Glycosylation at Asn-184 inhibits the conversion of single-chain to two-chain tissue-type plasminogen activator by plasmin

Tissue-type plasminogen activator (tPA) is a glycosylated serine protease which is an effective thrombolytic agent. Native single-chain tPA (sc-tPA) is converted to two-chain tPA (tc-tPA) by plasmin, the product of the reaction of plasminogen with tPA. Native sc-tPA occurs as two glycoforms. Type I sc-tPA is fully glycosylated, while type II lacks glycosylation at Asn-184. The rates at which type I and type II human melanoma sc-tPA were converted to type I and type II tc-tPA by plasmin were determined by two different methods. In each case, the second-order rate constant (kcat/Km) for type II sc-tPA (approximately 8 microM-1 s-1) was about twice that for type I sc-tPA (approximately 4 microM-1 s-1). These results indicate that glycosylation at Asn-184 hinders the conversion of sc-tPA to tc-tPA and suggest that under physiological conditions type I sc-tPA may persist in the single-chain form longer than type II sc-tPA. Previous studies have shown that type I tc-tPA has a lower activity than type II tc-tPA and that sc-tPA has a lower activity and susceptibility to inhibition when compared to tc-tPA. The present work provides further evidence that tPA glycosylation serves to modulate activity. The two major glycoforms may represent more persistent but slow acting (type I) and less persistent but faster acting (type II) variants of tPA.     

Conformations of tissue plasminogen activator (tPA) orchestrate neuronal survival by a crosstalk between EGFR and NMDAR

Tissue-type plasminogen activator (tPA) is a pleiotropic serine protease of the central nervous system (CNS) with reported neurotrophic and neurotoxic functions. Produced and released under its single chain form (sc), the sc-tPA can be cleaved by plasmin or kallikrein in a two chain form, tc-tPA. Although both sc-tPA and tc-tPA display a similar fibrinolytic activity, we postulated here that these two conformations of tPA (sc-tPA and tc-tPA) could differentially control the effects of tPA on neuronal survival. Using primary cultures of mouse cortical neurons, our present study reveals that sc-tPA is the only one capable to promote N-methyl-D-aspartate receptor (NMDAR)-induced calcium influx and subsequent excitotoxicity. In contrast, both sc-tPA and tc-tPA are capable to activate epidermal growth factor receptors (EGFRs), a mechanism mediating the antiapoptotic effects of tPA. Interestingly, we revealed a tPA dependent crosstalk between EGFR and NMDAR in which a tPA-dependent activation of EGFRs leads to downregulation of NMDAR signaling and to subsequent neurotrophic effects.Tissue-type plasminogen activator (tPA) is secreted by endothelial cells and promotes fibrinolysis via the conversion of fibrin-bound plasminogen into plasmin.¹ Neurons and some glial cells also secrete tPA.^{2, 3, 4, 5} tPA is secreted as a single-chain form (sc-tPA), which can be processed into a two-chain form (tc-tPA) by plasmin or kallikreins.^{6, 7} Interestingly, sc-tPA is proteolytically active even without proteolytic processing. In addition to its vascular functions, tPA displays critical roles in the brain parenchyma with roles in cell migration, neuronal plasticity and survival,^{8, 9, 10, 11, 12, 13, 14} acting either as an enzyme or as a cytokine-like molecule. Among its actions, tPA is well described to promote neurotoxicity, likely through promotion of N-methyl-D-aspartate receptor (NMDAR) activity.^{15, 16, 17} Recently, we reported that only sc-tPA can promote NMDAR signaling and neurotoxicity.¹⁸ Interestingly, data from wild-type mice,¹⁹ transgenic mice overexpressing tPA in neurons²⁰ or in vitro²¹ also report neuroprotective effects of tPA.^{9, 10} The proposed mechanisms involved a tPA-dependent and non-proteolytic activation of either epidermal growth factor receptors (EGFRs)²² on oligodendrocytes or NMDARs.²⁰Here we explored a link between tPA conformations (sc-tPA and tc-tPA), EGFR- and NMDAR-dependent signaling pathways. Our findings identify sc-tPA as a selective positive modulator of NMDAR signaling in neurons when present at high concentrations and both sc-tPA and tc-tPA as positive modulators of EGFR signaling, this even at low concentrations. We also reveal a crosstalk between these two families of receptors, with the tPA-dependent activation of EGFRs reducing NMDAR signaling. By these mechanisms, sc-tPA and tc-tPA control neuronal death and survival.     

The mechanism of the reaction between human plasminogen-activator inhibitor 1 and tissue plasminogen activator.

The structural events taking place during the reaction between PAI-1 (plasminogen-activator inhibitor 1) and the plasminogen activators sc-tPA (single-chain tissue plasminogen activator) and tc-tPA (two-chain tissue plasminogen activator) were studied. Complexes were formed by mixing sc-tPA or tc-tPA with PAI-1 in slight excess (on an activity basis). The complexes were purified from excess PAI-1 by affinity chromatography on fibrin-Sepharose. Examination of the purified complexes by SDS/polyacrylamide-gel electrophoresis (SDS/PAGE) and N-terminal amino acid sequence analysis demonstrated that a stoichiometric 1:1 complex is formed between PAI-1 and both forms of tPA. Data obtained from both complexes revealed the amino acid sequences of the parent molecules and, in addition, a new sequence: Met-Ala-Pro-Glu-Glu-. This sequence is found in the C-terminal portion of the intact PAI-1 molecule and thus locates the reactive centre of PAI-1 to Arg346-Met347. The proteolytic activity of sc-tPA is demonstrated by its capacity to cleave the 'bait' peptide bond in PAI-1. The complexes were inactive and dissociated slowly at physiological pH and ionic strength, but rapidly in aq. NH3 (0.1 mol/l). Amidolytic tPA activity was generated on dissociation of the complexes, corresponding to 0.4 mol of tPA/mol of complex. SDS/PAGE of the dissociated complexes indicated a small decrease in the molecular mass of PAI-1, in agreement with proteolytic cleavage of the 'bait' peptide bond during complex-formation.     

Expression of a Novel Chimeric-Truncated tPA in Pichia pastoris with Improved Biochemical Properties

Thrombolytic therapy by plasminogen activators (PAs) has been a main goal in the treatment of acute myocardial infarction. Despite improved outcomes of currently available thrombolytic therapies, all these agents have different drawbacks that may result in less than optimal outcomes. In order to make tissue plasminogen activator (tPA) more potent, while being more resistant to plasminogen activator inhibitor-1 (PAI-1) and having a higher affinity to fibrin, a new chimeric-truncated form of tPA (CT tPA) was designed and expressed in Pichia pastoris. This novel variant consists of a finger domain of Desmoteplase, an epidermal growth factor (EGF) domain, a kringle 1 (K1) domain, a kringle 2 (K2) domain, in which the lysine binding site (LBS) was deleted, and a protease domain, where the four amino acids lysine 296, arginine 298, arginine 299, and arginine 304 were substituted by aspartic acid. The chimera CT tPA showed 14-fold increase in its activity in the presence of fibrin compared to the absence of fibrin. Furthermore, CT tPA showed about 10-fold more potency than commercially available full-length tPA (Actylase^®) and provided 1.2-fold greater affinity to fibrin. A residual activity of only 68 % was observed after incubation of Actylase^® with PAI-1, however, 91 % activity remained for CT tPA. These promising findings suggest that the novel CT tPA variant might be an acceptable PA with superior characteristics and properties.     

The effect of polymerised fibrin on the catalytic activities of one-chain tissue-type plasminogen activator as revealed by an analogue resistant to plasmin cleavage

A one-chain recombinant tissue-type plasminogen activator (EC 2.4.31.-) (tPA) analogue was constructed in which Arg-275 of the activation site was changed to Gly by site-directed mutagenesis. This analogue, tPA-Gly275, was very resistant to plasmin (EC 2.4.21.5) cleavage. It has been used to gain information about the activity of the uncleaved one-chain tPA form, also when plasmin is generated as a result of a plasminogen activation reaction. The amidolytic activity of tPA-Gly275 with less than Glu-Gly-Arg-pNA was investigated and compared to that of one-chain and two-chain wild-type recombinant tPA. A small but significant intrinsic amidolytic activity was observed with the analogue as well as the wild-type one-chain tPA form. However, it was much lower than that of two-chain tPA. Polymerised fibrin enhanced the amidolytic activity of both one-chain tPA forms but not of two-chain tPA. Measurements of the plasminogen activation kinetics in the absence of fibrin revealed that tPA-Gly275 possessed a significant intrinsic activity. However, it was 30-fold lower than that of two-chain tPA. Addition of polymerised fibrin profoundly enhanced the plasminogen activation rate of both tPA-Gly275 and wild-type one- and two-chain tPA to approximately the same maximal level. The results were interpreted to mean that fibrin binding can induce an activated state of the intact tPA one-chain form.     

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.     

Inhibitory effect of angiostatins on activity of the plasminogen/plasminogen activator system

Angiostatins, kringle-containing fragments of plasminogen, are potent inhibitors of angiogenesis. Effects of three angiostatin forms, K1–3, K1–4, and K1-4.5 (0–2 μM), on the rate of native Glu-plasminogen activation by its physiological activators in the absence or presence of soluble fibrin were investigated in vitro. Angiostatins did not affect the intrinsic amidolytic activities of plasmin and plasminogen activators of tissue type (tPA) and urokinase type (single-chain scuPA and two-chain tcuPA), but inhibited conversion of plasminogen to plasmin in a dose-dependent manner. All three angiostatins suppressed Glu-plasminogen activation by tcuPA independently of the presence of fibrin, and the inhibitory effect increased in the order: K1-3 < K1-4 < K1-4.5. The inhibitory effects of angiostatins on the scuPA activator activity were lower and further decreased in the presence of fibrin. Angiostatin K1-3 (up to 2 μM) had no effect, while 2 μM angiostatins K1-4 and K1-4.5 inhibited the fibrin-stimulated Glu-plasminogen activation by tPA by 50 and 100%, respectively. The difference in effects of the three angiostatins on the Glu-plasminogen activation by scuPA, tcuPA, and tPA in the absence or presence of fibrin is due to the differences in angiostatin structures, mechanisms of action, and fibrin-specificity of plasminogen activators, as well as due to the influence of fibrin on the Glu-plasminogen conformation. Angiostatins in vivo, which mimic plasminogen-binding activity, can inhibit plasminogen activation stimulated by various proteins (including fibrin) of extracellular matrix, thereby blocking cell migration and angiogenesis. The data of this work indicate that the inhibition of Glu-plasminogen activation under the action of physiological plasminogen activators by angiostatins can be implicated in the complex mechanism of their antiangiogenic and antitumor action.     

Expression of Active Human Tissue-Type Plasminogen Activator in Escherichia coli

The formation of native disulfide bonds in complex eukaryotic proteins expressed in Escherichia coli is extremely inefficient. Tissue plasminogen activator (tPA) is a very important thrombolytic agent with 17 disulfides, and despite numerous attempts, its expression in an active form in bacteria has not been reported. To achieve the production of active tPA in E. coli, we have investigated the effect of cooverexpressing native (DsbA and DsbC) or heterologous (rat and yeast protein disulfide isomerases) cysteine oxidoreductases in the bacterial periplasm. Coexpression of DsbC, an enzyme which catalyzes disulfide bond isomerization in the periplasm, was found to dramatically increase the formation of active tPA both in shake flasks and in fermentors. The active protein was purified with an overall yield of 25% by using three affinity steps with, in sequence, lysine-Sepharose, immobilized Erythrina caffra inhibitor, and Zn-Sepharose resins. After purification, approximately 180 μg of tPA with a specific activity nearly identical to that of the authentic protein can be obtained per liter of culture in a high-cell-density fermentation. Thus, heterologous proteins as complex as tPA may be produced in an active form in bacteria in amounts suitable for structure-function studies. In addition, these results suggest the feasibility of commercial production of extremely complex proteins in E. coli without the need for in vitro refolding.     

Tissue Plasminogen Activator Coating on Implant Surfaces Reduces Staphylococcus aureus Biofilm Formation

Staphylococcus aureus biofilm infections of indwelling medical devices are a major medical challenge because of their high prevalence and antibiotic resistance. As fibrin plays an important role in S. aureus biofilm formation, we hypothesize that coating of the implant surface with fibrinolytic agents can be used as a new method of antibiofilm prophylaxis. The effect of tissue plasminogen activator (tPA) coating on S. aureus biofilm formation was tested with in vitro microplate biofilm assays and an in vivo mouse model of biofilm infection. tPA coating efficiently inhibited biofilm formation by various S. aureus strains. The effect was dependent on plasminogen activation by tPA, leading to subsequent local fibrin cleavage. A tPA coating on implant surfaces prevented both early adhesion and later biomass accumulation. Furthermore, tPA coating increased the susceptibility of biofilm infections to antibiotics. In vivo, significantly fewer bacteria were detected on the surfaces of implants coated with tPA than on control implants from mice treated with cloxacillin. Fibrinolytic coatings (e.g., with tPA) reduce S. aureus biofilm formation both in vitro and in vivo, suggesting a novel way to prevent bacterial biofilm infections of indwelling medical devices.     

The Omptins of Yersinia pestis and Salmonella enterica Cleave the Reactive Center Loop of Plasminogen Activator Inhibitor 1

Plasminogen activator inhibitor 1 (PAI-1) is a serine protease inhibitor (serpin) and a key molecule that regulates fibrinolysis by inactivating human plasminogen activators. Here we show that two important human pathogens, the plague bacterium Yersinia pestis and the enteropathogen Salmonella enterica serovar Typhimurium, inactivate PAI-1 by cleaving the R346-M347 bait peptide bond in the reactive center loop. No cleavage of PAI-1 was detected with Yersinia pseudotuberculosis, an oral/fecal pathogen from which Y. pestis has evolved, or with Escherichia coli. The cleavage and inactivation of PAI-1 were mediated by the outer membrane proteases plasminogen activator Pla of Y. pestis and PgtE protease of S. enterica, which belong to the omptin family of transmembrane endopeptidases identified in Gram-negative bacteria. Cleavage of PAI-1 was also detected with the omptins Epo of Erwinia pyrifoliae and Kop of Klebsiella pneumoniae, which both belong to the same omptin subfamily as Pla and PgtE, whereas no cleavage of PAI-1 was detected with omptins of Shigella flexneri or E. coli or the Yersinia chromosomal omptins, which belong to other omptin subfamilies. The results reveal a novel serpinolytic mechanism by which enterobacterial species expressing omptins of the Pla subfamily bypass normal control of host proteolysis.Plasminogen activator inhibitor 1 (PAI-1) is a key regulator of the mammalian fibrinolytic/plasminogen system (29, 37). The fibrinolytic system comprises the serine protease zymogen plasminogen, urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), PAI-1, and plasmin inhibitor α₂-antiplasmin (α₂AP) (for a review, see reference 52). Plasminogen is converted to plasmin, which is a broad-spectrum serine protease that dissolves fibrin in blood clots, degrades laminin of basement membranes, and activates matrix metalloproteinases that degrade collagens and gelatins in tissue barriers. Herewith, plasmin controls physiological processes such as fibrinolysis/coagulation, cell migration and invasion, and tumor metastasis (29, 37). PAI-1 maintains normal hemostasis by inhibiting the function of the plasminogen activators tPA and uPA, which are serine proteases and highly specific for cleavage of the plasminogen molecule. tPA binds to fibrin and is associated with plasmin-mediated breakdown of fibrin clots, whereas uPA has low affinity for fibrin and associates with cell surface proteolysis, cellular migration, and damage of tissue barriers (52).The mammalian fibrinolytic and coagulation systems are targeted by invasive bacterial pathogens during infection (reviewed in references 6, 11, 34, and 61). In bacterial sepsis, increased production of fibrin clots at a damaged endothelium results from enhanced thrombin-catalyzed fibrin generation and from an increased serum level of PAI-1. Coagulation can protect the host by activating immune systems or by physically restraining the bacteria (6, 15, 25, 41). On the other hand, several invasive bacterial pathogens enhance fibrinolysis either through direct plasminogen activation or by immobilizing plasminogen/plasmin on the surface (6, 34, 61). Activation of the plasminogen system by bacteria enhances bacterial dissemination and invasiveness through release of bacteria from fibrin deposits and through degradation of tissue barriers. Bacterial plasminogen activators and receptors have been under extensive structural and functional studies, but much less is known about interactions of bacteria with the regulatory proteins of fibrinolysis.PAI-1 is present in a large variety of tissues and is secreted by several human cells (37). In healthy individuals, the level of PAI-1 antigen in human plasma is low (6 to 85 ng/ml), but synthesis and secretion of PAI-1 are strongly elevated in disease states and induced by, e.g., inflammatory cytokines and endotoxin of Gram-negative bacteria (37). PAI-1 is a serine protease inhibitor (serpin), which exists in two forms. In its active form, PAI-1 rapidly inactivates both tPA and uPA by forming a covalent bond between the hydroxyl group of a catalytic serine residue of tPA/uPA and the carboxyl group of the residue R346 at the reactive center loop (RCL) of PAI-1 (52). The RCL of PAI-1 is a 19-amino-acid-long flexible loop which inserts into the catalytic center of tPA/uPA and contains the “bait” residues R346 and M347, which mimic the normal target of tPA/uPA. PAI-1 induces distortion of the active site of tPA/uPA, which prevents completion of the catalytic cycle (70). The active form of PAI-1 is unstable, with a half-life of 2 to 3 h at 37°C, and it changes spontaneously and irreversibly into a latent form, where the RCL is incorporated into a central β-sheet of the PAI-1 molecule and therefore cannot react with tPA or uPA. This conformational change takes place also after proteolytic cleavage of PAI-1 at the R346-M347 bond. The active form of PAI-1 binds with high affinity to vitronectin (Vn), and PAI-1/Vn complex formation increases the half-life of PAI-1 2- to 4-fold (10, 46, 69). Most circulating PAI-1 is thought to be in a complex with Vn, and the complex serves as the reservoir of physiologically active PAI-1 (44).Plague disease caused by Yersinia pestis is associated with imbalance of the fibrinolytic system, and decreased fibrin(ogen) deposition has been observed in both bubonic and pneumonic plague (11, 36). The plasminogen activator Pla, which is encoded by a Y. pestis-specific 9.5-kb virulence plasmid, pPCP1 (59), does not degrade fibrin directly but mimics the action of tPA and uPA in converting plasminogen to plasmin by cleavage at R561-V562. Pla also degrades the serpin α₂AP and thus creates uncontrolled plasmin activity (32, 60). Pla belongs to the omptin superfamily of bacterial β-barrel outer membrane proteases (for reviews of omptins, see references 21 and 23). The omptins share molecular size and transmembrane fold but differ markedly in their substrate selectivities. In their catalytic centers, omptins combine structural features of aspartic and serine proteases (66).Increased fibrinolysis observed in plague led us to investigate whether Y. pestis increases plasminogen activation also indirectly by controlling the activity of PAI-1. We compared Y. pestis to Salmonella enterica serovar Typhimurium and Yersinia pseudotuberculosis, and the study also included omptins of other enterobacterial species.     

Cloning,Expression and Characterization of a Gene from Earthworm Eisenia fetida Encoding a Blood-Clot Dissolving Protein

A lumbrokinase gene encoding a blood-clot dissolving protein was cloned from earthworm (Eisenia fetida) by RT-PCR amplification. The gene designated as CST1 (GenBank No. {"type":"entrez-nucleotide","attrs":{"text":"AY840996","term_id":"56718408","term_text":"AY840996"}}AY840996) was sequence analyzed. The cDNA consists of 888 bp with an open reading frame of 729 bp, which encodes 242 amino acid residues. Multiple sequence alignments revealed that CST1 shares similarities and conserved amino acids with other reported lumbrokinases. The amino acid sequence of CST1 exhibits structural features similar to those found in other serine proteases, including human tissue-type (tPA), urokinase (uPA), and vampire bat (DSPAα1) plasminogen activators. CST1 has a conserved catalytic triad, found in the active sites of protease enzymes, which are important residues involved in polypeptide catalysis. CST1 was expressed as inclusion bodies in Escherichia coli BL21(DE3). The molecular mass of recombinant CST1 (rCST) was 25 kDa as estimated by SDS–PAGE, and further confirmed by Western Blot analysis. His-tagged rCST1 was purified and renatured using nickel-chelating resin with a recovery rate of 50% and a purity of 95%. The purified, renatured rCST1 showed fibrinolytic activity evaluated by both a fibrin plate and a blood clot lysis assay. rCST1 degraded fibrin on the fibrin plate. A significant percentage (65.7%) of blood clot lysis was observed when blood clot was treated with 80 mg/mL of rCST1 in vitro. The antithrombotic activity of rCST1 was 912 units/mg calculated by comparison with the activity of a lumbrokinase standard. These findings indicate that rCST1 has potential as a potent blood-clot treatment. Therefore, the expression and purification of a single lumbrokinase represents an important improvement in the use of lumbrokinases.     

Mechanism of the inhibitory effect of angiostatin on plasminogen activation by its physiologic activators

The influence of angiostatin K1-4.5, a fragment of the heavy chain of plasmin and a powerful inhibitor of angiogenesis, on kinetic parameters (k _Pg and K _Pg) of human Glu-plasminogen activation under the action of urokinase (uPA) not having affinity for fibrin and fibrin-specific tissue plasminogen activator (tPA) was investigated. Angiostatin does not affect on the k _Pg value, but increases the value of K _Pg plasminogen activation by urokinase. A decrease in the k _Pg value and an increase in the K _Pg value were found for fibrin-stimulated plasminogen activation by tPA with increasing concentrations of angiostatin. The obtained results show that angiostatin is a competitive inhibitor of the uPA activator activity, while it inhibits the activator activity of tPA with a mixed type. Such an influence of angiostatin on the kinetic constants of the plasminogen activation by urokinase suggests that angiostatin dose-dependent manner replaces plasminogen in the binary enzyme-substrate complex uPA-Pg. In the case of fibrin-stimulated plasminogen activation by tPA, both zymogen and tPA are bound to fibrin with the formation of the effective triple tPA-Pg-fibrin complex. Angiostatin replaces plasminogen both from the fibrin surface and from the enzyme-substrate tPA-Pg complex, which leads to a decrease in k _Pg and an increase in K _Pg of the plasminogen activation. Inhibition constants by angiostatin (K _i) of plasminogen-activator activities of uPA and tPA determined by the Dixon method were found to be 0.59 ± 0.04 and 0.12 ± 0.05 μM, respectively.     

Fibrinogen–fibrin conversion. The mechanism of fibrin-polymer formation in solution

The fibrin polymers formed in solution during the earliest phase of the fibrinogen–fibrin conversion are shown to be stable soluble molecules at pH7.4 and 0.15m- or 0.3m-NaCl. The various sequential soluble fibrin polymers produced from the fibrinogen–thrombin reaction can be observed by gel chromatography and can be isolated for characterization. The mechanism of fibrin polymerization proposed from the present studies suggests that the initial event is the thrombin activation at only one of the Aα-chains in fibrinogen. The resulting highly reactive intermediate is the true fibrin monomer and it rapidly, and irreversibly, self-associates to form the stable fibrin dimer (s_20.w=12S). Fibrin dimer possesses the N-terminal pattern alanine/glycine/tyrosine (1:1:2) per 340000 molecular weight, and possesses the chain structure [(α)Aα)(Bβ)₂(γ)₂]₂. The fibrin dimer is a soluble inert molecule, but additional thrombin activation of its remaining intact Aα-chains leads to new associations into larger inert soluble fibrin polymers. In this manner progressively larger fibrin oligomers are constructed with thrombin continually in control of the process because of the necessity to repeatedly re-activate the various fibrin polymers in solution. The inert character of the soluble fibrin polymers can be explained by the reciprocal alignment of the associating molecules, which mutually consumes their active surfaces and leaves an intact Aα-chain at either end of each fibrin oligomer. The soluble fibrin polymers will proceed to further association only if thrombin activates these remaining Aα-chains, otherwise the fibrin molecules are stable indefinitely. The intermolecular associations within the soluble fibrin polymers are essentially irreversible under these nearly physiological conditions. However, the bonding is not covalent. This mechanism accounts for the clinical observations of stable fibrinogen-derived polymers in the plasma from patients undergoing thrombotic processes. Since it is shown that the intermediate fibrin polymers, themselves, are stable soluble molecules, it is no longer necessary, nor warranted, to invoke hypothetical `fibrinogen–fibrin complexes' to explain observations of fibrin solubility.     

Secondary-site binding of Glu-plasmin, Lys-plasmin and miniplasmin to fibrin

Active-site-inhibited plasmin was prepared by inhibition with d-valyl-l-phenylalanyl-l-lysylchloromethane or by bovine pancreatic trypsin inhibitor (Kunitz inhibitor). Active-site-inhibited Glu-plasmin binds far more strongly to fibrin than Glu-plasminogen [native human plasminogen with N-terminal glutamic acid (residues 1–790)]. This binding is decreased by α₂-plasmin inhibitor and tranexamic acid, and is, in the latter case, related to saturation of a strong lysine-binding site. In contrast, α₂-plasmin inhibitor and tranexamic acid have only weak effects on the binding of Glu-plasminogen to fibrin. This demonstrates that its strong lysine-binding site is of minor importance to its binding to fibrin. Active-site-inhibited Lys-plasmin and Lys-plasminogen (Glu-plasminogen lacking the N-terminal residues Glu¹–Lys⁷⁶, Glu¹–Arg⁶⁷ or Glu¹–Lys⁷⁷)display binding to fibrin similar to that of active-site inhibited Glu-plasmin. In addition, α₂-plasmin inhibitor or tranexamic acid similarly decrease their binding to fibrin. Glu-plasminogen and active-site-inhibited Glu-plasmin have the same gross conformation, and conversion into their respective Lys- forms produces a similar marked change in conformation [Violand, Sodetz & Castellino (1975) Arch. Biochem. Biophys. 170, 300–305]. Our results indicate that this change is not essential to the degree of binding to fibrin or to the effect of α₂-plasmin inhibitor and tranexamic acid on this binding. The conversion of miniplasminogen (Glu-plasminogen lacking the N-terminal residues Glu¹–Val⁴⁴¹) into active-site-inhibited miniplasmin makes no difference to the degree of binding to fibrin, which is similarly decreased by the addition of tranexamic acid and unaffected by α₂-plasmin inhibitor. Active-site-inhibited Glu-plasmin, Lys-plasmin and miniplasmin have lower fibrin-binding values in a plasma system than in a purified system. Results with miniplasmin(ogen) indicate that plasma proteins other than α₂-plasmin inhibitor and histidine-rich glycoprotein decrease the binding of plasmin(ogen) to fibrin.     

Y box-binding protein-1 binds preferentially to single-stranded nucleic acids and exhibits 3'-->5' exonuclease activity

We have previously shown that Y box-binding protein-1 (YB-1) binds preferentially to cisplatin-modified Y box sequences. Based on structural and biochemical data, we predicted that this protein binds single-stranded nucleic acids. In the present study we confirmed the prediction and also discovered some unexpected functional features of YB-1. We found that the cold shock domain of the protein is necessary but not sufficient for double-stranded DNA binding while the C-tail domain interacts with both single-stranded DNA and RNA independently of the cold shock domain. In an in vitro translation system the C-tail domain of the protein inhibited translation but the cold shock domain did not. Both in vitro pull-down and in vivo co-immunoprecipitation assays revealed that YB-1 can form a homodimer. Deletion analysis mapped the C-tail domain of the protein as the region of homodimerization. We also characterized an intrinsic 3′→5′ DNA exonuclease activity of the protein. The region between residues 51 and 205 of its 324-amino acid extent is required for full exonuclease activity. Our findings suggest that YB-1 functions in regulating DNA/RNA transactions and that these actions involve different domains.     

Stimulation of human 8-oxoguanine-DNA glycosylase by AP-endonuclease: potential coordination of the initial steps in base excision repair

8-Oxoguanine-DNA glycosylase 1 (OGG1), with intrinsic AP lyase activity, is the major enzyme for repairing 7,8-dihydro-8-oxoguanine (8-oxoG), a critical mutagenic DNA lesion induced by reactive oxygen species. Human OGG1 excised the damaged base from an 8-oxoG·C-containing duplex oligo with a very low apparent k_cat of 0.1 min^–1 at 37°C and cleaved abasic (AP) sites at half the rate, thus leaving abasic sites as the major product. Excision of 8-oxoG by OGG1 alone did not follow Michaelis–Menten kinetics. However, in the presence of a comparable amount of human AP endonuclease (APE1) the specific activity of OGG1 was increased ~5-fold and Michaelis–Menten kinetics were observed. Inactive APE1, at a higher molar ratio, and a bacterial APE (Nfo) similarly enhanced OGG1 activity. The affinity of OGG1 for its product AP·C pair (K_d ~ 2.8 nM) was substantially higher than for its substrate 8-oxoG·C pair (K_d ~ 23.4 nM) and the affinity for its final β-elimination product was much lower (K_d ~ 233 nM). These data, as well as single burst kinetics studies, indicate that the enzyme remains tightly bound to its AP product following base excision and that APE1 prevents its reassociation with its product, thus enhancing OGG1 turnover. These results suggest coordinated functions of OGG1 and APE1, and possibly other enzymes, in the DNA base excision repair pathway.     

High resolution analysis of functional determinants on human tissue-type plasminogen activator

Sixty-four variants of human tissue-type plasminogen activator (tPA) were produced using recombinant DNA techniques. Charged residues were converted to alanine in clusters of from one to four changes per variant; these clusters spanned all the domains of the molecule. The variants were expressed by mammalian cells and were analyzed for a variety of properties. Variants of tPA were found that had reduced activity with respect to each tested property; in a few cases increased activity was observed. Analysis of these effects prompted the following conclusions: 1) charged residues in the nonprotease domains are less involved in fibrin stimulation of tPA activity than those in the protease domain, and it is possible to increase the fibrin specificity (i.e. the stimulation of tPA activity by fibrin compared to fibrinogen) by mutations at several sites in the protease domain; 2) the difference in enzymatic activity between the one- and two-chain forms of tPA can be increased by mutations at several sites on the protease domain; 3) binding of tPA to lysine-Sepharose was affected only by mutations to kringle-2, whereas binding to fibrin was affected most by mutations in the other domains; 4) clot lysis was influenced by mutations in all domains except kringle-2; 5) sensitivity to plasminogen activator inhibitor-1 seems to reside exclusively in the region surrounding residue 300. A model of the tPA protease domain has been used to map some of the critical residues and regions.     

Fibrin Targeting of Echogenic Liposomes with Inactivated Tissue Plasminogen Activator

Fibrin-specific molecular targeting strategies are desirable for site-specific imaging and treatment of late stage atheroma, but fibrin-specific antibodies are difficult to produce and present immunogenicity problems. Tissue plasminogen activator (tPA) is an endogenous protein that has been shown to bind fibrin with high affinity and may circumvent antibody difficulties. Use of tPA-derived proteins or peptides, however, requires that the plasminogen-activating proteolytic activity be neutralized or removed. As an initial step in determining the feasibility of this targeting strategy, human recombinant tPA (Activase®) was irreversibly inhibited with D-phe-L-pro-L-arg-chloromethyl ketone (PPACK) and conjugated to intrinsically echogenic liposomes (ELIP) by a thioether coupling protocol. Fibrin-binding affinities were assessed with a novel two‐stage fibrin pad ELISA. We achieved 95–99% inactivation, while retaining both tPA fibrin-binding activities of K_D ～ 2 nM and 33 nM. Thermodynamic analysis of the PPACK-inactivated tPA (tPA(P)) revealed highly exothermic interactions, indicative of ionic associations, especially for the higher affinity. The conjugation efficiency of tPA(P) to ELIP was within the range of that previously achieved for IgG and exhibited satisfactory fibrin targeting, characterized by striking increases of enthalpy and entropy increments. Evidence for coupling of noncovalent association energetics with the phosphatidylethanolamine major phase transition, observed in previous IgG antibody conjugations, was also evident in this case, but the nature of the transduction mechanism was different. These results demonstrate that tPA-derived components lacking proteolytic activity can be employed as fibrin-targeting agents for delivery of therapeutic and diagnostic formulations.     

Rat oocyte tissue plasminogen activator is a catalytically efficient enzyme in the absence of fibrin. Endogenous potentiation of enzyme activity

Rat oocytes synthesize tissue plasminogen activator (tPA) in response to stimuli which initiate meiotic maturation. Purified tPA exhibits optimal activity only in the presence of fibrin or fibrin substitutes. Because oocytes are not exposed to fibrin in situ, we investigated the possible stimulation of rat oocyte tPA activity by other endogenous factor(s). Oocytes were obtained from immature female rats which were induced to ovulate with gonadotropins. tPA activity was measured by the plasminogen-dependent cleavage of a chromogenic substrate. Measurements of kinetic parameters with Glu- or Lys-plasminogen revealed a Km for the rat oocyte enzyme of 1.3-2.1 microM compared with 23-24 microM for purified human tPA. Inclusion of the soluble fibrin substitute polylysine lowered the Km of human tPA by 30-fold (0.8 microM) but had no effect on the oocyte tPA Km. Polylysine had no significant effect on the Vmax values. The rate of plasminogen activation catalyzed by oocyte tPA was increased only 4.3-fold by fibrin while fibrin stimulated purified human tPA activity by 15.2-fold. After fractionation of oocyte extract by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, polylysine enhanced oocyte tPA activity as seen by casein zymography. tPA activity in the conditioned medium of a rat insulinoma cell line was also not stimulated with polylysine prior to fractionation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These data suggest that extravascular cells which elaborate tPA may produce stimulatory factor(s) which allow for full tPA activity at physiological concentrations of plasminogen in the absence of fibrin.     

Tissue‐type plasminogen activator induces plasmin‐dependent proteolysis of intracellular neuronal nitric oxide synthase

Background information. Despite its pro‐fibrinolytic activity, tPA (tissue plasminogen activator) is a serine protease known to influence a number of physiological and pathological functions in the central nervous system. Accordingly, tPA was reported to mediate some of its functions in the central nervous system through NMDA (N‐methyl‐d ‐aspartate) receptors, LRP (low‐density lipoprotein receptor‐related protein) or annexin II. Results. We provide here both in vitro and in vivo evidence that tPA could mediate proteolysis and subsequent delocalization of neuronal nitric oxide synthase, thereby reducing endogenous neuronal nitric oxide release. We also demonstrate that although this effect is independent of NMDA receptors, LRP signalling and calpain‐mediated proteolysis, it is dependent on the ability of tPA to promote the conversion of plasminogen into plasmin. Conclusion. Altogether, these results demonstrate a new function for tPA in the central nervous system, which most likely contributes to its pleiotropic functions.