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。     

肝素对组织型纤溶酶原激活剂的作用

重组组织型纤溶酶原激活剂 ( rt PA)经肝素处理后与未经处理的 rt PA比较 ,结果显示 ,rt PA在体外的溶纤活性提高 5 0 %～ 90 % ,在兔体内的半衰期延长 1 min,同时也提高了 rt PA对热的稳定性     

Isolation and functional characterization of the heavy and light chains of human tissue-type plasminogen activator

Two-chain tissue-type plasminogen activator (t-PA), which consists of a heavy chain (Mr congruent to 38,000) and a light chain (Mr congruent to 31,000) connected by a disulfide bridge, was reduced with 2-mercaptoethanol and then air-reoxidized at a low protein concentration and carboxamidomethylated. The two chains were separated by means of zinc chelate-agarose, which was found to bind the light chain selectively. The light chain was fully active on the tripeptide substrate H-D-isoleucyl-L-prolyl-L-arginine p-nitroanilide (S-2288) and partially active on plasminogen. The plasminogen activator activity of the light chain was, in contrast to that of two-chain t-PA, not stimulated by fibrin or fibrinogen fragments. Fibrin-agarose chromatography of radiolabeled chains showed that only the heavy chain bound to fibrin. These results indicate that the active site-containing light chain in t-PA needs the heavy chain for fibrin stimulation of its plasminogen activator activity.     

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.     

Isolation and characterization of three different carbohydrate chains from melanoma tissue plasminogen activator

Electrophoretic analysis of endoglycosidase-treated tissue plasminogen activator obtained from human melanoma cells showed that the heterogeneity observed for the protein in these preparations is caused by an N-glycosidically linked N-acetyllactosamine type of carbohydrate chain which is present in about 50% of the molecules. An oligomannose type and an N-acetyllactosamine type of glycan is present in all molecules. Three glycopeptides were isolated and characterized by 1H-NMR, sugar determination, methylation analysis and amino acid determination. The exact attachment site for each of the three glycans could be deduced from the amino acid compositions of the glycopeptides. Asn-117 carries the oligomannose type of glycan, the structure of which was completely determined. Asn-184 is the site where the presence or absence of a biantennary N-acetyllactosamine type of glycan causes the size heterogeneity. The third N-glycosylation site, Asn-448, was found to carry a triantennary or tetraantennary N-acetyllactosamine type of carbohydrate chain.     

The role of carbohydrate side chains of plasminogen in its activation by staphylokinase

Kinetic parameters (k(Pg) and K(Pg)) were determined for activation of Glu-plasminogen (Glu-Pg) and Lys-plasminogen (Lys-Pg) type I (with N-linked carbohydrate chain at Asn-289) and type II (with unsubstituted Asn-289) by plasmin-staphylokinase (Pm-STA) complex. The K(Pg) values for Glu-Pg I and Lys-Pg I (17.1 and 11.2 microM, respectively) were higher than those for Glu-Pg II and Lys-Pg II (14.9 and 5.4 microM, respectively), while only minor differences in the k(Pg) values were observed between plasminogens type I and type II. Soluble fibrin significantly increased the k(Pg)/K(Pg) values for activation of all four plasminogens due to a decrease in the K(Pg) values but did not alter the k(Pg) values. However, the activation of plasminogens type I was stimulated by fibrin lesser degree than that of plasminogens type II. These findings indicate that N-glycosylation of kringle 3 of plasminogen decreases the stability of Pm-STA-Pg ternary enzyme-substrate complex in solution as well as interferes with its formation and rearrangement on the fibrin surface.     

Interaction of tissue-type plasminogen activator and plasminogen activator inhibitor 1 on the surface of endothelial cells

The site of the reaction between plasminogen activators and plasminogen activator inhibitor 1 (PAI-1) was investigated in cultures of human umbilical vein endothelial cells. In conditioned medium from endothelial cells, two forms of a plasminogen activator-specific inhibitor can be demonstrated: an active form that readily binds to and inhibits plasminogen activators and an immunologically related quiescent form which has no anti-activator activity but which can be activated by denaturation. In conditioned medium, only a few percent of PAI-1 is the active form. However, the addition of increasing concentrations of tissue-type plasminogen activator (t-PA) or urokinase to confluent endothelial cells produced a saturable (3.0 pmol/5 x 10(5) cells), dose-dependent increase of the activator-PAI-1 complex in the conditioned medium even in the presence of actinomycin D or cycloheximide. This resulted also in a dose-dependent decrease of the residual PAI activity measured by reverse fibrin autography both in the conditioned medium and cell extracts. Short-time exposure of endothelial cells to a large amount of t-PA caused almost complete depletion of all cell-associated PAI activity. Although there was no detectable PAI activity even after activation of PAI by denaturants or antigen in the culture medium at 4 degrees C without the addition of t-PA, the addition of t-PA at 4 degrees C not only resulted in the formation of 70% of the amount of the t-PA.PAI complex in conditioned medium at 37 degrees C, but also induced PAI-1 antigen in a time and dose-dependent manner in the conditioned medium. Moreover, 125I-labeled t-PA immobilized on Sepharose added directly to endothelial cells formed a complex with PAI-1 in a dose-dependent manner. On the other hand, no detectable complex was formed with PAI-1 when Sepharose-immobilized 125I-labeled t-PA was added to endothelial cells under conditions in which the added t-PA could not contact the cells directly but other proteins could pass freely by the use of a Transwell. All these results suggest that a "storage pool" on the surface of endothelial cells or the extracellular matrix produced by endothelial cells contains almost all the active PAI-1, and reaction between PA and PAI-1 mainly occurs on the endothelial cell membranes, resulting in a decrease of the conversion of active PAI-1 to the quiescent form.     

Clearance of the heavy and light polypeptide chains of human tissue-type plasminogen activator in rats.

In order to assess which part of the tissue-type plasminogen activator (t-PA) molecule should be (genetically) modified to obtain more-slowly-clearing mutants, two-chain t-PA and its isolated heavy and light chains were radiolabelled and injected into rats. The vast majority of t-PA and the heavy chain disappeared from the blood circulation with half-lives of 2.3 and 1.0 min respectively. The clearance of the light chain was biphasic, owing to complex-formation with plasma proteinase inhibitors. The disappearance of di-isopropylphospho-light chain, which has a blocked active site, was nearly monophasic, with a half-life of 5.7 min. Organ distribution studies showed that hepatic clearance constituted the major pathway in all cases. These results strongly suggest that t-PA is recognized by the liver primarily through the heavy chain.     

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.     

Construction and expression of hybrid plasminogen activators prepared from tissue-type plasminogen activator and urokinase-type plasminogen activator genes

Recent data from several studies have suggested that the non-protease domains in tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) determine their biological specificities, including binding to fibrin clots and survival in the circulatory system (Van Zonneveld, A.-J., Veerman, H., and Pannekoek, H. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 4670-4674; Rijken, D. C., and Emeis, J. J. (1986) Biochem. J. 238, 643-646). Structural manipulations (e.g. deletions, additions, or substitutions) in these domains can thus be utilized to maximize the desired biological effects. Using recombinant DNA technology, we constructed a number of hybrid molecules from the t-PA and u-PA genes. In hybrid A, the epidermal growth factor and finger domains of t-PA (residues 1-91) were replaced by the epidermal growth factor and kringle of u-PA (residues 1-131). In hybrids B and C, the u-PA kringle (residues 50-131) was inserted either before (residue 92) or after (residue 261) the double-kringle region of t-PA. All these hybrid PAs containing three kringles were expressed in mouse fibroblast cells (C-127). The hybrid proteins were synthesized in predominantly a single-chain form with molecular weights of 70,000-80,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were enzymatically active as assayed by the fibrin-agar plate method. In vitro studies on the binding of hybrid PAs to fibrin showed that hybrid B, like t-PA, possesses affinity toward fibrin, while hybrid A shows lower binding. This suggests that the finger domain, which is not present in hybrid A, plays a role in conferring fibrin affinity to the hybrid PAs. The enzymatic activities of the hybrids were compared with that of recombinant t-PA (rt-PA) expressed in the same vector/host system and found to be similar in activity toward a chromogenic peptide substrate. In addition, plasminogen activation with all the hybrid-PAs, as with rt-PA, was stimulated by fibrin, with the order of activity being rt-PA greater than or equal to hybrid B greater than hybrid C greater than hybrid A. This study shows the feasibility of shuffling functional domain(s) of known specificity in plasminogen activators which may lead to the design of a superior thrombolytic agent.     

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.     

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.     

Dexamethasone inhibition of tissue-type plasminogen activator (tPA) activity: paradoxical induction of both tPA antigen and plasminogen activator inhibitor

Incubation of HTC rat hepatoma cells with the synthetic glucocorticoid dexamethasone rapidly inhibits plasminogen activator (PA) activity and reveals the presence of a specific PA inhibitor (PAI-1). To determine whether the hormonal inhibition of PA activity reflects a decrease in the amount of PA or an increased amount of the inhibitor, or both, we have assayed PA and PAI-1 immunologically. HTC PA was determined to be entirely of the tissue type (tPA), and both free and complexed antigen was quantified by a RIA using rabbit antirat tPA, with rat insulinoma tPA as tracer and standard. PAI-1 was quantified by a Western blot assay using rabbit anti-HTC PAI-1 antibody and purified HTC PAI-1 as standard. Under conditions in which dexamethasone inhibited PA activity by 90%, there was no decrease in the cellular content of tPA antigen. Paradoxically, dexamethasone increased tPA antigen approximately 1.5-fold. Under these same conditions, dexamethasone increased PAI-1 antigen 4- to 5-fold. We conclude that the glucocorticoid inhibition of tPA activity in HTC cells is not secondary to a decrease in the amount of tPA but is secondary to the induction of a specific PA inhibitor.     

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.     

Characterization of interaction of active-site serine mutants of tissue-type plasminogen activator with plasminogen activator inhibitor-1

To define determinants of interactions of tissue-type plasminogen activator (t-PA) with plasminogen activator inhibitor type-1 (PAI-1), we utilized site-directed mutagenesis to substitute either threonine or glycine for the active-site serine of tissue-type plasminogen activator. Assays of conditioned media of transfected cells demonstrated that the threonine substitution markedly decreased but did not entirely abolish plasminogen activating activity. In contrast, the glycine substitution yielded a mutant with absolutely no detectable plasminogen activating activity. Wild-type t-PA formed stable complexes with PAI-1. However, even when exogenous inhibitor was present in the medium or purified mutant was added to plasma that had been rendered PAI-1-rich in vivo, the mutants were present in the free form exclusively judging from results of fibrin autography and Western blot analysis. Thus, despite maintenance of some residual plasminogen-activating activity associated with preservation of the hydroxyl group at the active site, the threonine mutant did not form stable complexes with inhibitor. The glycine mutant, developed so that steric hindrance or other unfavorable interactions at the modified active site would be minimal, was similarly incapable of forming complexes with PAI-1. These results show that the presence of an active site serine residue is necessary for formation of stable complexes between t-PA and PAI-1.     

A protease-hypersensitive deletion derivative of human tissue-type plasminogen activator

We have constructed amplified Chinese hamster ovary cell lines constitutively synthesizing human tissue-type plasminogen activator (t-PA) or a derivative in which the domains homologous to epidermal growth factor and kringle 1 have been removed [delta(G + K1)]. The properties of the secreted proteins were investigated when synthesized in the presence or absence of the serine protease inhibitor aprotinin in the medium. t-PA in the culture supernatants was either single-chain or two-chain protein. The protease activity of both forms was stimulated by fibrin. The biochemical properties of delta(G + K1) were significantly different when harvested from cells grown under different culturing conditions. Protease activity of delta(G + K1) was stimulated ten- to 20-fold by fibrin when harvested from medium with aprotinin, but was stimulated only two- to three-fold when aprotinin was absent from the serum. Characterization of the secreted proteins revealed that the heavy-chain equivalent of delta(G + K1) is degraded when serine protease inhibitor is absent in the culture medium. These results indicate that the functional and biochemical properties of restructured versions of t-PA may depend on the presence of protease(s) in the culture supernatants.     

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.     

Reaction of tissue-type plasminogen activator with 4-methylumbelliferyl-p-guanidinobenzoate hydrochloride

It has recently been reported that the fluorogenic serine proteolytic active site titrant, 4-methyl-umbelliferyl-p-guanidinobenzoate (MUGB), cannot be employed in this capacity for tissue-type plasminogen activator (TPA) [Geiger, M., and Binder, B.R. (1987) Biochim. Biophys. Acta 912, 34-40]. Since this observation has such important ramifications in this area of research, we have studied the reaction of MUGB with recombinant (rec)TPA under a variety of experimental conditions and find that MUGB is indeed an effective titrant of rec-two chain TPA (recTCTPA) at 4 degrees, a condition under which the deacylation rate constant is diminished to the point that acylation can be readily observed. The KS for the interaction of MUGB with recTCTPA is 43 microM-46 microM, the acylation rate constant, k2, is approximately 3.6 min-1-4.2 min-1, and the rate constant for deacylation of p-guanidinobenzoyl-recTCTPA is 0.084 min-1-0.110 min-1. This same recTCTPA, after treatment with diisopropylfluorophosphate, does not react with MUGB. Single-chain TPA (recSCTPA) has been found to acylate more slowly than its two-chain counterpart and to exhibit a higher degree of turnover of the acyl-enzyme with this reagent. These results demonstrate that the active site concentration of TCTPA can be accurately determined by titration with MUGB, a consideration which is essential to the proper kinetic evaluation of this agent and its genetic variants. On the other hand, the presteady state kinetic characteristics for MUGB toward SCTPA are not favorable for its use as a titrant with this form of the enzyme.