trans-5-prostaglandin E2 stimulates plasminogen activation by tissue-type plasminogen activator.

The effect of trans-5-prostaglandin E2 (trans-PGE2) on fibrinolysis was examined in vitro using synthetic chromogenic substrate S-2251. trans-PGE2 was found to enhance plasminogen (PLG) activation mediated by tissue-type plasminogen activator (tPA). The enhancing effect was dependent on the concentration of trans-PGE2. cis-PGE2 and the other PGs (PGE1 and PGI2) did not show such an effect as trans-PGE2, despite to the fact that their structures are similar to that of trans-PGE2. trans-Configuration around the double bond at the 5-position seems to be important in the enhancement of the fibrinolytic activity.     

Cytokine activation of vascular endothelium. Effects on tissue-type plasminogen activator and type 1 plasminogen activator inhibitor

Regulation of the fibrinolytic system of cultured human umbilical vein endothelial cells (HUVECs) by recombinant interleukin 1 beta (rIL-1 beta) and tumor necrosis factor alpha (rTNF alpha) was investigated. Functional and immunologic assays indicated that both cytokines decreased HUVEC tissue-type plasminogen activator (tPA) and increased type 1 plasminogen activator inhibitor (PAI-1) in a dose- and time-dependent manner. Maximal effects (50% decrease in tPA antigen; 300-400% increase in PAI-1 activity) were achieved with 2.5 units/ml rIL-1 beta and 200 units/ml rTNF alpha. Combinations of rIL-1 beta and rTNF alpha were not additive at these maximal concentrations. After a 24-h pretreatment with rIL-1 beta, HUVECs secreted tPA at one-quarter of the rate of control cells and released PAI-1 at a rate that was 5-fold higher than controls. Neither the basal rate of PAI-1 release nor the increased rate of release of PAI-1 in response to rIL-1 beta was affected by subsequently treating the cells with secretagogues (e.g. phorbol myristate acetate) suggesting that PAI-1 is not contained within a rapidly releasable, intracellular storage pool. Northern blot analysis using a PAI-1 cDNA probe indicated that the cytokines increased the steady-state levels of the 3.2- and 2.3-kb PAI-1 mRNA species, but with a preferential increase in the larger mRNA form. The fact that both rIL-1 beta and rTNF alpha act in a similar manner strengthens the hypothesis that the local development of inflammatory/immune processes could reduce endothelial fibrinolytic activity.     

Interleukin 1 preferentially stimulates the production of tissue-type plasminogen activator by human articular chondrocytes

Interleukin 1, derived from human placenta, stimulates plasminogen activator activity in human articular chondrocytes. The stimulation of plasminogen activator activity can be abolished by preincubation of placental interleukin 1 with an antiserum to homogeneous 22K factor, a species of interleukin 1 beta, indicating that the stimulation of plasminogen activator activity is due to interleukin 1 and not contaminating factors. Chondrocytes produce three species of plasminogen activator, with apparent Mr approximately 50,000, 65,000 and 100,000 as determined after sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis with gels containing casein and plasminogen. Both placental interleukin 1 and 22K factor enhance the production of the species of Mr approximately 65,000 and 100,000. Comparison of the mobility of the plasminogen activator species on SDS-polyacrylamide gel electrophoresis with human urokinase (u-PA) and human melanoma tissue-type plasminogen activator (t-PA) and studies with antibodies to these enzymes indicate that the Mr approximately 50,000 species is a u-PA and the Mr approximately 65,000 a t-PA. The Mr approximately 100,000 species is possibly an enzyme-inhibitor complex. Interleukin 1 therefore appears to enhance the production of t-PA and a putative enzyme-inhibitor complex. Abolition of plasminogen activator activity in the fibrin plate assay with antibodies to t-PA and u-PA also confirms enhanced t-PA production on interleukin 1 stimulation, though there is also evidence for increased cell-associated production of u-PA.     

Kinetics of the activation of plasminogen by natural and recombinant tissue-type plasminogen activator

The kinetics of the activation of plasminogen by tissue-type plasminogen activator were studied in the presence and the absence of CNBr-digested fibrinogen as a soluble cofactor. Michaelis-Menten kinetics applied and the kinetic parameters obtained were very similar to those previously reported for the activation in the presence of solid phase fibrin (Hoylaerts, M., Rijken, D. C., Lijnen, H. R., and Collen, D. (1982) J. Biol. Chem. 257, 2912-2919). The affinity of the enzyme for plasminogen dramatically increases in the presence of the soluble cofactor while the catalytic rate constant does not change significantly (KM drops from 83 to 0.18 microM and kcat increases from 0.07 to 0.28 s-1 for tissue-type plasminogen activator of melanoma origin). Fragments containing the lysine-binding sites of plasminogen compete with plasminogen for interaction with CNBr-digested fibrinogen. The dissociation constant of this interaction was found to be 4.5 microM for the high affinity lysine-binding site. No difference was found in the kinetic parameters for the activation of plasminogen by either tissue-type plasminogen activator of melanoma origin or by glycosylated forms of tissue-type plasminogen activator obtained by recombinant DNA technology. The present findings obtained in a homogenous liquid milieu support the previously proposed mechanism of the activation of plasminogen by tissue-type plasminogen activator in the presence of fibrin. This mechanism involves binding of both tissue-type plasminogen activator and plasminogen to fibrin.     

Vitronectin governs the interaction between plasminogen activator inhibitor 1 and tissue-type plasminogen activator.

The "serpin" plasminogen activator inhibitor 1 (PAI-1) is the fast acting inhibitor of plasminogen activators (tissue-type (t-PA) and urokinase type-PA) and is an essential regulatory protein of the fibrinolytic system. Its P1-P1' reactive center (R346 M347) acts as a "bait" for tight binding to t-PA/urokinase-type PA. In vivo, PAI-1 is encountered in complex with vitronectin, an interaction known to stabilize its activity but not to affect the second-order association rate constant (k1) between PAI-1 and t-PA. Nevertheless, by using PAI-1 reactive site variants (R346M, M347S, and R346M M347S), we show that the binding of vitronectin to the PAI-1 mutant proteins improves plasminogen activator inhibition. In the absence of vitronectin the PAI-1 R346M mutants are virtually inactive toward t-PA (k1 less than 1 x 10(3) M-1 s-1). In contrast, in the presence of vitronectin the rate of association increases about 1,000-fold (k1 of 6-8 x 10(5) M-1 s-1). This inhibition coincides with the formation of serpin-typical, sodium dodecyl sulfide-stable t-PA.PAI-1 R346M (R346M M347S) complexes. As evidenced by amino acid sequence analysis, the newly created M346-M/S347 peptide bond is susceptible to attack by t-PA, similar to the wild-type R346-M347 peptide bond, indicating that in the presence of vitronectin M346 functions as an efficient P1 residue. In addition, we show that the inhibition of t-PA and urokinase-type PA by PAI-1 mutant proteins is accelerated by the presence of the nonprotease A chains of the plasminogen activators.     

Kinetic analysis of the interaction between plasminogen activator inhibitor-1 and tissue-type plasminogen activator.

The kinetics of inhibition of tissue-type plasminogen activator (t-PA) by the fast-acting plasminogen activator inhibitor-1 (PAI-1) was investigated in homogeneous (plasma) and heterogeneous (solid-phase fibrin) systems by using radioisotopic and spectrophotometric analysis. It is demonstrated that fibrin-bound t-PA is protected from inhibition by PAI-1, whereas t-PA in soluble phase is rapidly inhibited (K1 = 10(7) M-1.s-1) even in the presence of 2 microM-plasminogen. The inhibitor interferes with the binding of t-PA to fibrin in a competitive manner. As a consequence the Kd of t-PA for fibrin (1.2 +/- 0.4 nM) increases and the maximal velocity of plasminogen activation by fibrin-bound t-PA is not modified. From the plot of the apparent Kd versus the concentration of PAI-1 a Ki value of 1.3 +/- 0.3 nM was calculated. The quasi-similar values for the dissociation constants between fibrin and t-PA (Kd) and between PAI-1 and t-PA (Ki), as well as the competitive type of inhibition observed, indicate that the fibrinolytic activity of human plasma may be the result of an equilibrium distribution of t-PA between both the amount of fibrin generated and the concentration of circulating inhibitor.     

On the reversible interaction of plasminogen activator inhibitor-1 with tissue-type plasminogen activator and with urokinase-type plasminogen activator

The reaction between plasminogen activators and plasminogen activator inhibitor-1 is characterized by an initial rapid formation of an inactive reversible complex. The second-order association rate constant (k1) of complex formation of recombinant two-chain tissue-type plasminogen activator (rt-PA) or recombinant two-chain urokinase-type plasminogen activator (rtcu-PA) by recombinant plasminogen activator inhibitor-1 (rPAI-1) is 2.9 +/- 0.4 x 10(7) M-1 s-1 (mean +/- S.D., n = 30) and 2.0 +/- 0.6 x 10(7) M-1 s-1 (n = 12), respectively. Different molecular forms of tissue- or urokinase-type plasminogen activator which do not form covalent complexes with rPAI-1, including rt-PA-Ala478 (rt-PA with the active-site Ser478 mutagenized to Ala) and anhydro-urokinase (rtcu-PA with the active-site Ser356 converted to dehydroalanine) reduced k1 in a concentration-dependent manner, compatible with 1:1 stoichiometric complex formation between rPAI-1 and these ligands. The apparent dissociation constant (KD) of the complex between rPAI-1 and rt-PA-Ala478, determined as the concentration of rt-PA-Ala478 which reduced k1 to 50% of its control value, was 3-5 nM. Corresponding concentrations of active-site-blocked two-chain rt-PA were 150-250-fold higher. The concentration of anhydro-urokinase which reduced k1 to 50% was 4-6 nM, whereas that of active-site-blocked rtcu-PA was 100-250-fold higher. Recombinant single-chain urokinase-type plasminogen activator had an apparent KD of about 2 microM. These results suggest that inhibition of rt-PA or rtcu-PA by rPAI-1 proceeds via a reversible high affinity interaction which does not require a functional active site but which is markedly reduced following inactivation of the enzymes with active-site titrants.     

Structural basis of the cofactor function of denatured albumin in plasminogen activation by tissue-type plasminogen activator

Certain denatured proteins function as cofactors in the activation of plasminogen by tissue-type plasminogen activator. The present study approached the structural requirements for the cofactor activity of a model protein (human serum albumin). Heat denaturation of 100-230 microM albumin (80 degrees C and 60-90 min) reproducibly yielded aggregates with radius in the range of 10-150 nm. The major determinant of the cofactor potency was the size of the aggregates. The increase of particle size correlated with the cofactor activity, and there was a minimal requirement for the size of the cofactor (about 10 nm radius). Similar to other proteins, the molecular aggregates with cofactor function contained a significant amount of antiparallel intermolecular beta-sheets. Plasmin pre-digestion increased the cofactor efficiency (related to C-terminal lysine exposure) and did not affect profoundly the structure of the aggregates, suggesting a long-lasting and even a self-augmenting cofactor function of the denatured protein.     

Structure and function of human tissue-type plasminogen activator (t-PA)

Full-length tissue-type plasminogen activator (t-PA) cDNA served to construct deletion mutants within the N-terminal "heavy" (H)-chain of the t-PA molecule. The H-chain cDNA consists of an array of structural domains homologous to domains present on other plasma proteins ("finger," "epidermal growth factor," "kringles"). These structural domains have been located on an exon or a set of exons. The endpoints of the deletions nearly coincide with exon-intron junctions of the chromosomal t-PA gene. Recombinant t-PA deletion mutant proteins were obtained after transient expression in mouse Ltk- cells, transfected with SV40-pBR322-derived t-PA cDNA plasmids. It is demonstrated that the serine protease moiety of t-PA and its substrate specificity for plasminogen is entirely contained within the C-terminal "light" (L)-chain of the protein. The presence of cDNA, encoding the t-PA signal peptide preceding the remaining portion of t-PA, suffices to achieve secretion of (mutant) t-PA into the medium. The stimulatory effect of fibrin on the plasminogen activator activity of t-PA was shown to be mediated by the kringle K2 domain and, to a lesser extent, by the finger domain. The other domains on the H-chain, kringle K1, and the epidermal growth-factor-like domain, do not contribute to this property of t-PA. These findings correlate well with the fibrin-binding properties of the rt-PA deletion-mutant proteins, indicating that stimulation of the activity is based on aligning of the substrate plasminogen and its enzyme t-PA on the fibrin matrix. The primary target for endothelial plasminogen activator inhibitor (PAI) is located within the L-chain of t-PA. Deleting specific segments of t-PA H-chain cDNA and subsequent transient expression in mouse Ltk- cells of t-PA deletion-mutant proteins did not affect the formation of a stable complex between mutant t-PA and PAI.     

Identification of a site in fibrin(ogen) which is involved in the acceleration of plasminogen activation by tissue-type plasminogen activator

The rate of activation of plasminogen by tissue-type plasminogen activator is greatly increased by fibrin, but not by fibrinogen. A possible explanation for this phenomenon could be that conformational changes take place during the transformation of fibrinogen to fibrin which lead to exposure of sites involved in the accelerated plasmin formation. This is also supported by our recent observation that some enzymatically prepared fragments of fibrinogen and fibrin (D EGTA, D-dimer, Y) and also CNBr fragment 2 from fibrinogen have this property. CNBr fragment 2 consists of amino acid residues A alpha (148-207), B beta (191-224) + (225-242) + (243-305) and gamma 95-265, kept together by disulphide bonds. In order to study the localization of a stimulating site within this structure we purified the chain remnants of CNBr fragment 2 after reduction and carboxymethylation, and found that only A alpha 148-207 was stimulating. This was further confirmed by digesting pure A alpha-chains with CNBr and purifying the resulting A alpha-chain fragments. CNBr digests of B beta- and gamma-chains were not stimulatory. The A alpha-chain remnant (residues 111-197) in D EGTA and D-dimer also comprise the major part (residues A alpha 148-197) of the CNBr A alpha-chain fragment. We conclude that a site capable of accelerating the plasminogen activation by tissue-type plasminogen activator preexists in fibrinogen, that this site becomes exposed upon fibrin formation or disruption of fibrinogen by plasmin or CNBr and that this site is within the stretch A alpha 148-197, which is retained in the A alpha-chain remnants of fibrinogen degradation products.     

Catabolism of tissue-type plasminogen activator by the human hepatoma cell line Hep G2. Modulation by plasminogen activator inhibitor type 1

Catalytic activity of tissue-type plasminogen activator (t-PA) in plasma is regulated in part by formation of complexes with specific inhibitors as well as by hepatic clearance. Potential interaction of these two regulatory mechanisms was examined in the human hepatoma cell line Hep G2. These cells secrete plasminogen activator inhibitor type-1 (PAI-1) and initiate catabolism of exogenous t-PA by receptor-mediated endocytosis. Specific binding of 125I-t-PA to cells at 4 degrees C results in dose-dependent formation of a 95-kDa species recognized by monospecific anti-PAI-1 and anti-t-PA antibodies and stable in the presence of low (0.2%) concentrations of sodium dodecyl sulfate (SDS). Specific binding of 125I-t-PA and formation of the 95-kDa SDS-stable species are inhibited in a concentration-dependent manner following preincubation of cells with anti-PAI-1 antibodies. High and low molecular weight forms of urokinase plasminogen activator (u-PA) capable of forming specific complexes with PAI-1 complete for 125I-t-PA binding sites. However, the proenzyme form of u-PA (scu-PA), incapable of forming complexes with PAI-1, does not compete for 125I-t-PA binding sites. The role of the serine protease active site of t-PA in mediating both interaction with PAI-1 and specific binding was examined using 125I-t-PA that had been functionally inactivated with D-phenylalanyl-L-propyl-L-arginyl-chloromethyl ketone (PPACK). 125I-t-PA-PPACK, despite a 6-fold lower affinity than active 125I-t-PA, exhibited specific binding to cells without detectable formation of SDS-stable complexes with PAI-1. Both surface-bound 125I-t-PA and 125I-t-PA-PPACK are internalized and degraded by cells at 37 degrees C. 125I-t-PA is internalized as a stable complex with PAI-1, whereas 125I-t-PA-PPACK is internalized with similar kinetics but without the presence of an SDS-stable complex. Thus, PAI-1 appears capable of modulating t-PA catabolism in the human hepatocyte.     

Oncostatin M induces tissue-type plasminogen activator and plasminogen activator inhibitor-1 in Calu-1 lung carcinoma cells

Oncostatin M (OSM) is a glycoprotein cytokine that is produced by activated T-lymphocytes, monocytes, and macrophages. In a DNA synthesis assay, OSM reduced tritiated thymidine incorporation by 53% in Calu-1 lung carcinoma cells. Radiolabeled cDNAs from untreated Calu-1 cells and 30-h OSM-treated cells were used to probe duplicate nylon membrane cDNA expression arrays. This study revealed OSM-mediated expression of mRNAs encoding tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1). Northern blot analysis showed that the steady-state level of tPA mRNA is nearly undetectable in Calu-1 cells. Exposure of these cells to OSM for 30 h increased tPA mRNA expression by 20-fold and PAI-1 mRNA expression by 5-fold. Exposure of these cells to other gp130 receptor family cytokines, including leukemia inhibitory factor (LIF), interleukin-6 (IL-6), and IL-11, do not significantly affect DNA synthesis or induction of tPA/PAI-1. Western blot studies demonstrated that OSM mediates a marked increase in secretion of the tPA protein. Secreted tPA was present in the conditioned medium almost exclusively as tPA/PAI-1 complexes. Inhibitor studies demonstrated that OSM-mediated induction of tPA and PAI-1 mRNAs is largely dependent upon activation of the MEK1/2 pathway. The JAK3/STAT3 pathway potentially serves a secondary role in these regulatory events.     

Heterogeneity of human tissue-type plasminogen activator

Tissue-type plasminogen activator (t-PA) from human melanoma cells (Bowes) was purified by immunosorbent chromatography on affinospecific polyclonal antibodies and gel filtration in the presence of KSCN. The immunosorbent eluate contained three major components of greater than 200, 85 and 65 kDa, respectively. The 65 kDa t-PA component could be separated by gel filtration on Ultrogel AcA44 in the presence of KSCN to a pure preparation yielding a unique N-terminal amino acid sequence. Immunoblot analysis, using affinospecific antibodies against t-PA, was a specific and sensitive method to identify different types of t-PA (I-IV), as well as t-PA-inhibitor complexes and degradation products in unstimulated melanoma cell culture fluids. Furthermore, the t-PA preparations, produced by phorbol ester-treated melanoma cells, were free of type IV and thus differed physiochemically from the constitutively produced t-PA preparations. The composition of t-PA from mammalian cell cultures is thus more complex than hitherto described.     

组织型纤溶酶原激活剂的纯化制备

简述了用于大规模生产组织型纤溶酶原激活剂(tPA)的重组动物细胞及其培养工艺。从重组tPA的大规模、快速纯化的角度考虑,对tPA的纯化制备方法进行了简要评述。     

Proteolytic activation of tissue-type plasminogen activator by the culture media of mouse cancer cells

Human single-chain tissue-type plasminogen activator (tPA) was activated by the culture media of mouse B16 melanoma and Lewis lung carcinoma cells. This activation was due to the proteolytic conversion of single-chain tPA to two-chain tPA. Typical serine proteinase inhibitors, such as diisopropylfluorophosphate and aprotinin, strongly inhibited the proteolytic activation, suggesting that the enyzme responsible for this activation is a serine proteinase. Through a process of gel filtration, the molecular weight of the putative tPA-activating enzyme was estimated to be approximately 35 kDa. Expression of the tPA mRNA was demonstrated by Northern blot analysis both for the melanoma and carcinoma cells. Zymographic experiments showed that the culture medium of the melanoma cells contained active two-chain tPA. These results indicate that a common serine proteinase may be involved in the proteolytic activation of single-chain tPA in these cancer cells.     

Interactions between the finger and kringle-2 domains of tissue-type plasminogen activator and plasminogen activator inhibitor-1.

We have shown that plasminogen activator inhibitor-1 (PAI-1) inhibits the fibrin binding of both the single chain and two chain forms of tissue-type plasminogen activator (tPA) through two different mechanisms. PAI-1 inhibits the finger domain-dependent fibrin binding of diisopropylfluorophosphate-inactivated single chain tPA and the kringle-2 domain-dependent fibrin binding of diisopropylfluorophosphate-inactivated two chain tPA. In accordance with the data, preformed complexes of single chain tPA/PAI-1 and of two chain tPA/PAI-1 lost the fibrin binding abilities mediated by the finger and kringle-2 domains, respectively. These effects of PAI-1 appear to be mediated by steric hindrance of the fibrin binding sites after PAI-1 binding to adjacent regions in the functional domains of tPA. We thus propose a model in which a PAI-1 binding site resides in the finger domain of a single chain, and plays a role in the reversible association of single chain tPA and PAI-1. Conformational changes may take place during the conversion of single chain tPA to two chain tPA, resulting in burying of the original PAI-1 binding site and exposure of an alternate PAI-1 binding site on the surface of the kringle-2 domain.     

The plasminogen activator inhibitor-1 binding site in the kringle-2 domain of tissue-type plasminogen activator

We have shown that synthetic peptides containing the amino acid sequence Asn-Arg-Arg-Leu, derived from the amino acid sequence of the inner loop of the kringle-2 domain of tissue-type plasminogen activator (tPA), inhibited complex formation between two chain tPA and plasminogen activator inhibitor-1 (PAI-1) by binding to PAI-1. This binding was reversible and was inhibited by not only tPA but also by enzymatically inactive tPA. Quantitative analyses of the interaction of PAI-1 with the peptide containing the Asn-Arg-Arg-Leu sequence indicated that the PAI-1 binding site residues in the inner loop of the kringle-2 domain and is preferentially expressed in two chain tPA.     

Binding of tissue-type plasminogen activator by the mannose receptor

Previous studies have shown that tissue-type plasminogen activator (t-PA) in blood is cleared by the liver partially through a mannose-specific uptake system. The present study was undertaken to investigate, in a purified system, whether t-PA is recognized by the mannose receptor which is expressed on macrophages and liver sinusoidal cells. The mannose receptor was isolated and purified from bovine alveolar macrophages and migrated as a single protein band at Mr 175,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Ligand blotting revealed that this protein specifically bound t-PA. The t-PA-receptor interaction was further characterized in a binding assay, which showed saturable binding with an apparent dissociation constant of 1 nM. t-PA binding required calcium ions and was negligible in the presence of EDTA or at acid pH. Mannose-albumin was an effective inhibitor, whereas galactose-albumin did not have a significant effect. From a series of monosaccharides tested, D-mannose and L-fucose were the most potent inhibitors, N-acetyl-D-glucosamine was a moderate inhibitor, whereas D-galactose and N-acetyl-D-galactosamine were ineffective. t-PA, deglycosylated by endoglycosidase H, did not interact with the receptor. It is concluded that the mannose receptor specifically binds t-PA, probably through its high mannose-type oligosaccharide.     

An active-site titrant for human tissue-type plasminogen activator.

The reaction of recombinant tissue-type plasminogen activator with the inverse substrate 4-amidino-2-nitrophenyl 4'-anisate results in the rapid release of the chromogen 4-amidino-2-nitrophenol and the accumulation of the relatively stable 4-anisoyl-enzyme. Spectrophotometric monitoring of the reaction enables the operational molarity of the enzyme to be determined.