Identification of annexin II heterotetramer as a plasmin reductase.

Annexin II heterotetramer (AIIt) is a Ca(2+)- and phospholipid-binding protein that consists of two copies of a p36 and p11 subunit. AIIt regulates the production and autoproteolysis of plasmin at the cell surface. In addition to its role as a key cellular protease, plasmin also plays a role in angiogenesis as the precursor for antiangiogenic proteins. Recently we demonstrated that the primary antiangiogenic plasmin fragment, called A(61) (Lys(78)-Lys(468)) was released from cultured cells. In the present study we report for the first time that AIIt possesses an intrinsic plasmin reductase activity. AIIt stimulated the reduction of the plasmin Cys(462)-Cys(541) bond in a time- and concentration-dependent manner, which resulted in the release of A(61) from plasmin. Mutagenesis of p36 C334S and either p11 C61S or p11 C82S inactivated the plasmin reductase activity of the isolated subunits, suggesting that specific cysteinyl residues participated in the plasmin reductase activity of each subunit. Furthermore, we demonstrated that the loss of AIIt from the cell surface of HT1080 cells transduced with a retroviral vector encoding p11 antisense dramatically reduced the cellular production of A(61) from plasminogen. This is the first demonstration that AIIt regulates the cellular production of the antiangiogenic plasminogen fragment, A(61).     

The p11 subunit of annexin II heterotetramer is regulated by basic carboxypeptidase

The Ca(2+)-dependent phospholipid-binding protein annexin II heterotetramer (AIIt) is composed of two copies of annexin II and a p11 dimer. The interaction of the carboxyl-terminal lysine residues of the p11 subunit of AIIt with the lysine-binding kringle domains of plasminogen is believed to play a key role in plasminogen binding and stimulation of the tPA-catalyzed cleavage of plasminogen to plasmin. In the current report, we show that AIIt-stimulated plasminogen activation is regulated by basic carboxypeptidases, in vitro. The incubation of AIIt with a 1/400 molar ratio of carboxypeptidase B for periods as short as 2 min resulted in a significant loss in AIIt-stimulated plasminogen activation. Carboxypeptidase B (CpB) as well as thrombin-activated fibrinolysis inhibitor (TAFIa) and carboxypeptidase N (CpN) rapidly reduced AIIt-stimulated plasminogen activation by 80%. The molar ratio of carboxypeptidase/AIIt for half-maximal inhibition of AIIt was 1/4700, 1/700, and 1/500 for CpB, TAFIa, and CpN, respectively. Treatment of AIIt with carboxypeptidase resulted in loss of both carboxyl-terminal lysine residues from the p11 subunit, which correlated with a decrease in the k(cat) and an increase in the K(m) for plasminogen activation. The data reveal a novel mechanism for the regulation of AIIt-stimulated plasminogen activation.     

Phospholipid-associated annexin A2-S100A10 heterotetramer and its subunits: characterization of the interaction with tissue plasminogen activator,plasminogen, and plasmin

Annexin A2 (p36) is a highly alpha-helical molecule that consists of two opposing sides, a convex side that contains the phospholipid-binding sites and a concave side, which faces the extracellular milieu and contains multiple ligand-binding sites. The amino-terminal region of annexin A2 extends along the concave side of the protein and contains the binding site for the S100A10 (p11) subunit. The interaction of these subunits results in the formation of the heterotetrameric form of the protein, annexin A2-S100A10 heterotetramer (AIIt). To simulate the orientation of AIIt on the plasma membrane we bound AIIt to a phospholipid bilayer that was immobilized on a BIAcore biosensor chip. Surface plasmon resonance was used to observe in real time the molecular interactions between phospholipid-associated AIIt or its annexin A2 subunit and the ligands, tissue-type plasminogen activator (t-PA), plasminogen, and plasmin. AIIt bound t-PA (Kd = 0.68 microm), plasminogen (Kd = 0.11 microm), and plasmin (Kd = 75 nm) with moderate affinity. Contrary to previous reports, the phospholipid-associated annexin A2 subunit failed to bind t-PA or plasminogen but bound plasmin (Kd = 0.78 microm). The S100A10 subunit bound t-PA (Kd = 0.45 microm), plasminogen (Kd = 1.81 microm), and plasmin (Kd = 0.36 microm). Removal of the carboxyl-terminal lysines from the S100A10 subunit attenuated t-PA and plasminogen binding to AIIt. These results show that the carboxyl-terminal lysines of S100A10 form t-PA and plasminogen-binding sites. In contrast, annexin A2 and S100A10 contain distinct binding sites for plasmin.     

Photoaffinity labeling of functionally different lysine-binding sites in human plasminogen and plasmin

Photoaffinity labeling of human plasmin using 4-azidobenzoylglycyl-L-lysine inhibits clot lysis activity, while the activity toward the active-site titrant, p-nitrophenyl-p'-guanidinobenzoate, or alpha-casein are maintained. Photoaffinity labeling of native Glu-plasminogen with the same reagent causes incorporation of approximately 1.5 mol label per mol plasminogen. This labeled plasminogen can be activated to plasmin by either urokinase or streptokinase. The resulting plasmin has full clot lysis activity and can be subsequently photoaffinity labeled with a loss of clot lysis activity. The rate of activation of labeled plasminogen by urokinase is increased relative to that of native plasminogen. epsilon-Aminocaproic acid blocks incorporation of photoaffinity label into both plasminogen and plasmin, indicating that the labeling is specific to the lysine-binding sites. The labels are located in the kringle 1+2+3 fragment in either photoaffinity-labeled plasminogen or plasmin. These results indicate that the specific lysine-binding site blocked in plasmin acts in concert with the active-site in binding and using fibrin as a substrate. This clot lysis regulating site is not available for labeling in plasminogen, but is exposed or changed upon activation to plasmin. The different lysine-binding sites labeled in plasminogen may regulate the conformation of the molecule as evidence by an enhanced rate of activation to plasmin.     

Tissue plasminogen activator and urokinase mediate the binding of Glu-plasminogen to plasma fibrin I. Evidence for new binding sites in plasmin-degraded fibrin I

The effect of tissue plasminogen activator (TPA) or urokinase on the specific binding of human Glu-plasminogen to fibrin I formed in plasma by clotting with Reptilase was studied using 125I-plasminogen and 131I-fibrinogen. In the absence of TPA, small amounts of plasminogen were bound to fibrin I. TPA induced binding of plasminogen to plasma fibrin I that was dependent upon the concentrations of TPA and plasminogen as well as upon the time of incubation. Plasminogen binding occurred in association with fibrin clot lysis and the formation in the clot supernatant of alpha 2-plasmin inhibitor-plasmin complexes. Urokinase also induced binding of plasminogen to plasma fibrin I that was concentration- and time-dependent. The molecular form of plasminogen bound to the fibrin I plasma clot was identified as Glu-plasminogen by dodecyl sulfate-polyacrylamide gel electrophoresis and by fast performance liquid chromatography. Further studies demonstrated that fibrin I formed from fibrinogen that had been progressively degraded by plasmin-bound Glu-plasminogen. The mole ratio of plasminogen bound increased with the time of plasmin digestion. Glu-plasminogen did not bind to fibrin I formed from fibrinogen progressively digested by human leukocyte elastase, thereby demonstrating the specificity of plasmin. These studies demonstrate that plasminogen activators regulate the binding of Glu-plasminogen to fibrin I by catalyzing plasmin-mediated modifications in the fibrin substrate.     

Thrombin-activable fibrinolysis inhibitor zymogen does not play a significant role in the attenuation of fibrinolysis

Activated thrombin-activable fibrinolysis inhibitor (TAFIa) plays a significant role in the prolongation of fibrinolysis. During fibrinolysis, plasminogen is activated to plasmin, which lyses a clot by cleaving fibrin after selected arginine and lysine residues. TAFIa attenuates fibrinolysis by removing the exposed C-terminal lysine residues. It was recently reported that TAFI zymogen possesses sufficient carboxypeptidase activity to attenuate fibrinolysis through a mechanism similar to TAFIa. Here, we show with a recently developed TAFIa assay that when thrombin is used to clot TAFI-deficient plasma supplemented with TAFI, there is some TAFI activation. The extent of activation was dependent upon the concentration of zymogen present in the plasma, and lysis times were prolonged by TAFIa in a concentration-dependent manner. Potato tuber carboxypeptidase inhibitor, an inhibitor of TAFIa but not TAFI, abolished the prolongation of lysis in TAFI-deficient plasma supplemented with TAFI zymogen. In addition, TAFIa but not TAFI catalyzed release of plasminogen bound to soluble fibrin degradation products. The data presented confirm that TAFI zymogen is effective in cleaving a small substrate but does not play a role in the attenuation of fibrinolysis because of its inability to cleave plasmin-modified fibrin degradation products.     

Regulation of plasmin activity by annexin II tetramer

Annexin II tetramer (AIIt) is a major Ca(2+)-binding protein of the endothelial cell surface which has been shown to stimulate the tissue plasminogen activator (t-PA)-dependent conversion of plasminogen to plasmin. In the present report, we have examined the regulation of plasmin activity by AIIt. The incubation of plasmin with AIIt resulted in a 95% loss in plasmin activity. SDS-PAGE analysis established that AIIt stimulated the autoproteolytic digestion of plasmin heavy and light chains. The kinetics of AIIt-stimulated plasmin autoproteolysis were first-order, suggesting that binding of plasmin to AIIt resulted in the spontaneous autoproteolysis of the bound plasmin. AIIt did not affect the activity of other serine proteases such as t-PA or urokinase-type plasminogen activator. Furthermore, other annexins such as annexin I, II, V, or VI did not stimulate plasmin autoproteolysis. Increasing the concentration of AIIt on the surface of human 293 epithelial cells increased cell-mediated plasmin autoproteolysis. Thus, in addition to stimulating the formation of plasmin, AIIt also promotes plasmin inactivation. These results therefore suggest that AIIt may function to provide the cell surface with a transient pulse of plasmin activity.     

Enhanced fibrinolysis by proteolysed coagulation factor Xa

We previously showed that coagulation factor Xa (FXa) enhances activation of the fibrinolysis zymogen plasminogen to plasmin by tissue plasminogen activator (tPA). Implying that proteolytic modulation occurs in situ, intact FXa (FXaα) must be sequentially cleaved by plasmin or autoproteolysis, producing FXaβ and Xa33/13, which acquire necessary plasminogen binding sites. The implicit function of Xa33/13 in plasmin generation has not been demonstrated, nor has FXaα/β or Xa33/13 been studied in clot lysis experiments. We now report that purified Xa33/13 increases tPA-dependent plasmin generation by at least 10-fold. Western blots confirmed that in situ conversion of FXaα/β to Xa33/13 correlated to enhanced plasmin generation. Chemical modification of the FXaα active site resulted in the proteolytic generation of a product distinct from Xa33/13 and inhibited the enhancement of plasminogen activation. Identical modification of Xa33/13 had no effect on tPA cofactor function. Due to its overwhelming concentration in the clot, fibrin is the accepted tPA cofactor. Nevertheless, at the functional level of tPA that circulates in plasma, FXaα/β or Xa33/13 greatly reduced purified fibrin lysis times by as much as 7-fold. This effect was attenuated at high levels of tPA, suggesting a role when intrinsic plasmin generation is relatively low. FXaα/β or Xa33/13 did not alter the apparent size of fibrin degradation products, but accelerated the initial cleavage of fibrin to fragment X, which is known to optimize the tPA cofactor activity of fibrin. Thus, coagulation FXaα undergoes proteolytic modulation to enhance fibrinolysis, possibly by priming the tPA cofactor function of fibrin.     

Annexin A2-S100A10 heterotetramer, a novel substrate of thioredoxin

The binding of plasminogen activators and plasminogen to the cell surface results in the rapid generation of the serine protease plasmin. Plasmin is further degraded by an autoproteolytic reaction, resulting in the release of an angiostatin, A61 (Lys78-Lys468). Previously, we demonstrated that the annexin A2-S100A10 heterotetramer (AIIt) stimulates the release of A61 from plasmin by promoting the autoproteolytic cleavage of the Lys468-Gly469 bond and reduction of the plasmin Cys462-Cys541 disulfide (Kwon, M., Caplan, J. F., Filipenko, N. R., Choi, K. S., Fitzpatrick, S. L., Zhang, L., and Waisman, D. M. (2002) J. Biol. Chem. 277, 10903-10911). Mechanistically, it was unclear if AIIt promoted a conformational change in plasmin, resulting in contortion of the plasmin disulfide, or directly reduced the plasmin disulfide. In the present study, we show that AIIt thiols are oxidized during the reduction of plasmin disulfides, establishing that AIIt directly participates in the reduction reaction. Incubation of HT1080 cells with plasminogen resulted in the rapid loss of thiol-specific labeling of AIIt by 3-(N-maleimidopropionyl)biocytin. The plasminogen-dependent oxidation of AIIt could be attenuated by thioredoxin. Thioredoxin reductase catalyzed the transfer of electrons from NADPH to the oxidized thioredoxin, thus completing the flow of electrons from NADPH to AIIt. Therefore, we identify AIIt as a substrate of the thioredoxin system and propose a new model for the role of AIIt in the redox-dependent processing of plasminogen and generation of an angiostatin at the cell surface.     

Molecular Aspects of Fibrin Clot Solubilization

THE human plasma protein, fibrinogen, is a disulphide bonded¹ dimer², each unit containing an Aα, Bβ and 8 chain^*, interconnected by disulphide bridges³. Thrombin (E.C.3.4.4.-13) releases fibrinopeptides A and B from the Aα and Bβ chains respectively⁴ to form fibrin monomer (α₂β₂γ₂) ? which polymerizes to form fibrin polymer or clotted fibrin. This polymer, following factor XIII (plasma transglutaminase, fibrin stabilizing factor) mediated crosslinking among the α chains and among the γ chains⁵, is one of the major and initiating constituents of a thrombus. Fibrinolytic activators, for example, streptokinase (SK) and urokinase (UK), are of thrombolytic value as they convert the thrombus plasminogen to plasmin (E.C.3.4.4.14) which by fibrinolytic action dissolves the thrombus. Whereas the interaction of fibrinogen and plasmin has been well studied^6–9, little is known concerning the mechanism of plasmin mediated fibrin clot lysis. I report here on the mechanism of non-cross-linked fibrin clot solubilization in near physiological conditions.     

Tissue-type plasminogen activator is a multiligand cross-beta structure receptor

Tissue-type plasminogen activator (tPA) regulates fibrin clot lysis by stimulating the conversion of plasminogen into the active protease plasmin. Fibrin is required for efficient tPA-mediated plasmin generation and thereby stimulates its own proteolysis. Several fibrin regions can bind to tPA, but the structural basis for this interaction is unknown. Amyloid beta (Abeta) is a peptide aggregate that is associated with neurotoxicity in brains afflicted with Alzheimer's disease. Like fibrin, it stimulates tPA-mediated plasmin formation. Intermolecular stacking of peptide backbones in beta sheet conformation underlies cross-beta structure in amyloid peptides. We show here that fibrin-derived peptides adopt cross-beta structure and form amyloid fibers. This correlates with tPA binding and stimulation of tPA-mediated plasminogen activation. Prototype amyloid peptides, including Abeta and islet amyloid polypeptide (IAPP) (associated with pancreatic beta cell toxicity in type II diabetes), have no sequence similarity to the fibrin peptides but also bind to tPA and can substitute for fibrin in plasminogen activation by tPA. Moreover, the induction of cross-beta structure in an otherwise globular protein (endostatin) endows it with tPA-activating potential. Our results classify tPA as a multiligand receptor and show that cross-beta structure is the common denominator in tPA binding ligands.     

Incorporation of fragment X into fibrin clots renders them more susceptible to lysis by plasmin

Bleeding, the most serious complication of thrombolytic therapy with tissue-type plasminogen activator (t-PA), is thought to result from lysis of fibrin in hemostatic plugs and from the systemic lytic state caused by unopposed plasmin. One mechanism by which systemic plasmin can impair hemostasis is by partially degrading fibrinogen to fragment X, a product that retains clottability but forms clots with reduced tensile strength that stimulate plasminogen activation by t-PA more than fibrin clots. The purpose of this study was to elucidate potential mechanisms by which fragment X accelerates t-PA-mediated fibrinolysis. In the presence of t-PA, clots containing fragment X were degraded faster than fibrin clots and exhibited higher rates of plasminogen activation. Although treatment with carboxypeptidase B, an enzyme that reduces plasminogen binding to fibrin, prolonged the lysis times of fragment X and fibrin clots, clots containing fragment X still were degraded more rapidly. Furthermore, plasmin or trypsin also degraded clots containing fragment X more rapidly than fibrin clots, suggesting that this effect is largely independent of plasminogen activation. Fragment X-derived degradation products were not preferentially released by plasmin from clots composed of equal concentrations of fibrinogen and fragment X, indicating that fragment X does not constitute a preferential site for proteolysis. These data suggest that structural changes resulting from incorporation of fragment X into clots promote their lysis. Thus, attenuation of thrombolytic therapy-induced fragment X formation may reduce the risk of bleeding.     

On the biological significance of the specific interaction between fibrin,plasminogen and antiplasmin

The influence of antiplasmin on the interaction between fibrin and plasminogen was studied in plasma and in a purified system. The amount of plasminogen bound to fibrin was quantitated using trace amounts of ¹²⁵I-labeled Glu-plasminogen (plasminogen with NH₂-terminal glutamic acid) or ¹²⁵I-labeled Lys-plasminogen (NH₂-terminal lysine).When whole plasma was clotted, 5.2% of Glu-plasminogen was associated with the fibrin clot. In plasma clotted in the presence of 20 mM 6-amino-hexanoic acid only 1.4% of the plasminogen was bound to fibrin, indicating that about 4% of the plasma plasminogen specifically binds to fibrin. With Lys-plasminogen these values were approximately twice as high.When antiplasmin-depleted plasma was used, only slightly higher amounts of both types of plasminogen were associated with the fibrin. The adsorbed plasminogen was not significantly eluted with plasma or with purified antiplasmin at physiological concentrations.These findings indicate that antiplasmin does not play a significant role in the inhibition of the binding of plasminogen to fibrin or the dissociation of the plasminogen · fibrin complex.These observations in conjunction with previous findings on the kinetics of the plasmin-antiplasmin reaction suggest that the lysine-binding site of plasminogen, which is responsible both for its interaction with fibrin and its interaction with antiplasmin, plays an important role in the very fast neutralization of plasmin formed in circulating blood and serves to attach plasminogen to fibrin and thereby sequestrate plasmin formed in loco from circulating antiplasmin.     

Structure and function of plasminogen/plasmin system

The main physiological function of plasmin is blood clot fibrinolysis and restoration of normal blood flow. To date, however, it became apparent that in addition to thrombolysis, the plasminogen/plasmin system plays an important physiological and pathological role in a number of other essential processes: degradation of the extracellular matrix, embryogenesis, cell migration, tissue remodeling, wound healing, angiogenesis, inflammation, and tumor cell migration. This review focuses on structural features of plasminogen, regulation of its activation by physiological plasminogen activators, inhibitors of plasmin, and plasminogen activators, and the role of plasminogen binding to fibrin, cellular receptors, and extracellular ligands in various functions performed by plasmin thus formed.     

The C terminus of annexin II mediates binding to F-actin

Annexin II heterotetramer (AIIt) is a multifunctional Ca(2+)-binding protein composed of two 11-kDa subunits and two annexin II subunits. The annexin II subunit contains the binding sites for anionic phospholipids, heparin, and F-actin, whereas the p11 subunit provides a regulatory function. The F-actin-binding site is presently unknown. In the present study we have utilized site-directed mutagenesis to create annexin II mutants with truncations in the C terminus of the molecule. Interestingly, a mutant annexin II lacking its C-terminal 16, 13, or 9 amino acids was unable to bind to F-actin but still retained its ability to interact with both anionic phospholipids and heparin. Recombinant AIIt, composed of wild-type p11 subunits and the mutant annexin II subunits, was also unable to bundle F-actin. This loss of F-actin bundling activity was directly attributable to the inability of mutant AIIt to bind F-actin. These results establish for the first time that the annexin II C-terminal amino acid residues, LLYLCGGDD, participate in F-actin binding.     

The effect of kringles K1-3, K4 and K5 on lysis of fibrin clots caused by the activation of Glu- and Lys-plasminogen by a tissue activator

Kringles K1-3, K4 and K5 are studied for their effect on tissue plasminogen activator-induced fibrin clot lysis in the presence of Glu- and Lys-plasminogen. It is established that kringles K4 and K5 inhibit fibrinolysis of Glu-plasminogen, and K1-3--that of Lys-plasminogen. The role of plasminogen molecule kringles in the plasminogen interaction with fibrin polymer is discussed.     

A fast-acting,modular-structured staphylokinase fusion with Kringle-1 from human plasminogen as the fibrin-targeting domain offers improved clot lysis efficacy

To develop a fast-acting clot dissolving agent, a clot-targeting domain derived from the Kringle-1 domain in human plasminogen was fused to the C-terminal end of staphylokinase with a linker sequence in between. Production of this fusion protein in Bacillus subtilis and Pichia pastoris was examined. The Kringle domain in the fusion protein produced from B. subtilis was improperly folded because of its complicated disulfide-bond profile, whereas the staphylokinase domain produced from P. pastoris was only partially active because of an N-linked glycosylation. A change of the glycosylation residue, Thr-30, to alanine resulted in a non-glycosylated biologically active fusion. The resulting mutein, designated SAKM3-L-K1, was overproduced in P. pastoris. Each domain in SAKM3-L-K1 was functional, and this fusion showed fibrin binding ability by binding directly to plasmin-digested clots. In vitro fibrin clot lysis in a static environment and plasma clot lysis in a flow-cell system demonstrated that the engineered fusion outperformed the non-fused staphylokinase. The time required for 50% clot lysis was reduced by 20 to 500% under different conditions. Faster clot lysis can potentially reduce the degree of damage to occluded heart tissues.     

Fucoidan-dependent conformational changes in annexin II tetramer

Fucoidan, a sulfated fucopolysaccharide, mimics the fucosylated glycans of glycoproteins and has therefore been used as a probe for investigating the role of membrane polysaccharides in cell-cell adhesion. In the present report we have characterized the interaction of fucoidan with the Ca(2+)- and phospholipid-binding protein annexin II tetramer (AIIt). AIIt bound to fucoidan with an apparent K(d) of 1.24 +/- 0.69 nM (mean +/- SD, n = 3) with a stoichiometry of 0.010 +/- 0.001 mol of fucoidan/mol of AIIt (mean +/- SD, n = 3). The binding of fucoidan to AIIt was Ca(2+)-independent. Furthermore, in the presence but not the absence of Ca(2+), the binding of fucoidan to AIIt caused a decrease in the alpha-helical content from 32% to 7%. A peptide corresponding to a region of the p36 subunit of AIIt, F(306)-S(313), which contains a Cardin-Weintraub consensus sequence for heparin binding, was shown to undergo a conformational change upon fucoidan binding. This suggests that heparin and fucoidan bound to this region of AIIt. The binding of fucoidan but not heparin by AIIt also inhibited the ability of AIIt to bind to and aggregate phospholipid liposomes. These results suggest that the binding of AIIt to the carbohydrate conjugates of certain membrane glycoproteins may have profound effects on the structure and biological activity of AIIt.     

Relationship of Plasminogen Activator to Fibrin

THERE are two principal theories of the mechanism of thrombus dissolution by the fibrinolytic system. Alkjaersig et al.¹ suggested that as fibrin polymerizes, plasminogen is adsorbed preferentially to the fibrin and is available in large quantities within a thrombus which is comparatively free of antiplasmin. When an activator enters the circulation it diffuses into the clot converting the plasminogen to plasmin in situ and so promotes lysis. Ambrus and Markus², however, proposed that when plasmin forms in the circulation naturally or during infusion of an activator it is normally bound to the excess antiplasmin present in blood. They suggested that this plasmin/antiplasmin complex is reversible and dissociates in the presence of fibrin, its preferred substrate, so allowing the plasmin to bring about fibrin dissolution by “external lysis”. Neither of these theories, however, is supported by an observed phenomena.