Regulation of Plasmin-dependent Fibrin Clot Lysis by Annexin II Heterotetramer*

Kyu-Sil ChoiDagger , Sandra L. FitzpatrickDagger , Nolan R. FilipenkoDagger , Darin K. FoggDagger , Geetha KassamDagger , Anthony M. Magliocco§, and David M. WaismanDagger ||

From the  Cancer Biology Research Group, Dagger  Department of Biochemistry and Molecular Biology, and § Department of Pathology, University of Calgary, Calgary, Alberta T2N 4N1, Canada

Received for publication, February 14, 2001, and in revised form, April 20, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In a previous report we showed that plasmin-dependent lysis of a fibrin polymer, produced from purified components, was totally blocked if annexin II heterotetramer (AIIt) was present during fibrin polymer formation. Here, we show that AIIt inhibits fibrin clot lysis by stimulation of plasmin autodegradation, which results in a loss of plasmin activity. Furthermore, the C-terminal lysine residues of its p11 subunit play an essential role in the inhibition of fibrin clot lysis by AIIt. We also found that AIIt binds to fibrin with a Kd of 436 nM and a stoichiometry of about 0.28 mol of AIIt/mol of fibrin monomer. The binding of AIIt to fibrin was not dependent on the C-terminal lysines of the p11 subunit. Furthermore, in the presence of plasminogen, the binding of AIIt to fibrin was increased to about 1.3 mol of AIIt/mol of fibrin monomer, suggesting that AIIt and plasminogen do not compete for identical sites on fibrin. Immunohistochemical identification of p36 and p11 subunits of AIIt in a pathological clot provides important evidence for its role as a physiological fibrinolytic regulator. These results suggest that AIIt may play a key role in the regulation of plasmin activity on the fibrin clot surface.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The formation of a fibrin polymer from its soluble precursor, fibrinogen, results from the hydrolytic catalysis of fibrinogen by thrombin, the terminal proteolytic enzyme in the coagulation cascade (1-3). The (patho-) physiological existence of fibrin is linked to its homeostatic roles, such as temporary matrix formation in wound healing and the formation of a hemostatic plug. Accumulation of fibrin is a hallmark of a variety of diseases, such as cancer and arteriosclerosis (4). The polymerization of the fibrin monomer and the degradation of the fibrin polymer are physiologically balanced by the coagulation and fibrinolysis systems, respectively (5-9). Dissolution of a fibrin clot is mainly conducted by plasmin, the terminal enzyme of the fibrinolytic cascade. Plasmin, an 85-kDa serine protease, is involved in a variety of physiological and pathological processes, including fibrinolysis, wound healing, tissue remodeling, embryogenesis, and the invasion and spread of transformed tumor cells (5, 8, 10-16). Active plasmin is produced from two serial activation processes. First, [Glu]plasminogen is converted to the more reactive [Lys]plasminogen by plasmin itself through proteolytic removal of the N-terminal 77 amino acids. Second, plasmin also activates the plasminogen activators, tissue-type plasminogen activator (tPA)1 and urokinase-type plasminogen activator, which, in turn, convert [Lys]- or [Glu]plasminogen to plasmin (13).

It is well established that fibrin stimulates the rate of tPA-dependent plasminogen activation by at least two orders of magnitude due to the localization of plasminogen and tPA to the fibrin surface via their lysine-binding kringle domains (17, 18). Other proteins, such as the histidine-proline rich glycoproteins or certain extracellular matrix proteins that interact with the kringle domains of plasminogen, have also been shown to stimulate plasminogen activation (19-21). Typically, these interactions involve the binding of the kringle domains with the C-terminal lysine residues of the plasminogen-binding protein. Hajjar's group recently reported that the Ca2+-binding protein, annexin II, stimulated the tPA-dependent formation of plasmin from [Glu]plasminogen or [Lys]plasminogen 20-fold or 14-fold, respectively, in vitro. They also reported that plasminogen activation was inhibited on human umbilical vein endothelial cell surfaces after transfection of these cells with antisense oligonucleotides directed against annexin II mRNA, suggesting that annexin II may function as a fibrinolytic receptor for plasminogen on the surface of endothelial cells (23-27).

Annexin II was originally described as an intracellular Ca2+- and phospholipid-binding protein, and subsequent studies suggested that this protein could be involved in regulating membrane trafficking events such as exocytosis or endocytosis (28). Surprisingly, annexin II does not contain a C-terminal lysine residue, and the mechanism by which this protein interacted with plasminogen was unclear. Annexin II also exists in cells as a heterotetramer, called annexin II heterotetramer (AIIt), which consists of two annexin II molecules referred to as the p36 subunit and two molecules of an 11-kDa regulatory subunit referred to as the p11 light chain. Importantly, p11 contains two lysine residues at its C terminus. Ninety to ninety-five percent of the total cellular annexin II is present in the heterotetrameric form in many cells such as Madin-Darby canine kidney cells, bovine intestinal epithelial cells, and calf pulmonary arterial endothelial cells (29, 30).

Recently, it was established that AIIt binds tPA, plasminogen, and plasmin and stimulates tPA-dependent plasminogen activation about 300-fold in vitro (31). This activation was mediated by binding of plasminogen kringle domains to the C-terminal lysine residue of the p11 subunit, suggesting that the p11 subunit facilitates the majority of the AIIt-mediated stimulation of tPA-dependent plasminogen activation. Furthermore, immunofluorescence and coimmunoprecipitation experiments established the colocalization of p11 and annexin II on the surface of human umbilical vein endothelial cells (31), BT20 human breast carcinoma cells, U87 human glioblastoma cells (32, 33), and fibrosarcoma cells.2 Taken together, these data provided evidence that AIIt played a key role in plasminogen activation on the cell surface.

We recently reported that AIIt inhibited the fibrinolytic activity of plasmin (34). These experiments were conducted using fibrin clots generated from purified components in a cell-free system (34). We also demonstrated that AIIt associated with the fibrin clot in vitro (34). In a separate series of experiments, we showed that preincubation of AIIt and plasmin resulted in the stimulation of the autocatalytic digestion of the plasmin heavy and light chains, resulting in a decrease in plasmin activity (31). It was therefore unclear from our results if the antifibrinolytic activity of AIIt was due to its stimulation of plasmin autodegradation or due to its ability to associate with fibrin thereby sterically hindering the interaction of plasmin with fibrin.

In the present report, we demonstrate that the antifibrinolytic activity of AIIt is due to its stimulation of plasmin autodegradation by a mechanism that requires the C-terminal lysine residues of the p11 subunit.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reagents-- Bovine intestinal mucosa heparin (3 kDa, 50 USP units/mg), epsilon -aminocaproic acid (epsilon -ACA), and thrombin (2300 NIH units/mg) were obtained from Sigma Chemical Co. Human recombinant tPA was obtained from Genentech, was purified by chromatography on benzamidine-Sepharose (35), and was an 80-90% single chain as determined by SDS-PAGE. [Glu]plasminogen, [Lys]plasminogen, plasmin, the amidolytic plasmin specific substrate, Spectrozyme 251 (H-D-norleucyl-hexahydrotyrosyl-lysine-p-nitroanilide), and the tPA amidolytic substrate Spectrozyme 444 (methyl-D-cyclohexatyrosyl-glycyl-arginine paranitroaniline acetate) were obtained from American Diagnostica. Fibrinogen was obtained from Sigma and further purified by chromatography on Superose 12 (Amersham Pharmacia Biotech) to remove contaminating plasminogen.

Annexin II tetramer (AIIt) was prepared from bovine lung as previously described (36). Recombinant annexin II monomer and p11 subunits were expressed in Escherichia coli as described previously (37, 38). Purification of recombinant annexin II from bacterial lysates involved chromatography on hydroxyapatite (Bio-Rad), affinity chromatography on heparin-Sepharose, and gel permeation chromatography on Superose 12. Purification of recombinant p11 involved ion-exchange chromatography on Fast-Q and Fast-S and gel permeation chromatography on Superose 12. Recombinant AIIt was formed by mixing equimolar annexin II and p11 followed by gel permeation chromatography on Superose 12. All proteins were stored at -70 °C in 40 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, 0.1 mM EGTA, and 150 mM NaCl. Anti-p11 and annexin II monoclonal antibodies were purchased from Transduction Laboratories.

Mutagenesis of Annexin II-- The bacterial expression vector (pDS10) encoding the cDNA of human total calcium mutant annexin II was kindly provided by Dr. Volker Gerke. This vector has all five Ca2+-binding sites of annexin II inactivated and has the mutation A65E in the annexin II sequence to provide an epitope for antibody recognition (39). This site was reverted back to the wild-type sequence (E65A) before bacterial transformation (40). The mutant annexin II cDNA sequence was excised from the pDS10 vector using PCR (40). The PCR reaction resulted in a blunt-ended cDNA encoding the exact amino acid sequence of annexin II and therefore did not encode for the six extra amino acids as previously reported. A BamHI site was added to the 3'-end using PCR with the appropriate primers. This cDNA sequence was then ligated into an NdeI-cut, Klenow-filled-in, BamHI-digested pAED4.91 vector. These vectors were then transformed into E. coli BL21(DE3) and grown as previously described (37).

Production of the p11 Subunit Deletion Mutant-- The cDNA of the p11 subunit deletion mutant (lacking the two C-terminal lysine residues) was produced by PCR mutagenesis as previously described (38). The cDNA expression construct, pAED4.91-p11del KK was confirmed by DNA sequencing to lack Lys-95 and Lys-96. The expression of the p11del KK in BL21(DE3) E. coli and the subsequent purification were performed as previously described (37).

Fibrin Polymer Lysis Assay-- Fibrin polymer lysis experiments were performed in a volume of 400 µl at 25 °C using a Beckman DU 640 spectrophotometer to monitor changes in turbidity at 450 nm (34). Initially, fibrin clots were formed by incubation of 8 µM fibrinogen with 0.83 NIH units/ml thrombin in buffer A (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, and 5 mM CaCl2) at room temperature. After turbidity had reached a stable level, 100 µl of buffer A containing 42 nM plasmin and test substances such as glycerol, epsilon -ACA, or other proteins were gently layered on top of the fibrin polymer reaction mixture, and turbidity was measured as described previously (34).

Radioiodination of Protein-- AIIt and [Glu]plasminogen were radioiodinated as previously described (41). Briefly, either AIIt or plasminogen (10 µM) was incubated for 3 min at room temperature with 37 MBq (= 1 mCi) of Na125I and three IODO-BEADs (Pierce Chemical Co.) in phosphate-buffered saline. Free I125 and protein were separated using a PD-10 desalting column (Sephadex G-25, Amersham Pharmacia Biotech), equilibrated, and eluted with buffer A. The specific activity of the protein preparations ranged from 1000 to 2000 cpm/pmol of protein.

Plasmin Autodegradation during Fibrinolysis-- Fibrin clots were formed as described above for the fibrin polymer lysis assay. After turbidity had reached a stable level, fibrin clot lysis was triggered by gently layering 200 nM radioiodinated plasminogen and 25 nM tPA with or without 1 µM AIIt. After 12 h of incubation at 25 °C, 10 µl of each reaction was subjected to SDS-PAGE, and the pattern of plasminogen or plasmin degradation was detected by autoradiography. The autoradiographs were quantitated by densitometric analysis using a Hewlett Packard ScanJet 4c flatbed scanner and Adobe Photoshop 5.5.

Fibrin Binding Assay-- Fibrin polymerization was initiated by addition of thrombin (5 NIH units/ml) to a series of Eppendorf tubes, preheated to 37 °C, containing 5 µM fibrinogen, 4.2 nM 125I-labeled AIIt, and various concentrations of unlabeled AIIt in buffer A. To examine the binding of AIIt to fibrin in the presence of plasminogen, fibrin was formed by incubation of thrombin (5 NIH units/ml), 5 µM fibrinogen, 4.2 nM 125I-labeled AIIt, 2 µM unlabeled AIIt, and various concentrations of plasminogen in the absence or presence of 10 mM epsilon -ACA (42). Fibrin clots were incubated for 1 h at 37 °C and pelleted by centrifugation at 10,000 × g for 30 min at 4 °C. The clots were washed with buffer A three times, and AIIt bound to the fibrin clot was calculated from radioactivity of the fibrin pellets. Radioactivity of the pelleted clots in the thrombin-untreated tubes was subtracted from that of the pellet in the thrombin-treated tubes. Nonspecific binding was typically less than 10% of total binding.

Plasminogen Activation Assay on Fibrin Surface-- Fibrin polymers were formed in 24-well plates by incubation of 8 µM fibrinogen with 0.83 NIH unit/ml thrombin in buffer A as described above. The kinetics of tPA-mediated plasminogen activation in the absence or presence of AIIt on fibrin was determined by measuring amidolytic activity of the plasmin generated during activation of plasminogen. The reaction was performed with the substrate H-D-norleucyl-hexahydrotyrosyl-lysine-p-nitroanilide (Spectrozyme 251) at a final concentration of 100 µM, and in buffer A containing 2 nM tPA in the absence or presence of 1 µM AIIt. Alternatively, AIIt-stimulated plasmin formation on the fibrin clot was examined in the presence of various concentrations of AIIt. The reaction was initiated by the addition of 0.1 µM [Glu]plasminogen and was monitored at 405 nm in a PerkinElmer Life Sciences HTS 7000 Bioassay reader.

Data Analysis-- Initial rates of plasmin generation were calculated using linear regression analysis of plots of A405 nm versus time squared as previously outlined (43). Linear regression was performed with the computer program SigmaPlot (Jandel Scientific). For time course data, the data were fit to the equation, A405 nm B + K*t2, where K is the rate constant for the acceleration of p-NA generation and B is the y intercept. This equation describes the rate of p-nitroanilide (p-NA) production from a mixture of tPA, plasminogen, and spectrozyme 251. Under our experimental conditions, K was proportional to the initial rate of plasmin formation from plasminogen. Typically, the initial rates of plasmin generation were reported in units of A405 nm/min2 × 10-3.

Immunohistochemistry-- A lung tissue specimen was obtained from an autopsy of a patient who had suffered a pulmonary thromboembolism. The lung tissue was fixed overnight in 4% phosphate-buffered formaldehyde solution, processed, and embedded in paraffin. Three-micron-thick sections were cut from the paraffin blocks and placed on coated glass slides. The sections were deparaffinized and rehydrated. No antigen retrieval technique was necessary. Sections were stained using a Ventana Nexes immunostainer with a Ventana DAB detection kit. Antibody was applied manually at an appropriate time in the cycle. Anti-p11 monoclonal antibody was diluted with Dako antibody diluting buffer to a working concentration of 1:40, whereas the anti-annexin II monoclonal antibody was diluted to a working dilution of 1:25. Antibodies were incubated with samples for 30 min. The slides were counterstained with Ventana hematoxylin and bluing agent. Coverslips were placed over the slides, which were then examined using a Zeiss microscope fitted with a SPOT digital camera.

Miscellaneous Techniques-- Nonlinear least-squares fitting was performed using SigmaPlot (Jandel Scientific). Results are representative of a minimum of three experiments. AIIt, fibrinogen, and [Glu]plasminogen concentrations were determined spectrophotometrically (A280) using the extinction coefficients (Emg/ml) 0.68, 1.51, and 1.68, respectively.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effects of Glycerol on the AIIt-mediated Inhibition of Plasmin Fibrinolytic Activity-- We previously reported that the plasmin-dependent lysis of a fibrin polymer, produced from purified components, was totally blocked if AIIt was present during fibrin polymer formation (34). It was unclear from these studies if, in the presence of fibrin, AIIt inhibited plasmin activity directly or indirectly inhibited plasmin activity by promoting plasmin autodegradation. We therefore used an in vitro fibrin polymer lysis assay to address this question. In this assay, fibrin was formed from fibrinogen by thrombin and fibrin polymer dissolution was initiated by addition of plasmin in the presence or absence of AIIt. Fibrin clot polymerization and subsequent lysis were measured with a simple turbidity assay (A450).

As shown in Fig. 1, the two respective processes of fibrin polymerization (clotting) and fibrinolysis are indicated by an initial rapid increase in turbidity to a plateau value (polymer formation) and, after addition of plasmin to the fibrin polymer, a subsequent return to the value of nonpolymerized material (fibrinolysis). Lysis of the fibrin clot was completely abolished in the presence of AIIt. Interestingly, the AIIt-mediated inhibition of plasmin fibrinolytic activity was blocked by glycerol in a dose-dependent manner (Fig. 1). Glycerol has been shown to inhibit plasmin autodegradation but not plasmin-dependent clot lysis (44). Our data therefore suggested that AIIt blocked fibrin clot lysis by accelerating plasmin autodegradation, thereby reducing the availability of plasmin for fibrin clot lysis.


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Fig. 1.   Glycerol blocks the inhibition of plasmin-dependent fibrin polymer lysis by AIIt. Thrombin (0.83 NIH units/ml) and fibrinogen (8 µM) were incubated at 25 °C in buffer A containing 5 mM CaCl2, 50 mM Tris, pH 7.5, and 100 mM NaCl. After fibrin polymerization was complete (30 min), 100 µl of buffer A alone (filled squares) or buffer A containing 1 µM plasmin and the following additions were layered on top of the fibrin polymer; 25% glycerol (filled circles); 25% glycerol and 1 µM AIIt (open circles); 50% glycerol and 1 µM AIIt (filled triangles); 1 µM AIIt (open triangles). Fibrinolysis was measured as described under "Experimental Procedures."

Effects of AIIt on Plasmin Degradation during Fibrinolysis-- It has been documented that plasmin autodegradation results in a loss in plasmin activity and degradation of the plasmin heavy (A) and light (B) chains. To establish if AIIt blocked fibrin clot lysis via stimulation of plasmin autodegradation, we analyzed the plasmin reaction products during fibrinolysis. Fibrinogen was incubated with thrombin, and, after 30 min, a stable fibrin clot was formed. Radioiodinated plasminogen was added to the clot, and fibrinolysis was initiated by addition of tPA. After 12 h, the reaction products were analyzed by SDS-PAGE. As shown in Fig. 2, in the presence of fibrin, tPA converted plasminogen into plasmin as illustrated by the appearance of the 55-kDa A chain and 25-kDa B chain of plasmin. In contrast, when AIIt was added to the reaction, degradation of both the plasmin A (H-Pm) and B (L-Pm) chain was apparent (Fig. 2). Densitometric quantitation of the autoradiogram showed that 11.8% of the A chain and 43.6% of the B chain of plasmin remained after incubation with 1 µM AIIt. Furthermore, in the presence of 5 µM AIIt, only 5.4% of the A chain and 35.1% of the B chain of plasmin remained (Fig. 2, inset). These results therefore establish that the inhibition of fibrin clot lysis by AIIt is due to its acceleration of plasmin autodegradation.


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Fig. 2.   Effects of AIIt on plasmin degradation during fibrinolysis. Thrombin (0.83 NIH units/ml) and fibrinogen (8 µM) were incubated at 25 °C in buffer A. After fibrin polymerization was complete (30 min), fibrin polymer lysis was initiated by the addition of 100 nM radioiodinated plasminogen and 2 nM tPA as described under "Experimental Procedures." Reactions were conducted in the presence of 1 µM (+) and 5 µM AIIt (++), respectively. After a 12-h incubation at 25 °C, 10 µl of each reaction mixture containing fibrin clot was dissolved with SDS sample buffer and subjected to SDS-PAGE, and the gel was vacuum-dried. Upper panel, pattern of plasminogen or plasmin degradation was detected by autoradiography. Lower panel, the band intensity of the autoradiogram was quantitated using a Hewlett Packard ScanJet 4c flatbed scanner and Adobe Photoshop 5.5. The lanes of the autoradiogram were aligned with a densitometric histogram.

Domains of AIIt Responsible for the Inhibition of Plasmin-dependent Fibrin Clot Lysis-- AIIt is a Ca2+-binding protein, and binding of Ca2+ to the p36 subunit regulates many of its biological activities (28, 46). The p36 subunit of AIIt contains five established Ca2+-binding sites. We utilized a recombinant AIIt mutant to establish if the Ca2+-binding sites of AIIt played a role in the inhibition of fibrin clot lysis. TCM AIIt, which has all five Ca2+-binding sites of the p36 subunit inactivated, significantly blocked plasmin-dependent fibrin clot lysis in a similar fashion to wild-type AIIt (Fig. 3A). This suggested that the five Ca2+-binding sites in the p36 subunit of AIIt do not play a role in the inhibitory effect of AIIt on fibrin clot lysis.


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Fig. 3.   Role of the Ca2+-binding sites of the p36 subunit and C-terminal lysines of the p11 subunit of AIIt in the inhibition of plasmin fibrinolytic activity. A, effects of Ca2+-binding site deletion mutant AIIt (TCM) on plasmin-dependent fibrin polymer lysis. Thrombin (0.83 NIH units/ml) and fibrinogen (8 µM) were incubated at 25 °C in buffer A. After fibrin polymerization was complete (30 min), 100 µl of buffer A without (open squares) or buffer A containing 1 µM plasmin was layered on top of the fibrin polymer in the absence (open circles) or presence of 2 µM wild-type AIIt (filled triangles), 2 µM TCM AIIt (open triangles), 5 µM TCM AIIt (filled squares). Fibrinolysis was measured as described under "Experimental Procedures." B, effects of p11 C terminus K-K deletion mutant AIIt (AIIt(K-K)-) on plasmin-dependent fibrin polymer lysis. Thrombin (0.83 NIH units/ml) and fibrinogen (8 µM) were incubated at 25 °C in buffer A. After fibrin polymerization was complete (30 min), 100 µl of buffer A without (filled circles) or buffer A containing 1 µM plasmin was layered on top of the fibrin polymer in the absence (open circles) or presence of 5 µM wild-type AIIt (open squares), 5 µM AIIt(K-K)- (open triangles) or 10 µM AIIt(K-K)- (filled squares). Fibrinolysis was measured as described under "Experimental Procedures."

AIIt binds plasminogen via the C-terminal lysine residues of its p11 subunit (38). Removal of the C-terminal lysines blocks the ability of AIIt to accelerate tPA-dependent plasminogen conversion to plasmin (38). To examine if the C-terminal lysines of the p11 subunit of AIIt participate in the antifibrinolytic activity of AIIt, we carried out plasmin-dependent fibrin clot lysis in the presence of recombinant mutant AIIt (AIIt(K-K)-) whose two p11 C-terminal lysine residues were deleted (Fig. 3B). The results show that, unlike the wild-type and TCM AIIt, AIIt(K-K)- did not block plasmin-dependent fibrin clot lysis. Therefore, these data suggested that the C-terminal lysine residues of AIIt play an important role in the inhibitory effect of AIIt on fibrin clot lysis.

Fibrin Binding Properties of AIIt-- We previously demonstrated that AIIt pelleted with a fibrin clot (34). This result prompted us to characterize the binding of AIIt to the fibrin polymer. A fibrin polymer was formed by incubation of fibrinogen and thrombin with radioiodinated AIIt. The fibrin clot was centrifuged, and the AIIt binding to the fibrin clot was quantitated. Fig. 4 shows the binding isotherm for the binding of AIIt to fibrin. AIIt bound fibrin polymer in a specific and saturable manner with a Kd of 269.4 ± 60.0 nM (mean ± S.D., n = 3). The binding approached a plateau at about 1.6 µM AIIt, which corresponded to about 0.28 mol of AIIt/mol of fibrin monomer. Scatchard analysis (Fig. 4, inset) established that the binding of AIIt to fibrin was mediated by a single, moderate affinity-binding site. The low stoichiometric binding of AIIt to fibrin could be due to steric hindrance. For example, it is possible that the binding sites in the dense areas of the clot may not be accessible to AIIt.


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Fig. 4.   AIIt binding to fibrin. AIIt was radioiodinated as described "Experimental Procedures." Fibrin polymerization was initiated by addition of thrombin (5 NIH units/ml) to a series of Eppendorf tubes, preheated to 37 °C, which contained 648 nM fibrinogen, 4.2 nM 125I-labeled AIIt, and various concentrations of non-labeled AIIt in buffer A. After incubation at 37 °C for 1 h, fibrin clots were pelleted by centrifugation at 10,000 rpm for 30 min at 4 °C. After three washes with buffer A, AIIt bound to the fibrin clot was calculated from radioactivity of clot. Inset, Scatchard plot analysis of the binding isotherm.

It was previously reported that heparin binds AIIt (47) and that it inhibits the association of AIIt with the fibrin polymer (34). As shown in Fig. 5A, heparin blocks the binding of AIIt to fibrin polymer. Inhibition of AIIt binding to fibrin by heparin was dose-dependent, and half-maximal inhibition occurred at 1.1 ± 0.2 µM heparin (mean ± S.D., n = 3).


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Fig. 5.   Analysis of the interaction of AIIt with fibrin. Fibrin was polymerized by incubation of 648 nM fibrinogen with thrombin (5 NIH units/ml) in the presence of 4.2 nM 125I-labeled AIIt and various concentrations of heparin (A), TCM AIIt (B), and AIIt(K-K)- (C). The binding was assessed as described under "Experimental Procedures."

To determine if the Ca2+-binding sites of the p36 subunit or the C-terminal lysines of the p11 subunit of AIIt played a role in fibrin binding, we investigated the ability of the AIIt mutants, TCM and AIIt(K-K)-, to dissociate radiolabeled wild-type AIIt from the fibrin clot. It was therefore expected that the recombinant mutant AIIt would compete with the radiolabeled wild-type AIIt, unless the mutation rendered the protein unable to bind to fibrin. Fig. 5, B and C, shows that both TCM AIIt and AIIt(K-K)- competitively inhibited wild-type AIIt binding to fibrin polymer with an IC50 values of about 260 or 330 nM, respectively. Therefore, neither the Ca2+-binding sites of AIIt nor the C-terminal lysine residues of the p11 subunit of AIIt mediate the binding of AIIt to the fibrin polymer.

Effects of Plasminogen on AIIt Binding to the Fibrin Polymer-- Plasminogen exists at micromolar concentration in blood. Because plasminogen binds to both fibrin and AIIt, we examined the binding of AIIt to the fibrin clot in the presence of plasminogen (Fig. 6). The binding of AIIt to fibrin polymer was increased by addition of plasminogen in a dose-dependent manner and approached a plateau at about 1 µM plasminogen. Half-maximal binding was obtained at ~200 nM plasminogen (Fig. 6, filled circles). This binding event was also examined in the presence of a lysine analogue, epsilon -ACA, which inhibits the interaction between AIIt and plasminogen. The addition of 10 mM epsilon -ACA blocked the increased AIIt binding to fibrin polymer mediated by plasminogen (Fig. 6, open circles). This suggests that the p11 C-terminal lysine residues of AIIt are involved in plasminogen-facilitated binding of AIIt to fibrin. This data also implies that binding of plasminogen to fibrin may influence the interaction of AIIt with fibrin polymer.


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Fig. 6.   Effects of plasminogen on AIIt binding to fibrin in the absence or presence of epsilon -ACA. Fibrin was polymerized by incubation of 648 nM fibrinogen with thrombin (5 NIH units/ml) in buffer A containing 4.2 nM 125I-labeled AIIt, 2 µM unlabeled AIIt, and various concentrations of plasminogen in the absence (filled circles) or presence (open circles) of 10 mM epsilon -ACA. The binding was assessed as described under "Experimental Procedures."

The interaction of AIIt with the fibrin polymer may therefore physiologically occur in at least one of two ways; its intrinsic binding ability (independent of the C-terminal lysine as shown in Fig. 5C), or through plasminogen-mediated additional binding capacity, which is governed by its p11 C-terminal lysine residues.

Acceleration of Plasminogen Activation by AIIt on Fibrin Surface-- AIIt has been reported to stimulate both the tPA-dependent conversion of plasminogen to plasmin as well as plasmin autodigestion in vitro. Although our data shows that, in the presence of fibrin stimulation of plasmin autodigestion is the predominant reaction, we could not rule out the possibility that AIIt might also stimulate tPA-dependent plasmin formation. Fig. 7A shows that fibrin accelerated tPA-dependent plasmin formation 7.8-fold. This fibrin-accelerated tPA-dependent plasmin formation was increased further 6.4 times by AIIt (Fig. 7A, inset). Analysis of the dose dependence of the stimulation of tPA-dependent plasmin formation by AIIt showed that about 0.58 ± 0.10 µM AIIt was required for half-maximal stimulation of plasmin formation (Fig. 7B). This concentration range of AIIt was similar to that required for half-maximal inhibition of plasmin-dependent fibrin polymer lysis. Collectively, our data suggest that AIIt may support the turnover of plasmin at the fibrin clot surface by stimulating both plasmin formation and destruction, although under our experimental conditions the latter reaction predominates.


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Fig. 7.   Stimulation of tPA-dependent plasminogen activation by AIIt on fibrin surface. A, time course of AIIt-dependent stimulation of plasmin formation on the fibrin clot. After a stable fibrin polymer was formed, tPA-dependent plasminogen (Pg) activation was examined in the absence (open circles) or presence (triangles) of 1 µM AIIt as described under "Experimental Procedures." Potential plasmin contamination in the plasminogen preparation was also measured (closed circles). Inset, the rate constant for the plasminogen activation was calculated from the slope of curve fitting of the data according to the equation A405 nm = B + K*t2, where K is the slope and B is the y intercept.t-PA-dependent plasminogen activation in the absence of fibrin was also examined as described above, and the rate of activation was compared with that obtained in the presence of fibrin. B, dose dependence of AIIt-stimulated plasmin formation on the fibrin clot. Initial rates of tPA-dependent Pg activation were determined in the presence of various concentrations of AIIt as described under "Experimental Procedures."

Immunohistochemical Identification of AIIt in a Physiological Blood Clot-- AIIt or annexin II has been shown by several groups to localize to the extracellular surface of variety of cells (31-33). However, whether or not AIIt associates with fibrin under physiological conditions has not been established. Accordingly, lung tissue obtained from a patient with a pulmonary thromboembolism was stained for annexin II and p11 (Fig. 8). The results show that both annexin II and p11 localize at contact areas of the fibrin clot and cell components. Thus, the association of AIIt with fibrin observed in vitro appears to be of physiological relevance.


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Fig. 8.   Immunohistochemical staining of p36 and p11 in a pathological blood clot. Human lung tissue was processed for immunohistochemical staining as described under "Experimental Procedures." The presence of the fibrin clots in the lung tissue is indicated by arrows and immunostaining of the p36 (A) and p11 (B) subunits of AIIt are indicated by arrowheads.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The formation of a fibrin polymer subsequent to blood vessel injury not only conserves blood constituents but also provides temporary matrices for blood vessel growth. However, the accumulation of a fibrin clot due to unbalanced hemostasis may lead to a variety of pathological disorders, including inflammation, arteriosclerosis, or stroke. During the formation of a solid blood clot, plasmin-mediated fibrinolysis is triggered by the actions of blood vessel endothelial cells growing on the fibrin substratum during the wound healing process. This process relies on the spatial and temporal regulation of plasmin activity within the fibrin clots.

Recently, annexin II, the 36-kDa subunit of AIIt, was shown to function as a receptor for both plasminogen and tPA on the surface of endothelial cells (22). Furthermore, it has recently been shown that AIIt, but not annexin II, inhibits plasmin-dependent fibrinolysis (34) as well as accelerates both tPA-dependent plasminogen activation (31) and subsequent plasmin autodegradation (45). In this report, we demonstrate that AIIt can bind to the fibrin clot in vitro and associates with the fibrin clot in vivo. Furthermore, we show that AIIt inhibits plasmin-dependent fibrin clot lysis by stimulation of plasmin autodegradation in vitro.

The ability of AIIt to inhibit the fibrinolytic activity of plasmin severely diminishes the concentration of active plasmin that is normally targeted for fibrin clot dissolution. This notion is supported by the finding that glycerol, an inhibitor of the plasmin autodegradation, inhibits the antifibrinolytic activity of AIIt (Fig. 1). The inhibition of plasmin-dependent fibrinolysis by AIIt also corresponded with plasmin heavy and light chain degradation (Fig. 2), suggesting a relationship between the antifibrinolytic activity of AIIt and AIIt-stimulated plasmin autodegradation. Although the stimulation of plasmin breakdown is the predominant reaction observed under our experimental condition, we have also shown that, in the presence of the fibrin clot, AIIt stimulates tPA-dependent plasmin formation. This suggests that the physiological role of AIIt may be to provide a transient pulse of plasmin activity on the fibrin surface.

We also examined two AIIt mutants to determine the potential role of the Ca2+-binding sites of the p36 subunit of AIIt or of the C-terminal lysine residues of p11 subunit of AIIt in its antifibrinolytic activity (Fig. 3). We first examined a mutant AIIt in which all five Ca2+-binding sites of the p36 subunit were inactivated (TCM AIIt). This mutant was chosen because Ca2+ is an essential cofactor in the coagulation cascade, and it was, therefore, reasonable to suspect that the Ca2+-binding sites of AIIt might play a role in its inhibition of fibrin clot lysis. However, as observed in Fig. 3A, the TCM AIIt also inhibited plasmin-dependent fibrinolysis. Thus, the Ca2+-binding sites of AIIt do not play a direct role in mediating its antifibrinolytic activity.

The second mutational analysis of AIIt involved the removal of the C-terminal lysine residues of the p11 subunit (AIIt(K-K)-). We have previously shown that removal of these key residues disables the ability of AIIt to stimulate tPA-dependent plasminogen activation (38). These residues are believed to be involved in the binding of kringle domains of plasminogen, plasmin, and tPA. As shown in Fig. 3B, this mutant AIIt does not inhibit plasmin-dependent fibrin clot lysis. This suggests that the C-terminal lysine residues of the p11 subunit of AIIt participate in both the stimulation of plasmin formation and destruction by AIIt. In the absence of fibrin the former reaction is favored, whereas, in the presence of fibrin, the latter reaction is favored.

The profound effect of fibrin on the regulatory properties of AIIt led us to examine in detail the interaction of AIIt with fibrin. First we found that AIIt bound fibrin with a Kd of 269.4 ± 60.0 nM (S.D., n = 1) (Fig. 4). Scatchard analysis reveals that a single saturable binding site is involved in this interaction (Fig. 4, inset). The low stoichiometry (0.28 mol of AIIt/mol of fibrin monomer) probably indicates that all of the AIIt binding sites in the artificial fibrin polymer formed in test tubes are not accessible to AIIt. Second, we found that the interaction of AIIt with fibrin was inhibited by heparin with an IC50 of 1.1 ± 0.2 µM (S.D., n = 1). The heparin binding region is located at residues 306-313 of the p36 subunit and may, therefore, be involved in the interaction of AIIt with fibrin (Fig. 5A). Third, using the TCM AIIt and the AIIt(K-K)- mutants, we found that the binding of AIIt to fibrin does not involve the Ca2+-binding sites of the p36 subunit nor the C-terminal lysine residue of the p11 subunit (Fig. 5, B and C). Fourth, we found that in the presence of plasminogen the binding of AIIt to fibrin was actually increased (Fig. 6). The simplest explanation for this observation was that AIIt bound to fibrin and to fibrin-associated plasminogen. This was an important finding, because it ruled out the possibility that AIIt and plasminogen competed for similar sites on the fibrin polymer. Fifth, we demonstrated the localization of AIIt to a thrombus in vivo. This established that under physiological conditions AIIt associated with fibrin (Fig. 8).

The binding of these components to the p11 subunit of AIIt localizes tPA, plasminogen, plasmin, and AIIt to the fibrin surface. It is therefore reasonable to assume that the mechanism by which the C-terminal lysines of the p11 subunit of AIIt stimulates plasmin autodegradation is due to the spatial localization of plasmin to the C-terminal lysine binding sites of AIIt thus resulting in a localized concentration of plasmin at these sites. It is also possible that the interaction of plasmin with the C-terminal lysines of the p11 subunit of AIIt may induce a conformational change in plasmin resulting in stimulation of plasmin autodegradation.

Our results suggest that the inhibition of plasmin-dependent fibrinolytic activity by AIIt requires both the interaction of AIIt with the plasminogen activation system (enzyme system) and with the fibrin polymer (substrate components). These binding events appear to localize tPA, plasminogen, plasmin, and AIIt to the fibrin surface. The demonstration of AIIt-stimulated tPA-dependent plasminogen activation on the fibrin surface suggests that AIIt may transiently increase plasmin activity on the fibrin surface (Fig. 7). It is clear that, in the presence of fibrin, AIIt stimulates both plasmin formation and destruction. However, under our experimental conditions, the AIIt-mediated destruction of plasmin predominates. We cannot, however, rule out the possibility that other regulators of AIIt may be present at or in the vicinity of the fibrin clot. These putative regulators could influence whether or not plasmin formation or destruction is the predominant reaction stimulated by AIIt.

The above results, along with previous findings, form the basis for a model of the mechanism of plasmin regulation by AIIt (Fig. 9). AIIt bound to fibrin is capable of accelerating the tPA-dependent formation of plasmin from plasminogen bound either to fibrin or AIIt itself. The ability of AIIt to bind fibrin, plasminogen, and tPA allows recruitment of plasminogen and tPA to the fibrin surface and thus spatially regulates plasmin production. Moreover, AIIt also accelerates the autodegradation of plasmin subsequent to its activation. The result is a tight regulation of plasmin activity on the fibrin surface. It is possible that the dual role of AIIt in plasmin formation and destruction provides the fibrin surface with a transient pulse of proteolytic activity. This allows for dissolution of the fibrin clot and prevents constitutive activation of plasminogen, which is also detrimental to hemostatic balance.


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Fig. 9.   Hypothetical scheme for regulation of plasmin activity on the fibrin surface. AIIt forms a complex with fibrin, plasminogen, and tPA and initially accelerates tPA-dependent plasminogen activation on the fibrin surface. Subsequently, AIIt accelerates plasmin autodegradation resulting in a loss of plasmin fibrinolytic activity.

In summary, the present report establishes that the ability of AIIt to inhibit plasmin-dependent fibrin clot lysis is due to its acceleration of plasmin autocatalytic activity and therefore the destruction of plasmin. Additionally, AIIt accelerates tPA-dependent plasmin formation, although under our experimental conditions plasmin destruction is the predominant reaction stimulated by AIIt. Furthermore, AIIt and plasminogen form a complex on the fibrin polymer, in vitro. The data also establish for the first time that AIIt is present on the surface of a fibrin clot in vivo. These novel findings suggest that AIIt may play a central role in regulation of plasmin activity on the fibrin surface.

    FOOTNOTES

* This work was supported by a grant from the Alberta Heart and Stroke Foundation.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.

|| To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, 3330 Hospital Dr. NW, Calgary, Alberta T2N 4N1, Canada. Tel.: 403-220-3022; Fax: 403-283-4841; E-mail: waisman@acs.ucalgary.ca.

Published, JBC Papers in Press, April 23, 2001, DOI 10.1074/jbc.M101426200

2 K.-S. Choi et al., manuscript in preparation..

    ABBREVIATIONS

The abbreviations used are: tPA, tissue-type plasminogen activator; AIIt, annexin II heterotetramer; epsilon -ACA, epsilon -aminocaproic acid; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; p-NA, p-nitroanilide.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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