin activity. J Biol Chem. 1983;258:12171-12177. [Abstract/Free Full Text]
  9. Takada A, Takada Y, Sugawara Y. The activation of Glu- and Lys-plasminogens by streptokinase: effects of fibrin, fibrinogen and their degradation products. Thromb Res. 1985;37:465-475. [Medline] [Order article via Infotrieve]
  10. Fears R, Hibbs MJ, Smith RAG. Kinetic studies on the interaction of streptokinase and other plasminogen activators with plasminogen and fibrin. Biochem J. 1985;229:555-558. [Medline] [Order article via Infotrieve]
  11. Wu H, Chang B, Wu D, Chang L, Gong C, Lou K, Shi G. Interaction of plasminogen and fibrin in plasminogen activation. J Biol Chem. 1990;265:19658-19684. [Abstract/Free Full Text]
  12. Pannel R, Black J, Gurewich V. Complementary modes of action of tissue-type plasminogen activator and pro-urokinase by which their synergistic effect on clot lysis may be explained. J Clin Invest. 1988;81:853-859.
  13. Suenson E, Lutzen O, Thorsen S. Initial plasmin-degradation of fibrin as a basis of a positive feed-back mechanism in fibrinolysis. Eur J Biochem. 1984;140:513-522. [Medline] [Order article via Infotrieve]
  14. Suenson E, Bjerrum P, Holm A, Lind B, Meldal M, Selmer J, Petersen LC. The role of fragment X polymers in the fibrin enhancement of tissue plasminogen activator-catalyzed plasmin formation. J Biol Chem. 1990;265:22228-22237. [Abstract/Free Full Text]
  15. Tran-Thang C, Kruithof EKO, Bachmann F. Tissue-type plasminogen activator increases the binding of glu-plasminogen to clots. J Clin Invest. 1984;74:2009-2016.
  16. Harpel PC, Chang TS, Verderber E. Tissue plasminogen activator and urokinase mediate the binding of Glu-plasminogen to plasma fibrin I. J Biol Chem. 1985;260:4432-4440. [Abstract/Free Full Text]
  17. Bok RA, Mangel WF. Quantitative characterization of the binding of plasminogen to intact fibrin clots, lysine-Sepharose, and fibrin cleaved by plasmin. Biochemistry. 1985;24:3279-3286. [Medline] [Order article via Infotrieve]
  18. Tran-Thang C, Kruithof EKO, Atkinson J, Bachmann F. High-affinity binding sites for human Glu-plasminogen unveiled by limited plasmic degradation of human fibrin. Eur J Biochem. 1986;160:599-604. [Medline] [Order article via Infotrieve]
  19. Fleury V, Anglés-Cano E. Characterization of the binding of plasminogen to fibrin surfaces: the role of carboxy-terminal lysines. Biochemistry. 1991;30:7630-7638. [Medline] [Order article via Infotrieve]
  20. Verstraete M. New thrombolytic drugs in acute myocardial infarction: theoretical and practical considerations. Circulation. 1987;76(suppl II):II-31-II-38.
  21. The GUSTO Investigators. An international randomized trial comparing four thrombolytic strategies for acute myocardial infarction. N Engl J Med. 1993;329:673-682. [Abstract/Free Full Text]
  22. The GUSTO Angiographic Investigators. The effects of tissue plasminogen activator, streptokinase, or both on coronary-artery patency, ventricular function, and survival after acute myocardial infarction. N Engl J Med. 1993;329:1615-1622. [Abstract/Free Full Text]
  23. Deutsch DG, Mertz ET. Plasminogen: purification from human plasma by affinity chromatography. Science. 1970;170:1095-1096. [Abstract/Free Full Text]
  24. Loscalzo J. Structural and kinetic comparison of recombinant human single- and two-chain tissue plasminogen activator. J Clin Invest. 1988;82:1391-1397.
  25. Novokhatny VV, Ingham KC, Medved LV. Domain structure and domain-domain interactions of recombinant tissue plasminogen activator. J Biol Chem. 1991;266:12994-13002. [Abstract/Free Full Text]
  26. Verheijen JH, Mullaart E, Chang GTG, Kluft C, Wijngaards G. A simple, sensitive spectrophotometric assay for extrinsic (tissue-type) plasminogen activator applicable to measurements in plasma. Thromb Haemost. 1982;48:266-269. [Medline] [Order article via Infotrieve]
  27. Sakharov DV, Domogatsky SP, Bos R, Rijken DC. An experimental system for investigation of penetration of components of the fibrinolytic system into a plasma clot. Fibrinolysis. 1994;8(suppl 2):116-119.
  28. Blomback B, Carlsson K, Hessel B, Liljeborg A, Procyk R, Aslund N. Native fibrin gel networks observed by 3D microscopy, permeation and turbidity. Biochim Biophys Acta. 1989;997:96-110. [Medline] [Order article via Infotrieve]
  29. Kluft C, Vellenga E, Brommer EJP, Wijngaards G. A familial hemorrhagic diathesis in a Dutch family: an inherited deficiency of {alpha}2-antiplasmin. Blood. 1982;59:1169-1180. [Abstract/Free Full Text]
  30. Blinc A, Planinsic G, Keber D, Jarh O, Lahajnar G, Zidansek A, Demsar F. Dependence of blood clot lysis on the mode of transport of urokinase into the clot: a magnetic resonance imaging study in vitro. Thromb Haemost. 1991;65:549-552. [Medline] [Order article via Infotrieve]
  31. Blinc A, Keber D, Lahajnar G, Stegnar M, Zidansek A, Demsar F. Lysing patterns of retracted blood clots with diffusion or bulk flow transport of plasma with urokinase into clots: a magnetic resonance imaging study in vitro. Thromb Haemost. 1992;68:667-671. [Medline] [Order article via Infotrieve]
  32. Diamond SL, Anand S. Inner clot diffusion and permeation during fibrinolysis. Biophys J. 1993;65:2622-2643. [Abstract/Free Full Text]
  33. Wu JH, Siddiqui K, Diamond SL. Transport phenomena and clot dissolving therapy: an experimental investigation of diffusion-controlled and permeation-enhanced fibrinolysis. Thromb Haemost. 1994;72:105-112. [Medline] [Order article via Infotrieve]
  34. Blinc A, Kennedy SD, Bryant RG, Marder VJ, Francis CV. Flow through clots determines the rate and pattern of fibrinolysis. Thromb Haemost. 1994;71:230-235. [Medline] [Order article via Infotrieve]
  35. Sakata Y, Aoki N. Significance of crosslinking of {alpha}2-plasmin inhibitor to fibrin in inhibition of fibrinolysis and in hemostasis. J Clin Invest. 1982;69:536-542.
  36. Sakata Y, Mimuro J, Aoki N. Differential binding of plasminogen to crosslinked and noncrosslinked fibrins: its significance in hemostatic defect in factor XIII deficiency. Blood. 1984;63:1393-1401. [Abstract/Free Full Text]
  37. Sabovic M, Lijnen HR, Keber D, Collen D. Effect of retraction on the lysis of human clots with fibrin specific and non-fibrin specific plasminogen activators. Thromb Haemost. 1989;62:1083-1087. [Medline] [Order article via Infotrieve]
  38. Nishino N, Kakkar VV, Scully MF. Influence of intrinsic and extrinsic plasminogen upon the lysis of thrombi in vitro. Thromb Haemost. 1991;66:672-677. [Medline] [Order article via Infotrieve]
  39. Bugelski PJ, Kopia GA, Kopaciewicz L, Cadogan AS, Morgan DG. Ultrastructural analysis of thrombolysis by streptokinase and tissue-type plasminogen activator of experimental coronary arterial thrombosis. Fibrinolysis. 1989;3:137-145.
  40. Marcus G, DePasquale JL, Wissler FC. Quantitative determination of the binding of {varepsilon}-aminocaproic acid to native plasminogen. J Biol Chem. 1978;253:727-732. [Abstract/Free Full Text]
  41. Marcus G, Priore RL, Wissler FC. The binding of tranexamic acid to native (glu) and modified (lys) human plasminogen and its effect on conformation. J Biol Chem. 1979;254:1211-1216. [Free Full Text]
  42. Iga Y, Greenwald SR, Chandler AB. Uptake of 125I-lys-plasminogen by in vitro thrombi. Haemostasis. 1983;13:25-35. [Medline] [Order article via Infotrieve]
  43. Collen D. On the regulation and control of fibrinolysis. Thromb Haemost. 1980;43:77-89. [Medline] [Order article via Infotrieve]
  44. Sabovic M, Lijnen HR, Keber D, Collen D. Correlation between progressive adsorption of plasminogen to blood clots and their sensitivity to lysis. Thromb Haemost. 1990;64:450-454. [Medline] [Order article via Infotrieve]
  45. Garman AJ, Smith RAG. The binding of plasminogen to fibrin: evidence for plasminogen-bridging. Thromb Res. 1982;27:311-320. [Medline] [Order article via Infotrieve]
  46. Carr ME. Fibrin formed in plasma is composed of fibers more massive than those formed from purified plasminogen. Thromb Haemost. 1988;59:535-539. [Medline] [Order article via Infotrieve]
  47. Binnema DJ, van Iersel JJL, Dooijewaard G. Quantitation of urokinase antigen in plasma and culture media by use of an ELISA. Thromb Res. 1986;43:569-577. [Medline] [Order article via Infotrieve]
  48. Rijken DC, Juhan-Vague I, de Cock F, Collen D. Measurement of human tissue-type plasminogen activator by a two-site immunoradiometric assay. J Lab Clin Med. 1983;101:274-279. [Medline] [Order article via Infotrieve]
  49. Westlund LE, Andersson LO. Studies on the influence of reactants and buffer environment on clot lysis induced by human plasminogen activators. Thromb Res. 1985;37:213-223. [Medline] [Order article via Infotrieve]
  50. Sobel BE, Nachowiak DA, Fry ETA, Bergmann SR, Torr SR. Paradoxical attenuation of fibrinolysis attributable to `plasminogen steal' and its implications for coronary thrombolysis. Coron Artery Dis. 1990;1:111-119.
  51. Onundarson PT, Francis CW, Marder V. Depletion of plasminogen in vitro or during thrombolytic therapy limits fibrinolytic potential. J Lab Clin Med. 1992;120:120-128. [Medline] [Order article via Infotrieve]
  52. Andrew M, Brooker L, Leaker M, Paes B, Weitz J. Fibrin clot lysis by thrombolytic agents is impaired in newborns due to a low plasminogen concentration. Thromb Haemost. 1992;68:325-330. [Medline] [Order article via Infotrieve]
  53. Onundarson PT, Haraldsson HM, Bergmann L, Francis CW, Marder VJ. Plasminogen depletion during streptokinase treatment or two-chain urokinase incubation correlates with decreased clot lysability ex vivo and in vitro. Thromb Haemost. 1993;70:998-1004. [Medline] [Order article via Infotrieve]
  54. Kakkar VV, Scully MF. Intermittent plasminogen-streptokinase treatment of deep vein thrombosis. Haemostasis. 1988;18(suppl 1):127-138.
  55. Anderle K, Frohlich A. Review of studies with plasminogen concentrates and proposals for further therapeutic strategies with plasminogen concentrates. Haemostasis. 1988;18(suppl 1):165-175.



This article has been cited by other articles:


Home page
 BloodHome page
B.-S. Ding, N. Hong, J.-C. Murciano, K. Ganguly, C. Gottstein, M. Christofidou-Solomidou, S. M. Albelda, A. B. Fisher, D. B. Cines, and V. R. Muzykantov
Prophylactic thrombolysis by thrombin-activated latent prourokinase targeted to PECAM-1 in the pulmonary vasculature
Blood, February 15, 2008; 111(4): 1999 - 2006.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
A. H.C. Guimaraes, N. Laurens, E. M. Weijers, P. Koolwijk, V. W.M. van Hinsbergh, and D. C. Rijken
TAFI and Pancreatic Carboxypeptidase B Modulate In Vitro Capillary Tube Formation by Human Microvascular Endothelial Cells
Arterioscler. Thromb. Vasc. Biol., October 1, 2007; 27(10): 2157 - 2162.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
K. Danielyan, B.-S. Ding, C. Gottstein, D. B. Cines, and V. R. Muzykantov
Delivery of Anti-Platelet-Endothelial Cell Adhesion Molecule Single-Chain Variable Fragment-Urokinase Fusion Protein to the Cerebral Vasculature Lyses Arterial Clots and Attenuates Postischemic Brain Edema
J. Pharmacol. Exp. Ther., June 1, 2007; 321(3): 947 - 952.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
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]

Home page
 Mol. Interv.Home page
B.-S. Ding, T. Dziubla, V. V. Shuvaev, S. Muro, and V. R. Muzykantov
Advanced drug delivery systems that target the vascular endothelium.
Mol. Interv., April 1, 2006; 6(2): 98 - 112.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
K. Ganguly, M. S. Goel, T. Krasik, K. Bdeir, S. L. Diamond, D. B. Cines, V. R. Muzykantov, and J.-C. Murciano
Fibrin Affinity of Erythrocyte-Coupled Tissue-Type Plasminogen Activators Endures Hemodynamic Forces and Enhances Fibrinolysis in Vivo
J. Pharmacol. Exp. Ther., March 1, 2006; 316(3): 1130 - 1136.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
B.-S. Ding, C. Gottstein, A. Grunow, A. Kuo, K. Ganguly, S. M. Albelda, D. B. Cines, and V. R. Muzykantov
Endothelial targeting of a recombinant construct fusing a PECAM-1 single-chain variable antibody fragment (scFv) with prourokinase facilitates prophylactic thrombolysis in the pulmonary vasculature
Blood, December 15, 2005; 106(13): 4191 - 4198.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
K. Ganguly, T. Krasik, S. Medinilla, K. Bdeir, D. B. Cines, V. R. Muzykantov, and J. C. Murciano
Blood Clearance and Activity of Erythrocyte-Coupled Fibrinolytics
J. Pharmacol. Exp. Ther., March 1, 2005; 312(3): 1106 - 1113.
[Abstract] [Full Text] [PDF]

Home page